Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Sep 17.
Published in final edited form as: Dev Biol. 2025 Sep 17;528:217–227. doi: 10.1016/j.ydbio.2025.09.014

Genetic knockout suggests Isl1 enhancer redundancy in mouse hindlimb development

Seth Olson 1, Hiroko Kawakami 1,2,3, Alexandra Maria Nichitean 1, Corinne Boerner 1, Amel Awad 1, Dylan Corcoran 1, Cailin McMahon 1, Ryuichi Nishinakamura 4, Maureen Cetera 1,3, Yasuhiko Kawakami 1,2,3
PMCID: PMC12624849  NIHMSID: NIHMS2113717  PMID: 40972968

Abstract

Isl1 encodes a LIM homeodomain transcription factor, which is expressed in hindlimb progenitor cells in the lateral plate mesoderm and is required for initiating hindlimb development. Previous studies by lacZ transgenesis in mouse embryos identified a cis-element located 3’ to the Isl1 gene, which could drive lacZ reporter expression in hindlimb progenitor cells and the branchial arch ectoderm. We refer to this cis-element as the Isl1 hindlimb progenitor enhancer (HLPE). Our previous study also showed that SALL4, a transcription factor, is enriched at the Isl1 HLPE, suggesting that SALL4 may regulate Isl1 expression through Isl1 HLPE. We sought to determine whether Isl1 HLPE regulates Isl1 expression and created mutant mice that lack the Isl1 HLPE sequence by CRISPR/Cas9. The Isl1 HLPE−/− mouse lines, established after breeding with wild-type mice, did not exhibit gross morphological defects, except that their long bones are shorter than those of wild type. The shorter long bone phenotype was observed in the mid-gestation stage and was associated with misregulation of the expression of several chondrogenic genes, suggesting that the deletion of HLPE affects chondrogenesis. Although Sall4 regulation of Isl1 through Isl1 HLPE was suspected, skeletal analysis did not exhibit any synergy between Isl1 HLPE−/− and conditional Sall4 mutation. In situ hybridization showed seemingly normal expression of Isl1 and its downstream gene Tbx4 in Isl1 HLPE−/−, TCre; Sall4fl/fl mutants, and Isl1 HLPE−/−; TCre; Sall4fl/fl mutants. Finally, by quantitative gene expression analysis, Isl1 expression is reduced but not abolished in Isl1 HLPE−/− and Isl1+/−; Isl1 HLPE+/− embryos, compared to wild-type embryos. Similarly, quantitative imaging analysis after immunostaining showed reduced ISL1 signals in the branchial arch ectoderm in Isl1 HLPE−/− embryos. These results support our notion that the Isl1 HLPE sequence functions as an enhancer for Isl1 expression in hindlimb progenitor cells and branchial arch ectoderm cells and suggest that multiple redundant enhancers co-regulate Isl1 expression.

Keywords: Isl1, hindlimb progenitor cells, cis-regulatory elements, enhancer

Graphical Abstract

graphic file with name nihms-2113717-f0007.jpg

Introduction

Vertebrate limb development begins with the specification of cells that give rise to limb bud mesenchyme (limb progenitor cells) in the lateral plate mesoderm at specific locations along the anterior-posterior body axis (Tickle, 2015). During the initiation of limb bud development, limb progenitors start to express fibroblast growth factor 10 (Fgf10), which encodes a secreted signaling molecule (Ohuchi et al., 1997). Concomitantly, limb progenitors maintain active proliferation, while cells in the flank tissue reduce proliferation (Searls and Janners, 1971). These two events are spatially and temporally linked to each other. Thereafter, Fgf10 forms a positive feedback loop with ectodermal Fgf8, which maintains limb bud outgrowth (Ohuchi et al., 1997; Xu et al., 1998). Two different classes of transcription factors act upstream of Fgf10 expression and the active proliferation of limb progenitor cells; Tbx5 (encoding a T-box transcription factor) and Isl1 (encoding a LIM homeodomain protein) are required for initiating the development of the forelimb bud and hindlimb bud, respectively (Agarwal et al., 2003; Kawakami et al., 2011; Rallis et al., 2003).

Isl1 is transiently expressed in the hindlimb progenitor cells in the posterior part of the lateral plate mesoderm at E9.5, a stage just before the initiation of hindlimb bud outgrowth (Kawakami et al., 2011). Several studies have been conducted to investigate the regulation of Isl1 expression through cis-regulatory elements or enhancers. A previous study did not find any possible Isl1 enhancers within 95 kb upstream of the Isl1 transcription start site in the mouse genome; instead, the study found that a 10 kb sequence, spanning from 1.5 kb upstream of exon 6 (the most 3’ exon) to 7kb downstream of the transcription termination site, can drive lacZ reporter expression in the hindlimb-forming region in the lateral plate mesoderm (Kappen and Salbaum, 2009). Independent analyses identified that 3 evolutionarily conserved sequences located 3’ to the Isl1 gene, within the 10 kb sequence, can drive lacZ reporter expression in the hindlimb progenitor cells in the lateral plate mesoderm (Jurberg et al., 2013; Kang et al., 2009; Tahara et al., 2018). The three putative Isl1 enhancers are termed CR1, CR2, and CR3 (Kang et al., 2009), with the evolutionary conservation of CR2 and CR3 being the highest and lowest, respectively. Our lacZ transgenesis experiments in mouse embryos demonstrated that a 1.3 kb sequence that contains both CR1 and CR2 can drive LacZ reporter expression in cardiac progenitors, the mandibular arch, and the caudal part of the lateral plate mesoderm, including hindlimb progenitors (Tahara et al., 2018). The LacZ expression pattern essentially recapitulated the Isl1 mRNA expression pattern in these tissues. Hereafter, we refer to this 1.3 kb element as the Isl1 hindlimb progenitor enhancer (HLPE).

We previously reported that SALL4 binds to the CR2 sequence by ChIP-seq in the posterior trunk mesoderm of mouse embryos (Chen et al., 2024; Tahara et al., 2019). Sall4 encodes a zinc finger transcription factor (Al-Baradie et al., 2002; de Celis and Barrio, 2009; Sakaki-Yumoto et al., 2006; Sweetman and Munsterberg, 2006) and is involved in the development of the posterior trunk mesoderm, including the hindlimb-forming region (Akiyama et al., 2015; Chen et al., 2024; Pappas et al., 2024; Tahara et al., 2019). Conditional knockout of Sall4 in mesoderm progenitors by TCre leads to the development of hindlimbs with defects in the femur, tibia, and anterior digits (Akiyama et al., 2015), indicating that Sall4 is dispensable for the initiation of hindlimb development. However, our recent study showed that Sall1, another Sall family member, partially compensates for Sall4 functions (Chen et al., 2024). TCre; Sall1fl/fl; Sall4fl/fl and TCre; Sall1+/fl; Sall4fl/fl mice lack hindlimbs, while TCre; Sall1fl/fl; Sall4+/fl mice developed hindlimbs with a fraction of embryos showing fusion of the proximal part of metatarsals. Thus, Sall1 and Sall4 are redundant, and there is a greater requirement for Sall4 than Sall1 in hindlimb development.

In this study, we investigate the function of Isl1 HLPE. We deleted Isl1 HLPE by CRISPR/Cas9, and the HLPE−/− mice are viable and fertile. The gross morphology of mutant mice appears to be normal, but the long bone lengths are shorter than wild type. Gene expression and immunofluorescence analyses support our notion that the HLPE sequence acts as an Isl1 enhancer in hindlimb progenitors and the branchial arch ectoderm; however, we did not observe synergy between Isl1 HLPE−/− and conditional Sall4 inactivation in hindlimb development. Our data suggest that Isl1 expression in hindlimb progenitors and the branchial arch ectoderm is regulated by multiple enhancers, including HLPE.

2. Materials and Methods

2.1. Generation of Isl1 HLPE knockout mice

In order to delete the Isl1 HLPE (Tahara et al., 2018), we used the following two sgRNA from Synthego: Isl1 enhancer 5’ sgRNA: A*G*U*AGAACUCAAGCCUAAUU + Synthego modified EZ scaffold, and Isl1 enhancer 3’ sgRNA: U*G*C*UUUAUGCCACCAGCGCU + Synthego modified EZ scaffold. The asterisks denote modification by 2’-O-methyl analogs and 3’-phosphorothioate internucleotide linkages. TrueCut Cas9 (Thermo Fisher Scientific, A36498) and sgRNAs were combined at a 1:1 molar ratio and incubated for 10 minutes at room temperature to generate ribonucleoprotein complexes. C57BL/6 zygotes were electroporated with the ribonucleoprotein complexes, cultured, and transferred into pseudopregnant mice as previously described (Rollins et al., 2023). Mice were genotyped by genomic PCR with the primers in Table S1. The deleted sequence was confirmed by cloning the knockout PCR product and Sanger sequencing (Fig. S1). Mice are maintained on the C57BL/6 background.

2.2. Animal breeding, whole mount in situ hybridization, and skeletal preparation

The following mouse lines are used in this study: Sall4fl (Sakaki-Yumoto et al., 2006; Yuri et al., 2009), TCre (Perantoni et al., 2005). The Isl1− allele was generated by germline deletion of Isl1fl (Song et al., 2009; Sun et al., 2008) as previously described (Chen et al., 2024). Sall4fl/fl and TCreTg/Tg; Sall4+/fl mice were backcrossed more than 7 generations to C57BL/6 mice before experimental breeding. Embryos for in situ hybridization were collected by timed mating, fixed in 4% paraformaldehyde in PBS + 0.1% Tween 20 overnight, washed and dehydrated in a graded series of PBS + 0.1% Tween 20/methanol, and stored in 100% methanol. Whole mount in situ hybridization was performed as previously described (Akiyama et al., 2015). Three to five embryos per probe per stage were examined. For skeletal analysis, P0 neonates were euthanized, fixed in 50% ethanol, and processed for Alcian Blue-Alizarin Red staining as previously described (Akiyama et al., 2015). Animal breeding was performed according to the approval by the Institutional Animal Care and Use Committee of the University of Minnesota.

2.3. Femur and humerus length analysis

The length of the femur and humerus was measured by using FIJI as previously described (Chen et al., 2022). Lines were drawn parallel to the long axis of the bone, then perpendicular to those lines such that the lines touched the edges of the bone. The length of the bone was measured by using the distance between the edges of the line parallel to the long axis. The measurement was performed by two independent individuals, and the average value was used for statistical examination.

2.4. Quantitative gene expression analysis

For qRT-PCR, the tissue posterior to the 24th somite was dissected from the E9.5 embryo using a carbon steel scalpel (10316–14, Fine Science Tools). Hindlimb buds from E12.5 embryos were dissected from the trunk using fine forceps. Total RNA was isolated using the Direct-zol RNA MicroPrep kit (R2062, Zymo Research) and cDNA was synthesized using iScript cDNA synthesis kit (#1708890, BioRad) according to the manufacturers’ instructions. qPCR was performed using PowerUp SYBR Green Master Mix (A25742, Thermo Fisher). Actb was used as a reference gene. The primer sequences are shown in Table S2, including previously published primers for Actb, Isl1(exon 2-exon 3), Sox9, and Tbx4 (Beverdam et al., 2010; Chen et al., 2024).

2.5. Immunofluorescence and image acquisition

For histological examination, 10 μm thickness cryosections (longitudinal hindlimb sections from E14.5 embryos or coronal sections through the branchial arch of E9.5 embryos) were processed for immunofluorescence as previously described (Akiyama et al., 2014; Chen et al., 2020). Briefly, sections were washed with PBS with 0.1% Triton X-100, and heat-induced antigen retrieval was performed with citrate buffer (pH 6.0) by boiling, followed by a standard immunofluorescence procedure. The boiling times for branchial arch analysis (E9.5, anti-ISL1and anti-TRP63) and cartilage analysis (E14.5, anti-pHH3 and anti-active caspase 3) were 60 minutes and 10 minutes, respectively. The information of antibodies used in this study are listed in Table S3. Images were captured using a Nikon Eclipse Ti2 widefield microscope using a 20x objective (NA 0.75) and an Orca-fusion BT Digital camera C15440. Exposure and laser power were maintained for each experiment.

2.6. Quantification of proliferation and cell death in chondrocytes

Z stacks were acquired to capture all cells in the section based on the DAPI signal. Maximum projections were analyzed using ImageJ. The hypertrophic and proliferative zones were manually segmented based on the morphology of the tissue in the DAPI channel. Nuclei within the segmented regions were identified and counted using the StarDist2D plugin on the DAPI channel. The total number of nuclei and the area of the selection were measured. Using the multi-point tool, pHH3 and active caspase3 expressing cells were counted in each section. The density of nuclei in the proliferative cartilage area, the ratio of pHH3-positive cells over DAPI-positive nuclei in the proliferative cartilage area, and the ratio of active caspase 3-positive cells over DAPI-positive nuclei in the proliferative cartilage area were calculated and plotted. Measurements were plotted and statistical analysis was performed using GraphPad Prism (version 10.5).

2.7. Quantification of the ISL1 signal intensity in the branchial arch ectoderm

Image analysis was performed using single Z plane images and ImageJ. A macro was used to make a mask of the TRP63 expressing ectoderm region by applying a maximum filter and a Gaussian blur to a duplicated TRP63 image. Auto thresholding (Yen white method) was used to create a binary image, and the wand tool was used to select the region of interest. The mask was then applied to the original ISL1, TRP63, and DAPI channels using the restore selection option, and the mean grey value of the region of interest was measured. For each channel, the background grey value was subtracted from the fluorescent grey value. Masks were hand corrected using only the DAPI and TRP63 channels when necessary. Measurements were plotted and statistical analysis was performed using GraphPad Prism.

2.8. Statistical examination

Statistical examination was performed by one-way ANOVA with post hoc Tukey test for multiple comparisons using GraphPad Prism. For 1:1 comparison, two-tailed unpaired t-test with equal variance was performed. For the hindlimb defect comparison between TCre; Sall4fl/fl neonates and Isl1 HLPE−/−; TCre; Sall4fl/fl neonates, Fisher’s exact test and Boschloo’s test were performed (Agresti, 2002; Boschloo, 1970). Details are described in the results section.

3. Results

Isl1 HLPE−/− neonates do not exhibit gross morphological defects.

To examine whether Isl1 HLPE is required for hindlimb development, we deleted the Isl1 HLPE sequence by CRISPR/Cas9 in C57BL/6 zygotes. The F0 mice were bred with wild-type C57BL/6 mice, and we confirmed germline transmission of the deleted allele at the F1 generation. By genomic DNA PCR and Sanger sequencing, we confirmed deletion of both CR1 and CR2 (Fig. S1). By crossing Isl1 HLPE+/− F1 mice, we obtained wild type, Isl1 HLPE+/−, and Isl1 HLPE−/− postnatal day 0 (P0) neonates (line 1). Isl1 HLPE−/− P0 neonates developed hindlimbs and did not exhibit obvious limb defects.

In order to exclude the possibility of potential off-target effects of the CRISPR/Cas9 system, we used an Isl1 HLPE+/− line that is derived from an independent F0 mouse (line 2). The line 2 mice also possess the same deletion of the Isl1 HLPE sequence as the line 1 mice. We crossed line 2 mice three times with wild-type C57 BL/6 mice. Then, we crossed male and female Isl1 HLPE+/− mice, which generated wild type, Isl1 HLPE+/−, and Isl1 HLPE−/−. The Isl1 HLPE−/− line 2 mice also developed hindlimbs without obvious defects (Fig. 1A–B”). Isl1 HLPE−/− mice are viable and fertile.

Fig. 1. Isl1 HLPE is dispensable for hindlimb skeletal development.

Fig. 1

Open in a new tab

(A-E) Lateral views of P0 neonates with the indicated genotype. Hindlimbs are removed. Scale bar: 5 mm.

(A’-E’) Lateral views of hindlimbs with the indicated genotype. The asterisks in D’ and E’ indicate a lack of tibia. Arrows in D’ and E’ point to the cartilage condensation in the position of the femur. fe: femur, fi: fibula, pg: pelvic girdle, t: tibia. Scale bar: 1 mm.

(A”-E”) Dorsal views of the hindlimb autopod with the indicated genotype. Scale bar: 1 mm. In A’-E’ and A”-E”, not all panels are labeled for simplicity of images. Digits are labeled as 1 to 5 in A”. In D” and E”, # indicates digits whose identities are unknown, and 5 indicates digit 5.

The long bones are shorter in Isl1 HLPE−/− mice

To assess whether deletion of Isl1 HLPE has any impact on hindlimb development, we examined the length of the femur. Compared to wild-type P0 neonates, both Isl1 HLPE−/− line 1 and line 2 exhibited shorter femur length (Fig. 2A). To further examine the influence of Isl1 HLPE deletion, we crossed Isl1 HLPE−/− mice (line 2) with Isl1+/− mice, which generated viable Isl1+/−; Isl1 HLPE+/− neonates (Fig. 1C–C”). The femur length of Isl1+/−; Isl1 HLPE+/− P0 neonates was shorter than wild-type neonates (Fig. 2A). Among two Isl1 HLPE−/− lines and Isl1+/−; Isl1 HLPE+/− mice, there was no significant difference in the femur length (Fig. 2A). While examining the femur length, we noticed that the body of Isl1 HLPE mutant mice seemed to be smaller than wild type. Because Isl1 is not expressed in forelimb progenitors (Kawakami et al., 2011), deletion of the Isl1 HLPE sequence would not affect forelimb development. Thus, we used the humerus length to normalize the body size of each neonate. The ratio of the length of the femur to the humerus did not exhibit differences among wild-type, Isl1 HLPE−/−, and Isl1+/−; Isl1 HLPE+/− neonates (Fig. 2B), indicating that the short femur length in Isl1 HLPE mutant mice reflects their smaller body size. Given that the hindlimb morphologies are not affected in Isl1 HLPE−/− and Isl1+/−; Isl1 HLPE+/− mice, this result suggests that Isl1 HLPE is dispensable for hindlimb development.

Fig. 2. Quantitative examination of the skeletal phenotypes.

Fig. 2

Open in a new tab

(A) Femur length of wild-type (WT), HLPE−/− line 1 (HLPE KO 1), HLPE−/− line 2 (HLPE KO 2), and Isl1+/−; HLPE+/− P0 neonates.

(B) The ratio of the femur length and humerus length of wild-type, HLPE−/− line 1, HLPE−/− line 2, and Isl1+/−; HLPE+/− P0 neonates.

(C) Frequency of femur phenotypes of wild-type, TCre; Sall4fl/fl (Sall4 cKO), and HLPE−/−; TCre; Sall4fl/fl P0 neonates. Condensation: A cartilage condensation is formed in the position of the femur. Short: The femur was formed with less than 2/3 of the length of the wild-type femur.

(D) The number of digits in the hindlimb of wild-type, TCre; Sall4fl/fl, and HLPE−/−; TCre; Sall4fl/fl P0 neonates.

The sample number for each genotype is shown in the panel. In A and B, p values are shown in each panel. p≥0.05 (not significant) is not shown for the simplicity of presentation.

Isl1 HLPE contributes to chondrogenesis

To assess whether the shorter long bones and smaller bodies in Isl1 HLPE−/− mutants occur earlier in development, we examined the femur length at E14.5, a stage prior to the onset of vascularization and endochondral bone formation in the limb (Kozhemyakina et al., 2015). The femur length was shorter in Isl1 HLPE−/− compared to wild-type embryos (Fig. 3A), similar to neonates (Fig. 2A). Proliferation of chondrocytes contributes to the elongation of the cartilage (Fowler and Larsson, 2020; Kozhemyakina et al., 2015). Given that chondrocyte hypertrophy begins by E14.5 and that hypertrophic chondrocytes decline proliferation (Kozhemyakina et al., 2015; Long and Ornitz, 2013), we examined whether cell proliferation and/or cell death were altered in the proliferating cartilage area. We visually identified the cartilage area in each section, where the nuclei density is lower than the surrounding connective tissues. Then, we analyzed the area with higher nuclei density (proliferating cartilage) and excluded the hypertrophic area in the center of the long bone, where the nuclei density is lower (Fig. 3B). The nuclei density of the proliferating cartilage area we examined was similar between wild-type and Isl1 HLPE−/− hindlimbs at E14.5 (Fig. 3C), confirming that we chose similar cartilage areas between the two genotypes. Within the. proliferative cartilage area, we did not observe differences in cell proliferation (labelled by pHH3) and cell death (labelled by active caspase 3) between wild-type and Isl1 HLPE−/− hindlimbs (Fig. 3D, E). This data suggests that changes that lead to shorter long bones in Isl1 HLPE−/− mutants occurred at earlier stages.

Fig. 3. Misregulation of the chondrogenic program in Isl1 HLPE−/− hindlimbs.

Fig. 3

Open in a new tab

(A) Femur length of wild-type (WT) and Isl1 HLPE−/− embryos at E14.5. The p value by unpaired t-test is shown in the panel.

(B) Representative image of a wild-type E14.5 hindlimb longitudinal section stained with DAPI (cyan), active caspase 3 (green), and pHH3 (magenta). The white dotted line surrounds the cartilage; the hypertrophic area is in the middle (sparse cell density area), and the proliferative areas are on either side of the hypertrophic area (yellow brackets, higher cell density areas within the cartilage). Scale bar: 200 μm. Single-color images are shown in grayscale.

(C-E) Comparison of chondrocyte nuclei density (C), chondrocyte proliferation (D), and chondrocyte death (E) in the proliferating cartilage area of wild-type and Isl1 HLPE−/− E14.5 hindlimbs. The hypertrophic area (the middle region in panel B) is excluded from the analysis. N=4 animals/genotype, with 5–17 sections/embryo. In C-E, each column represents one embryo, and each data point represents one section. Mean and SEM were calculated based on the means of each embryo within a genotype. A nested two-tailed t-test with equal variance was performed on the means of each animal. The p value is shown in each panel.

(C) Cell density in the proliferative area of E14.5 hindlimb cartilage in wild type and Isl1 HLPE−/− calculated as the ratio of nuclei/area (μm2).

(D) Ratio of the number of pHH3-positive cells over the total number of cells in the proliferative cartilage area.

(E) Ratio of the number of active caspase 3-positive cells over the total number of cells in the proliferative area.

(F-O) Relative expression levels of Sox9 (F), Sox5 (G), Sox6 (H), Col9a1 (I), Vcan (J), Col2a1 (K), Acan (L), Runcx2 (M), Mef2c (N), and Col10a1 (O) between wild-type (n=7) and Isl1 HLPE−/− (n=9) hindlimbs at E12.5 by qRT-PCR. The p value is shown in each panel.

To investigate this hypothesis, we examined the expression of genes involved in early processes of chondrogenesis (Kozhemyakina et al., 2015; Liu et al., 2017) between wild-type and Isl1 HLPE−/− hindlimbs at E12.5, a stage of active chondrogenesis, by qRT-PCR. The expression of Sox9, a master regulator of chondrogenesis (Lefebvre et al., 2019), was reduced in Isl1 HLPE−/− hindlimbs (Fig. 3F). In contrast, the expression of Sox5, Sox6, Col9a1, and Vcan was elevated in Isl1 HLPE−/− hindlimbs (Fig. 3G–J), and the expression of Co2a1 and Acan was at similar levels in wild-type and Isl1 HLPE−/− hindlimbs (Fig. 3K, L). We also examined the expression of genes involved in chondrocyte hypertrophy (Kozhemyakina et al., 2015; Liu et al., 2017). We detected elevated expression of Runx2 and Mef2c, while the expression of another hypertrophy marker Col10a1 did not exhibit differences (Fig. 3M–O). These results suggest misregulation of the chondrogenic differentiation program in Isl1 HLPE−/− hindlimbs, which might have contributed to the short length of the femur in Isl1 HLPE−/− hindlimbs.

Mutations in both Isl1 HLPE and Sall4 do not exhibit synergy in hindlimb patterning

Our ChIP-seq experiments showed SALL4 enrichment in the CR2 sequence within Isl1 HLPE (Chen et al., 2024; Tahara et al., 2019), suggesting that SALL4 may regulate Isl1 through Isl1 HLPE. If Isl1 is regulated by multiple redundant enhancers, SALL4 may regulate those enhancers as well. If so, deleting Sall4 and Isl1 HLPE may cause hindlimb defects that were not observed in Isl1 HLPE−/− mutants or Sall4 mutants. Therefore, we generated mice that lack both Isl1 HLPE and Sall4. The Isl1 HLPE−/−; TCre; Sall4fl/fl hindlimbs are smaller than the TCre; Sall4fl/fl hindlimbs (Fig. 1D’, E’), which is consistent with the shorter femur in Isl1 HLPE−/− mutants than the wild-type femur (Fig. 2A). In both TCre; Sall4fl/fl neonates and Isl1 HLPE−/−; TCre; Sall4fl/fl neonates, the femur formed as a cartilage aggregate or was shorter than wild type (<2/3 of wild-type femur length) (Fig. 1D–E”, Fig. 2C, Fig. S2). All the hindlimbs of TCre; Sall4fl/fl and Isl1 HLPE−/−; TCre; Sall4fl/fl neonates possessed two or three digits, except one Isl1 HLPE−/−; TCre; Sall4fl/fl hindlimb with four digits (Fig. 1D”, E”, Fig. 2D). These observations suggest that there was no phenotypic change in hindlimb patterning between TCre; Sall4fl/fl and Isl1 HLPE−/−; TCre; Sall4fl/fl.

We further compared the hindlimb patterning defects between TCre; Sall4fl/fl neonates and Isl1 HLPE−/−; TCre; Sall4fl/fl neonates. Using the 2×2 contingency table for the femur defects (Table S4) and a 2×3 contingency table for digit numbers (Table S5), marked by thick lines in the tables, we tested a null hypothesis by Fisher’s exact test: The ratio of phenotypes in two genotypes is the same. The p values are 0.469 and 0.788 for Table S4 and Table S5, respectively; we failed to reject the null hypothesis at confidence level 0.05. Therefore, the null hypothesis is supported; the severity ratio between the two genotypes is not different.

However, all marginal distributions are fixed in a strictly performed Fisher’s exact test (Agresti, 2002); thus, we tested the null hypothesis by Boschloo’s test (Boschloo, 1970). We chose to use Boschloo’s test because of the low sample size, which excludes the use of the chi-square test. In addition, Boschloo’s test is suitable because only one of two marginal distributions (i.e., the number of samples for each genotype) is fixed. Because Boschloo’s test is performed on a 2×2 contingency table, we converted Table S5 into Table S6 by combining sample numbers for 4-digit and 3-digit to determine if the ratio for a more severe phenotype (2-digit) is different between the 2 genotypes. The p values are 0.391 and 1.00 for Table S4 and Table S6, respectively; we failed to reject the null hypothesis at confidence level 0.05. Therefore, the severity of hindlimb patterning defects between the two genotypes is not different.

Isl1 HLPE contributes to Isl1 expression in hindlimb progenitor cells

In order to assess whether Isl1 HLPE has a role in Isl1 expression, we examined Isl1 expression by whole mount in situ hybridization at E9.5. We observed a similar expression pattern of Isl1 in wild-type and Isl1 HLPE−/− embryos (Fig. 4A, B). Tbx4 expression is downregulated in the hindlimb-forming region in TCre; Isl1 mutant embryos (Kawakami et al., 2011). The expression pattern of Tbx4 was also similar in wild-type and Isl1 HLPE−/− embryos (Fig. 4E, F).

Fig. 4. Expression pattern of Isl1 and Tbx4 by whole mount in situ hybridization.

Fig. 4

Open in a new tab

(A-D) Expression pattern of Isl1 in embryos with indicated genotypes at E9.5.

(E-H) Expression pattern of Tbx4 in embryos with indicated genotypes at E9.5.

Arrows point to the expression in hindlimb progenitors.

In order to assess their expression more quantitatively, we performed qRT-PCR using a primer set used in a previous study of Isl1 expression (Chen et al., 2024). In this analysis, we included Isl1+/− and Isl1+/−; Isl1 HLPE+/− embryos to assess cis-regulation by Isl1 HLPE. Isl1 expression in Isl1 HLPE−/− was approximately 50% downregulated compared to wild type (Fig. 5A). Tbx4 transcript levels are similar between wild type and Isl1 HLPE−/− (Fig. 5E), suggesting that approximately 50% downregulation of Isl1 transcript levels does not impact Tbx4 expression. In Isl1+/−; Isl1 HLPE+/− embryos, Isl1 expression was downregulated compared to wild-type and Isl1+/− embryos (Fig. 5A). The previously used Isl1 primer pair is designed in the sequence of exon 2 and exon 3, and the Isl1− allele was generated by germline deletion of the Isl1 exon 4-floxed allele. The exon 2-exon 3 primer pair detects residual Isl1 transcripts in Isl1−/− embryos (Fig. S3A). Therefore, we designed a new primer pair that anneals to the sequence in exon 4 and exon 5, which does not detect Isl1 transcripts from the Isl1− allele (Fig. S3B). With the exon 4-exon 5 primer pair, downregulation of Isl1 was also observed in Isl1+/−; Isl1 HLPE+/− embryos compared to wild-type embryos (Fig. 5B). Similar to Isl1 HLPE−/− embryos, Tbx4 expression in Isl1+/−; Isl1 HLPE+/− embryos did not show significant differences compared to wild-type and Isl1+/− embryos (Fig. 5C). These results support the notion that Isl1 HLPE acts as an enhancer for Isl1 in hindlimb progenitor cells. Given that Isl1 expression is not abolished in Isl1 HLPE−/− and Isl1+/−; Isl1 HLPE+/− embryos, these results also suggest the presence of a functionally redundant enhancer(s) that regulates Isl1 expression in hindlimb progenitor cells.

Fig. 5. Isl1 HLPE contributes to Isl1 expression in the posterior trunk.

Fig. 5

Open in a new tab

(A) Relative levels of Isl1 expression in the posterior trunk tissue of wild-type (WT), HLPE−/−, Isl1+/−, and Isl1+/−; HLPE+/− embryos at E9.5, determined by a primer set for exon 2 and exon 3.

(B) Relative levels of Isl1 expression in the posterior trunk tissue of wild-type and Isl1+/−; HLPE+/− embryos at E9.5, determined by a primer set for exon 4 and exon 5.

(C) Relative levels of Tbx4 expression in the posterior trunk tissue of wild-type, HLPE−/−, Isl1+/−, and Isl1+/−; HLPE−/− embryos at E9.5.

(D) Relative levels of Isl1 expression in the posterior trunk tissue of wild-type, HLPE−/−, TCre; Sall4fl/fl, and HLPE−/−; TCre; Sall4fl/fl embryos at E9.5, determined by a primer set for exon 2 and exon 3.

(E) Relative levels of Tbx4 expression in the posterior trunk tissue of wild-type, HLPE−/−, TCre; Sall4fl/fl, and HLPE−/−; TCre; Sall4fl/fl embryos at E9.5.

The expression level is normalized to the average expression level of wild-type samples. The sample number for each genotype and p values are shown in the panel. p≥0.05 (not significant) is not shown for the simplicity of presentation.

Mutations in both Isl1 HLPE and Sall4 do not exhibit synergy in the expression of Isl1 and Tbx4

Although TCre; Sall4fl/fl and Isl1 HLPE−/−; TCre; Sall4fl/fl neonates do not show phenotypic differences (Fig. 1, 2, Table S4–S6), we examined whether there is a difference in gene expression without affecting hindlimb morphogenesis. TCre; Sall4fl/fl and Isl1 HLPE−/−; TCre; Sall4fl/fl embryos exhibit similar expression patterns of Isl1 and Tbx4 (Fig. 4C, D, G, H). Analysis by qRT-PCR showed that Isl1 expression was approximately 50% downregulated in Isl1 HLPE−/− and Isl1 HLPE−/−; TCre; Sall4fl/fl embryos compared to wild type; however, there was no significant difference between TCre; Sall4fl/fl and Isl1 HLPE−/−; TCre; Sall4fl/fl embryos (Fig. 5D). This result suggests that Sall4 and Isl1 HLPE do not exhibit synergy in Isl1 expression. Expression of Tbx4 is significantly downregulated in both TCre; Sall4fl/fl and Isl1 HLPE−/−; TCre; Sall4fl/fl embryos compared to wild-type embryos (Fig. 5E). Similar to Isl1 expression levels, Tbx4 expression levels are similar between these two mutants. These results suggest that Sall4 regulates Tbx4 expression. The results also suggest that Isl1 HLPE and Sall4 do not show synergy in the expression of Isl1 and Tbx4, similar to hindlimb morphogenesis (Fig. 1)

Isl1 HLPE contributes to ISL1 expression in the branchial arch ectoderm

Isl1 is expressed in the branchial arch ectoderm (Akiyama et al., 2014), and the HLPE-lacZ reporter expression was detected in the branchial arch in mouse embryos (Tahara et al., 2018), suggesting that Isl1 HLPE also contributes to Isl1 expression in the branchial arch. Compared to posterior trunk tissue, consistently dissecting the brachial arch for qRT-PCR assays was difficult. Thus, we took a histological approach, similar to the study of Isl1 enhancer activities in the pacemaker cells (Galang et al., 2020). Coronal sections of E9.5 embryos were stained with antibodies against ISL1 and TRP63, a pan-ectoderm marker (Yang et al., 1999) (Fig. 6A–A’”, B–B’”). We performed quantitative image analyses to evaluate whether the ISL1 signal intensity exhibits differences between wild-type and Isl1 HLPE−/− embryos. The DAPI signal intensity and TRP63 signal intensity in the branchial arch ectoderm were similar between wild-type and Isl1 HLPE−/− embryos (Fig. 6C, D), confirming the integrity of sections and proper staining procedure. In contrast, the ISL1 signal intensity was reduced in Isl1 HLPE−/− branchial arch ectoderm compared to wild type (Fig. 6E). These data support the notion that Isl1 HLPE contributes to ISL1 expression in the branchial arch ectoderm, in addition to hindlimb progenitor cells in the lateral plate mesoderm.

Fig. 6. Isl1 HLPE contributes to ISL1 expression in the branchial arch ectoderm.

Fig. 6

Open in a new tab

(A) Representative images of the wild-type (A-A’”) and Isl1 HLPE−/− (B-B’”) E9.5 branchial arch stained with DAPI (A, B), anti-TRP63 antibody (A’, B’), and anti-ISL1 antibody (A”, B”). Panels A’” and B’” show merged images. The white dotted line in A’” and B’” represents the region of interest where fluorescent intensity was analyzed. Scale bar: 100 μm. Single-color images are shown in grayscale.

(C-E) Quantification of the fluorescent signal intensity of DAPI (C), TRP63 (D), and ISL1 (E) in the E9.5 branchial arch ectoderm of wild type and Isl1 HLPE−/−. N=4 animals/genotype, with 3–10 sections/embryo. Each column represents a single embryo; each dot is the mean grey value of a section. Mean and SEM were calculated based on the means of each animal within a genotype. A nested two-tailed t-test with equal variance was performed on the means of each animal. The p value is shown in each panel.

4. Discussion

4.1. Regulation of Isl1 expression in hindlimb progenitor cells through Isl1 HLPE

Isl1 is required for the development of a variety of cell types, including the hindlimb progenitor cells (Kawakami et al., 2011), the motor neuron (Pfaff et al., 1996; Song et al., 2009), and the pacemaker cells of the sinoatrial node (Liang et al., 2015), among others. Enhancers for Isl1 expression in hindlimb progenitor cells in the lateral plate mesoderm (Jurberg et al., 2013; Tahara et al., 2018), the motor neuron (Kim et al., 2015), and the pacemaker cells (Galang et al., 2020) have been reported. Activities of these enhancers were examined by cell/tissue-specific expression of a lacZ reporter by transgenesis in mice. However, functional assessment by deleting the corresponding enhancer sequence was performed only for the sinoatrial node enhancer, which is located approximately 120 kb downstream of the Isl1 gene (Galang et al., 2020). Deletion of the Isl1 sinoatrial node enhancer resulted in reduced but detectable levels of ISL1 protein in the pacemaker cells.

In Isl1 HLPE−/− embryos, the Isl1 gene is intact, but Isl1 expression was reduced by approximately 50% compared to wild-type embryos (Isl1+/+; HLPE+/+). The Isl1+/−; HLPE+/− embryo possesses an Isl1 null allele, and the other wild-type Isl1 allele lacks HLPE in the cis position. The Isl1 expression in Isl1+/−; HLPE+/− embryos was also downregulated by approximately 50% compared to wild-type embryos. These results support the idea that the HLPE sequence acts as a cis-regulatory element for Isl1 expression in the hindlimb progenitors.

Our previous study showed SALL4 enrichment in CR2 of the Isl1 HLPE sequence (Chen et al., 2024), suggesting SALL4 regulation of Isl1 expression. If other enhancers act redundantly with the Isl1 HLPE to regulate Isl1 expression, and if SALL4 also regulates those putative redundant enhancers, Isl1 HLPE−/−; TCre; Sall4fl/fl mutants would exhibit further reduction of Isl1 expression compared to Isl1 HLPE−/− embryos. However, Isl1 expression in TCre; Sall4fl/fl, and Isl1 HLPE−/−; TCre; Sall4fl/fl mutants did not show significant differences. A possible reason is compensation of the loss of Sall4 by Sall1. Our recent study demonstrated redundancy between Sall1 and Sall4 in hindlimb development (Chen et al., 2024). TCre; Sall1fl/fl; Sall4fl/fl mutants failed to express Isl1 and lacked the hindlimbs. In TCre; Sall4fl/fl mutants, SALL1 may regulate Isl1 expression through Isl1 HLPE and other putative enhancers.

4.2. Isl1 HLPE also contributes to ISL1 expression in the branchial arch ectoderm

Our imaging analysis shows that ISL1 protein expression is reduced in the branchial arch ectoderm in Isl1 HLPE−/− embryos compared to wild-type embryos. Similar to Isl1 expression in hindlimb progenitor cells, the ISL1 signals were reduced but detectable, suggesting that other functionally redundant enhancers may simultaneously regulate ISL1 expression in the branchial arch ectoderm. Numerous studies have suggested that the development of the jaw and the development of the limb share molecular mechanisms (Gillis et al., 2009; Schneider et al., 1999; Tucker et al., 1999). Isl1 might be in a shared regulatory system for branchial arches and hindlimbs, and HLPE and other redundant enhancers may regulate Isl1 expression in both branchial arch ectoderm and hindlimb progenitors in the lateral plate mesoderm.

4.3. Isl1 HLPE contributes to normal chondrogenesis

Isl1 HLPE−/− neonates exhibit smaller bodies compared to wild type, and the femur length is shorter at E14.5. Given that skeletal growth by E14.5 is driven by cartilage growth (Fowler and Larsson, 2020; Kozhemyakina et al., 2015), this observation suggests that cartilage growth is reduced in Isl1 HLPE−/− mutants. However, chondrocyte proliferation and death did not exhibit differences between wild-type and Isl1 HLPE−/− femur at E14.5, suggesting molecular or cellular events in earlier stages contribute to shorter femur in HLPE−/− mutants. Gene expression analysis of the hindlimb at E12.5 shows misregulation of several genes involved in early chondrogenesis as well as chondrocyte hypertrophy in Isl1 HLPE−/− mutants. This data suggests that the Isl1 HLPE sequence contributes to normal chondrogenesis. Since Isl1 expression was not observed in the cartilage (Zhuang et al., 2013), the Isl1 HLPE sequence might contribute to regulation of other genes involved in cartilage development or growth.

4.4. Multiple factors regulate Tbx4 expression in hindlimb progenitors

In addition to previously reported Tbx4 regulation by Pitx1 (DeLaurier et al., 2006; Lanctot et al., 1999; Logan and Tabin, 1999; Szeto et al., 1999) and Isl1 (Kawakami et al., 2011), our study clarified that Sall4 regulates Tbx4 in hindlimb progenitor cells. SALL4 ChIP-seq using mouse embryo trunk tissues showed that SALL4 is enriched in a Tbx4 hindlimb enhancer (Chen et al., 2024). Downregulation of Tbx4 expression in TCre; Sall4fl/fl mutants in this study suggests that SALL4 directly regulates Tbx4 expression in hindlimb progenitors. Inducible knockout of Tbx4 in mice causes femur hypoplasia (Naiche and Papaioannou, 2007), which is also observed in Sall4 mutants (Akiyama et al., 2015). The phenotypic similarity suggests that part of the hindlimb defects in Sall4 mutants is caused by significant reduction of Tbx4.

4.5. Enhancer redundancy in gene regulation

Accumulating evidence supports the idea that cell type-specific expression of many developmental regulators is controlled by shadow enhancers, which are redundant cis-regulatory elements that control spatial-temporal expression of the same gene in an overlapping manner (Kvon et al., 2021). Even with the advancement of technologies, identifying and validating functional enhancers remains challenging (Zaugg et al., 2022). If multiple enhancers regulate the expression of a cognate gene, deletion of multiple enhancers on a sensitized background may be needed to assess their functions (Osterwalder et al., 2018). Even if multiple enhancers are simultaneously deleted, phenotypic changes may not be detectable (Cunningham et al., 2018; Duester, 2019). Such a multiple-enhancer system could create a buffer against perturbation by environmental and/or genetic factors, thus contributing to the overall robustness of gene expression during embryonic development. In the case of Isl1, a recent study of sinoatrial node enhancer suggested a presence of shadow enhancers in pacemaker cells (Galang et al., 2020). Similarly, our study suggests a presence of shadow enhancers for Isl1 expression in the hindlimb progenitors and the branchial arch ectoderm. Isl1 is located in a 1.4 Mbp gene desert (Touceda-Suárez et al., 2020). Gene deserts are large regions of chromosomes devoid of protein-coding sequences, and some of them have been shown to contain regulatory sequences for nearby developmental regulators (Abassah-Oppong et al., 2024; Ovcharenko et al., 2005). It is conceivable to speculate that other Isl1 regulatory elements are located in the gene desert and redundantly regulate Isl1 expression in hindlimb progenitor cells and the branchial arch ectoderm. Future research should strive to map these potential redundant enhancer elements to more completely understand the Isl1 regulatory network.

Supplementary Material

1
2
3
4
5
6
7
8
9
10

Highlights.

  • A cis element is previously reported to be an Isl1 hindlimb progenitor enhancer (HLPE).

  • Isl1 HLPE−/− mice are viable and fertile and do not show gross morphological hindlimb defects.

  • Gene expression analysis shows reduced expression of Isl1 in the posterior trunk in Isl1 HLPE−/− and Isl1+/−; Isl1 HLPE+/− embryos compared to wild-type embryos.

  • Imaging analysis shows reduced expression of ISL1 in the branchial arch ectoderm in Isl1 HLPE−/− embryos compared to wild-type embryos.

  • Isl1 HLPE acts as an enhancer, and multiple redundant enhancers likely regulate Isl1 expression in hindlimb progenitor cells and the branchial arch ectoderm.

Acknowledgements

We are grateful to Lauren Brackney, Sofiia Fursa, Nhi Huynh, Yeon-Ji Kim, Jackson Moorman, Mira Schrandt, Will Sullivan, and Matthew Walters for their excellent technical support. We are also grateful to Dr. Nobuaki Kikyo for sharing materials, Dr. Sho Kawakami for the help in statistical analysis, and Dr. Yun You and the Mouse Genetics Laboratory of the University of Minnesota for the excellent service in generating Isl1 HLPE mutant mice.

Funding

This study was supported by a grant from the National Institutes of Health to YK (R01AR064195). The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

CRediT authorship contribution statement

SO: Writing – original draft, Investigation, Formal analysis. HK: Writing – review & editing, Investigation, Formal analysis. AMN: Writing – review & editing, Investigation, Formal analysis. CB: Writing – review & editing, Investigation, Formal analysis. AA: Writing – review & editing, Investigation, Formal analysis. DC: Writing – review & editing, Investigation, Formal analysis. CM: Writing – review & editing, Investigation. RN: Writing – review & editing. MC: Writing – review & editing, Methodology, Investigation, Formal analysis, Supervision. YK: Writing – original draft, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization, Supervision, Resources, Project administration.

Declaration of competing interest

The authors have no competing interest.

References

  1. Abassah-Oppong S, Zoia M, Mannion BJ, Rouco R, Tissières V, Spurrell CH, Roland V, Darbellay F, Itum A, Gamart J, Festa-Daroux TA, Sullivan CS, Kosicki M, Rodríguez-Carballo E, Fukuda-Yuzawa Y, Hunter RD, Novak CS, Plajzer-Frick I, Tran S, Akiyama JA, Dickel DE, Lopez-Rios J, Barozzi I, Andrey G, Visel A, Pennacchio LA, Cobb J, Osterwalder M, 2024. A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival. Nat Commun 15, 8793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agarwal P, Wylie JN, Galceran J, Arkhitko O, Li C, Deng C, Grosschedl R, Bruneau BG, 2003. Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo. Development (Cambridge, England) 130, 623–633. [DOI] [PubMed] [Google Scholar]
  3. Agresti A, 2002. Inference for Contingency Tables, Categorical Data Analysis, Second edition ed. John Wiley & Sons, Inc., pp. 91–101. [Google Scholar]
  4. Akiyama R, Kawakami H, Taketo MM, Evans SM, Wada N, Petryk A, Kawakami Y, 2014. Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the beta-catenin pathway. Developmental biology 387, 37–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Akiyama R, Kawakami H, Wong J, Oishi I, Nishinakamura R, Kawakami Y, 2015. Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements. Proceedings of the National Academy of Sciences of the United States of America 112, 5075–5080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Al-Baradie R, Yamada K, St Hilaire C, Chan WM, Andrews C, McIntosh N, Nakano M, Martonyi EJ, Raymond WR, Okumura S, Okihiro MM, Engle EC, 2002. Duane radial ray syndrome (Okihiro syndrome) maps to 20q13 and results from mutations in SALL4, a new member of the SAL family. American journal of human genetics 71, 1195–1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Beverdam A, Svingen T, Bagheri-Fam S, McClive P, Sinclair AH, Harley VR, Koopman P, 2010. Protein tyrosine kinase 2 beta (PTK2B), but not focal adhesion kinase (FAK), is expressed in a sexually dimorphic pattern in developing mouse gonads. Dev Dyn 239, 2735–2741. [DOI] [PubMed] [Google Scholar]
  8. Boschloo RD, 1970. Raised Conditional Level of Significance for the 2×2-table when Testing the Equality of Two Probabilities. Statistica Neerlandica 24, 1–35. [Google Scholar]
  9. Chen KQ, Anderson A, Kawakami H, Kim J, Barrett J, Kawakami Y, 2022. Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4. PloS one 17, e0267273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen KQ, Kawakami H, Anderson A, Corcoran D, Soni A, Nishinakamura R, Kawakami Y, 2024. Sall genes regulate hindlimb initiation in mouse embryos. Genetics 227, iyae029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen KQ, Tahara N, Anderson A, Kawakami H, Kawakami S, Nishinakamura R, Pandolfi PP, Kawakami Y, 2020. Development of the Proximal-Anterior Skeletal Elements in the Mouse Hindlimb Is Regulated by a Transcriptional and Signaling Network Controlled by Sall4. Genetics 215, 129–141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cunningham TJ, Lancman JJ, Berenguer M, Dong PDS, Duester G, 2018. Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development. Cell Rep 23, 3146–3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. de Celis JF, Barrio R, 2009. Regulation and function of Spalt proteins during animal development. The International journal of developmental biology 53, 1385–1398. [DOI] [PubMed] [Google Scholar]
  14. DeLaurier A, Schweitzer R, Logan M, 2006. Pitx1 determines the morphology of muscle, tendon, and bones of the hindlimb. Developmental biology 299, 22–34. [DOI] [PubMed] [Google Scholar]
  15. Duester G, 2019. Knocking Out Enhancers to Enhance Epigenetic Research. Trends Genet 35, 89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fowler DA, Larsson HCE, 2020. The tissues and regulatory pattern of limb chondrogenesis. Developmental biology 463, 124–134. [DOI] [PubMed] [Google Scholar]
  17. Galang G, Mandla R, Ruan H, Jung C, Sinha T, Stone NR, Wu RS, Mannion BJ, Allu PKR, Chang K, Rammohan A, Shi MB, Pennacchio LA, Black BL, Vedantham V, 2020. ATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and Function. Circ Res 127, 1502–1518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gillis JA, Dahn RD, Shubin NH, 2009. Shared developmental mechanisms pattern the vertebrate gill arch and paired fin skeletons. Proceedings of the National Academy of Sciences of the United States of America 106, 5720–5724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jurberg AD, Aires R, Varela-Lasheras I, Novoa A, Mallo M, 2013. Switching axial progenitors from producing trunk to tail tissues in vertebrate embryos. Developmental cell 25, 451–462. [DOI] [PubMed] [Google Scholar]
  20. Kang J, Nathan E, Xu SM, Tzahor E, Black BL, 2009. Isl1 is a direct transcriptional target of Forkhead transcription factors in second-heart-field-derived mesoderm. Developmental biology 334, 513–522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kappen C, Salbaum JM, 2009. Identification of regulatory elements in the Isl1 gene locus. The International journal of developmental biology 53, 935–946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kawakami Y, Marti M, Kawakami H, Itou J, Quach T, Johnson A, Sahara S, O’Leary DD, Nakagawa Y, Lewandoski M, Pfaff S, Evans SM, Izpisua Belmonte JC, 2011. Islet1-mediated activation of the beta-catenin pathway is necessary for hindlimb initiation in mice. Development (Cambridge, England) 138, 4465–4473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim N, Park C, Jeong Y, Song MR, 2015. Functional Diversification of Motor Neuron-specific Isl1 Enhancers during Evolution. PLoS Genet 11, e1005560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kozhemyakina E, Lassar AB, Zelzer E, 2015. A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation. Development (Cambridge, England) 142, 817–831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kvon EZ, Waymack R, Gad M, Wunderlich Z, 2021. Enhancer redundancy in development and disease. Nat Rev Genet 22, 324–336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lanctot C, Moreau A, Chamberland M, Tremblay ML, Drouin J, 1999. Hindlimb patterning and mandible development require the Ptx1 gene. Development (Cambridge, England) 126, 1805–1810. [DOI] [PubMed] [Google Scholar]
  27. Lefebvre V, Angelozzi M, Haseeb A, 2019. SOX9 in cartilage development and disease. Current opinion in cell biology 61, 39–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liang X, Zhang Q, Cattaneo P, Zhuang S, Gong X, Spann NJ, Jiang C, Cao X, Zhao X, Zhang X, Bu L, Wang G, Chen HS, Zhuang T, Yan J, Geng P, Luo L, Banerjee I, Chen Y, Glass CK, Zambon AC, Chen J, Sun Y, Evans SM, 2015. Transcription factor ISL1 is essential for pacemaker development and function. The Journal of clinical investigation 125, 3256–3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu CF, Samsa WE, Zhou G, Lefebvre V, 2017. Transcriptional control of chondrocyte specification and differentiation. Seminars in cell & developmental biology 62, 34–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Logan M, Tabin CJ, 1999. Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity. Science (New York, N.Y 283, 1736–1739. [DOI] [PubMed] [Google Scholar]
  31. Long F, Ornitz DM, 2013. Development of the endochondral skeleton. Cold Spring Harb Perspect Biol 5, a008334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Naiche LA, Papaioannou VE, 2007. Tbx4 is not required for hindlimb identity or post-bud hindlimb outgrowth. Development (Cambridge, England) 134, 93–103. [DOI] [PubMed] [Google Scholar]
  33. Ohuchi H, Nakagawa T, Yamamoto A, Araga A, Ohata T, Ishimaru Y, Yoshioka H, Kuwana T, Nohno T, Yamasaki M, Itoh N, Noji S, 1997. The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor. Development (Cambridge, England) 124, 2235–2244. [DOI] [PubMed] [Google Scholar]
  34. Osterwalder M, Barozzi I, Tissieres V, Fukuda-Yuzawa Y, Mannion BJ, Afzal SY, Lee EA, Zhu Y, Plajzer-Frick I, Pickle CS, Kato M, Garvin TH, Pham QT, Harrington AN, Akiyama JA, Afzal V, Lopez-Rios J, Dickel DE, Visel A, Pennacchio LA, 2018. Enhancer redundancy provides phenotypic robustness in mammalian development. Nature 554, 239–243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ovcharenko I, Loots GG, Nobrega MA, Hardison RC, Miller W, Stubbs L, 2005. Evolution and functional classification of vertebrate gene deserts. Genome Res 15, 137–145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pappas MP, Kawakami H, Corcoran D, Chen KQ, Scott EP, Wong J, Gearhart MD, Nishinakamura R, Nakagawa Y, Kawakami Y, 2024. Sall4 regulates posterior trunk mesoderm development by promoting mesodermal gene expression and repressing neural genes in the mesoderm. Development (Cambridge, England) 151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Perantoni AO, Timofeeva O, Naillat F, Richman C, Pajni-Underwood S, Wilson C, Vainio S, Dove LF, Lewandoski M, 2005. Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development. Development (Cambridge, England) 132, 3859–3871. [DOI] [PubMed] [Google Scholar]
  38. Pfaff SL, Mendelsohn M, Stewart CL, Edlund T, Jessell TM, 1996. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84, 309–320. [DOI] [PubMed] [Google Scholar]
  39. Rallis C, Bruneau BG, Del Buono J, Seidman CE, Seidman JG, Nissim S, Tabin CJ, Logan MP, 2003. Tbx5 is required for forelimb bud formation and continued outgrowth. Development (Cambridge, England) 130, 2741–2751. [DOI] [PubMed] [Google Scholar]
  40. Rollins MR, Raynor JF, Miller EA, Butler JZ, Spartz EJ, Lahr WS, You Y, Burrack AL, Moriarity BS, Webber BR, Stromnes IM, 2023. Germline T cell receptor exchange results in physiological T cell development and function. Nat Commun 14, 528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sakaki-Yumoto M, Kobayashi C, Sato A, Fujimura S, Matsumoto Y, Takasato M, Kodama T, Aburatani H, Asashima M, Yoshida N, Nishinakamura R, 2006. The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development (Cambridge, England) 133, 3005–3013. [DOI] [PubMed] [Google Scholar]
  42. Schneider RA, Hu D, Helms JA, 1999. From head to toe: conservation of molecular signals regulating limb and craniofacial morphogenesis. Cell Tissue Res 296, 103–109. [DOI] [PubMed] [Google Scholar]
  43. Searls RL, Janners MY, 1971. The initiation of limb bud outgrowth in the embryonic chick. Developmental biology 24, 198–213. [DOI] [PubMed] [Google Scholar]
  44. Song MR, Sun Y, Bryson A, Gill GN, Evans SM, Pfaff SL, 2009. Islet-to-LMO stoichiometries control the function of transcription complexes that specify motor neuron and V2a interneuron identity. Development (Cambridge, England) 136, 2923–2932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sun Y, Dykes IM, Liang X, Eng SR, Evans SM, Turner EE, 2008. A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs. Nature neuroscience 11, 1283–1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sweetman D, Munsterberg A, 2006. The vertebrate spalt genes in development and disease. Developmental biology 293, 285–293. [DOI] [PubMed] [Google Scholar]
  47. Szeto DP, Rodriguez-Esteban C, Ryan AK, O’Connell SM, Liu F, Kioussi C, Gleiberman AS, Izpisua-Belmonte JC, Rosenfeld MG, 1999. Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development. Genes & development 13, 484–494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tahara N, Akiyama R, Theisen JWM, Kawakami H, Wong J, Garry DJ, Kawakami Y, 2018. Gata6 restricts Isl1 to the posterior of nascent hindlimb buds through Isl1 cis-regulatory modules. Developmental biology 434, 74–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tahara N, Kawakami H, Chen KQ, Anderson A, Yamashita Peterson M, Gong W, Shah P, Hayashi S, Nishinakamura R, Nakagawa Y, Garry DJ, Kawakami Y, 2019. Sall4 regulates neuromesodermal progenitors and their descendants during body elongation in mouse embryos. Development (Cambridge, England) 146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tickle C, 2015. How the embryo makes a limb: determination, polarity and identity. Journal of anatomy 227, 418–430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Touceda-Suárez M, Kita EM, Acemel RD, Firbas PN, Magri MS, Naranjo S, Tena JJ, Gómez-Skarmeta JL, Maeso I, Irimia M, 2020. Ancient Genomic Regulatory Blocks Are a Source for Regulatory Gene Deserts in Vertebrates after Whole-Genome Duplications. Mol Biol Evol 37, 2857–2864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tucker AS, Al Khamis A, Ferguson CA, Bach I, Rosenfeld MG, Sharpe PT, 1999. Conserved regulation of mesenchymal gene expression by Fgf-8 in face and limb development. Development (Cambridge, England) 126, 221–228. [DOI] [PubMed] [Google Scholar]
  53. Xu X, Weinstein M, Li C, Naski M, Cohen RI, Ornitz DM, Leder P, Deng C, 1998. Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction. Development (Cambridge, England) 125, 753–765. [DOI] [PubMed] [Google Scholar]
  54. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F, 1999. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718. [DOI] [PubMed] [Google Scholar]
  55. Yuri S, Fujimura S, Nimura K, Takeda N, Toyooka Y, Fujimura Y, Aburatani H, Ura K, Koseki H, Niwa H, Nishinakamura R, 2009. Sall4 is essential for stabilization, but not for pluripotency, of embryonic stem cells by repressing aberrant trophectoderm gene expression. Stem Cells 27, 796–805. [DOI] [PubMed] [Google Scholar]
  56. Zaugg JB, Sahlén P, Andersson R, Alberich-Jorda M, de Laat W, Deplancke B, Ferrer J, Mandrup S, Natoli G, Plewczynski D, Rada-Iglesias A, Spicuglia S, 2022. Current challenges in understanding the role of enhancers in disease. Nat Struct Mol Biol 29, 1148–1158. [DOI] [PubMed] [Google Scholar]
  57. Zhuang S, Zhang Q, Zhuang T, Evans SM, Liang X, Sun Y, 2013. Expression of Isl1 during mouse development. Gene Expr Patterns 13, 407–412. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

1
NIHMS2113717-supplement-1.pdf (36.3KB, pdf)
2
NIHMS2113717-supplement-2.tif (920.8KB, tif)
3
NIHMS2113717-supplement-3.tif (1.8MB, tif)
4
NIHMS2113717-supplement-4.docx (16.9KB, docx)
5
NIHMS2113717-supplement-5.docx (15.6KB, docx)
6
NIHMS2113717-supplement-6.docx (17.9KB, docx)
7
NIHMS2113717-supplement-7.docx (16.4KB, docx)
8
NIHMS2113717-supplement-8.docx (17.4KB, docx)
9
NIHMS2113717-supplement-9.docx (17.2KB, docx)
10
NIHMS2113717-supplement-10.docx (17.3KB, docx)

RESOURCES