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. 2025 Jun 13;13:RP100497. doi: 10.7554/eLife.100497

BCAS2 promotes primitive hematopoiesis by sequestering β-catenin within the nucleus

Guozhu Ning 1,2,†, Yu Lin 3,†, Haixia Ma 4,5, Jiaqi Zhang 4,5, Liping Yang 1, Zhengyu Liu 1, Lei Li 4,5,✉, Xinyu He 1,✉, Qiang Wang 1,✉
Editors: Eirini Trompouki6, Didier YR Stainier7
PMCID: PMC12165693  PMID: 40511787

Abstract

Breast carcinoma amplified sequence 2 (BCAS2), a core component of the hPrP19 complex, plays crucial roles in various physiological and pathological processes. However, whether BCAS2 has functions other than being a key RNA-splicing regulator within the nucleus remains unknown. Here, we show that BCAS2 is essential for primitive hematopoiesis in zebrafish and mouse embryos. The activation of Wnt/β-catenin signaling, which is required for hematopoietic progenitor differentiation, is significantly decreased upon depletion of bcas2 in zebrafish embryos and mouse embryonic fibroblasts. Interestingly, BCAS2 deficiency has no obvious impact on the splicing efficiency of β-catenin pre-mRNA, while significantly attenuating β-catenin nuclear accumulation. Moreover, we find that BCAS2 directly binds to β-catenin via its coiled-coil domains, thereby sequestering β-catenin within the nucleus. Thus, our results uncover a previously unknown function of BCAS2 in promoting Wnt signaling by enhancing β-catenin nuclear retention during primitive hematopoiesis.

Research organism: Mouse, Zebrafish

Introduction

Hematopoiesis refers to the lifelong process by which all blood cell lineages are generated. It begins at the early stage of embryonic development, providing the growing embryo with sufficient oxygen and nutrients (Galloway and Zon, 2003). Evolutionarily conserved across vertebrate species, hematopoiesis consists of two successive and partially overlapping waves: primitive and definitive. In mammals, the first wave of hematopoiesis occurs in the yolk-sac blood islands, producing primitive erythroid, megakaryocyte, and macrophage progenitors, which can be observed in mouse embryos as early as embryonic day 7.25 (E7.25) (Murry and Keller, 2008; Ferkowicz and Yoder, 2005; Palis, 2016). In zebrafish, primitive hematopoiesis initiates at around 11 hours post fertilization (hpf), when hemangioblasts emerge from the anterior lateral mesoderm (ALM) and posterior lateral mesoderm (PLM) and later differentiate into both hematopoietic and endothelial cells (Paik and Zon, 2010; Detrich et al., 1995; Leung et al., 2005).

Breast cancer amplified sequence 2 (BCAS2), also known as pre-mRNA splicing factor SPF27, is a 26 kDa nuclear protein containing two coiled-coil (CC) domains (Kuo et al., 2009). It was initially found to be overexpressed and amplified in human breast cancer cell lines (Neubauer et al., 1998; Nagasaki et al., 1999; Qi et al., 2005). Further studies have identified BCAS2 as a vital component of the human Prp19/CDC5L complex, which forms the catalytic ribonucleoprotein (RNP) core of spliceosome and is required for the activation of pre-mRNA splicing (Neubauer et al., 1998; Ajuh et al., 2000; Grote et al., 2010). In Drosophila, the function of BCAS2 in RNA splicing is essential for cell viability (Chen et al., 2013). In mouse, disruption of Bcas2 in male germ cells impairs mRNA splicing and leads to a failure of spermatogenesis (Liu et al., 2017). Additionally, BCAS2 has been shown to be a negative regulator of p53 by directly interacting with p53 or modulating alternative splicing of Mdm4, a major p53 inhibitor (Kuo et al., 2009; Yu et al., 2019). Zebrafish bcas2 transcripts were enriched in the sites of both primitive and definitive hematopoiesis during embryonic development (Yu et al., 2019). However, a previous study showed that p53 overactivation induced by zebrafish bcas2 depletion did not affect primitive hematopoiesis, but impaired definitive hematopoiesis (Yu et al., 2019). In recent years, several studies have highlighted the importance of regulating the expression and activity of p53 in primitive erythroid cell differentiation in both mouse and zebrafish embryos (Bissinger et al., 2018; Yang et al., 2023; Stanic et al., 2019). Thus, it is necessary to reexamine the exact function of BCAS2 in primitive hematopoiesis.

Wnt signaling, usually categorized into canonical and non-canonical pathways, is involved in the process of hematopoiesis (Richter et al., 2017; Krimpenfort and Nethe, 2021; Kokolus and Nemeth, 2010). Notably, the canonical Wnt signaling pathway, which is dependent on the nuclear accumulation of β-catenin to regulate gene transcription, controls primitive hematopoietic progenitor formation and promotes definitive hematopoietic stem cell (HSC) specification (Tarafdar et al., 2013; Nostro et al., 2008; Sturgeon et al., 2014). For instance, it has been demonstrated in Xenopus that Wnt4-mediated activation of Wnt/β-catenin signaling plays a critical role in the induction and maintenance of primitive hematopoiesis (Tran et al., 2010). Moreover, transient inhibition of canonical Wnt signaling in zebrafish embryos impairs embryonic blood formation (Lengerke et al., 2008). However, previous studies utilizing human pluripotent stem cells revealed an opposite role of Wnt/β-catenin pathway in primitive progenitor generation (Sturgeon et al., 2014; Paluru et al., 2014). Therefore, the impact of Wnt/β-catenin signaling on primitive hematopoiesis remains elusive and even controversial. Moreover, it has been suggested that BCAS2 is important for neural stem cell proliferation and dendrite growth in mice by regulating β-catenin pre-mRNA splicing (Chen et al., 2022; Huang et al., 2016). As a nuclear protein, it is unclear whether BCAS2 can modulate Wnt/β-catenin signaling in a splicing-independent manner.

In this study, we generated two zebrafish bcas2 mutant lines, both of which exhibited defects in male fertility and embryonic HSC formation, similar to what was previously reported in mice and zebrafish (Liu et al., 2017; Yu et al., 2019). More importantly, loss-of-function experiments suggest that BCAS2 is necessary for primitive hematopoiesis in both zebrafish and mouse embryos. We further find that bcas2 is dispensable for the survival and proliferation of hematopoietic cells, but plays a crucial role in the differentiation of the hematopoietic lineage from hemangioblasts. Using a comprehensive approach, we reveal that BCAS2 is a nuclear retention factor for β-catenin during primitive hematopoiesis. Subsequent biochemical and functional experiments demonstrate that BCAS2 directly binds to β-catenin and suppresses its nuclear export to promote Wnt signal activation and hematopoietic progenitor differentiation. Furthermore, the CC domains on BCAS2 and the Armadillo (ARM) repeats on β-catenin are responsible for their interaction. Collectively, we have uncovered a novel function of BCAS2 in regulating Wnt/β-catenin signaling by sequestering β-catenin within the nucleus during primitive hematopoiesis.

Results

BCAS2 is necessary for primitive hematopoiesis

To confirm that bcas2 is expressed in the posterior intermediate cell mass (ICM) where primitive hematopoiesis occurs in zebrafish, we first examined the spatiotemporal expression pattern of bcas2 during zebrafish embryo development by performing whole-mount in situ hybridization (WISH). The results showed that bcas2 was ubiquitously expressed from 1-cell stage to 10-somite stage (Figure 1—figure supplement 1). Its expression in the ICM became detectable at 18 hpf and was significantly elevated at 22 hpf (Figure 1A). We further observed that bcas2 was co-expressed with the primitive erythropoietic marker gata1 in the ICM at 22 hpf by fluorescence in situ hybridization (FISH) (Figure 1B). In contrast, bcas2 was hardly detectable in the ICM in cloche-/- mutants that lack both endothelial and hematopoietic cells (Figure 1C). These results demonstrate a dynamic expression of bcas2 in the ICM and imply a potential role of this gene in primitive hematopoiesis.

Figure 1. bcas2 is expressed in the intermediate cell mass (ICM) and required for primitive hematopoiesis.

(A) Whole-mount in situ hybridization (WISH) assay showing bcas2 expression in the ICM at the 18-somite stage and 22 hpf. The dotted lines represent the section position and the black arrowheads indicate the ICM region. n, notochord. (B) Double fluorescence in situ hybridization (FISH) assay showing the expression pattern of bcas2 and gata1 in the ICM at 22 hpf. (C, D) Comparison of bcas2 expression in cloche mutants (C) or bcas2 heterozygous mutants (D) along with their corresponding siblings. (E, F) Expression analysis of gata1 and hbbe3 in bcas2+/Δ7 and bcas2+/Δ14 embryos. (G) Hemoglobin detection using o-dianisidine staining in bcas2 homozygous mutant at 36 and 48 hpf. (H) Representative images of yolk sac from the hemangioblast-specific Bcas2 knockout mice and their siblings. Bcas2F/F females were crossed with Bcas2F/+;Kdr-Cre males to induce the deletion of Bcas2 in hemangioblasts. Scale bars, 100 μm (A, C–G), 50 μm (B), 1 mm (H).

Figure 1.

Figure 1—figure supplement 1. Expression patterns of bcas2 in wild-type embryos during development.

Figure 1—figure supplement 1.

Expression of bcas2 in wild-type embryos at the indicated developmental stages was analyzed using whole-mount in situ hybridization. Scale bars, 100 μm.
Figure 1—figure supplement 2. Zebrafish bcas2 mutants are generated by using CRISPR/Cas9 system.

Figure 1—figure supplement 2.

(A) Schematic showing generation of bcas2 mutants. Two mutant lines were obtained with mutations that resulted in premature translation termination, resulting in truncated Bcas2 proteins lacking the C-terminal CC domains. (B) Identification of bcas2 mutations using DNA sequencing. (C) The bcas2+/Δ7 and bcas2+/Δ14 mutants were identified via T7 endonuclease (the upper panel) or FspI restriction enzyme (the lower panel) digestions. (D) Bright-field images of embryos derived from crossing indicated females with heterozygous male mutants. Black arrows refer to the embryos that exhibited an abnormal cleavage. The ratio of viable embryos was indicated. Scale bars, 1 mm (D).
Figure 1—figure supplement 2—source data 1. PDF file containing original gel images for Figure 1—figure supplement 2C with the relevant bands and treatments indicated.
Figure 1—figure supplement 2—source data 2. Original gel images in Figure 1—figure supplement 2C.
Figure 1—figure supplement 3. bcas2 is essential for definitive hematopoiesis.

Figure 1—figure supplement 3.

(A, B) Expression changes of cymb and rag1 in bcas2+/Δ7 (A) and bcas2+/Δ14 (B) embryos compared with their siblings. Arrowheads on the left panels indicate the caudal hematopoietic tissue, and those on the right panels indicate the thymus. Scale bars, 200 μm (A, B).
Figure 1—figure supplement 4. Knockdown of bcas2 impairs primitive hematopoiesis.

Figure 1—figure supplement 4.

(A) Western blot analysis showing the expression changes of Bcas2 protein in wild-type embryos and embryos injected with 8 ng control MO (cMO) or bcas2 MO. (B, C) Expression of gata1 (B) and hbbe3 (C) in bcas2 morphants and control embryos. (D) Detection of hemoglobin levels by o-dianisidine staining in bcas2 morphants and control embryos. Scale bars, 100 μm (B–D).
Figure 1—figure supplement 4—source data 1. Original western blots for Figure 1—figure supplement 4A with the relevant bands and treatments indicated.
Figure 1—figure supplement 4—source data 2. Original western blot images in Figure 1—figure supplement 4A.
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To gain insight into the developmental function of bcas2, we employed CRISPR/Cas9 system to generate bcas2 mutants. Two mutant lines were obtained, designated bcas2Δ7 (with a 7-base deletion) and bcas2Δ14 (with a 14-base deletion). These mutations led to premature translation termination, which resulted in truncated Bcas2 proteins lacking the C-terminal CC domains (Figure 1—figure supplement 2A and B). bcas2+/Δ7 and bcas2+/Δ14 mutants were identified by T7 endonuclease I assay or restriction enzyme analysis (FspI) (Figure 1—figure supplement 2C). We found that nearly 85% of the embryos derived from crossing bcas2+/- males with bcas2+/- females did not develop to the cleavage stage (Figure 1—figure supplement 2D). Only 3% of the living embryos were homozygotes. In contrast, embryos obtained by crossing between wild-type males and bcas2+/- females were viable and showed normal morphology, with a heterozygosity rate consistent with Mendelian inheritance. This could be explained by male infertility as previously documented in Bcas2 knockout mice (Liu et al., 2017). Combining the above findings, we propose that Bcas2 may have an evolutionarily conserved role in spermatogenesis.

Given the difficulty of obtaining homozygous mutants, embryos lacking one copy of bcas2 gene were produced from crosses between heterozygous females and wild-type males. We observed a significant decrease of bcas2 expression in the ICM region in bcas2+/Δ7 or bcas2+/Δ14 mutants, likely resulting from nonsense-mediated RNA decay (Figure 1D). In line with a previous report (Yu et al., 2019), a marked reduction in the expression of the HSC marker cmyb and T-cell marker rag1 was found in bcas2+/Δ7 or bcas2+/Δ14 embryos at 5 dpf, indicating an essential role of bcas2 in definitive hematopoiesis (Figure 1—figure supplement 3A and B). These findings suggest that bcas2+/Δ7 and bcas2+/Δ14 mutants can be used to examine the involvement of bcas2 in primitive hematopoiesis.

To explore whether bcas2 is required for primitive hematopoiesis, we first examined the expression of primitive erythropoietic markers gata1 and hbbe3 in bcas2+/Δ7 and bcas2+/Δ14 embryos at 22 hpf, and observed a marked decrease in the expression of these genes in the mutants (Figure 1E and F). Surprisingly, o-dianisidine staining showed similar hemoglobin contents in the bcas2+/Δ7 and bcas2+/Δ14 embryos at 48 hpf compared with control embryos, suggesting that the defect in primitive hematopoiesis induced by haploinsufficiency of bcas2 was alleviated at later developmental stages. In order to further explore the role of BCAS2 in primitive hematopoiesis, we identified several bcas2Δ14/Δ14 mutants from about 100 embryos. These homozygous mutants display a severe decrease in hemoglobin (Figure 1G). Moreover, injection of a translation-blocking MO into wild-type embryos to downregulate bcas2 expression resulted in severe defects in erythropoiesis at 22 hpf and 48 hpf (Figure 1—figure supplement 4A–D). These results indicate that bcas2 is indispensable for primitive hematopoiesis in zebrafish. In addition, transgenic mice expressing Cre recombinase under the control of the Kdr promoter were crossed to Bcas2F/F animals to induce the deletion of Bcas2 in endothelial/hematopoietic cells. We found that red blood cells were eliminated in the yolk sac of Bcas2F/F;Kdr-Cre mice at E12.5 despite the presence of vessels (Figure 1H). Therefore, Bcas2 has a conserved role in vertebrates to regulate primitive hematopoiesis.

bcas2 deficiency impairs hematopoietic progenitor differentiation

The decrease in primitive hematopoietic cells in bcas2 deficient animals may be attributed to a number of possible causes: excessive apoptosis, hampered proliferation of hematopoietic cells, or impaired differentiation of hematopoietic progenitor cells. To shed light on this issue, we first performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay in Tg(gata1:GFP) embryos at the 10-somite stage to examine DNA fragmentations in apoptotic cells and found no obvious apoptotic signal in the gata1+ cells in either bcas2+/Δ14 embryos or their wild-type siblings (Figure 2—figure supplement 1A). Meanwhile, BrdU incorporation assay revealed no significant difference in hematopoietic cell proliferation between bcas2+/Δ14 mutants and their corresponding wild-types (Figure 2—figure supplement 1B and C). These data suggest that bcas2 is dispensable for the survival and proliferation of hematopoietic cells.

In the developing embryo, hemangioblasts are derived from the ventral mesoderm at early somite stage and then differentiate into both hematopoietic and endothelial lineages (Vogeli et al., 2006; Reischauer et al., 2016). To test whether bcas2 functions in cell fate decision during primitive hematopoietic cell development, the expression of hemangioblast markers npas4l, scl, and gata2 in bcas2+/Δ14 embryos was examined at the 1- to 2-somite stage. As shown in Figure 2A, haploinsufficiency of bcas2 did not affect the emergence of the hemangioblast population. Then we extended our analysis to include the markers of hematopoietic and endothelial progenitors. Consistent with the decrease in primitive hematopoietic cells in bcas2 deficient mutants, a marked reduced expression of erythrocyte progenitor markers gata1 and hbbe3 was observed in the PLM of bcas2+/Δ14 embryos at the 10-somite stage (Figure 2B). Interestingly, the expression of myeloid progenitor marker pu.1 was also dramatically decreased (Figure 2C). Moreover, overexpression of human BCAS2 enhanced the expression of gata1 in both wild-type and mutant embryos at the 10-somite stage (Figure 2D). In contrast, the endothelial progenitor marker fli1a was expressed at a similar level in bcas2+/Δ14 embryos as in wild-type animals (Figure 2E). Consistently, blood vessels appeared normal in bcas2+/Δ14 mutants with Tg(kdrl:GFP) background at 54 hpf (Figure 2F). These data provide convincing evidence that bcas2 is required for the differentiation of the hematopoietic lineage from hemangioblasts during primitive hematopoiesis.

Figure 2. bcas2 is required for hematopoietic progenitor differentiation.

(A–C) Expression analysis of hemangioblast markers npas4l, scl, gata2 (A), erythroid progenitor markers gata1, hbbe3 (B), and myeloid marker pu.1 (C) in bcas2+/Δ14 embryos and their wild-type siblings at indicated stages. (D) Expression changes of gata1 in bcas2+/Δ14 embryos overexpressing BCAS2 at the 10-somite stage. The indicated embryos were injected with or without 300 pg of human BCAS2 mRNA at the one-cell stage. (E) Expression of endothelial marker fli1a in bcas2+/Δ14 and sibling embryos at the 10-somite stage. (F) Confocal imaging of bcas2+/Δ14 and control sibling Tg(kdrl:GFP) embryos at 54 hpf. Scale bars, 100 μm (A–E), 500 μm (F).

Figure 2.

Figure 2—figure supplement 1. bcas2 is dispensable for the survival and proliferation of hematopoietic cells.

Figure 2—figure supplement 1.

(A) Cell apoptosis was detected by TUNEL assay in bcas2+/Δ14 embryos and their siblings with Tg(gata1:GFP) background. (B, C) Cell proliferation was detected by BrdU staining in bcas2+/Δ14 embryos and their siblings with Tg(gata1:GFP) background. The percentage of BrdU+/GFP+ cells (% over the total number of GFP+ cells) was quantified in (C) (n=6). ns, not significant (Student’s t-test). Scale bars, 50 μm (A), 20 μm (B).
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BCAS2 functions in primitive hematopoiesis by activating Wnt signaling

Previous studies have shown that Wnt/β-catenin plays a key role in primitive hematopoiesis (Tran et al., 2010; Lengerke et al., 2008; Sun et al., 2018). As both BCAS2 and β-catenin-like 1 (CTNNBL1) are members of the Prp19/CDC5L complex, which is a major building block of the spliceosome’s catalytic RNP core (Grote et al., 2010), we speculate that BCAS2 may be a regulator of Wnt signaling through interaction with β-catenin during hematopoiesis. To test our hypothesis, human BCAS2 was overexpressed in HEK293T cells and mouse embryonic fibroblasts (MEFs). Ectopic expression of BCAS2 enhanced the Wnt3a-induced expression of the TOPflash luciferase reporter in a dose-dependent manner (Figure 3A and B). Importantly, Wnt3a-induced luciferase activity in HEK293T cells could be effectively reduced by knockdown of BCAS2 using two shRNAs targeting different regions of human BCAS2 (Figure 3C, Figure 3—figure supplement 1). Similar results were also observed in conditional Bcas2 knockout (Bcas2-cKO) MEFs in the presence of tamoxifen (Figure 3D). Furthermore, the expression of cdx4 and hoxa9a, which are targets of canonical Wnt signaling in the regulation of hematopoietic development (Pilon et al., 2006, Sun et al., 2018), were downregulated in the lateral plate mesoderm of bcas2+/- embryos at the six-somite stage (Figure 3—figure supplement 2). These findings support that BCAS2 promotes Wnt signaling activation.

Figure 3. BCAS2 promotes primitive hematopoiesis via activating Wnt signaling.

(A, B) Overexpression of BCAS2 increases Wnt3a-induced TOPflash activity in HEK293T cells (A) and mouse embryonic fibroblasts (MEFs) (B). Different amounts of plasmid expressing BCAS2 (0, 80, 160, or 320 ng/well) were transfected into cells, together with the super-TOPflash luciferase and Renilla luciferase vectors. After 36 h of transfection, cells were treated with or without Wnt3a CM for 12 h and harvested for luciferase assays (n=3). *p<0.05; **p<0.01 (Student’s t-test). (C) The Wnt3a-induced TOPflash activity is decreased in BCAS2-deficient cells. HEK293T cells were transfected with shRNA plasmids, along with indicated plasmids, and harvested for luciferase reporter assay (n=3). *p<0.05 (Student’s t-test) (D) Bcas2-cKO MEFs prepared from Bcas2F/F mouse embryos were incubated in medium containing 100 μM tamoxifen for 72 h and then subjected to western blotting and luciferase reporter assay (n=3). **p<0.01 (Student’s t-test). (E, F) Expression analysis of gata1 (E) and hbbe3 (F) in Tg(hsp70l:dkk1b-GFP) embryos after heat shock at 16 hpf. (G, H) Immunofluorescence staining of β-catenin in Tg(gata1:GFP) embryos at 16 hpf. The embryos were injected with 8 ng of the indicated MO at the one-cell stage. The dotted lines show the GFP-positive hematopoietic progenitor cells. The relative fluorescence intensity of nuclear β-catenin was quantified in (H) (n=6). **p<0.01 (Student’s t-test). (I, J) Expression of hbbe3 in bcas2 morphants (I) and bcas2+/Δ14 mutants (J) overexpressing ΔN-β-catenin. Embryos were injected with the indicated MO together with ΔN-β-catenin mRNA at the 1-cell stage and harvested at the 10-somite stage for in situ hybridization. Scale bars, 100 μm (E, F, I, J), 5 μm (G).

Figure 3—source data 1. Original western blots for Figure 3D, indicating the relevant bands and treatments.
Figure 3—source data 2. Original western blot images in Figure 3D.

Figure 3.

Figure 3—figure supplement 1. Western blot analysis of HEK293T cells transfected with corresponding shRNA constructs.

Figure 3—figure supplement 1.

Figure 3—figure supplement 1—source data 1. Original western blots for Figure 3—figure supplement 1 with the relevant bands labeled.
Figure 3—figure supplement 1—source data 2. Uncropped immunoblotting images in Figure 3—figure supplement 1.
Figure 3—figure supplement 2. Expression patterns of cdx4 and hoxa9a in bcas2+/Δ14 embryos and their siblings at the 6-somite stage.

Figure 3—figure supplement 2.

Black arrows indicate the posterior lateral mesoderm. Scale bars, 100 μm.
Figure 3—figure supplement 3. Inhibition of Wnt signaling does not affect the generation of hemangioblasts or their endothelial differentiation, but impairs their hematopoietic differentiation.

Figure 3—figure supplement 3.

Expression of hemangioblast markers npas4l, scl, gata2 (A), endothelial marker fli1a (B) and erythroid progenitor marker gata1 (C) at the indicated stages. Wild-type embryos were treated with 10 µM CCT036477 from 9 hpf and then collected for whole-mount in situ hybridizations. DMSO treated wild-type embryos were regarded as control groups. Scale bars, 100 μm (A–C).
Figure 3—figure supplement 4. Knockdown of bcas2 significantly reduces nuclear β-catenin in the primitive myeloid cells.

Figure 3—figure supplement 4.

(A) Immunofluorescence staining of β-catenin in Tg(coro1a:GFP) embryos at 17 hpf. The embryos were injected with 8 ng of the indicated MOs at the one-cell stage and then collected for immunofluorescence staining. The dotted lines refer to the GFP-positive primitive myeloid cells. The relative fluorescence intensity of β-catenin was quantified in (B) (n=6). **p<0.01 (Student’s t-test). Scale bars, 10 μm (A).
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To confirm that Wnt signaling was required for zebrafish embryonic hematopoiesis, we induced the expression of canonical Wnt inhibitor Dkk1 by heat-shocking Tg(hsp70l:dkk1-GFP)w32 embryos at the bud stage (Stoick-Cooper et al., 2007). As expected, diminished expression of gata1 and hbbe3 was detected in the resulting embryos at the 10-somite stage (Figure 3E and F). In addition, treatment with a small molecule β-catenin antagonist CCT036477 from 9 hpf did not affect the expression of hemangioblast markers npas4l, (Krause et al., 2014) scl, and gata2 or endothelial marker fli1a (Figure 3—figure supplement 3A and B), yet significantly reduced the expression of erythroid progenitor marker gata1 in wild-type embryos (Figure 3—figure supplement 3C), suggesting that canonical Wnt signaling may not be required for the generation of hemangioblasts or their endothelial differentiation, but is pivotal for their hematopoietic differentiation. To further validate that bcas2 functions in primitive hematopoiesis via Wnt/β-catenin signaling, the expression pattern of β-catenin was examined in bcas2 morphants with Tg(gata1:GFP) background at the 10-somite stage by immunofluorescence staining. The signal of nuclear β-catenin was substantially decreased in hematopoietic progenitor cells (Figure 3G and H) and primitive myeloid cells (Figure 3—figure supplement 4A and B). Moreover, overexpression of ΔN-β-catenin, a constitutively active form of β-catenin, effectively restored the expression of hbbe3 in bcas2 morphants and mutants (Figure 3I and J). All these data suggest that BCAS2 functions in primitive hematopoiesis by regulating Wnt/β-catenin signaling.

BCAS2 promotes β-catenin nuclear accumulation independently of protein stability regulation

To investigate how BCAS2 regulates Wnt/β-catenin signaling, HEK293T cells were treated with LiCl, a canonical Wnt agonist that inhibits GSK-3β activity and stabilizes cytosolic β-catenin (Meijer et al., 2003). The results showed that TOPflash activity was significantly elevated in LiCl-treated cells (Figure 4A). BCAS2 overexpression further upregulated, whereas shRNA-mediated knockdown of BCAS2 downregulated LiCl-induced TOPflash activity (Figure 4A and B). Likewise, HEK293T cells transfected with S37A-β-catenin, a constitutively active form of β-catenin that is resistant to GSK-3β-mediated degradation (Easwaran et al., 1999), displayed a much higher level of TOPflash activity, which was reduced by BCAS2 knockdown (Figure 4C). These results strongly imply that BCAS2 regulates Wnt signaling downstream of β-catenin stability control.

Figure 4. BCAS2 is essential for β-catenin nuclear accumulation.

Figure 4.

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(A–C) BCAS2 enhances LiCl-induced TOPflash activity in HEK293T cells. Cells were transfected with BCAS2 expression plasmids (A), shRNA plasmids (B), or S37A-β-catenin expression plasmids (C), together with the TOPflash luciferase and Renilla luciferase vectors. After transfection, cells were subsequently treated with or without 100 ng/ml LiCl for 12 h and assayed for luciferase activity (n=3). *p<0.05; **p<0.01 (Student’s t-test). (D, E) Bcas2-cKO mouse embryonic fibroblasts (MEFs) were incubated with tamoxifen for 24 h and then treated with or without 100 ng/mL LiCl. The nuclear accumulation of β-catenin was analyzed using immunofluorescence (D) and western blotting (E). (F) SW480 cells were transfected with the indicated shRNA constructs, and the endogenous β-catenin protein was detected using immunofluorescence 48 h after transfection. The expression of GFP served as a transfection control. The arrowheads indicate the cells transfected with indicated shRNA constructs. (G) Bcas2-cKO MEFs were cultured in the presence of tamoxifen for 24 h and then treated with 20 μM MG132 for 6 h. The expression of BCAS2 and β-catenin was measured by immunofluorescence. Scale bars, 10 μm (D, F, G).

Figure 4—source data 1. Original western blots for Figure 4E with the relevant bands and treatments labeled.
Figure 4—source data 2. Original western blot images in Figure 4E.

To test the above hypothesis, we evaluated nuclear β-catenin level by performing immunofluorescence staining and immunoblotting experiments. Upon tamoxifen exposure, nuclear accumulation of β-catenin induced by LiCl was greatly inhibited in Bcas2-cKO MEFs, while nuclear/cytoplasmic fractionation suggested that cytoplasmic β-catenin level remained relatively unchanged (Figure 4D and E). Similarly, silencing BCAS2 with shRNA led to reduced nuclear β-catenin in the human colon cancer cell line SW480, in which β-catenin was activated because of mutations in the adenomatous polyposis coli protein (APC), an integral component of the β-catenin destruction complex (Figure 4F, Rosin-Arbesfeld et al., 2003). Next, MG132, a proteasome inhibitor, was applied to activate Wnt/β-catenin signaling in Bcas2-cKO MEFs by inhibiting β-catenin degradation. In the absence of tamoxifen and MG132, endogenous β-catenin was localized almost exclusively in the cytoplasm; MG132 treatment dramatically triggered β-catenin accumulation in the nuclei (Figure 4G). However, in Bcas2-cKO MEFs exposed to tamoxifen, MG132 treatment was not able to induce nuclear accumulation of β-catenin (Figure 4G). These findings indicate that BCAS2 promotes β-catenin nuclear accumulation in a manner that is independent of β-catenin stability regulation.

BCAS2 sequesters β-catenin within the nucleus

In addition to be affected by protein stability, the nuclear level of β-catenin is also fine-tuned by the opposing actions of nuclear export and import (Lu et al., 2017; Henderson and Fagotto, 2002; Henderson, 2000). To examine the effect of BCAS2 on the nuclear import and export of β-catenin, fluorescent recovery after photobleaching (FRAP) experiments were carried out in HeLa cells expressing GFP-tagged S37A-β-catenin. After photobleaching the nucleus, no significant difference was found in the recovery of nuclear GFP signals between the cells with and without overexpression of BCAS2 (Figure 5—figure supplement 1A, A’ and C), suggesting that BCAS2 does not regulate β-catenin nuclear import. Conversely, after photobleaching the cytoplasm, BCAS2 overexpressed cells showed a much slower recovery of cytoplasmic fluorescence (Figure 5—figure supplement 1B, B’, and C), indicating that BCAS2 inhibits β-catenin nuclear export.

It has been suggested that the nuclear exit of β-catenin can be either dependent or independent on CRM1, a major nuclear export receptor (Xu and Massagué, 2004). To shed light on the mechanism underlying BCAS2 mediated β-catenin nuclear retention, we treated Bcas2-cKO MEFs with the CRM1-specific export inhibitor leptomycin B (LMB) (Wolff et al., 1997). Regardless of the presence or absence of endogenous BCAS2, LMB treatment could effectively increase the level of β-catenin in the nucleus (Figure 5A). Consistently, treatment of LMB was able to rescue the impaired nuclear accumulation of β-catenin in BCAS2-deficient SW480 cells (Figure 5B). Moreover, when bcas2 morphants in Tg (gata1:GFP) background were treated with LMB from bud stage to 10 somite stage, the level of nuclear β-catenin was partially recovered (Figure 5C and D). Importantly, the expression of gata1 was also restored in bcas2 mutants upon LMB treatment (Figure 5E). We further tested if BCAS2 specifically regulates CRM1-mediated nuclear export of β-catenin by analyzing the nucleocytoplasmic distribution of other known CRM1 cargoes, such as ATG3 and CDC37L (Kirlı et al., 2015). Intriguingly, BCAS2 overexpression in HeLa cells only slightly enhanced the nuclear localization of CDC37L and had no significant impact on that of ATG3 (Figure 5—figure supplement 2), indicating the specificity of BCAS2-mediated inhibition of CRM1-dependent nuclear export of β-catenin. Taken together, these findings suggest that BCAS2 negatively regulates CRM1-mediated nuclear export of β-catenin.

Figure 5. BCAS2 functions in CRM1-mediated nuclear export of β-catenin.

(A) Tamoxifen-treated Bcas2-cKO mouse embryonic fibroblasts (MEFs) were incubated with 20 nM LMB for 3 h. The expression of Bcas2 and β-catenin was analyzed using immunofluorescence. The arrowheads show the cells with nuclear β-catenin accumulation. (B) SW480 cells were transfected with the indicated shRNA constructs and then treated with LMB for 3 h before immunostaining. GFP was regarded as a transfection control. The arrowheads indicate the transfected cells. (C, D) Immunofluorescence staining of β-catenin in bcas2 morphants with Tg(gata1:GFP) background at 16 hpf. Embryos were exposed to 20 nM LMB from the bud stage. The dotted lines indicate the GFP-positive hematopoietic progenitor cells. The relative fluorescence intensity of nuclear β-catenin was quantified in (D) (n=6). ns, not significant; **p<0.01 (Student’s t-test). (E) bcas2+/Δ14 embryos were treated with 20 nM LMB for 6 h and then subjected to WISH assay to analyze the expression of gata1 at the indicated stages. Scale bars, 10 μm (A, B), 5 μm (C), 100 μm (E).

Figure 5.

Figure 5—figure supplement 1. BCAS2 inhibits the nuclear export of β-catenin.

Figure 5—figure supplement 1.

(A–B’) GFP-tagged S37A-β-catenin was co-expressed with or without BCAS2 in HeLa cells. The entire nucleus (A) or entire cytoplasm (B) was bleached. (A, A’) Representative images at the indicated timepoints (A) and the related fluorescence recovery curves (A’, n=7) showing the kinetics of nuclear import of S37A-β-catenin. (B, B’) Time-lapse imaging (B) and recovery curves (B’, n=7) from cytoplasmic photobleaching experiments reflecting the kinetics of nuclear export of S37A-β-catenin. (C) Quantitative analysis of fluorescence recoveries after photobleaching (n=7). ns, not significant, **p<0.01 (Student’s t-test). Scale bars, 10 μm (A, B).
Figure 5—figure supplement 2. Overexpression of BCAS2 slightly enhances the nuclear accumulation of CDC37L and has no influence on the distribution of ATG3.

Figure 5—figure supplement 2.

HeLa cells were transfected with the indicated plasmids and then subjected to immunostaining using anti-Flag and anti-HA antibodies. The nuclei were labeled with DAPI. Scale bar, 10 μm.
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BCAS2 directly interacts with β-catenin in the nucleus

To investigate whether BCAS2 inhibits the nuclear export of β-catenin through physical binding, HEK293T cells were transfected with Flag-tagged β-catenin and HA-tagged BCAS2 constructs. Co-immunoprecipitation (Co-IP) experiments showed that Flag-β-catenin was precipitated with HA-BCAS2 as well as endogenous BCAS2, indicating an interaction between these two proteins (Figure 6A and B). In addition, the interaction was enhanced upon Wnt3a stimulation (Figure 6C). Given that Wnt ligand stimulation ultimately induces β-catenin nuclear accumulation, this enhanced interaction implies that BCAS2 associates with β-catenin within the nucleus. Therefore, we performed the bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of BCAS2 and β-catenin in living cells. In this assay, the N-terminal fragment of yellow fluorescent protein (YFP) was fused to BCAS2 (YN-BCAS2), while the C-terminal fragment was fused to β-catenin (YC-β-catenin) (Figure 6D). As expected, the YFP fluorescence was specifically observed in the nucleus (Figure 6E).

Figure 6. BCAS2 interacts with β-catenin.

(A–C) Flag-tagged β-catenin was co-transfected with or without HA-tagged BCAS2 into HEK293T cells. Cell lysates were immunoprecipitated using anti-Flag antibody. Eluted proteins were analyzed by western blotting using indicated antibodies. In (C), for Wnt signaling activation, cells were treated with Wnt3a CM for 5 h before harvest. (D, E) YN-BCAS2 and YC-β-catenin were either individually or collectively transfected into HeLa cells. The expression of YN-BCAS2 and YC-β-catenin was analyzed with anti-GFP antibody (D). The reconstituted YFP fluorescence in living cells was detected by confocal laser scanning microscopy with excitation at 488 nm (E). (F) Schematics of full-length and deletion mutants of β-catenin. (G) HEK293T cells were transfected with HA-tagged BCAS2 and Flag-tagged deletion mutants of β-catenin. Cell lysates were then immunoprecipitated using anti-Flag antibody followed by western blot analysis. (H) GST pull-down assays were performed using bacterially expressed GST, GST-ARM1-12, and His-BCAS2. Scale bars, 10 μm (D, E).

Figure 6—source data 1. Original western blots for Figure 6A–C, G and H with the relevant bands and treatments indicated.
Figure 6—source data 2. Uncropped immunoblotting images in Figure 6A–C, G and H.

Figure 6.

Figure 6—figure supplement 1. The interaction between β-catenin and TCF4 remains unaffected in the presence of BCAS2.

Figure 6—figure supplement 1.

HEK293T cells were transfected with the indicated constructs. Cell lysates were immunoprecipitated using anti-c-Myc antibody and the eluted proteins were analyzed by western blotting.
Figure 6—figure supplement 1—source data 1. Original western blots for Figure 6—figure supplement 1, indicating the relevant bands and treatments.
Figure 6—figure supplement 1—source data 2. Original files for western blot analysis in Figure 6—figure supplement 1.
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Previous studies have divided the β-catenin protein into three distinct domains, including the N-terminal domain (residues 1–133), the central domain with 12 ARM repeats (residues 134–670), and the C-terminal domain (residues 671–781) (Dimitrova et al., 2010). To identify the BCAS2 binding site, constructs expressing various truncated forms of β-catenin were generated and co-transfected with BCAS2 into HEK293T cells (Figure 6F). Co-IP assays revealed that deletion of the N-terminal or C-terminal domain of β-catenin did not alter the interaction between β-catenin and BCAS2 (Figure 6G). In contrast, when the ARM repeats 1–12 of β-catenin were deleted, the resulting deletion mutant showed virtually no interaction with BCAS2 (Figure 6G). GST pull-down assay also demonstrated a direct interaction between BCAS2 and the ARM repeats of β-catenin (Figure 6H). These results indicate that BCAS2 physically binds to the ARM repeats of β-catenin. Furthermore, we found that the ARM repeats 9–12, but not 1–8, bound to BCAS2 (Figure 6G).

Transcriptional activation of the canonical Wnt target genes depends on β-catenin nuclear localization and its physical association with TCF/LEF family members. As the binding sites for TCF have been located in the ARM repeats 3–10 of β-catenin, (Graham et al., 2000) it is likely that BCAS2-mediated nuclear sequestration of β-catenin through interacting with the ARM repeats 9–12 would be compatible with the initiation of gene transcription by allowing for the association of β-catenin and TCF. To validate this possibility, co-IP assays were performed and we found that β-catenin still bound with TCF4 in the presence of BCAS2 (Figure 6—figure supplement 1), confirming that the binding of BCAS2 to β-catenin would not interfere with the formation of β-catenin/TCF complex.

BCAS2 enhances β-catenin nuclear accumulation through its CC domains

To determine which domain of BCAS2 binds to β-catenin, we constructed a series of deletion mutants of BCAS2 (Figure 7A). Notably, we observed that among these truncated mutants, only the one lacking both CC1 and CC2 domains lost the ability to interact with β-catenin (Figure 7B). Moreover, these two CC domains alone or together could interact with β-catenin (Figure 7C). Therefore, we conclude that BCAS2 binds to β-catenin via its CC domains.

Figure 7. BCAS2 sequesters β-catenin in the nucleus via its CC domains.

(A) Schematics of full length and deletion mutants of BCAS2. (B, C) HEK293T cells were transfected with Flag-β-catenin and indicated deletion mutants of BCAS2. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Eluted proteins were immunoblotted using anti-HA (B) or anti-GFP antibodies (C) for BCAS2 detection. (D) HEK293T cells transfected with the indicated plasmids were treated with 100 ng/ml LiCl for 12 h, and then subjected to luciferase assay (n=3). ns, not significant; **p<0.01 (Student’s t-test). (E, F) Immunofluorescence staining of β-catenin in Tg(gata1:GFP) embryos at 16 hpf. The embryos were injected with 8 ng bcas2 MO and 300 pg of full-length BCAS2 mRNA or ΔCC1-2 mRNA at the one-cell stage. The relative fluorescence intensity of nuclear β-catenin was quantified in (F) (n=5). ns, not significant; **p<0.01; ****p<0.0001 (Student’s t-test). (G) Transcripts of gata1 were evaluated by WISH in bcas2+/Δ14 embryos injected with 300 pg of BCAS2 mRNA or ΔCC1-2 mRNA. Scale bars, 5 μm (E), 100 μm (G).

Figure 7—source data 1. Original western blots for Figure 7B and C, indicating the relevant bands and treatments.
Figure 7—source data 2. Original files for western blot analysis in Figure 7B and C.

Figure 7.

Figure 7—figure supplement 1. Overexpression of the CC domains of BCAS2 restores nuclear β-catenin accumulation in bcas2 morphants.

Figure 7—figure supplement 1.

Immunofluorescence staining of β-catenin in Tg(gata1:GFP) embryos at 16 hpf. The embryos were injected with 8 ng bcas2 MO and 300 pg of BCAS2 CC1-2 mRNA at the one-cell stage. The dotted lines show the GFP-positive hematopoietic progenitor cells. The relative fluorescence of β-catenin was quantified in (B) (n=6). *p<0.05 (Student’s t-test). Scale bars, 10 μm (A).
Figure 7—figure supplement 2. Haploinsufficiency of bcas2 does not affect pre-mRNA splicing during primitive hematopoiesis.

Figure 7—figure supplement 2.

(A) The number of five major types of alternative splicing events was analyzed from RNA sequencing data. Embryos were lysed at the 10-somite stage and subjected to reverse transcription. The cDNA library was prepared, sequenced and then analyzed using rMATS. The five main alternative splicing types refer to exon skipping (SE), alternative 3’ splicing site (A3SS), alternative 5’ splicing site (A5SS), intron retention (RI), and mutually exclusive exons (MEX). (B) Examples of different types of alternative splicing were analyzed by reverse transcription PCR using total RNA from sibling and bcas2+/Δ14 embryos at the 10-somite stage. (C) Reverse transcription PCR analysis of total mdm4-FL and mdm4-S isoforms in bcas2+/Δ14 and sibling embryos at the 10-somite stage. (D) Analysis the pre-mRNA and mature mRNA of β-catenin in bcas2+/Δ14 and sibling embryos at the 10-somite stage.
Figure 7—figure supplement 2—source data 1. PDF file containing original gel images for Figure 7—figure supplement 2B, C, D with the relevant bands indicated.
Figure 7—figure supplement 2—source data 2. Original gel images in Figure 7—figure supplement 2B, C, D.
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We next examined whether the CC domains are required for BCAS2 to promote Wnt/β-catenin signaling. As shown in Figure 7D, overexpression of BCAS2 without the CC domains failed to increase LiCl-induced TOPflash activity in HEK-293T cells. Likewise, overexpression of the full-length or the CC domains alone, but not BCAS2 lacking the CC domains, restored the nuclear accumulation of β-catenin in bcas2 morphants (Figure 7E-F, Figure 7—figure supplement 1A and B). The expression of gata1 in bcas2 mutants was also recovered by overexpression of the full-length BCAS2, but not the truncated form without the CC-domains (Figure 7G). Collectively, these findings indicate that BCAS2 positively regulates Wnt signaling through sequestering β-catenin within the nucleus via its CC domains during primitive hematopoiesis.

As BCAS2 is involved in the Prp19-CDC5L spliceosome complex that regulates RNA splicing during spermiogenesis, neurogenesis, and definitive hematopoiesis, (Liu et al., 2017; Yu et al., 2019) we wondered if this protein participates in primitive hematopoiesis via mRNA alternative splicing. To this end, we performed RNA sequencing of 10-somite stage embryos to identify abnormal events in alternative splicing in bcas2+/Δ14 mutants. However, upon haploinsufficiency of bcas2, neither the number of five major types of alternative splicing events, nor the typical forms of alternative splicing were significantly affected (Figure 7—figure supplement 2A and B). Additionally, haploinsufficiency of bcas2 did not result in the alternative splicing of mdm4 that predisposes cells to undergo p53-mediated apoptosis in definitive hematopoiesis, as reported previously by Yu et al. (Figure 7—figure supplement 2C; Yu et al., 2019; Rallapalli et al., 1999). Furthermore, the splicing efficiency of β-catenin pre-mRNA remained almost unchanged in bcas2+/Δ14 mutants (Figure 7—figure supplement 2D). These results demonstrate that the defects in primitive hematopoiesis of bcas2+/Δ14 mutants are independent of the regulatory role of Bcas2 in pre-mRNA splicing.

Discussion

BCAS2 is a 26 kDa nuclear protein involved in a multitude of developmental processes, such as Drosophila wing development, dendritic growth, and spermatogenesis (Kuo et al., 2009; Chen et al., 2013; Liu et al., 2017; Huang et al., 2016; Xu et al., 2015; Zhang et al., 2022). In our study, we generated bcas2 knockout zebrafish. The heterozygotes not only showed male infertility, resembling the phenotype of Bcas2 germ cell-specific knockout mice reported previously (Liu et al., 2017), but also exhibited impaired definitive hematopoiesis, consistent with the earlier study (Yu et al., 2019). Importantly, we found a marked decrease in the expression of the primitive erythroid progenitor markers gata1 and hbbe3 in these heterozygous mutants, which was rescued by overexpression of BCAS2. Moreover, the defective primitive hematopoiesis in mutant zebrafish was phenocopied in hemangioblast-specific Bcas2 knockout mice. While the reason(s) for the discrepancy between our data and the observations made by Yu et al. regarding the role of bcas2 in the development of primitive erythroid and myeloid cells remains to be determined (Yu et al., 2019), our findings in zebrafish and mouse embryos provide solid evidence that BCAS2 plays a conserved role in primitive hematopoiesis.

As demonstrated in previous studies, BCAS2 is involved in various developmental events by regulating pre-mRNA splicing (Chen et al., 2013; Liu et al., 2017; Yu et al., 2019; Chen et al., 2022; Huang et al., 2016). However, our data showed that haploinsufficiency of bcas2 did not affect alternative splicing during primitive hematopoiesis. These results imply that one copy of the bcas2 gene is sufficient to support mRNA splicing in zebrafish. Instead, we find that Bcas2 promotes primitive hematopoiesis by sequestering β-catenin within the nucleus. It has been reported that the bcas2 deletion in zebrafish embryos induces alternative splicing of Mdm4 that predisposes cells to undergo p53-mediated apoptosis in HSPCs during definitive hematopoiesis (Yu et al., 2019). Intriguingly, we found that the loss of one copy of bcas2 gene in zebrafish also resulted in severe impairment of HSPCs and their derivatives. It is possible that Bcas2 might also have a role in definitive hematopoiesis independent of its splicing regulatory function.

For the past decades, given the contradictory conclusions obtained from various in vitro and in vivo studies, the function of Wnt/β-catenin signaling in primitive hematopoiesis remains elusive and controversial (Sturgeon et al., 2014; Tran et al., 2010; Lengerke et al., 2008; Paluru et al., 2014). In the present study, we have provided several lines of evidence supporting that Wnt/β-catenin signaling positively regulates primitive hematopoiesis: (1) inhibition of Wnt/β-catenin by overexpression of the canonical Wnt inhibitor Dkk1 disrupts the formation of erythrocyte progenitors at the 10-somite stage. (2) Defects in primitive hematopoiesis in bcas2 morphants and mutants are readily restored by overexpression of ΔN-β-catenin, a constitutively active β-catenin. (3) Overexpression of the full-length BCAS2, but not the CC domain-deleted BCAS2, restores the formation of the primitive erythroid progenitor in bcas2 mutants. (4) BCAS2 overexpression enhances the development of primitive blood cells in wild-type embryos. All these data suggest that BCAS2-mediated Wnt/β-catenin signal activation is necessary for primitive hematopoiesis.

In addition, Wnt/β-catenin signaling has been known as an important pathway involved in the regulation of axis determination and neural patterning during gastrulation (Yamaguchi, 2001; Kozmikova and Kozmik, 2020; Brafman and Willert, 2017; Lickert et al., 2005). In our study, neither the heterozygous bcas2 mutant embryos nor the very few homozygous ones exhibited any morphological defects typically associated with inhibition of Wnt signaling, such as ventralization or brain anteriorization. This may be due to the presence of maternal Bcas2 in heterozygous and homozygous mutant embryos which were derived from crossing bcas2 heterozygous adult zebrafish.

CRM1 can facilitate β-catenin nuclear export in distinct ways (Morgan et al., 2014). For example, CRM1 usually recognizes and binds with the nuclear export signal (NES) sequences in chaperon proteins, such as APC, Axin, and Chibby (Neufeld et al., 2000; Cong and Varmus, 2004; Li et al., 2008), to mediate the nuclear export of β-catenin. On the other hand, CRM1 can also bind directly to and function as an efficient nuclear exporter for β-catenin (Ki et al., 2008). Since BCAS2 has not been reported to contain any recognizable NES sequences, it will be interesting to investigate whether BCAS2 competitively inhibits β-catenin from associating with CRM1, or with the chaperone proteins.

In summary, we uncover a novel role of BCAS2 in primitive hematopoiesis through enhancing nuclear retention of β-catenin. Our study provides new insights into the mechanism of BCAS2-mediated Wnt signal activation during primitive hematopoiesis. Given that BCAS2 and Wnt signaling are well documented to contribute to cancer development (Murillo-Garzón and Kypta, 2017; Zhan et al., 2017; Yu et al., 2021; Salmerón-Hernández et al., 2019; Wang et al., 2020), it is appealing to further explore whether our findings can be applied to future cancer research.

Materials and methods

Animal models

Our studies, including animal maintenance and experiments, were performed in compliance with the guidelines of the Animal Care and Use Committee of the South China University of Technology (Permission Number: 2023092). Seven strains of zebrafish were used in this study, including Tübingen wild-type, bcas2 mutant, cloche mutant, Tg(gata1:GFP), Tg(coro1a:eGFP), Tg(kdrl:GFP), and Tg(hsp70l:dkk1b-GFP). cloche mutant, Tg(gata1:GFP) and Tg(kdrl:GFP) lines were provided by Professor Feng Liu (Chinese Academy of Sciences). Tg(coro1a:eGFP) was provided by Professor Yiyue Zhang (South China University of Technology). Tg(hsp70l:dkk1b-GFP) strain was purchased from the China Zebrafish Resource Center. Bcas2Floxed/Floxed (Bcas2F/F) mouse line was generated as previously described (Liu et al., 2017). Kdr-Cre mouse line was provided by Professor Dahua Chen (Yunnan University). Genotyping of Bcas2F/F mouse and Kdr-Cre mouse was performed using primers listed in Supplementary file 1. The mouse model with Bcas2 specifically disrupted in the hemangioblasts was derived from mating female Bcas2F/F mice with Kdr-Cre transgenic mice. All mouse lines were maintained on a mixed background (129/C57BL/6).

Cell lines and transfection

HEK293T (RRID:CVCL_0063), HeLa (RRID:CVCL_0030), SW480 (RRID:CVCL_0546), and L cells (RRID:CVCL_4536) were obtained from ATCC. All cell lines were authenticated by Short Tandem Repeat (STR) analysis, tested for mycoplasma contamination, and confirmed to be negative. Bcas2-cKO MEFs were prepared from Bcas2F/F embryos at E13.5. Cells were cultured in Dulbecco’s modified eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin (HyClone) at 37°C and 5% CO2. L cells expressing Wnt3a were maintained under similar conditions in the presence of 400 µg/ml G-418, from which Wnt3a conditioned medium (Wnt3a CM) was generated. Culture medium prepared from L cells was used as a control. To stimulate Wnt signaling, cells were treated with Wnt3a CM in a 1:1 ratio with normal media. To deplete Bcas2 expression, Bcas2-cKO MEFs were cultured in medium containing 2 μM tamoxifen for 72 h and the knockout efficiency was evaluated using western blot analysis. The same cells cultured without tamoxifen were used as a control. To silence BCAS2 expression, shRNA constructs in pLL 3.7-GFP plasmid were generated to target the following sequences: shRNA1, GAATGTGTAAACAATTCTA; shRNA2: GAAGGAACTTCAGAAGTTA. Transfection was performed with Lipofectamine 2000 (Invitrogen Cat# 11668019) according to the manufacturer’s instructions.

Generation of CRISPR-Cas9-mediated bcas2 knockout zebrafish

The bcas2 knockout zebrafish mutants were generated by CRISPR-Cas9 system as previously described (Chang et al., 2013). The guide RNA was designed to target the sequences 5′-GGCGCAGCTGGAGCATCAGG-3′ within exon 4 of bcas2. Humanized Cas9 mRNA and gRNA were co-injected into wild-type embryos at the one-cell stage. Embryos or adult fin clips were collected to prepare genomic DNA. To screen for mutant alleles, the genomic regions containing gRNA-targeted sequences were amplified by polymerase chain reaction (PCR) with primers listed in Supplementary file 1. The PCR products were sequenced or digested with T7 endonuclease or restriction enzyme FspI for genotyping.

RNA, morpholinos, and microinjection

Capped mRNAs for human BCAS2, BCAS2 △CC1-2, BCAS2 CC1-2, and mouse ΔN-β-catenin mRNA were synthesized from the corresponding linearized plasmids using an mMESSAGE mMACHINE T7 transcription kit (Ambion Cat# AM1344). Morpholino (MOs) were designed and purchased from Gene Tools: mismatch MO (cMO 5’-AGCCACTCATCCTGCTCCTCCCATC-3’), and bcas2 translation-blocking MO (tMO; 5’-AGCGACTGATGCTGGTCCTGCCATC-3’). The mRNAs and morpholinos were injected into embryos at the 1- to 2 cell stage.

Whole-mount in situ hybridization

Digoxigenin-labeled and fluorescein-labeled probes were synthesized using a RNA Labeling kit (Roche Cat# 11175025910). WISH and double FISH for zebrafish embryos were performed following previously published methods (Jia et al., 2008; Welten et al., 2006). Anti-digoxigenin-POD (Roche Cat# 11633716001) and anti-fluorescein-POD (Roche Cat# 11426346910) were used to detect digoxigenin-labeled probes and fluorescein-labeled probes, respectively. After WISH, the stained embryos were embedded in OCT and sections were prepared with a LEICA CM1900. The mouse yolk sac layers were separated as previously described (Wallingford and Giachelli, 2014).

o-Dianisidine staining

To evaluate hemoglobin level, embryos were harvested at 36 hpf or 48 hpf, then stained with o-dianisidine as previously described (Lieschke et al., 2001).

Proliferation and apoptosis assays

Embryos were incubated with 10 mM bromodeoxyuridine (BrdU) (Sigma-Aldrich Cat# B5002) for 20 min. The incorporated BrdU was detected with anti-BrdU (Sigma-Aldrich Cat# B2531, RRID:AB_476793) antibody. TUNEL staining was performed using In Situ Cell Death Detection Kit, TMR red (Roche Cat# 12156792910) according to the manufacturer’s recommendation.

Heat shock treatment

To induce dkk1 expression, Tg(hsp70l:dkk1b-GFP) embryos were subjected to heat shock (42°C) for 10 min at 10 hpf, and then collected at the indicated stage for WISH.

Dual reporter assay

HEK293T cells or MEFs were seeded in 24-well plates and transfected with a Super-TOPflash plasmid containing multimerized TCF-binding elements and a Renilla luciferase plasmid, along with the indicated vectors. Then cells were treated with 100 ng/ml LiCl and/or Wnt3a CM for 12 h and assayed for luciferase activity using the Dual luciferase system (Promega Cat# E1910).

Immunoprecipitation, GST pulldown, and western blotting

For immunoprecipitation, HEK293T cells were transfected with the indicated plasmids and collected 48 h after transfection. Subsequently, HEK293T cells were lysed in a lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40) containing protease inhibitors. Immunoprecipitation was performed in accordance with the standard protocols.

For GST pulldown assay, GST, GST tagged β-catenin ARM 1–12 and His tagged BCAS2 were expressed in Escherichia coli BL21, then purified using Glutathione-Sepharose 4B beads (GE Healthcare Cat# 71024800-GE) and HisPur Ni-NTA beads (Thermo Fisher Cat# 88831), respectively. GST and GST-β-catenin ARM 1–12 proteins were immobilized onto Glutathione-Sepharose 4B beads and incubated with purified His-BCAS2 at 4°C for 4 h. Beads were washed three times and analyzed using western blotting.

Cytoplasmic and nuclear extracts were separated with nuclear and cytoplasmic extraction kit (CWBIO Cat# CW0199). Cell lysates were subjected to immunoprecipitation with anti-Flag M2 affinity gel (Sigma-Aldrich Cat# A2220, RRID:AB_10063035) or anti-c-Myc agarose affinity gel (Sigma-Aldrich Cat# A7470, RRID:AB_10109522) antibodies. Proteins were analyzed by western blot using anti-Flag (Sigma-Aldrich Cat# F2555, RRID:AB_796202), anti-HA (CWBIO Cat# CW0092A), anti-β-catenin (Abmart Cat# M24002, RRID:AB_2920853), anti-BCAS2 (Proteintech Cat# 10414–1-AP, RRID:AB_2063400), anti-β-Tubulin (CWBIO Cat# CW0265A), anti-GFP (Thermo Fisher Scientific Cat# A-11120, RRID:AB_221568), anti-Histone H3 (Abcam Cat# ab1791, RRID:AB_302613), anti-GST (Sigma-Aldrich Cat# SAB4200237, RRID:AB_2858197), and anti-His Tag (Beyotime Cat# AF5060) antibodies.

Immunofluorescence staining

Cells on coverslips and embryos were processed for immunofluorescence staining as previously described (Wei et al., 2017; Yang et al., 2022). Before fixation, bcas2-deficient MEFs were treated with a concentration of 20 μM MG132 or 20 nM LMB for 6 h, while Tg(gata1:GFP) embryos were treated with 20 nM LMB from the bud stage to the 10-somite stage. The prepared samples were stained with anti-BCAS2 (Proteintech Cat# 10414-1-AP, RRID:AB_2063400), anti-β-catenin (Abmart Cat# M24002, RRID:AB_2920853), and anti-GFP (Thermo Fisher Scientific Cat# A-11122, RRID:AB_221569) antibodies. Meanwhile, 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma-Aldrich Cat# 10236276001) was used to label nuclei. Fluorescence imaging was performed using a Nikon A1R Confocal Laser Scanning Microscope (RRID:SCR_020317), and all images were captured with the same settings. The relative fluorescence intensity was calculated by dividing the fluorescence intensity of nuclear β-catenin by the fluorescence intensity of DAPI.

Bimolecular fluorescence complementation assay

To construct the plasmids for BiFC, BCAS2 was fused to the N-terminal half of yellow fluorescent protein (YN-BCAS2) and β-catenin to the C-terminal half (YC-β-catenin). YN-BCAS2 and YC-β-catenin were either individually or collectively transfected into HeLa cells. Fluorescence was detected 48 h after transfection using a Nikon A1R Confocal Laser Scanning Microscope (RRID:SCR_020317).

Fluorescence recovery after photobleaching

BCAS2 and GFP tagged S37A-β-catenin were co-transfected into HeLa cells. Fluorescence recovery after photobleaching (FRAP) assay was performed according to previously reported methods (Schmierer and Hill, 2005). The cells were bleached by the 488 nm laser line of the 20 mW argon laser at 100% power. About 90% of nuclear or cytoplasmic GFP signal was bleached. Images were acquired with 35 frames at 25 s intervals by a Zeiss LSM 510 Confocal Microscope (RRID:SCR_018062).

RNA sequencing

Embryos were collected at the 10-somite stage and gently transferred into lysis buffer. Reverse transcription was performed using a SMARTer Ultra Low RNA Kit (Clontech Cat# 634437) directly from the cell lysates. The cDNA library was prepared using an Advantage 2 PCR Kit (Clontech Cat# 639206) and then sequenced via the Illumina NovaSeq 6000 Sequencing System (RRID:SCR_016387). The difference in the number of alternative splicing events between groups was analyzed using rMATS (RRID:SCR_023485, version 4.1.0).

Reverse transcription PCR

Total RNA was isolated from wild-type and bcas2 mutant embryos at the 10-somite stage with MicroElute Total RNA kit (OMEGA Cat# R6831-01), followed by reverse transcription using ReverTra Ace qPCR RT Kit (Toyobo Cat# FsQ-101). The cDNA was amplified with the primers listed in Supplementary file 2.

Quantification and statistical analysis

Images were quantified with ImageJ (RRID:SCR_003070). Statistical data were analyzed using GraphPad Prism (RRID:SCR_002798). Comparisons between experimental groups were done using the Student’s t-test. Data are presented as mean ± SD. p<0.05, p<0.01, p<0.001, and p<0.0001 were considered statistically significant and marked with *, **, ***, and ****, respectively (Student’s t-test).

Materials availability statement

Further information and requests for reagents should be directed to the corresponding author, Qiang Wang (qiangwang@scut.edu.cn).

Acknowledgements

We acknowledge the financial support of the National Natural Science Foundation of China (32025014 and 32330029) and the National Key Research and Development Program of China (2018YFA0800200 and 2020YFA0804000).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Lei Li, Email: lil@ioz.ac.cn.

Xinyu He, Email: hexyu@scut.edu.cn.

Qiang Wang, Email: qiangwang@scut.edu.cn.

Eirini Trompouki, Howard Hughes Medical Institute, Boston's Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, United States.

Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany.

Funding Information

This paper was supported by the following grants:

  • National Natural Science Foundation of China 32025014 to Qiang Wang.

  • National Natural Science Foundation of China 32330029 to Qiang Wang.

  • National Key Research and Development Program of China 2018YFA0800200 to Qiang Wang.

  • National Key Research and Development Program of China 2020YFA0804000 to Qiang Wang.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft.

Data curation, Software, Formal analysis, Investigation, Visualization, Methodology.

Validation, Methodology.

Validation, Methodology.

Validation, Methodology.

Validation, Methodology.

Resources.

Writing – review and editing.

Supervision, Funding acquisition, Project administration.

Ethics

Our studies including animal maintenance and experiments were performed in compliance with the guidelines of the Animal Care and Use Committee of the South China University of Technology (Permission Number: 2023092).

Additional files

MDAR checklist
Supplementary file 1. Primers used for genotyping.
elife-100497-supp1.docx (16.1KB, docx)
Supplementary file 2. Primers used for reverse transcription-PCR.

Data availability

All data generated or analyzed during this study are included in the manuscript and/or supplementary materials. RNA sequencing data have been deposited in GEO under accession codes GSE297155. Original western blot images have been provided as source data.

The following dataset was generated:

Ning G, Lin Y. 2025. BCAS2 promotes primitive hematopoiesis by sequestering β-catenin within the nucleus. NCBI Gene Expression Omnibus. GSE297155

References

  1. Ajuh P, Kuster B, Panov K, Zomerdijk JC, Mann M, Lamond AI. Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry. The EMBO Journal. 2000;19:6569–6581. doi: 10.1093/emboj/19.23.6569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bissinger R, Lang E, Gonzalez-Menendez I, Quintanilla-Martinez L, Ghashghaeinia M, Pelzl L, Sukkar B, Bhuyan AAM, Salker MS, Singh Y, Fehrenbacher B, Fakhri H, Umbach AT, Schaller M, Qadri SM, Lang F. Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival. Apoptosis. 2018;23:641–650. doi: 10.1007/s10495-018-1481-8. [DOI] [PubMed] [Google Scholar]
  3. Brafman D, Willert K. Wnt/β-catenin signaling during early vertebrate neural development. Developmental Neurobiology. 2017;77:1239–1259. doi: 10.1002/dneu.22517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong J-W, Xi JJ. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Research. 2013;23:465–472. doi: 10.1038/cr.2013.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen PH, Lee CI, Weng YT, Tarn WY, Tsao YP, Kuo PC, Hsu PH, Huang CW, Huang CS, Lee HH, Wu JT, Chen SL. BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing. RNA. 2013;19:208–218. doi: 10.1261/rna.034835.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen HH, Lu HY, Chang CH, Lin SH, Huang CW, Wei PH, Chen YW, Lin YR, Huang HS, Wang PY, Tsao YP, Chen SL. Breast carcinoma-amplified sequence 2 regulates adult neurogenesis via β-catenin. Stem Cell Research & Therapy. 2022;13:160. doi: 10.1186/s13287-022-02837-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cong F, Varmus H. Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of beta-catenin. PNAS. 2004;101:2882–2887. doi: 10.1073/pnas.0307344101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Detrich HW, Kieran MW, Chan FY, Barone LM, Yee K, Rundstadler JA, Pratt S, Ransom D, Zon LI. Intraembryonic hematopoietic cell migration during vertebrate development. PNAS. 1995;92:10713–10717. doi: 10.1073/pnas.92.23.10713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dimitrova YN, Li J, Lee Y-T, Rios-Esteves J, Friedman DB, Choi H-J, Weis WI, Wang C-Y, Chazin WJ. Direct ubiquitination of beta-catenin by Siah-1 and regulation by the exchange factor TBL1. The Journal of Biological Chemistry. 2010;285:13507–13516. doi: 10.1074/jbc.M109.049411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Easwaran V, Song V, Polakis P, Byers S. The ubiquitin-proteasome pathway and serine kinase activity modulate adenomatous polyposis coli protein-mediated regulation of beta-catenin-lymphocyte enhancer-binding factor signaling. The Journal of Biological Chemistry. 1999;274:16641–16645. doi: 10.1074/jbc.274.23.16641. [DOI] [PubMed] [Google Scholar]
  11. Ferkowicz MJ, Yoder MC. Blood island formation: longstanding observations and modern interpretations. Experimental Hematology. 2005;33:1041–1047. doi: 10.1016/j.exphem.2005.06.006. [DOI] [PubMed] [Google Scholar]
  12. Galloway JL, Zon LI. Ontogeny of hematopoiesis: examining the emergence of hematopoietic cells in the vertebrate embryo. Current Topics in Developmental Biology. 2003;53:139–158. doi: 10.1016/s0070-2153(03)53004-6. [DOI] [PubMed] [Google Scholar]
  13. Graham TA, Weaver C, Mao F, Kimelman D, Xu WQ. Crystal structure of a beta-catenin/Tcf complex. Cell. 2000;103:885–896. doi: 10.1016/s0092-8674(00)00192-6. [DOI] [PubMed] [Google Scholar]
  14. Grote M, Wolf E, Will CL, Lemm I, Agafonov DE, Schomburg A, Fischle W, Urlaub H, Lührmann R. Molecular architecture of the human Prp19/CDC5L complex. Molecular and Cellular Biology. 2010;30:2105–2119. doi: 10.1128/MCB.01505-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henderson BR. Nuclear-cytoplasmic shuttling of APC regulates beta-catenin subcellular localization and turnover. Nature Cell Biology. 2000;2:653–660. doi: 10.1038/35023605. [DOI] [PubMed] [Google Scholar]
  16. Henderson BR, Fagotto F. The ins and outs of APC and beta-catenin nuclear transport. EMBO Reports. 2002;3:834–839. doi: 10.1093/embo-reports/kvf181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang CW, Chen YW, Lin YR, Chen PH, Chou MH, Lee LJ, Wang PY, Wu JT, Tsao YP, Chen SL. Conditional knockout of breast carcinoma amplified sequence 2 (BCAS2) in mouse forebrain causes dendritic malformation via β-catenin. Scientific Reports. 2016;6:34927. doi: 10.1038/srep34927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jia S, Ren Z, Li X, Zheng Y, Meng A. smad2 and smad3 are required for mesendoderm induction by transforming growth factor-beta/nodal signals in zebrafish. The Journal of Biological Chemistry. 2008;283:2418–2426. doi: 10.1074/jbc.M707578200. [DOI] [PubMed] [Google Scholar]
  19. Ki H, Oh M, Chung SW, Kim K. Beta-catenin can bind directly to CRM1 independently of adenomatous polyposis coli, which affects its nuclear localization and LEF-1/beta-catenin-dependent gene expression. Cell Biology International. 2008;32:394–400. doi: 10.1016/j.cellbi.2007.12.008. [DOI] [PubMed] [Google Scholar]
  20. Kirlı K, Karaca S, Dehne HJ, Samwer M, Pan KT, Lenz C, Urlaub H, Görlich D. A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning. eLife. 2015;4:e11466. doi: 10.7554/eLife.11466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kokolus K, Nemeth MJ. Non-canonical Wnt signaling pathways in hematopoiesis. Immunologic Research. 2010;46:155–164. doi: 10.1007/s12026-009-8116-7. [DOI] [PubMed] [Google Scholar]
  22. Kozmikova I, Kozmik Z. Wnt/β-catenin signaling is an evolutionarily conserved determinant of chordate dorsal organizer. eLife. 2020;9:e56817. doi: 10.7554/eLife.56817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krause U, Ryan DM, Clough BH, Gregory CA. An unexpected role for a Wnt-inhibitor: Dickkopf-1 triggers a novel cancer survival mechanism through modulation of aldehyde-dehydrogenase-1 activity. Cell Death & Disease. 2014;5:e1093. doi: 10.1038/cddis.2014.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krimpenfort RA, Nethe M. Canonical Wnt: a safeguard and threat for erythropoiesis. Blood Advances. 2021;5:3726–3735. doi: 10.1182/bloodadvances.2021004845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kuo PC, Tsao YP, Chang HW, Chen PH, Huang CW, Lin ST, Weng YT, Tsai TC, Shieh SY, Chen SL. Breast cancer amplified sequence 2, a novel negative regulator of the p53 tumor suppressor. Cancer Research. 2009;69:8877–8885. doi: 10.1158/0008-5472.CAN-09-2023. [DOI] [PubMed] [Google Scholar]
  26. Lengerke C, Schmitt S, Bowman TV, Jang IH, Maouche-Chretien L, McKinney-Freeman S, Davidson AJ, Hammerschmidt M, Rentzsch F, Green JBA, Zon LI, Daley GQ. BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway. Cell Stem Cell. 2008;2:72–82. doi: 10.1016/j.stem.2007.10.022. [DOI] [PubMed] [Google Scholar]
  27. Leung AYH, Mendenhall EM, Kwan TTF, Liang R, Eckfeldt C, Chen E, Hammerschmidt M, Grindley S, Ekker SC, Verfaillie CM. Characterization of expanded intermediate cell mass in zebrafish chordin morphant embryos. Developmental Biology. 2005;277:235–254. doi: 10.1016/j.ydbio.2004.09.032. [DOI] [PubMed] [Google Scholar]
  28. Li FQ, Mofunanya A, Harris K, Takemaru KI. Chibby cooperates with 14-3-3 to regulate beta-catenin subcellular distribution and signaling activity. The Journal of Cell Biology. 2008;181:1141–1154. doi: 10.1083/jcb.200709091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lickert H, Cox B, Wehrle C, Taketo MM, Kemler R, Rossant J. Dissecting Wnt/beta-catenin signaling during gastrulation using RNA interference in mouse embryos. Development. 2005;132:2599–2609. doi: 10.1242/dev.01842. [DOI] [PubMed] [Google Scholar]
  30. Lieschke GJ, Oates AC, Crowhurst MO, Ward AC, Layton JE. Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish. Blood. 2001;98:3087–3096. doi: 10.1182/blood.v98.10.3087. [DOI] [PubMed] [Google Scholar]
  31. Liu W, Wang F, Xu Q, Shi J, Zhang X, Lu X, Zhao Z-A, Gao Z, Ma H, Duan E, Gao F, Gao S, Yi Z, Li L. BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis. Nature Communications. 2017;8:14182. doi: 10.1038/ncomms14182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lu Y, Xie S, Zhang W, Zhang C, Gao C, Sun Q, Cai Y, Xu Z, Xiao M, Xu Y, Huang X, Wu X, Liu W, Wang F, Kang Y, Zhou T. Twa1/Gid8 is a β-catenin nuclear retention factor in Wnt signaling and colorectal tumorigenesis. Cell Research. 2017;27:1422–1440. doi: 10.1038/cr.2017.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meijer L, Skaltsounis A-L, Magiatis P, Polychronopoulos P, Knockaert M, Leost M, Ryan XP, Vonica CA, Brivanlou A, Dajani R, Crovace C, Tarricone C, Musacchio A, Roe SM, Pearl L, Greengard P. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chemistry & Biology. 2003;10:1255–1266. doi: 10.1016/j.chembiol.2003.11.010. [DOI] [PubMed] [Google Scholar]
  34. Morgan RG, Ridsdale J, Tonks A, Darley RL. Factors affecting the nuclear localization of β-catenin in normal and malignant tissue. Journal of Cellular Biochemistry. 2014;115:1351–1361. doi: 10.1002/jcb.24803. [DOI] [PubMed] [Google Scholar]
  35. Murillo-Garzón V, Kypta R. WNT signalling in prostate cancer. Nature Reviews. Urology. 2017;14:683–696. doi: 10.1038/nrurol.2017.144. [DOI] [PubMed] [Google Scholar]
  36. Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132:661–680. doi: 10.1016/j.cell.2008.02.008. [DOI] [PubMed] [Google Scholar]
  37. Nagasaki K, Maass N, Manabe T, Hanzawa H, Tsukada T, Kikuchi K, Yamaguchi K. Identification of a novel gene, DAM1, amplified at chromosome 1p13.3-21 region in human breast cancer cell lines. Cancer Letters. 1999;140:219–226. doi: 10.1016/s0304-3835(99)00091-9. [DOI] [PubMed] [Google Scholar]
  38. Neubauer G, King A, Rappsilber J, Calvio C, Watson M, Ajuh P, Sleeman J, Lamond A, Mann M. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nature Genetics. 1998;20:46–50. doi: 10.1038/1700. [DOI] [PubMed] [Google Scholar]
  39. Neufeld KL, Nix DA, Bogerd H, Kang Y, Beckerle MC, Cullen BR, White RL. Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm. PNAS. 2000;97:12085–12090. doi: 10.1073/pnas.220401797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nostro MC, Cheng X, Keller GM, Gadue P. Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood. Cell Stem Cell. 2008;2:60–71. doi: 10.1016/j.stem.2007.10.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Paik EJ, Zon LI. Hematopoietic development in the zebrafish. The International Journal of Developmental Biology. 2010;54:1127–1137. doi: 10.1387/ijdb.093042ep. [DOI] [PubMed] [Google Scholar]
  42. Palis J. Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo. FEBS Letters. 2016;590:3965–3974. doi: 10.1002/1873-3468.12459. [DOI] [PubMed] [Google Scholar]
  43. Paluru P, Hudock KM, Cheng X, Mills JA, Ying L, Galvão AM, Lu L, Tiyaboonchai A, Sim X, Sullivan SK, French DL, Gadue P. The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cells. Stem Cell Research. 2014;12:441–451. doi: 10.1016/j.scr.2013.12.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pilon N, Oh K, Sylvestre J-R, Bouchard N, Savory J, Lohnes D. Cdx4 is a direct target of the canonical Wnt pathway. Developmental Biology. 2006;289:55–63. doi: 10.1016/j.ydbio.2005.10.005. [DOI] [PubMed] [Google Scholar]
  45. Qi C, Zhu YT, Chang J, Yeldandi AV, Rao MS, Zhu YJ. Potentiation of estrogen receptor transcriptional activity by breast cancer amplified sequence 2. Biochemical and Biophysical Research Communications. 2005;328:393–398. doi: 10.1016/j.bbrc.2004.12.187. [DOI] [PubMed] [Google Scholar]
  46. Rallapalli R, Strachan G, Cho B, Mercer WE, Hall DJ. A novel MDMX transcript expressed in a variety of transformed cell lines encodes a truncated protein with potent p53 repressive activity. The Journal of Biological Chemistry. 1999;274:8299–8308. doi: 10.1074/jbc.274.12.8299. [DOI] [PubMed] [Google Scholar]
  47. Reischauer S, Stone OA, Villasenor A, Chi N, Jin S-W, Martin M, Lee MT, Fukuda N, Marass M, Witty A, Fiddes I, Kuo T, Chung W-S, Salek S, Lerrigo R, Alsiö J, Luo S, Tworus D, Augustine SM, Mucenieks S, Nystedt B, Giraldez AJ, Schroth GP, Andersson O, Stainier DYR. Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification. Nature. 2016;535:294–298. doi: 10.1038/nature18614. [DOI] [PubMed] [Google Scholar]
  48. Richter J, Traver D, Willert K. The role of Wnt signaling in hematopoietic stem cell development. Critical Reviews in Biochemistry and Molecular Biology. 2017;52:414–424. doi: 10.1080/10409238.2017.1325828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rosin-Arbesfeld R, Cliffe A, Brabletz T, Bienz M. Nuclear export of the APC tumour suppressor controls beta-catenin function in transcription. The EMBO Journal. 2003;22:1101–1113. doi: 10.1093/emboj/cdg105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Salmerón-Hernández Á, Noriega-Reyes MY, Jordan A, Baranda-Avila N, Langley E. BCAS2 enhances carcinogenic effects of estrogen receptor alpha in breast cancer cells. International Journal of Molecular Sciences. 2019;20:966. doi: 10.3390/ijms20040966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schmierer B, Hill CS. Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads. Molecular and Cellular Biology. 2005;25:9845–9858. doi: 10.1128/MCB.25.22.9845-9858.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stanic K, Reig G, Figueroa RJ, Retamal PA, Wichmann IA, Opazo JC, Owen GI, Corvalán AH, Concha ML, Amigo JD. The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish. Scientific Reports. 2019;9:7131. doi: 10.1038/s41598-019-43436-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stoick-Cooper CL, Weidinger G, Riehle KJ, Hubbert C, Major MB, Fausto N, Moon RT. Distinct Wnt signaling pathways have opposing roles in appendage regeneration. Development. 2007;134:479–489. doi: 10.1242/dev.001123. [DOI] [PubMed] [Google Scholar]
  54. Sturgeon CM, Ditadi A, Awong G, Kennedy M, Keller G. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nature Biotechnology. 2014;32:554–561. doi: 10.1038/nbt.2915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sun H, Wang Y, Zhang J, Chen Y, Liu Y, Lin Z, Liu M, Sheng K, Liao H, Tsang KS, Zhang X, Jiang X, Xu W, Mao M, Chan HC. CFTR mutation enhances Dishevelled degradation and results in impairment of Wnt-dependent hematopoiesis. Cell Death & Disease. 2018;9:275. doi: 10.1038/s41419-018-0311-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tarafdar A, Dobbin E, Corrigan P, Freeburn R, Wheadon H. Canonical Wnt signaling promotes early hematopoietic progenitor formation and erythroid specification during embryonic stem cell differentiation. PLOS ONE. 2013;8:e81030. doi: 10.1371/journal.pone.0081030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tran HT, Sekkali B, Van Imschoot G, Janssens S, Vleminckx K. Wnt/beta-catenin signaling is involved in the induction and maintenance of primitive hematopoiesis in the vertebrate embryo. PNAS. 2010;107:16160–16165. doi: 10.1073/pnas.1007725107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Vogeli KM, Jin S-W, Martin GR, Stainier DYR. A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula. Nature. 2006;443:337–339. doi: 10.1038/nature05045. [DOI] [PubMed] [Google Scholar]
  59. Wallingford MC, Giachelli CM. Loss of PiT-1 results in abnormal endocytosis in the yolk sac visceral endoderm. Mechanisms of Development. 2014;133:189–202. doi: 10.1016/j.mod.2014.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wang LP, Chen TY, Kang CK, Huang HP, Chen SL. BCAS2, a protein enriched in advanced prostate cancer, interacts with NBS1 to enhance DNA double-strand break repair. British Journal of Cancer. 2020;123:1796–1807. doi: 10.1038/s41416-020-01086-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wei S, Dai M, Liu Z, Ma Y, Shang H, Cao Y, Wang Q. The guanine nucleotide exchange factor Net1 facilitates the specification of dorsal cell fates in zebrafish embryos by promoting maternal β-catenin activation. Cell Research. 2017;27:202–225. doi: 10.1038/cr.2016.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Welten MCM, de Haan SB, van den Boogert N, Noordermeer JN, Lamers GEM, Spaink HP, Meijer AH, Verbeek FJ. ZebraFISH: fluorescent in situ hybridization protocol and three-dimensional imaging of gene expression patterns. Zebrafish. 2006;3:465–476. doi: 10.1089/zeb.2006.3.465. [DOI] [PubMed] [Google Scholar]
  63. Wolff B, Sanglier JJ, Wang Y. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chemistry & Biology. 1997;4:139–147. doi: 10.1016/s1074-5521(97)90257-x. [DOI] [PubMed] [Google Scholar]
  64. Xu L, Massagué J. Nucleocytoplasmic shuttling of signal transducers. Nature Reviews. Molecular Cell Biology. 2004;5:209–219. doi: 10.1038/nrm1331. [DOI] [PubMed] [Google Scholar]
  65. Xu Q, Wang F, Xiang Y, Zhang X, Zhao Z-A, Gao Z, Liu W, Lu X, Liu Y, Yu X-J, Wang H, Huang J, Yi Z, Gao S, Li L. Maternal BCAS2 protects genomic integrity in mouse early embryonic development. Development. 2015;142:3943–3953. doi: 10.1242/dev.129841. [DOI] [PubMed] [Google Scholar]
  66. Yamaguchi TP. Heads or tails: Wnts and anterior-posterior patterning. Current Biology. 2001;11:R713–R24. doi: 10.1016/s0960-9822(01)00417-1. [DOI] [PubMed] [Google Scholar]
  67. Yang S, Ning G, Hou Y, Cao Y, Xu J, Wu J, Zhang T, Wang Q. Myoneurin regulates BMP signaling by competing with Ppm1a for Smad binding. iScience. 2022;25:104495. doi: 10.1016/j.isci.2022.104495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yang S, Cao S, Xu X, Li Q, Li J, Guo J, Wang F, Bao Y, Jiang Z, Zhang T, Wang L, Sun S. adducin 1 is essential for the survival of erythroid precursors via regulating p53 transcription in zebrafish. iScience. 2023;26:107516. doi: 10.1016/j.isci.2023.107516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Yu S, Jiang T, Jia D, Han Y, Liu F, Huang Y, Qu Z, Zhao Y, Tu J, Lv Y, Li J, Hu X, Lu Z, Han S, Qin Y, Liu X, Xie S, Wang QK, Tang Z, Luo D, Liu M. BCAS2 is essential for hematopoietic stem and progenitor cell maintenance during zebrafish embryogenesis. Blood. 2019;133:805–815. doi: 10.1182/blood-2018-09-876599. [DOI] [PubMed] [Google Scholar]
  70. Yu F, Yu C, Li F, Zuo Y, Wang Y, Yao L, Wu C, Wang C, Ye L. Wnt/β-catenin signaling in cancers and targeted therapies. Signal Transduction and Targeted Therapy. 2021;6:701. doi: 10.1038/s41392-021-00701-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:1461–1473. doi: 10.1038/onc.2016.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Zhang J, Liu W, Li G, Xu C, Nie X, Qin D, Wang Q, Lu X, Liu J, Li L. BCAS2 is involved in alternative splicing and mouse oocyte development. The FASEB Journal. 2022;36:e22128. doi: 10.1096/fj.202101279R. [DOI] [PubMed] [Google Scholar]

eLife Assessment

Eirini Trompouki 1

This important work supports the role of breast carcinoma amplified sequence 2 (Bcas2) in positively regulating primitive wave hematopoiesis through amplification of beta-catenin-dependent (canonical) Wnt signaling. The study is convincing: it uses appropriate and validated methodology in line with the current state-of-the-art, and there is a first-rate analysis of a strong phenotype with highly supportive mechanistic data. The findings shed light on the controversial question of whether, when, and how canonical Wnt signaling may be involved in hematopoietic development. The work will be of interest to hematologists and developmental biologists.

Reviewer #1 (Public review):

Anonymous

Summary:

In this manuscript, Ning et al. reported that Bcas2 played an indispensable role in zebrafish primitive hematopoiesis via sequestering β-catenin in the nucleus. The authors showed that loss of Bcas2 caused primitive hematopoietic defects in zebrafish. They unraveled that Bcas2 deficiency promoted β-catenin nuclear export via a CRM1-dependent manner in vivo and in vitro. They further validated that BCAS2 directly interacted with β-catenin in the nucleus and enhanced β-catenin accumulation through its CC domains. They unveil a novel insight into Bcas2, which is critical for zebrafish primitive hematopoiesis via regulating nuclear β-catenin stabilization rather than its canonical pre-mRNA splicing functions. Overall, the study is impressive and well-performed, although there are also some issues to address.

Strengths:

The study unveils a novel function of Bcas2, which is critical for zebrafish primitive hematopoiesis by sequestering β-catenin. The authors validated the results in vivo and in vitro. Most of the figures are clear and convincing. This study nicely complements the function of Bcas2 in primitive hematopoiesis.

Comments on revisions:

The authors have nicely answered all my questions, I have no problem.

Reviewer #2 (Public review):

Anonymous

Summary:

Ning and colleagues present studies supporting a role for breast carcinoma amplified sequence 2 (Bcas2) in positively regulating primitive wave hematopoiesis through amplification of beta-catenin-dependent (canonical) Wnt signaling. The authors present compelling evidence that zebrafish bcas2 is expressed at the right time and place to be involved in primitive hematopoiesis, that there are primitive hematopoietic defects in hetero- and homozygous mutant and knockdown embryos, that Bcas2 mechanistically positively regulates canonical Wnt signaling, and that Bcas2 is required for nuclear retention of B-cat through physical interaction involving armadillo repeats 9-12 of B-cat and the coiled-coil domains of Bcas2. Overall, the data and writing are clean, clear, and compelling. This study is a first rate analysis of a strong phenotype with highly supportive mechanistic data. The findings shed light on the controversial question of whether, when, and how canonical Wnt signaling may be involved in hematopoietic development.

In the revised version of their previous work, they have included responses to some of our suggestions for minor experiments and edits. We previously suggested they examine the structural compatibility of a Bcas2/beta-catenin dimer with binding to the DNA-binding protein Tcf7l1 (previously Tcf3), which would be expected for a beta-catenin nuclear-retention factor that potentiates canonical Wnt signaling responses. Although the authors did not test compatibility of Bcas2 with Tcf3 binding to beta-catenin, they show that a three-way complex with the family member Tcf4 is possible (Fig. S12), which suggests that Lef/Tcf family binding in general is plausible.

The authors' acceptance of our suggestion to evaluate cdx and hox gene expression is welcome, as these genes have previously been defined as canonical Wnt targets (Lengerke et al., 2009) that regionalize the lateral plate mesoderm (LPM) and confer pre-hematopoietic identity there (Davidson et al., 2003; Davidson and Zon, 2004). The authors' finding that cdx4 and hoxa9a are diminished in the bcas2 mutants (Fig. S7) validates this suggestion and seem to imply that the primary defect here is specification of the early hematopoietic field in the LPM, however the results are a little confusing or surprising given that scl - which is unaffected in the bcas2 mutant (Fig. 2A) - is a downstream target of Cdx4 (Davidson et al., 2003, Fig. 1b, 3d). The results in the current submission imply that early maintenance of pre-hematopoietic competence in the LPM is a canonical-Wnt-directed phenomenon separable from the earliest specification of the hematopoietic field. We believe it would be of value to further evaluate regulation of cdx1, which has been shown to cooperate with cdx4 in regulation of the LPM hematopoietic field, as well as analyze some of the putative downstream hox family targets.

We previously reviewed the article as suitable for publication and we continue to support our prior assessment. The authors have presented strong data supporting a role for Bcas2 in hematopoietic development across phyla and a mechanistic involvement in promoting canonical Wnt signaling.

Strengths:

(1) The study features clear and compelling phenotypes and results.

(2) The manuscript narrative exposition and writing are clear and compelling.

(3) The authors have attended to important technical nuances sometimes overlooked, for example, focusing on different pools of cytosolic or nuclear b-catenin.

(4) The study sheds light on a controversial subject: regulation of hematopoietic development by canonical Wnt signaling and presents clear evidence of a role.

(5) The authors present evidence of phylogenetic conservation of the pathway.

Reviewer #3 (Public review):

Anonymous

Summary:

This manuscript utilized zebrafish bcas2 mutants to study the role of bcas2 in primitive hematopoiesis, and further confirms that it has a similar function in mice. Moreover, they showed that bcas2 regulates the transition of hematopoietic differentiation from angioblasts via activating Wnt signaling. By performing a series of biochemical experiments, they also showed that bcas2 accomplishes this by sequestering b-catenin within the nucleus, rather than through its known function in pre-mRNA splicing.

Strengths:

The work is well-performed, and the manuscript is well-written.

Comments on revisions:

The revised manuscript is substantially improved, and all my previous questions are now well addressed.

eLife. 2025 Jun 13;13:RP100497. doi: 10.7554/eLife.100497.3.sa4

Author response

Guozhu Ning 1, Yu Lin 2, Haixia Ma 3, Jiaqi Zhang 4, Liping Yang 5, Zhengyu Liu 6, Lei Li 7, Xinyu He 8, Qiang Wang 9

The following is the authors’ response to the original reviews

Reviewer #1 (Public Review):

Summary:

In this manuscript, Ning et al. reported that Bcas2 played an indispensable role in zebrafish primitive hematopoiesis via sequestering β-catenin in the nucleus. The authors showed that loss of Bcas2 caused primitive hematopoietic defects in zebrafish. They unraveled that Bcas2 deficiency promoted β-catenin nuclear export via a CRM1-dependent manner in vivo and in vitro. They further validated that BCAS2 directly interacted with β-catenin in the nucleus and enhanced β-catenin accumulation through its CC domains. They unveil a novel insight into Bcas2, which is critical for zebrafish primitive hematopoiesis via regulating nuclear β-catenin stabilization rather than its canonical pre-mRNA splicing functions. Overall, the study is impressive and well-performed, although there are also some issues to address.

Strengths:

The study unveils a novel function of Bcas2, which is critical for zebrafish primitive hematopoiesis by sequestering β-catenin. The authors validated the results in vivo and in vitro. Most of the figures are clear and convincing. This study nicely complements the function of Bcas2 in primitive hematopoiesis.

Weaknesses:

A portion of the figures were over-exposed.

Thank you for the time reviewing our manuscript. We agree with your suggestion and the exposure of Figure 5C and Figure 7E has been reduced. We hope that the revisions will meet your expectation.

Reviewer #2 (Public Review):

Summary:

Ning and colleagues present studies supporting a role for breast carcinoma amplified sequence 2 (Bcas2) in positively regulating primitive wave hematopoiesis through amplification of beta-catenin-dependent (canonical) Wnt signaling. The authors present compelling evidence that zebrafish bcas2 is expressed at the right time and place to be involved in primitive hematopoiesis, that there are primitive hematopoietic defects in hetero- and homozygous mutant and knockdown embryos, that Bcas2 mechanistically positively regulates canonical Wnt signaling, and that Bcas2 is required for nuclear retention of B-cat through physical interaction involving armadillo repeats 9-12 of B-cat and the coiled-coil domains of Bcas2. Overall, the data and writing are clean, clear, and compelling. This study is a first-rate analysis of a strong phenotype with highly supportive mechanistic data. The findings shed light on the controversial question of whether, when, and how canonical Wnt signaling may be involved in hematopoietic development. We detail some minor concerns and questions below, which if answered, we believe would strengthen the overall story and resolve some puzzling features of the phenotype. Notwithstanding these minor concerns, we believe this is an exceptionally well-executed and interesting manuscript that is likely suitable for publication with minor additional experimental detail and commentary.

Strengths:

(1) The study features clear and compelling phenotypes and results.

(2) The manuscript narrative exposition and writing are clear and compelling.

(3) The authors have attended to important technical nuances sometimes overlooked, for example, focusing on different pools of cytosolic or nuclear b-catenin.

(4) The study sheds light on a controversial subject: regulation of hematopoietic development by canonical Wnt signaling and presents clear evidence of a role.

(5) The authors present evidence of phylogenetic conservation of the pathway.

Weaknesses:

(1) The authors present compelling data that Bcas2 regulates nuclear retention of B-cat through physical association involving binding between the Bcas2 CC domains and B-cat arm repeats 9-12. Transcriptional activation of Wnt target genes by B-cat requires physical association between B-cat and Tcf/Lef family DNA binding factors involving key interactions in Arm repeats 2-9 (Graham et al., Cell 2000). Mutually exclusive binding by B-cat regulatory factors, such as ICAT that prevent Tcf-binding is a documented mechanism (e.g. Graham et al., Mol Cell 2002). It would appear - based on the arm repeat usage by Bcas2 (repeats 9-12)-that Bcas2 and Tcf binding might not be mutually exclusive, which would support their model that Bcas2 physical association with B-cat to retain it in the nucleus would be compatible with co-activation of genes by allowing association with Tcf. It might be nice to attempt a three-way co-IP of these factors showing that B-cat can still bind Tcf in the presence of Bcas2, or at least speculate on the plausibility of the three-way interaction.

We appreciate your assessment and generous comments for the manuscript. As you mentioned, the binding sites for TCF on β-catenin almost do not overlap with those for BCAS2. It is likely that BCAS2-mediated nuclear sequestration of β-catenin would be compatible with the initiation of gene transcription by allowing TCF to associate with β-catenin. To test this possibility, we have taken your suggestion and performed co-IP assays. The results showed that β-catenin still bound with TCF4 in the presence of BCAS2 (Supplemental Figure 12), confirming that the binding of BCAS2 to β-catenin would not interfere with the formation of β-catenin/TCF complex.

(2) A major way that canonical Wnt signaling regulates hematopoietic development is through regulation of the LPM hematopoietic competence territories by activating expression of cdx1a, cdx4, and their downstream targets hoxb5a and hoxa9a (Davidson et al., Nature 2003; Davidson et al., Dev Biol 2006; Pilon et al., Dev Biol 2006; Wang et al., PNAS 2008). Could the authors assess (in situ) the expression of cdx1a, cdx4, hoxb5a, and hoxa9a in the bcas2 mutants?

We agree with your suggestion and have examined the expression of cdx4 and hoxa9a by performing WISH. Diminished expression of cdx4 and hoxa9a was detected in the lateral plate mesoderm of bcas2+/- embryos at the 6-somite stage (Supplemental Figure 7).

(3) The authors show compellingly that even heterozygous loss of bcas2 has strong Wnt-inhibitory effects. If Bcas2 is required for canonical Wnt signaling and bcas2 is expressed ubiquitously from the 1-cell stage through at least the beginning of gastrulation, why do bcas2 KO embryos not have morphological axis specification defects consistent with loss of early Wnt signaling, like loss of head (early), or brain anteriorization (later)? Could the authors provide some comments on this puzzle? Or if they do see any canonical Wnt signaling patterning defects in het- or homozygous embryos, could they describe and/or present them?

You have raised an interesting question. In fact, we did not observe ventralization or axis determination defects in the early embryos of bcas2+/- mutants. Even in the very small number of homozygous mutant embryos, we did not find such morphological defects. Given that the homozygous and heterozygous mutant embryos were derived from crossing bcas2+/- males with bcas2+/- females, maternal Bcas2 might still remain and function in these embryos during gastrulation when axis determination and neural patterning took place. Accordingly, we have expanded our discussion to incorporate these insights (Line 565-572).

Reviewer #3 (Public Review):

Summary:

This manuscript utilized zebrafish bcas2 mutants to study the role of bcas2 in primitive hematopoiesis and further confirms that it has a similar function in mice. Moreover, they showed that bcas2 regulates the transition of hematopoietic differentiation from angioblasts via activating Wnt signaling. By performing a series of biochemical experiments, they also showed that bcas2 accomplishes this by sequestering b-catenin within the nucleus, rather than through its known function in pre-mRNA splicing.

Strengths:

The work is well-performed, and the manuscript is well-written.

Weaknesses:

Several issues need to be clarified.

(1) Is wnt signaling also required during hematopoietic differentiation from angioblasts? Can the authors test angioblast and endothelial markers in embryos with wnt inhibition? Also, can the authors add export inhibitor LMB to the mouse mutants to test if sequestering of b-catenin by bcas2 is conserved during primitive hematopoiesis in mice?

Thank you very much for your appreciation and detailed assessment. To test whether Wnt signaling is also required during hematopoietic differentiation from angioblasts, wild-type embryos were exposed to 10 µM CCT036477, a small molecule β-catenin antagonist, from 9 hpf and then collected for WISH experiments. As shown in Supplemental Figure 8, the expression of hemangioblast markers npas4l, scl, and gata2 and endothelial marker fli1a remained unchanged, but the expression of erythroid progenitor marker gata1 was significantly reduced. These results suggest that canonical Wnt pathway may not be required for the generation of hemangioblasts or their endothelial differentiation, but is pivotal for their hematopoietic differentiation.

It is quite difficult to validate the conserve role of BCAS2 during primitive hematopoiesis in mice, because the toxicity of LMB may cause severe adverse effects in mice.[1,2]

(2) Bcas2 is required for primitive myelopoiesis in ALM. Does bcas2 play a similar function in primitive myelopoiesis, or is bcas2/b-catenin interaction more important for hematopoietic differentiation in PLM?

You have raised an important question. In our study, we have demonstrated that the expression of myeloid progenitor marker pu.1 was significantly decreased in bcas2 mutants, hinting that Bcas2 is pivotal for primitive myelopoiesis. To further clarify the function of Bcas2 in primitive myelopoiesis, we injected 8 ng of bcas2 morpholino into Tg(coro1a:GFP) embryos at the 1-cell stage and examined β-catenin distribution at 17 hpf via immunostaining. We observed a significant decline of nuclear β-catenin in primitive myeloid cells (Supplemental Figure 9), indicating that Bcas2 is highly likely to play a similar role in sequestering β-catenin within the nucleus during primitive myelopoiesis.

(3) Is it possible that CC1-2 fragment sequester b-catenin? The different phenotypes between this manuscript and the previous article (Yu, 2019) may be due to different mutations in bcas2. Is it possible that the bcas2 mutation in Yu's article produces a complete CC1-2 fragment, which might sequester b-catenin?

This is an interesting perspective. To test the possibility that CC1-2 sequesters β-catenin, mRNA expressing the CC domains of BCAS2 has been co-injected with bcas2 morpholino into Tg(gata1:GFP) embryo at the one-cell stage. Increased nuclear β-catenin levels were detected in the GFP-positive hematopoietic progenitor cells at 16 hpf (Supplemental Figure 11). Our findings support that CC1-2 fragment of BCAS2 can sequester β-catenin within the nucleus.

In the previous article (Yu, 2019), a deletion 5 bases mutation in the third exon of BCAS2 was produced by TALEN, therefore the CC domains of this mutant should be affected. It is difficult to conclude that the mutant BCAS2 protein in Yu’s study still remains association with β-catenin.

(4) Can the author clarify what embryos the arrows point to in SI Figure 2D? In SI Figure 6B and B', can the author clarify how the nucleus and cytoplasm are bleached? In B, the nucleus also appears to be bleached.

Thank you for your query and suggestion. In our revisions, the corresponding clarifications have been supplemented (Line 239-242; Line 978-979).

We acknowledge that the nuclei in both the BCAS2 overexpression group and control group were slightly bleached. Given that we have performed real-time analysis for fluorescent recovery after photobleaching, and we have observed a much slower recovery of cytoplasmic fluorescence in BCAS2 overexpressed cells, the conclusion that BCAS2 inhibits the nuclear export of β-catenin but not its nuclear import, remains changed.

Reviewer #1 (Recommendations For The Authors):

Major concerns:

(1) In this study, the authors detected β-catenin distribution in erythrocytes (gata1-GFP+ cells). Estimating the β-catenin distribution in the myeloid cells is recommended.

Thank you for your assessment and we have taken your suggestion. Tg(coro1a:GFP) embryos, which is commonly used to track both macrophages and neutrophils,[3] were injected with 8 ng of bcas2 morpholino into at the 1-cell stage and collected for immunostaining to examine the β-catenin distribution at 17 hpf. We observed a significant decline of nuclear β-catenin in primitive myeloid cells (Supplemental Figure 9). This result indicates that Bcas2 is highly likely to play a similar role in sequestering β-catenin within the nucleus during primitive myelopoiesis.

(2) The reduced nuclear localization of β-catenin in Figure 3H required further evidence. It would be helpful if the authors quantified the fluorescence intensity in the cell nucleus and cytoplasm. Meanwhile, the figures (Figure 5C, Figure 7E) were over-exposed. Please validate these figures.

Thank you for your suggestions. We agree with you that the fluorescence intensity of β-catenin in the nucleus and cytoplasm should be quantified. However, as the nucleus comprises a large part of the cell, we believe it would be more appropriate to quantify the relative fluorescence intensity by dividing the fluorescence intensity of nuclear β-catenin by the fluorescence intensity of DAPI.

Such quantifications have been added for Figure 3G, 5C, 7E, S9A, and S13A. In addition, we have reduced the exposure of Figure 5C and Figure 7E. We hope that you will be satisfied with the revisions.

(3) The authors used cKO mice to validate that the erythrocytes were eliminated. It would be interesting to detect β-catenin distribution by immunofluorescent staining in primitive hematopoietic cells in cKO mice. Addressing this issue can provide further evidence to support the conservation of Bcas2.

We appreciate your suggestion. However, we found that red blood cells were almost eliminated in the yolk sac of Bcas2F/F;Flk1-Cre mice at E12.5. It is difficult to further detect β-catenin distribution in primitive erythroid cells in these mice.

(4) The authors discovered that Bcas2 mediated β-catenin nuclear export in a CRM1-dependent manner. CRM1 is a key regulator involved in the majority of factors of nuclear export via recognizing specific nuclear export signals (NES). Validating the NES of Bcas2 is recommended. Furthermore, I wonder about the relationship between Bcas2 and CRM1 in regulating β-catenin nuclear export. One possibility is that Bcas2 covers the NES to inhibit the interaction between CRM1 and β-catenin, thus leading to β-catenin accumulation in the cell nucleus. The authors should discuss this possibility accordingly.

Thank you for providing an interesting perspective. CRM1-mediated nuclear export of β-catenin usually requires CRM1 recognition and binding with the NES sequences in chaperon proteins, such as APC, Axin and Chibby.[4-6] Moreover, CRM1 can bind directly to and function as an efficient nuclear exporter for β-catenin.[7] Since BCAS2 has not been reported to contain any recognizable NES sequences, it will be interesting to investigate whether BCAS2 competitively inhibits β-catenin from associating with CRM1, or with the chaperone proteins. We have rewritten the discussion on CRM1-dependent nuclear export of β-catenin in line with your comments (Line 572-578).

(5) It would be interesting if the authors could answer the specificity in Bcas2-mediated protein nuclear export pathway. The authors should detect other classical factors (CRM1 mediated) distribution when loss of Bcas2.

Thank you for bringing up this point. To test whether BCAS2 specifically regulates CRM1-mediated nuclear export of β-catenin, we have investigated the nucleocytoplasmic distribution of other known CRM1 cargoes, such as ATG3 and CDC37L.[8] BCAS2 overexpression in HeLa cells slightly enhanced the nuclear localization of CDC37L, and had no significant impact on that of ATG3 (Supplemental Figure 11), indicating the specificity of BCAS2 in the regulation of CRM1-dependent nuclear export of β-catenin.

Minor concerns:

(1) The name "bcas2Δ7+/- and bcas2Δ14+/-" should be changed into "bcas2+/Δ7 and bcas2+/Δ14"(+/Δ7 or +/Δ14 should be superior on the right).

Thank you for your suggestion. We have changed the names of the mutants throughout the manuscript.

(2) The scale bar position in the figures should be unified.

We agree with your suggestion and have unified the scale bar position in all figures.

(3) In Figure 4E, "Nuclear" should be changed into "Nucleus".

We apologize for the mistake and Figure 4E has been revised.

(4) There are some unaesthetic issues in the figures. The figures need to be further edited. Figure 3H "β-catenin and Merge", Figure 4D "Merge". All these words should be centered in the figures.

Thank you. We have edited all the figures to ensure that the text is centered.

Reviewer #2 (Recommendations For The Authors):

(1) It would be nice to have whole blot images for the Westerns in Supplementary Info.

Thank you for your suggestion. Whole images for immunoblotting have been supplemented as Source data.

(2) Line 292 change 5 hpf to 5 dpf.

(3) Line 301 change "primary" to "primitive"?

We apologize for the mistakes. We have incorporated these suggestions in the revised manuscript and reexamined spelling throughout the paper.

(4) Figure S2C: is "Maker" a typographical error? Change to "ladder"?

We apologize for this typographical error and we have revised it in Figure S2C.

Reference

(1) Ishizawa J, Kojima K, Hail N, Tabe Y, Andreeff M. Expression, function, and targeting of the nuclear exporter chromosome region maintenance 1 (CRM1) protein. Pharmacology & Therapeutics. 2015;153:25-35.

(2) Li X, Feng Y, Yan MF, et al. Inhibition of Autism-Related Crm1 Disrupts Mitosis and Induces Apoptosis of the Cortical Neural Progenitors. Cerebral Cortex. 2020;30(7):3960-3976.

(3) Li L, Yan B, Shi YQ, Zhang WQ, Wen ZL. Live Imaging Reveals Differing Roles of Macrophages and Neutrophils during Zebrafish Tail Fin Regeneration. Journal of Biological Chemistry. 2012;287(30):25353-25360.

(4) Neufeld KL, Nix DA, Bogerd H, et al. Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(22):12085-12090.

(5) Li FQ, Mofunanya A, Harris K, Takemaru KI. Chibby cooperates with 14-3-3 to regulate β-catenin subcellular distribution and signaling activity. Journal of Cell Biology. 2008;181(7):1141-1154.

(6) Cong F, Varmus H. Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of β-catenin. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(9):2882-2887.

(7) Ki H, Oh M, Chung SW, Kim K. β-Catenin can bind directly to CRM1 independently of adenomatous polyposis coli, which affects its nuclear localization and LEF-1/β-catenin-dependent gene expression. Cell Biology International. 2008;32(4):394-400.

(8) Kirli K, Karaca S, Dehne HJ, et al. A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning. Elife. 2015;4.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Ning G, Lin Y. 2025. BCAS2 promotes primitive hematopoiesis by sequestering β-catenin within the nucleus. NCBI Gene Expression Omnibus. GSE297155 [DOI] [PMC free article] [PubMed]

    Supplementary Materials

    Figure 1—figure supplement 2—source data 1. PDF file containing original gel images for Figure 1—figure supplement 2C with the relevant bands and treatments indicated.
    elife-100497-fig1-figsupp2-data1.zip (382KB, zip)
    Figure 1—figure supplement 2—source data 2. Original gel images in Figure 1—figure supplement 2C.
    elife-100497-fig1-figsupp2-data2.zip (3.1MB, zip)
    Figure 1—figure supplement 4—source data 1. Original western blots for Figure 1—figure supplement 4A with the relevant bands and treatments indicated.
    elife-100497-fig1-figsupp4-data1.zip (127.2KB, zip)
    Figure 1—figure supplement 4—source data 2. Original western blot images in Figure 1—figure supplement 4A.
    elife-100497-fig1-figsupp4-data2.zip (389.7KB, zip)
    Figure 3—source data 1. Original western blots for Figure 3D, indicating the relevant bands and treatments.
    elife-100497-fig3-data1.zip (38.9KB, zip)
    Figure 3—source data 2. Original western blot images in Figure 3D.
    elife-100497-fig3-data2.zip (241.4KB, zip)
    Figure 3—figure supplement 1—source data 1. Original western blots for Figure 3—figure supplement 1 with the relevant bands labeled.
    elife-100497-fig3-figsupp1-data1.zip (84.8KB, zip)
    Figure 3—figure supplement 1—source data 2. Uncropped immunoblotting images in Figure 3—figure supplement 1.
    elife-100497-fig3-figsupp1-data2.zip (395.7KB, zip)
    Figure 4—source data 1. Original western blots for Figure 4E with the relevant bands and treatments labeled.
    elife-100497-fig4-data1.zip (150.5KB, zip)
    Figure 4—source data 2. Original western blot images in Figure 4E.
    elife-100497-fig4-data2.zip (527.5KB, zip)
    Figure 6—source data 1. Original western blots for Figure 6A–C, G and H with the relevant bands and treatments indicated.
    elife-100497-fig6-data1.zip (1.2MB, zip)
    Figure 6—source data 2. Uncropped immunoblotting images in Figure 6A–C, G and H.
    elife-100497-fig6-data2.zip (5.4MB, zip)
    Figure 6—figure supplement 1—source data 1. Original western blots for Figure 6—figure supplement 1, indicating the relevant bands and treatments.
    elife-100497-fig6-figsupp1-data1.zip (410KB, zip)
    Figure 6—figure supplement 1—source data 2. Original files for western blot analysis in Figure 6—figure supplement 1.
    elife-100497-fig6-figsupp1-data2.zip (4.7MB, zip)
    Figure 7—source data 1. Original western blots for Figure 7B and C, indicating the relevant bands and treatments.
    elife-100497-fig7-data1.zip (357.8KB, zip)
    Figure 7—source data 2. Original files for western blot analysis in Figure 7B and C.
    elife-100497-fig7-data2.zip (1.3MB, zip)
    Figure 7—figure supplement 2—source data 1. PDF file containing original gel images for Figure 7—figure supplement 2B, C, D with the relevant bands indicated.
    elife-100497-fig7-figsupp2-data1.zip (349.4KB, zip)
    Figure 7—figure supplement 2—source data 2. Original gel images in Figure 7—figure supplement 2B, C, D.
    elife-100497-fig7-figsupp2-data2.zip (29.9MB, zip)
    MDAR checklist
    elife-100497-mdarchecklist1.docx (88.7KB, docx)
    Supplementary file 1. Primers used for genotyping.
    elife-100497-supp1.docx (16.1KB, docx)
    Supplementary file 2. Primers used for reverse transcription-PCR.
    elife-100497-supp2.docx (16KB, docx)

    Data Availability Statement

    All data generated or analyzed during this study are included in the manuscript and/or supplementary materials. RNA sequencing data have been deposited in GEO under accession codes GSE297155. Original western blot images have been provided as source data.

    The following dataset was generated:

    Ning G, Lin Y. 2025. BCAS2 promotes primitive hematopoiesis by sequestering β-catenin within the nucleus. NCBI Gene Expression Omnibus. GSE297155

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