Influence of FGF4 on Digit Morphogenesis during Limb Development in the Mouse

Abstract

Much of what we currently know about digit morphogenesis during limb development is deduced from embryonic studies in the chick. In this study, we used ex utero surgical procedures to study digit morphogenesis during mouse embryogenesis. Our studies reveal some similarities; however, we have found considerable differences in how the chick and the mouse autopods respond to experimentation. First, we are not able to induce ectopic digit formation from interdigital cells as a result of wounding or TGFbeta-1 application in the mouse, in contrast to what is observed in the chick. Second, FGF4, which inhibits the formation of ectopic digits in the chick, induces a digit bifurcation response in the mouse. We demonstrate with cell marking studies that this bifurcation response results from a reorganization of the prechondrogenic tip of the digit rudiment. The FGF4 effect on digit morphogenesis correlates with changes in the expression of a number of genes, including Msx1, Igf2, and the posterior members of the HoxD cluster. In addition, the bifurcation response is digit-specific, being restricted to digit IV. We propose that FGF4 is an endogenous signal essential for skeletal branching morphogenesis in the mouse. This work stresses the existence of major differences between the chick and the mouse in how digit morphogenesis is regulated and is thus consistent with the view that vertebrate digit evolution is a relatively recent event. Finally, we discuss the relationship between the digit IV bifurcation restriction and the placement of the metapterygial axis in the evolution of the tetrapod limb.

Why five fingers? Evolutionary constraints on digit numbers

“…Therefore, in mice, the phylotypic stage can be specified as being from E7 to E11. In these animals, the development of the limb bud starts on E8, and mesenchymal digit condensations appear on E10 and E11, in the forelimb slightly before the hind limb 20 . The patterning of the limb up to the determination of the number of digits therefore occurs during a stage that is characterized by global inductive interactivity.…”

Digit regeneration is regulated byMsx1andBMP4in fetal mice

“…For in situ hybridization, digoxigenin-labeled riboprobes complementary to Msx1 (Ngo- Muller and Muneoka, 2000b), Msx2 (Ngo- Muller and Muneoka, 2000b), Bmp4 (Jones et al, 1991), Hoxc13 (Godwin and Capecchi, 1998) and Ihh (St-Jacques et al, 1999) were used in whole-mount or paraffin-sectioned preparations as described (Schaller and Muneoka, 2001;Omi et al, 2002). Fetal digit tissues were fixed by immersion in 4% paraformaldehyde and post-natal digits were fixed by injection of fixative into the digits followed by immersion in 4% paraformaldehyde.…”

“…At this stage, Msx1 and Msx2 are expressed in distinct domains that overlap in the distal mesenchyme. Msx1 is expressed in all mesenchymal cells at the digit tip and forms a domain that is complementary to the central chondrofying terminal phalangeal element (Ngo- Muller and Muneoka, 2000b) (Fig. 1G,H).…”

BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling

“…However, FGF-soaked beads can prevent interdigital PCD in chick embryos (Buckland et al, 1998;Ganan et al, 1996;Macias et al, 1996;Ngo-Muller and Muneoka, 2000), suggesting that interdigital PCD might be induced by the loss of AER-FGF signaling that occurs during AER regression (SalasVidal et al, 2001;Yokouchi et al, 1996;Zou and Niswander, 1996). On the other hand, application of FGF-soaked beads can also increase interdigital apoptosis, consistent with a model of cooperation between FGFs and BMPs acting as cell death triggers (Ganan et al, 1998;Montero et al, 2001).…”