An Fgf/Gremlin inhibitory feedback loop triggers termination of limb bud outgrowth

Abstract

During organ formation and regeneration a proper balance between promoting and restricting growth is critical to achieve stereotypical size. Limb bud outgrowth is driven by signals in a positive feedback loop involving fibroblast growth factor (Fgf) genes, sonic hedgehog (Shh) and Gremlin1 (Grem1). Precise termination of these signals is essential to restrict limb bud size. The current model predicts a sequence of signal termination consistent with that in chick limb buds. Our finding that the sequence in mouse limb buds is different led us to explore alternative mechanisms. Here we show, by analysing compound mouse mutants defective in genes comprising the positive loop, genetic evidence that FGF signalling can repress Grem1 expression, revealing a novel Fgf/Grem1 inhibitory loop. This repression occurs both in mouse and chick limb buds, and is dependent on high FGF activity. These data support a mechanism where the positive Fgf/Shh loop drives outgrowth and an increase in FGF signalling, which triggers the Fgf/Grem1 inhibitory loop. The inhibitory loop then operates to terminate outgrowth signals in the order observed in either mouse or chick limb buds. Our study unveils the concept of a self-promoting and self-terminating circuit that may be used to attain proper tissue size in a broad spectrum of developmental and regenerative settings.

Figure 1. Fgf8 repression of Fgf4 expression is dependent on Grem1 but not Shh

(a–n) Gene expression in mouse forelimb (FL) or hindlimb (HL) buds. (a–i) In wild-type mouse limb buds, Fgf4 expression terminates first, followed by Shh and then Grem1. In d and e, a combination of RNA probes is used to detect non-overlapping patterns of Shh (arrowhead) and Grem1 (arrow) expression. Both genes are expressed in the E12 hindlimb bud, which is at an earlier developmental stage than the E12 forelimb bud from the same embryo, where only Grem1 is expressed(n=4). Downregulation of Spry4 expression at E11.75 compared to E10.75 reflects decreased AER-FGF activity, consistent with loss of Fgf4 and reduced Fgf8 expression. (j–m) In E10.5 hindlimb buds, Fgf4 expression is detected in the posterior two-thirds of the AER in normal, expanded through the entire AER in the Fgf8;Shh-DKO mutant and absent in Fgf8;Grem1-DKO mutant. (n) Detection of the remaining exon 1 of the truncated Fgf8 mRNA indicates that the AER is present. (o–r) No forelimb or hindlimb elements are observed in Fgf8;Grem-DKO skeletons. Fgf8;Shh-DKO embryos were generated by mating Msx2cre;Fgf8^del/flox;Shh^+/− males to Fgf8^flox/flox;Shh^+/− females,. Fgf8;Grem-DKO embryos were generated by crossing Msx2cre;Fgf8^del/+;Grem1^+/− males to Fgf8^flox/flox;Grem1^+/− females,. sc, scapula; pg, pelvic girdle.

Figure 2. FGF signaling represses Grem1 expression

(a–k) Gene expression in mouse forelimb buds at (a,b) E10.75, (c–i) E11 and (j,k) E11.5. (a) The yellow bracket indicates distance between AER and high Grem1 expression. (c,d) Reduced Spry4 expression in the posterior mesenchyme delineates Fgfr-inactivated domain. Arrows in d, f, i, k indicate anterior boundary of Shh^cre-mediated receptor inactivation domain. (e–g) Grem1 is ectopically expressed in the distal portion of the Fgfr-inactivated domain. Limb buds shown in d and f are contralateral limb buds from the same embryo. Boxed region in f is magnified in g. (h,i) Bmp4 is reduced in Fgfr-inactivated domain, but is present in the overlying AER. (j,k) No ectopic Grem1 expression is detected in Shh^cre;Bmpr1a^fl/fl (Bmpr1a-KO) limb buds. (l) A diagram depicting gene expression regulation within the Fgfr-inactivated domain in Fgfr1;r2- DKO limb buds as shown in g. Bmps from the AER may be required to promote ectopic Grem1–, leading to higher Grem1 in the distal portion of the Fgfr inactivated domain as shown in Fig. 2g, Fig. 3l,n. Fgfr1;r2-DKO embryos were generated by mating Shh^cre;Fgfr1^co/co;Fgfr2^c/+ males to Fgfr1^co/co;Fgfr2^c/+ females–. Bmpr1a-DKO embryos were generated by mating Shh^cre;Bmpr1a^fl/+ males to Bmpr1a^fl/fl females,.

Figure 3. AER-FGF repression of Grem1 expression is dose-sensitive

(a–e) Correlation between Grem1 repression in the distal mesenchyme and increased AER-FGF signaling (yellow brackets in c–e). (f–i) Beads (circle) soaked in 1mg/ml FGF2 suppress Grem1 expression distal to the bead, possibly working in combination with FGFs expressed from the AER (n=4/6). No Grem1 suppression is observed with 0.1mg/ml FGF2 (n=7). Beads were implanted in stage 21 limb buds and gene expression was assayed after 12 hours of incubation. (j–n) While ectopic Grem1 expression is more intense in E11.5 Fgfr1;r2-DKO limb buds than in Fgfr1-DKO limb buds, Grem1 expression outside of the Fgfr-inactivated domain remains comparable. Boxed regions in k,m are magnified in l,n, respectively. (o,p) Though absent in E11.75 normal limb bud, Fgf4 expression persists in the posterior AER overlying the Fgfr-inactivated domain in Fgfr1;r2-DKO limb buds (delineated by yellow dashed line).

Figure 4. A model describing a self-promoting and self-terminating mechanism to control limb bud outgrowth signals

(a) A schematic of the inhibitory loop (outlined in red) in relation to the existing positive loop. Arrows indicate activation, while “T” lines indicate inhibition. BMP regulation of AER architecture indirectly affects Fgf8 expression,. Grem1 is also positively regulated by BMP signaling,,,. (b) A model explaining how the two loops are utilized to first promote (phase I) and then terminate (phase II) signals. Dashed lines represent diminishing regulation while dashed line with “X” emphasizes absence of regulation. In phase I, the positive regulatory loop operates to increase all signals. Transition to phase II occurs when AER-FGFs reach a level that confers efficient Grem1 repression (represented by “T” in both distal and posterior mesenchyme). Together with mesenchymal growth, the Grem1-negative domain expands. Increasing distance between Grem1-expressing cells and Fgf or Shh-expressing cells leads to inability of signals to maintain one another at the end of phase II.