Antagonism between retinoic acid and fibroblast growth factor signaling during limb development

Abstract

The vitamin A metabolite retinoic acid (RA) provides patterning information during vertebrate embryogenesis, but the mechanism through which RA influences limb development is unclear. During patterning of the limb proximodistal axis (upper limb to digits), avian studies suggest that a proximal RA signal generated in the trunk antagonizes a distal fibroblast growth factor (FGF) signal. However, mouse and zebrafish genetic studies suggest that loss of RA suppresses forelimb initiation. Here, using genetic and pharmacological approaches, we demonstrate that limb proximodistal patterning is not RA dependent, thus indicating that RA-FGF antagonism does not occur along the proximodistal axis of the limb. Instead, our studies show that RA-FGF antagonism acts prior to limb budding along the anteroposterior axis of the trunk lateral plate mesoderm to provide a patterning cue that guides formation of the forelimb field. These findings reconcile disparate ideas regarding RA-FGF antagonism and provide insight into how endogenous RA programs the early embryo.

Figure 1. Meis1/2 Expression Is Maintained During Limb Proximodistal Patterning following Loss of RA Signaling

Shown are in situ hybridization results for Meis1, Meis2, and RARb expression in E10.25 wild-type (WT) and Rdh10 mutant (Rdh10 mut) embryos cultured for 6 hr in DMEM/F-12 medium ± 10 μM BMS493; eye (e), neural (n), lateral plate mesoderm (lpm), and proximal hindlimb bud (h) expression are indicated. See also Figure S1.

Figure 2. Meis1/2 Expression Is Established during Limb Field Prepatterning in the Absence of RA Signaling

(A–D) In situ hybridization for Meis1 and Meis2 prior to and during limb development. (A and B) Meis1 and Meis2 expression at E9.5 in wild-type and Rdh10 mutant embryos; forelimb bud (f) expression is indicated. (C and D) Meis1 and Meis2 expression prior to budding at E8.5 in wild-type and Raldh2^−/− embryos. Lower panels represent transverse sections through the presumptive forelimb field revealing the lateral plate mesoderm (lpm). See also Figure S2.

Figure 3. The RARE-lacZ Reporter Transgene Is Sensitive to RA Signaling Down to 0.25 nM RA

Wild-type (WT) and Rdh10 mutant (Rdh10 mut) E8.5 embryos stained with X-gal to detect RARE-lacZ RA signaling activity following culture for 12 hr in DMEM/F-12 medium (control) or DMEM/F-12 plus 0.25 nM, 1 nM, or 2.5 nM RA. Neural (n) and lateral plate mesoderm (lpm) expression is indicated. See also Figure S3.

Figure 4. Spatiotemporal Tbx5 Deficiency Is Concomitant with Expanded Heart FGF Signaling and Reduced Heart RA Signaling

(A–C) In situ hybridization at E8.5 in wild-type (WT) and Rdh10 mutant (Rdh10 mut) embryos (lateral view unless stated). (A) Shown is Tbx5 expression in the presumptive forelimb field (f) in 13-somite (13s) and 17-somite (17s) embryos; embryos were costained for Uncx4.1 expression, which labels somites as previously described (Zhao et al., 2009). (B) Fgf8 expression preceding and during normal Tbx5 initiation at six-somite (6s; dorsal and lateral views), eight-somite (8s; ventral view), and ten-somite (10s) stages. Arrows represent the posterior boundary of the heart (h); arrowheads represent the anterior boundary of the caudal progenitor zone (CPZ) and node (n). (C) Spry2 expression during normal Tbx5 initiation at 8s (dorsal view) and nine-somite (9s) stages. Arrows represent the posterior boundary of the heart (h). (D and E) Wild-type (WT) and Rdh10 mutant embryos stained with X-gal to detect RARE-lacZ RA signaling activity preceding and immediately following normal Tbx5 initiation at 6s and 13s stages. (E) Transverse sections through the heart (middle panels) and presumptive forelimb field (lower panels) are shown. Activity in heart (h), neural tube (n), intermediate mesoderm (im), dorsal heart (dh), and lateral plate mesoderm (lpm) is indicated.

Figure 5. RA Controls Limb Tbx5 Independently of Heart Isl1

(A–C) E8.5 mouse embryos analyzed by in situ hybridization (A and C) or stained with X-gal to detect Isl1-nlacZ expression (B). (A) Isl1 mRNA in Rdh10 mutant (Rdh10 mut) versus wild-type (WT) embryos (ventral view). Arrows represent the posterior boundary of the heart field. (B) Isl1-nlacZ expression in Raldh2^−/−;Isl1-nlacZ^+/− versus Isl1-nlacZ^+/− embryos. Arrows represent the posterior boundary of the heart field (h). (C) Assessment of heart expansion (h) and forelimb field Tbx5 expression (f) in wild-type, Isl1-nlacZ^−/−, Raldh2^−/−, and Raldh2^−/−;Isl1-nlacZ^−/− embryos. See also Figures S4 and S5.

Figure 6. RA-FGF Antagonism Is Required to Generate the Forelimb Field

(A) Assessment of forelimb field Tbx5 expression (f) in control versus FGF8-treated wild-type (WT) embryos cultured in vitro for 12 hr. Heart Tbx5 mRNA (h) and somite Uncx4.1 mRNA (s) are also indicated. (B) Rescue of missing pectoral fins in raldh2 (nls) mutant zebrafish by crossing to heat-shock-inducible dn-fgfr1 transgenic fish (heat shock at 37°C; 8–10 hpf). Arrows indicate pectoral fins. See also Figure S6.

Figure 7. Model for Axial RA-FGF Antagonism prior to Limb Budding

Previous genetic studies support FGF8 repression of Meis1/2 in distal limb elements, but our genetic findings show that RA signaling is not required for Meis1/2 expression in the proximal limb. Thus, RA-FGF antagonism is not required along the limb proximodistal axis to provide Meis1/2 expression proximally in the stylopod (blue) and prevent distal expression in the zeugopod (orange) and autopod (green). Instead, our studies support a model in which RA is required earlier for axial antagonization of Fgf8 expression (in both the second heart field and caudal progenitor zone -CPZ) prior to forelimb budding for correct induction and positioning of Tbx5 expression in the forelimb field.