Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2019 Nov 1.
Published in final edited form as: FEBS J. 2018 Sep 20;285(21):4071–4081. doi: 10.1111/febs.14653

phlda3 overexpression impairs specification of hemangioblasts and vascular development

Xiaojing Wang 1,#, Jia Li 1,#, Zhongcheng Yang 1,#, Li Wang 1,#, Lei Li 1, Wenqing Deng 1, Juan Zhou 1, Longfei Wang 1, Chengqi Xu 1, Qiuyun Chen 2,*, Qing K Wang 1,2,*
PMCID: PMC6218282  NIHMSID: NIHMS988504  PMID: 30188605

Abstract

The phlda3 gene encodes a small, 127 amino acid protein with only a PH domain, and is involved in tumor suppression, proliferation of islet β-cells, insulin secretion, glucose tolerance, and liver injury. However, the role of phlda3 in vascular development is unknown. Here, we show that phlda3 overexpression decreases the expression levels of hemangioblast markers scl, fli1 and etsrp and intersegmental vessel (ISV) markers flk1 and cdh5, and disrupts ISV development in tg(flk1:GFP) and tg(fli1:GFP) zebrafish. Moreover, phlda3 overexpression inhibits the activation of AKT in zebrafish embryos, and the developmental defects of ISVs by phlda3 overexpression were reversed by the expression of a constitutively active form of AKT. These data suggest that phlda3 is a negative regulator of hemangioblast specification and ISV development via AKT signaling.

Keywords: phlda3, hemangioblast, vascular development, hematopoiesis, AKT

Description to the Graphic Abstract:

Hemangioblasts are multipotent progenitor cells that differentiate into hematopoietic and endothelial cells. Although several factors for hemangioblast differentiation, such as aggf1, scl and etsrp, were identified, most hemangioblast regulators remain unknown. Here, we show that overexpression of phlda3 impairs hemangioblast specification and development of intersegmental vessels by inhibiting AKT signaling, suggesting that phlda3 is a negative regulator of hemangioblast specification.

graphic file with name nihms-988504-f0001.jpg

INTRODUCTION

Hemangioblasts are multipotent precursor cells that are differentiated from the mesoderm, and can further differentiate into both hematopoietic and endothelial cells [1, 2]. The differentiation of hemangioblasts is regulated by many transcription factors, including scl/tal1 [3–5], fli1 and etsrp [6–9]. The differentiation of hematopoietic cells (hematopoiesis) in zebrafish includes either primitive hematopoiesis or definitive hematopoiesis [10–12]. Primitive hematopoiesis can be regulated and marked by two early transcription factors gata1 [13, 14] and pu.1 [15–17]. Definitive hematopoiesis is regulated by two critical transcription factors, c-myb [18] and runx1 [19], to generate hematopoietic stem cells (HSCs), which are subsequently differentiated into blood cells with erythroid, myeloid, and lymphoid lineages. In the differentiation of endothelial cells, hemangioblasts are first differentiated into angioblasts, which later develop into vascular endothelial cells, resulting in the development of the primary vascular plexus (de novo formation of vessels; vasculogenesis) [20]. Dramatic expansion of the vascular plexus leads to the development of capillaries, arteries, and veins (angiogenesis) [1].

Together with Qian et al [21], we cloned the novel human gene IPL while searching for the long QT syndrome gene on chromosome 11p15 (later referred to as PHLDA2) through cDNA selection by using a bacterial artificial chromosome (BAC) at the 11p15.1 chromosomal region. The IPL protein showed a high degree of homology to the TDAG51 protein, later referred to as PHLDA1 [21]. The human PHLDA3 gene is the third member of this family of genes and encodes a PH domain-only protein [22]. The PHLDA3 protein was shown to block the activation of AKT by competing with the PH domain of AKT for the binding of lipids on the cellular membrane in tumor cell lines [22]. Knockout mice deficient in PHLDA3 demonstrated islet hyperplasia, enhanced proliferation of β-cells and insulin secretion, and improved glucose tolerance via increased AKT activation [23]. PHLDA3 blocked the generation of induced pluripotent stem cells by activating the AKT-GSK3β pathway [24]. Moreover, PHLDA3 overexpression was shown to enhance liver injury by inhibiting AKT [25]. However, the role of PHLDA3 in vascular development is unknown. Here, we used the zebrafish system to characterize the role of phlda3 in the specification of hemangioblasts, vascular development, primitive hematopoiesis, and definitive hematopoiesis. We show that phlda3 is involved in the specification of hemangioblasts and development of ISVs by regulating the AKT signaling pathway, but it does not play a role in hematopoiesis.

RESULTS

Molecular cloning and expression profile of zebrafish phlda3 gene

Zebrafish phlda3 is located on chromosome 23 and encodes a PH domain-only protein with 127 amino acids. The protein sequence of zebrafish PHLDA3 protein (RefSeq peptide: NP_001002455) shows a high degree of homology to the human PHLDA3 protein (NP_036528) (52% identity, 69% homology) and mouse PHLDA3 protein (NP_038778) (52% identity, 69% homology) (Fig. 1A and 1B).

Figure 1.

Figure 1.

Open in a new tab

Sequence homology of PHLDA3 proteins and the expression profile of the phlda3 gene during zebrafish embryogenesis. (A) Alignment of amino acid sequences for human PHLDA3 (NCBI accession number NP_036528), mouse PHLDA3 (NCBI accession number NP_038778) and zebrafish PHLDA3 (NCBI accession number NP_001002455). “*”, identical amino acid residues; “:”, highly homologous amino acid residues; “.”, homologous amino acid residues. (B) Percentage of homology shared among PHLDA3 proteins from different species. (C) Whole-mount in situ hybridization for phlda3 at different developmental stages during embryogenesis. I, 2-cell stage, top view from animal pole; II, 4-cell stage, top view from animal pole; III, 2.5 hpf, lateral view; IV, 6 hpf, lateral view; V, 12 hpf, lateral view; VI, 24 hpf, lateral view; VII, 48 hpf, lateral view. Scale bar, 200 μm.

We determined the expression pattern of phlda3 during zebrafish embryogenesis using whole-mount in situ hybridization. The expression of phlda3 was detected in two-cell stage embryos (Fig. 1C, I) and four-cell stage embryos (Fig. 1C, II), which suggests that phlda3 is maternally expressed. At the 2.5 hours post fertilization (hpf) stage (the beginning of blastula stage), phlda3 was localized in the blastomeres (Fig. 1C, III). At the 6 hpf stage and 12 hpf stage, phlda3 was expressed ubiquitously in embryos (Fig. 1C, IV and V). At 24 hpf, phlda3 expression appeared to be restricted to the head region and dorsal aorta (Fig. 1C, VI arrows). By 48 hpf, the phlda3 mRNA was predominantly expressed in the head and fin bud (Fig. 1C, VII arrows).

phlda3 regulates specification of hemangioblasts

Hemangioblasts are developed from the mesoderm during early embryogenesis [26, 27]. They are multipotent progenitor cells that can differentiate into both hematopoietic and endothelial cells. Transcription factor genes scl, fli1 and etsrp are markers for hemangioblasts [4, 5, 7, 8]. To investigate the role of phlda3 in the specification of hemangioblasts, vascular development and hematopoiesis, we studied the effects of overexpression of phlda3. We amplified the full-length zebrafish phlda3 (RefSeqDNA: NM_001002455) by RT-PCR analysis and cloned it into an expression vector to generate phlda3 mRNA. We injected one-cell stage embryos with either control mCherry mRNA or phlda3 mRNA. At 12 hpf, we collected the embryos and performed whole-mount in situ hybridization with antisense probes for scl, fli1 and etsrp. Whole-mount in situ hybridization data showed that the expression levels of scl, fli1 and etsrp were markedly reduced in embryos with an overexpression of phlda3 as compared to control embryos injected with mCherry mRNA (Fig. 2). These data suggest that an overexpression of phlda3 disrupts the specification of hemangioblasts.

Figure 2.

Figure 2.

Open in a new tab

Overexpression of phlda3 causes abnormal specification of hemangioblasts. Zebrafish embryos at the one-cell stage were injected with the mCherry mRNA (110 pg; A, C, E) and phlda3 mRNA (110 pg; B, D, F), and collected at 12 hpf for whole-mount in situ hybridization with antisense RNA probes for scl (A, B), fli1 (C, D) and etsrp (E, F). The expression levels of scl, fli1, and etsrp were markedly reduced in the posterior lateral plate mesoderm (PLPM) by overexpression of phlda3. Embryos are shown in a dorsal view with the anterior at the top. Scale bar, 200 μm.

phlda3 is involved in regulation of vascular development in zebrafish

Since phlda3 is involved in the specification of hemangioblasts, and hemangioblasts are differentiated into either vascular endothelial cells or hematopoietic cells, we investigated whether or not phlda3 affects vascular development. We injected phlda3 mRNA into one-cell stage embryos to overexpress phlda3, and then we performed whole-mount in situ hybridization with antisense probes for flt4, dab2 and notch3 at 28 hpf and for flk1 and cdh5 at 24 hpf. flt4 and dab2 are markers of the posterior cardinal vein (PCV) and notch3 is a marker of the dorsal aorta (DA) [27–29]. flk1 and cdh5 are markers of intersegmental vessels (ISVs), which connect the dorsal aorta to the dorsal longitudinal anastomotic vessels (DLAVs) [30, 31]. The whole-mount in situ hybridization data showed that compared with uninjected embryos or embryos injected with control mCherry mRNA, embryos injected with phlda3 mRNA displayed abnormal development of ISVs (incompletely developed ISVs sprouting from DA) (Fig. 3A, VII-XI, arrows). However, the overexpression of phlda3 did not affect the development of the DA or PCV (Fig. 3A, I-VI).

Figure 3.

Figure 3.

Open in a new tab

Overexpression of phlda3 causes abnormal development of ISVs during zebrafish embryogenesis. (A) Whole-mount in situ hybridization. Zebrafish embryos at the one-cell stage were injected with either mCherry mRNA (110 pg; I, III, V, VII, X) or phlda3 mRNA (110 pg; II, IV, VI, VIII, XI), and used for whole-mount in situ hybridization at 28 hpf with PCV probes flt4 (I, II) and dab2 (III, IV), DA probe notch3 (V, VI), and at 24 hpf with ISVs probes flk1 (VII, VIII) and cdh5 (IX - XI). The black arrows in VIII and XI are used to show incompletely developed ISVs sprouting from DA. Scale bar for I-VI, 200 μm, scale bar for VII and VIII, 200 μm, Scale bar for IX - XI, 200 μm. (B) Direct visualization of ISV development in tg(flk1:GFP) and tg(fli1:GFP) transgenic zebrafish. When embryos at the one-cell stage were injected with either mCherry mRNA (110 pg; II, V) or phlda3 mRNA (110 pg; III, VI), and GFP signals at 24 hpf were directly observed under a florescence microscope. These results show that the overexpression of phlda3 mRNA inhibited the normal development of ISVs at 24 hpf. The white arrows in III and VI are used to show incompletely developed ISVs sprouting from DA. Scale bar, 100 μm.

We also injected mCherry mRNA or phlda3 mRNA into tg(flk1:GFP) and tg(fli1:GFP) embryos to observe the development of ISVs at 24 hpf. The data showed that the overexpression of phlda3 mRNA inhibited the normal development of ISVs compared to uninjected embryos or embryos injected with control mCherry mRNA (Fig. 3B). Taken together, these data demonstrated that an overexpression of phlda3 affects the development of ISVs during zebrafish embryogenesis.

phlda3 is not involved in either primitive or definitive hematopoiesis

Hematopoiesis in zebrafish includes two successive processes: primitive hematopoiesis and definitive hematopoiesis [10–12]. The two genes gata1 [13, 14] and pu.1 [15–17] encode two early transcription factors that regulate primitive hematopoiesis. They are markers for erythroid progenitors and myeloid progenitors, respectively. The other two genes, c-myb [18] and runx1 [19], are involved in definitive hematopoiesis to generate hematopoietic stem cells (HSCs) that further differentiate into erythroid, myeloid and lymphoid cells. We overexpressed phlda3 with an injection of phlda3 mRNA into embryos and performed whole mount in situ hybridization using antisense probes for gata1, pu.1, c-myb, and runx1. Whole mount in situ hybridization did not detect apparent differences in the expression levels of primitive and definitive hematopoiesis markers (gata1, pu.1, c-myb and runx1) between control embryos (mCherry mRNA) and embryos with an overexpression of phlda3 (Fig. 4A-H). These data suggest that phlda3 is not involved in the regulation of either primitive or definitive hematopoiesis during zebrafish embryogenesis.

Figure 4.

Figure 4.

Open in a new tab

The overexpression of phlda3 affects neither primitive nor definitive hematopoiesis during zebrafish embryogenesis. (A-H) The effects of overexpression of phlda3 on the development of primitive and definitive hematopoiesis in zebrafish. Zebrafish embryos at the one-cell stage were injected with either mCherry mRNA (110 pg; A, C, E, G) or phlda3 mRNA (110 pg; B, D, F, H), and used for whole-mount in situ hybridization with the primitive hematopoiesis probes gata1 (A, B) and pu.1 (C, D), and definitive hematopoiesis probes c-myb (E, F) and runx1 (G, H), at 28 hpf. The expression levels of gata1, pu.1, c-myb and runx1 were not affected by injection of phlda3 mRNA. Scale bar, 200 μm.

AKT rescues the developmental defects of ISVs induced by overexpression of phlda3

In 2009, Kawase et al showed that PHLDA3 blocks the activation of AKT [22]. Therefore, we investigated a possible molecular mechanism by which the overexpression of phlda3 regulates development of ISVs via its effect on the activation of AKT. We injected either control mCherry mRNA or phlda3 mRNA into zebrafish embryos and collected embryos at 24 hpf to prepare protein extracts for Western blot analysis with an anti-phosphorylated AKT antibody or a control antibody against total AKT [28]. Western blot analysis showed that compared with uninjected embryos or embryos injected with control mCherry mRNA, embryos injected with phlda3 mRNA exhibited a significantly reduced level of phosphorylated AKT (Fig. 5A). These data suggest that the overexpression of phlda3 mRNA inhibits the activation of AKT in zebrafish.

Figure 5.

Figure 5.

Open in a new tab

The overexpression of phlda3 mRNA inhibits the activation of AKT, and constitutively active AKT rescues the developmental defects of ISVs caused by phlda3 overexpression. (A) phlda3 overexpression inhibits activation of AKT. Western blot analysis was carried out to examine the activation/phosphorylation of zebrafish AKT in uninjected embryos (n=80), embryos injected with 200 pg of mCherry mRNA (n=80), and embryos injected with 200 pg of phlda3 mRNA (n=80) at 24 hpf. The images for Western blotting were quantified and the fold change of phosphorylated AKT (P-AKT) over the control total AKT was plotted on the right. An unpaired Student’s t-test was used to compare the means of two different groups. The data were shown as means ± SD from four independent experiments. A P-value of ≤ 0.05 was defined to be statistically significant. (B) Injection of human myristoylated-AKT mRNA can rescue the developmental defects of ISVs (flk1 signal) in embryos injected with phlda3 mRNA. Zebrafish embryos at the one-cell stage were injected with 200 pg of mCherry mRNA (II), 100 pg of mCherry mRNA plus 100 pg of human myristoylated-AKT mRNA (III), 100 pg of phlda3 mRNA (IV), or 100 pg of phlda3 mRNA plus 100 pg of human myristoylated-AKT mRNA (V), and then used for whole-mount in situ hybridization with ISVs probe flk1 at 24 hpf. Scale bar, 200 μm.

To determine whether reduced AKT activation by the overexpression of phlda3 is critical to the disrupted ISV development, we co-injected phlda3 mRNA and mRNA for a constitutively active form of AKT1 (human myristoylated-AKT1) into one-cell embryos and performed whole mount in situ hybridization for flk1 with 24 hpf embryos [28]. As shown in Fig. 5B, compared with uninjected embryos or embryos injected with control mCherry mRNA, embryos an injection of phlda3 mRNA showed dramatically inhibited development of ISVs. However, the effect was abolished by the overexpression of the constitutive active form of human myristoylated-AKT. These data suggest that an overexpression of phlda3 inhibits ISV development by blocking the activation of AKT.

DISCUSSION

Hemangioblasts are bi-potent precursor cells that can generate both endothelial cells (vascular differentiation and development) and blood cells. In this study, we showed that the overexpression of phlda3 impaired the specification of hemangioblasts, as demonstrated by the reduced expression levels of hemangioblasts markers scl, fli1 and etsrp (Fig. 2). With regard to vascular development, an overexpression of phlda3 inhibited the development of ISVs (reduced signal for ISVs markers flk1and cdh5) (Fig. 3A). Furthermore, compared to uninjected embryos or embryos injected with mCherry mRNA, tg(flk1:GFP) and tg(fli:GFP) transgenic embryos injected with phlda3 mRNA showed impaired development of ISVs (Fig. 3B). However, phlda3 overexpression had no effect on expression of the venous markers flt4 and dab2 (Fig. 3A). Moreover, overexpression of phlda3 did not affect expression levels of primitive and definitive hematopoiesis markers gata1, pu.1, c-myb and runx1 (Fig. 4). These data reveal a novel role of phlda3 in the specification of hemangioblasts and development of ISVs.

The molecular mechanism for the differentiation of hemangioblasts from the mesoderm is largely unknown. We previously reported that aggf1, a gene encoding an angiogenic factor essential for embryogenesis, is one of the earliest regulators involved in the differentiation of hemangioblasts [28]. Consequently, the knockdown of aggf1 expression had profound effects on the development of both ISVs and veins, primitive hematopoiesis, and definitive hematopoiesis [21, 29]. Although both aggf1 and phlda3 are involved in the differentiation of hemangioblasts, their effects on vascular development and hematopoiesis are clearly different. Specifically, phlda3 only affected the development of ISVs, and it did not have any overt effects on the development of veins, primitive or definitive hematopoiesis (Figs. 3 and 4). The data may suggest that hemangioblasts comprise multiple heterogeneous populations of cells. Some hemangioblasts can differentiate into vascular cells, whereas other hemangioblasts differentiate into hematopoietic cells. phlda3 may play a role in the differentiation of hemangioblasts targeted for vascular development and not hematopoiesis, thus its overexpression affects the development of ISVs, but not the differentiation of blood cells.

The PHLDA3 protein was found to block the activation of AKT signaling in various tumor cells [22] and β-cells [23]. Consistent with this, one molecular mechanism in which the overexpression of phlda3 impairs the development of ISVs is the inhibition of the AKT signaling pathway as shown in Fig. 5A [22]. Moreover, a constitutively active form of AKT successfully reversed the impaired ISV development by the overexpression of phlda3 (Fig. 5B). We previously reported that a) a knockdown of aggf1 expression inhibited vascular development by blocking AKT activation and b) a constitutively active form of AKT successfully rescued the impaired vascular development via knockdown of aggf1 expression [28]. Therefore, both phlda3 and aggf1 regulate hemangioblast specification and ISV development via the AKT signaling pathway, however, phlda3 acts as a negative regulator whereas aggf1 acts as a positive regulator of AKT activation.

Despite the marked effects of the overexpression of phlda3 on the specification of hemangioblasts and development of ISVs, we did not observe any effects with a phlda3 morpholino oligomer (MO) (data not shown). Moreover, we found that the phlda3 MO failed to rescue the effects of aggf1 MO on ISVs (data not shown). One possible explanation for the lack of effect for the phlda3 MO is that the knockdown of phlda3 expression with MO is expected to enhance the development of ISVs and specification of hemangioblasts, but such great enhancements above a normal level may be difficult to achieve. Alternatively, the effect of phlda3 expression knockdown may be masked by the activity of redundant genes or genes with similar functions, such as phlda1 and phlda2 [32–35]. Future studies with a knockdown of all three phlda1-3 genes may further validate the role of phlda3 in the specification of hemangioblasts and development of ISVs.

In conclusion, our observations suggest that similar to aggf1, phlda3 plays an important role in the specification of hemangioblasts and regulation of development of ISVs via the AKT signaling pathway. However, phlda3 differs from aggf1 in that it may not be involved in the specification of veins and regulation of primitive and definitive hematopoiesis.

MATERIALS AND METHODS

Zebrafish strain and care

Wild-type AB zebrafish strain tg(flk1:EGFP) (bought from China Zebrafish Resource Centre) and tg(fli1a:EGFP) (kindly provided by Prof. Jingxia Liu from Huazhong Agricultural University) were used in the study. Zebrafish and their embryos were raised at 28.5°C using standard protocols, as described previously [36], and collected at different stages for subsequent analysis [37, 38]. The study was approved by the ethics committee of Huazhong University of Science and Technology.

Preparation and microinjection of mRNA samples

The full-length coding sequence for zebrafish phlda3 gene was PCR-amplified from embryonic cDNA samples using two PCR primers (Forward Primer: 5’-CGCGGATCCGCCATGAACCAGTGTAAAGTAAT-3’; Reverse Primer: 5’-CCGGAATTCTCAAAGTTCGACGTCACCTT-3’). Then, the PCR product was subcloned into the pCS2+ vector, resulting in plasmid phlda3-pCS2+. The plasmid mCherry-pSP64 (negative control) and human myristoylated-AKT-pSP64 (myrAKT-pSP64) were described previously by us [28].

The phlda3-pCS2+, mCherry-pSP64, and myrAKT-pSP64 plasmids were linearized by restriction digestion and used for the generation of phlda3, mCherry, and myrAKT mRNA samples by in vitro transcription with SP6 RNA polymerase from the mMESSAGE mMACHINE kit (Ambion, Austin, TX).

Capped full-length mRNA samples were injected into the zebrafish embryos (one-cell stage) using a pneumatic picopump as described by Hyatt et al [39]. The embryos were then maintained and collected at different developmental stages for the subsequent experiments.

Synthesis of antisense RNA probes for whole mount in situ hybridization

To develop a probe for whole mount in situ hybridization for phlda3, we used RT-PCR analysis to amplify a fragment specific for phlda3 with a forward primer (5’-TGGAGTATAAACGGGGTCTG-3’) and a reverse primer (5’-GCAAAGTGAGGAGTGGAATC-3’). The PCR fragment was subcloned into the pGEM-T easy vector, generating the phlda3-pGEM-T easy plasmid.

The plasmids for making antisense RNA probes for scl, fli1, etsrp, gata1, pu.1, c-myb, runx1, flt4, dab2, notch3, flk1 and cdh5 were described previously by us [28, 40].

Each plasmid was linearized by restriction digestion and used for making antisense RNA probes for whole mount in situ hybridization using either T7 or SP6 polymerase (Promega) in the presence of DIG-labeled nucleotides (Roche, Mannheim, Germany).

Whole mount in situ hybridization was performed using standard protocols as described by Thisse et al [41]. The hybridization signals were detected using anti-digoxigenin-AP (Roche Mannheim, Germany) and staining with BCIP/NBT (Promega) as previously described [28, 36, 37, 40, 42].

Western blot analysis

Protein extracts from 24 hpf stage zebrafish embryos (n=80) were prepared as previously described [28], separated by 10% SDS-PAGE, and then transferred to PVDF membranes. The membranes were probed with either an anti-phosphorylated-AKT (Ser473) antibody (Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060) (1:1000) or an anti-AKT antibody (Akt Antibody #9272) (1:1000) from Cell Signaling Technology. Then, the membranes were incubated with a goat anti-rabbit HRP-conjugated secondary antibody (1:20,000). Signals were detected by incubation with a SuperSignal West Pico Chemiluminescent Substrate (Thermo) and ChemiDoc XRS (Bio-Rad).

Acknowledgements

We greatly appreciate Dr. Jingxia Liu at Huazhong Agricultural University for her generous sharing of tg(fli:GFP) transgenic zebrafish. We thank Isabel Z. Wang at Stanford University, Annabel Z. Wang in the Harvard/MIT M.D./Ph.D. program, and an anonymous expert reviewer for carefully proof-reading and revising the manuscript. This study was supported by the China National Natural Science Foundation grants (81630002, 31430047 and 91439129), 2016 Top-Notch Innovative Talent Development Project from the Bureau of Human Resources and Social Security of Wuhan City, Hubei Province Natural Science Programs (2016CFB224 and 2014CFA074), Chinese National Basic Research Programs (973 Programs 2013CB531101 and 2012CB517801), and NIH/NHLBI grants (R01 HL121358 and R01 HL126729).

ABBREVIATIONS:

ISVs

intersegmental vessels

AGGF1

AngioGenic factor with G-patch and FHA domains 1 or AnGioGenic Factor 1

PHLDA3

Pleckstrin Homology-Like Domain, family A, member 3

hpf

hours post fertilization

AKT

Protein kinase B

MOs

morpholinos

HSCs

hematopoietic stem cells

Footnotes

DISCLOSURES

None.

REFERENCES

  • 1.Childs S, Chen JN, Garrity DM & Fishman MC (2002) Patterning of angiogenesis in the zebrafish embryo, Development. 129, 973–82. [DOI] [PubMed] [Google Scholar]
  • 2.Vogeli KM, Jin SW, Martin GR & Stainier DY (2006) A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula, Nature. 443, 337–9. [DOI] [PubMed] [Google Scholar]
  • 3.Begley CG & Green AR (1999) The SCL gene: from case report to critical hematopoietic regulator, Blood. 93, 2760–70. [PubMed] [Google Scholar]
  • 4.Porcher C, Liao EC, Fujiwara Y, Zon LI & Orkin SH (1999) Specification of hematopoietic and vascular development by the bHLH transcription factor SCL without direct DNA binding, Development. 126, 4603–15. [DOI] [PubMed] [Google Scholar]
  • 5.Patterson LJ, Gering M & Patient R (2005) Scl is required for dorsal aorta as well as blood formation in zebrafish embryos, Blood. 105, 3502–11. [DOI] [PubMed] [Google Scholar]
  • 6.Pham VN, Lawson ND, Mugford JW, Dye L, Castranova D, Lo B & Weinstein BM (2007) Combinatorial function of ETS transcription factors in the developing vasculature, Dev Biol 303, 772–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Liu F & Patient R (2008) Genome-wide analysis of the zebrafish ETS family identifies three genes required for hemangioblast differentiation or angiogenesis, Circ Res 103, 1147–54. [DOI] [PubMed] [Google Scholar]
  • 8.Liu F, Walmsley M, Rodaway A & Patient R (2008) Fli1 acts at the top of the transcriptional network driving blood and endothelial development, Curr Biol 18, 1234–40. [DOI] [PubMed] [Google Scholar]
  • 9.Sumanas S & Lin S (2006) Ets1-related protein is a key regulator of vasculogenesis in zebrafish, PLoS Biol 4, e10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Orkin SH & Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology, Cell. 132, 631–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Paik EJ & Zon LI (2010) Hematopoietic development in the zebrafish, Int J Dev Biol. 54, 1127–37. [DOI] [PubMed] [Google Scholar]
  • 12.Yamauchi H, Hotta Y, Konishi M, Miyake A, Kawahara A & Itoh N (2006) Fgf21 is essential for haematopoiesis in zebrafish, EMBO Rep 7, 649–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Galloway JL, Wingert RA, Thisse C, Thisse B & Zon LI (2005) Loss of gata1 but not gata2 converts erythropoiesis to myelopoiesis in zebrafish embryos, Dev Cell. 8, 109–16. [DOI] [PubMed] [Google Scholar]
  • 14.Belele CL, English MA, Chahal J, Burnetti A, Finckbeiner SM, Gibney G, Kirby M, Sood R & Liu PP (2009) Differential requirement for Gata1 DNA binding and transactivation between primitive and definitive stages of hematopoiesis in zebrafish, Blood. 114, 5162–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Rhodes J, Hagen A, Hsu K, Deng M, Liu TX, Look AT & Kanki JP (2005) Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish, Dev Cell. 8, 97–108. [DOI] [PubMed] [Google Scholar]
  • 16.Ward AC, McPhee DO, Condron MM, Varma S, Cody SH, Onnebo SM, Paw BH, Zon LI & Lieschke GJ (2003) The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish, Blood. 102, 3238–40. [DOI] [PubMed] [Google Scholar]
  • 17.Hsu K, Traver D, Kutok JL, Hagen A, Liu TX, Paw BH, Rhodes J, Berman JN, Zon LI, Kanki JP & Look AT (2004) The pu.1 promoter drives myeloid gene expression in zebrafish, Blood. 104, 1291–7. [DOI] [PubMed] [Google Scholar]
  • 18.Zhang Y, Jin H, Li L, Qin FX & Wen Z (2011) cMyb regulates hematopoietic stem/progenitor cell mobilization during zebrafish hematopoiesis, Blood. 118, 4093–101. [DOI] [PubMed] [Google Scholar]
  • 19.Lam EY, Hall CJ, Crosier PS, Crosier KE & Flores MV (2010) Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells, Blood. 116, 909–14. [DOI] [PubMed] [Google Scholar]
  • 20.Risau W & Flamme I (1995) Vasculogenesis, Annu Rev Cell Dev Biol 11, 73–91. [DOI] [PubMed] [Google Scholar]
  • 21.Qian N, Frank D, O’Keefe D, Dao D, Zhao L, Yuan L, Wang Q, Keating M, Walsh C & Tycko B (1997) The IPL gene on chromosome 11p15.5 is imprinted in humans and mice and is similar to TDAG51, implicated in Fas expression and apoptosis, Hum Mol Genet. 6, 2021–9. [DOI] [PubMed] [Google Scholar]
  • 22.Kawase T, Ohki R, Shibata T, Tsutsumi S, Kamimura N, Inazawa J, Ohta T, Ichikawa H, Aburatani H, Tashiro F & Taya Y (2009) PH domain-only protein PHLDA3 is a p53-regulated repressor of Akt, Cell. 136, 535–50. [DOI] [PubMed] [Google Scholar]
  • 23.Ohki R, Saito K, Chen Y, Kawase T, Hiraoka N, Saigawa R, Minegishi M, Aita Y, Yanai G, Shimizu H, Yachida S, Sakata N, Doi R, Kosuge T, Shimada K, Tycko B, Tsukada T, Kanai Y, Sumi S, Namiki H, Taya Y, Shibata T & Nakagama H (2014) PHLDA3 is a novel tumor suppressor of pancreatic neuroendocrine tumors, Proceedings of the National Academy of Sciences of the United States of America. 111, E2404–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Qiao M, Wu M, Shi R & Hu W (2017) PHLDA3 impedes somatic cell reprogramming by activating Akt-GSK3beta pathway, Scientific reports. 7, 2832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Han CY, Lim SW, Koo JH, Kim W & Kim SG (2016) PHLDA3 overexpression in hepatocytes by endoplasmic reticulum stress via IRE1-Xbp1s pathway expedites liver injury, Gut 65, 1377–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Choi K (1998) Hemangioblast development and regulation, Biochem Cell Biol 76, 947–56. [PubMed] [Google Scholar]
  • 27.Thompson MA, Ransom DG, Pratt SJ, MacLennan H, Kieran MW, Detrich HW 3rd, Vail B, Huber TL, Paw B, Brownlie AJ, Oates AC, Fritz A, Gates MA, Amores A, Bahary N, Talbot WS, Her H, Beier DR, Postlethwait JH & Zon LI (1998) The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis, Dev Biol 197, 248–69. [DOI] [PubMed] [Google Scholar]
  • 28.Chen D, Li L, Tu X, Yin Z & Wang Q (2013) Functional characterization of Klippel-Trenaunay syndrome gene AGGF1 identifies a novel angiogenic signaling pathway for specification of vein differentiation and angiogenesis during embryogenesis, Hum Mol Genet 22, 963–76. [DOI] [PubMed] [Google Scholar]
  • 29.Lawson ND, Scheer N, Pham VN, Kim CH, Chitnis AB, Campos-Ortega JA & Weinstein BM (2001) Notch signaling is required for arterial-venous differentiation during embryonic vascular development, Development. 128, 3675–83. [DOI] [PubMed] [Google Scholar]
  • 30.Habeck H, Odenthal J, Walderich B, Maischein H & Schulte-Merker S (2002) Analysis of a zebrafish VEGF receptor mutant reveals specific disruption of angiogenesis, Curr Biol 12, 1405–12. [DOI] [PubMed] [Google Scholar]
  • 31.Larson JD, Wadman SA, Chen E, Kerley L, Clark KJ, Eide M, Lippert S, Nasevicius A, Ekker SC, Hackett PB & Essner JJ (2004) Expression of VE-cadherin in zebrafish embryos: a new tool to evaluate vascular development, Dev Dyn 231, 204–13. [DOI] [PubMed] [Google Scholar]
  • 32.Johnson EO, Chang KH, de Pablo Y, Ghosh S, Mehta R, Badve S & Shah K (2011) PHLDA1 is a crucial negative regulator and effector of Aurora A kinase in breast cancer, Journal of cell science. 124, 2711–22. [DOI] [PubMed] [Google Scholar]
  • 33.Fearon AE, Carter EP, Clayton NS, Wilkes EH, Baker AM, Kapitonova E, Bakhouche BA, Tanner Y, Wang J, Gadaleta E, Chelala C, Moore KM, Marshall JF, Chupin J, Schmid P, Jones JL, Lockley M, Cutillas PR & Grose RP (2018) PHLDA1 Mediates Drug Resistance in Receptor Tyrosine Kinase-Driven Cancer, Cell reports. 22, 2469–2481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Tunster SJ, Creeth HDJ & John RM (2016) The imprinted Phlda2 gene modulates a major endocrine compartment of the placenta to regulate placental demands for maternal resources, Dev Biol 409, 251–260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Sikora KM, Magee DA, Berkowicz EW, Lonergan P, Evans AC, Carter F, Comte A, Waters SM, MacHugh DE & Spillane C (2012) PHLDA2 is an imprinted gene in cattle, Animal genetics. 43, 587–90. [DOI] [PubMed] [Google Scholar]
  • 36.Su Z, Si W, Li L, Zhou B, Li X, Xu Y, Xu C, Jia H & Wang QK (2014) MiR-144 regulates hematopoiesis and vascular development by targeting meis1 during zebrafish development, The international journal of biochemistry & cell biology. 49, 53–63. [DOI] [PubMed] [Google Scholar]
  • 37.Zhou J, Wang L, Zuo M, Wang X, Ahmed AS, Chen Q & Wang QK (2016) Cardiac sodium channel regulator MOG1 regulates cardiac morphogenesis and rhythm, Scientific reports. 6, 21538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kimmel CB, Ballard WW, Kimmel SR, Ullmann B & Schilling TF (1995) Stages of embryonic development of the zebrafish, Dev Dyn 203, 253–310. [DOI] [PubMed] [Google Scholar]
  • 39.Hyatt TM & Ekker SC (1999) Vectors and techniques for ectopic gene expression in zebrafish, Methods Cell Biol 59, 117–26. [DOI] [PubMed] [Google Scholar]
  • 40.Li L, Chen D, Li J, Wang X, Wang N, Xu C & Wang QK (2014) Aggf1 acts at the top of the genetic regulatory hierarchy in specification of hemangioblasts in zebrafish, Blood. 123, 501–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Thisse C & Thisse B (2008) High-resolution in situ hybridization to whole-mount zebrafish embryos, Nat Protoc 3, 59–69. [DOI] [PubMed] [Google Scholar]
  • 42.Lu Q, Yao Y, Hu Z, Hu C, Song Q, Ye J, Xu C, Wang AZ, Chen Q & Wang QK (2016) Angiogenic Factor AGGF1 Activates Autophagy with an Essential Role in Therapeutic Angiogenesis for Heart Disease, PLoS Biol 14, e1002529. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES