Sall genes regulate hindlimb initiation in mouse embryos

Abstract

Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10, which triggers molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5), and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4, zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing 1 allele of Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre-mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation.

Keywords: Isl1; Sall1; Sall4; hindlimb; limb initiation.

Fig. 1.

Sall4 promotes Isl1 function during initiation of hindlimb bud development. a) SALL4 enrichment around the Isl1 gene. SALL4 ChIP-seq track and evolutional conservation (Evol. cons) are shown. The numbers 1–6 indicate Isl1 exons. Bars immediately under the SALL4 ChIP-seq track indicate statistically significant SALL4 enrichment. The data represents 0–0.47 counts per million. b) Enlarged view of the boxed area in (a). The previously characterized Isl1 Enhancer is shown by the bar under CR1 and CR2. CR1 and CR2 indicate conserved regions 1 and 2 (Kang et al. 2009). c and d). Skeletal images of WT (c) and Hoxb6Cre; Isl1^fl/−; Sall4^fl/+ d) at E17.5. Arrows in (c) point to normal hindlimbs. Asterisks in (d) indicate a lack of hindlimbs. Images are at the same scale. Scale bar, 2 mm. e) Bar graph showing percentages of normal, hindlimbs with skeletal defects (defective), or lack of hindlimbs (no hindlimb) in Hoxb6Cre; Isl1; Sall4 compound mutants. The skeleton was examined at E13.5—P0. WT, cKO, and cHet indicate WT, conditional KO, and conditional heterozygous (fl/+), respectively. n indicates a number of hindlimbs.

Fig. 2.

Isl1 and Sall4 genetically interact upstream of Fgf10 expression in hindlimb progenitors. Dorsal views of expression patterns of Fgf10 a–h), Dusp6 i–l), Tbx4 m–p), and Pitx1 q–t) at E9.75 (a–d, i–t) and E10.0 (e–h) at indicated genotypes. Thick arrows in a, e, i, m, q–t indicate normal expression. Thin arrows in b, f, j, n indicate weaker expression than WT. Asterisks in c, d, g, h, k, l, o, p indicate loss of expression.

Fig. 3.

Combined function of Sall1 and Sall4 regulates hindlimb development. Lateral views of the body a, d, g, and j), dorsal views of forelimbs b, e, h, and k), dorsal views of hindlimbs c and f) and dorsal views around the sacral region i, l) of Alcian blue-stained E14.5 skeleton with the indicated genotypes. TCre; Sall1^fl/+; Sall4^fl/fl mutants lacked hindlimb skeleton with only hypoplastic pelvic girdle (i, arrowheads). TCre; Sall1^fl/fl; Sall4^fl/fl mutants possess only central digits in the forelimb (k, marked by #) and completely lack hindlimbs (l, asterisks). (l’) A dorsal view of vertebrae and ribs of TCre; Sall1^fl/fl; Sall4^fl/fl mutants, showing partial fusion of posterior ribs (pointed by arrows). a, d, g and j is at the same scale, scale bar, 2 mm. b, c, e, f, h, i, k, and l is at the same scale, scale bar, 1 mm. l’, scale bar, 1 mm. m) Bar graph showing percentages of normal hindlimbs with skeletal defects (defective), or lack of hindlimbs (no hindlimb) in TCre; Sall1; Sall4 mutants. The skeleton was examined at E13.5–15.5. cKO and cHet indicate conditional KO and conditional heterozygous (fl/+), respectively. n indicates number of hindlimbs. Abbreviations. fe: femur, fi: fibula, h: humerus, pg: pelvic girdle, r: radius, sg: shoulder girdle, ti: tibia, u: ulna. Digits are numbered as 1–5. For simplicity, only panels b and c are labeled.

Fig. 4.

Combined function of Sall1 and Sall4 act as master regulators of hindlimb initiation. Dorsal view a–d, f–i, k–n, p–s, and u–x) and lateral views f’–i’) of expression patterns of Fgf10 (a–d), Isl1 (f–i’), Lef1 (k–n), Tbx4 p–s), and Pitx1 u–x) at E9.75 at indicated genotypes. Thick arrows in a, f, f’, k, p, q, u, and v indicate normal expression. Thinner arrows in b, g, g’, l, r, and w indicate weaker expression than WT. Thinnest arrows in c, h, h’, m indicate faint expression. Asterisks in d, i, i’, n, s, and x indicate loss of expression. Black arrows in f”—i” indicate Isl1 expression in the ventral mesenchyme. Arrowheads in k and l indicate normal expression, while arrowheads in m and n denote reduced expression of Lef1 in the presomitic mesoderm. e, j, o, t, and y) Relative expression levels of Fgf10 (e), Isl1 (j), Lef1 (o), Tbx4 (t), and Pitx1 (y) in the LPM of WT (n = 6) and Sall1/Sall4 DKO (n = 5) embryos by qRT-PCR. Each dot represents an embryo.

Fig. 5.

A model contrasting genetic systems for initiation of forelimbs and hindlimbs. See details in the Discussion section.