Current research on mechanisms of limb bud development, and challenges for the next decade.

The developmental mechanisms of limb buds have been studied in developmental biology as an excellent model of pattern formation. Chick embryos have contributed to the discovery of new principles in developmental biology, as it is easy to observe live embryos and manipulate embryonic tissues. Herein, I outline recent findings and future issues over the next decade regarding three themes, based on my research: limb positioning, proximal-distal limb elongation and digit identity determination. First, how hindlimb position is determined at the molecular level is described, with a focus on the transforming growth factor-ß signaling molecule GDF11. Second, I explain how the cell population in the limb bud deforms with developmental progress, shaping the limb bud with elongation along the proximal-distal axis. Finally, I describe the developmental mechanisms that determine digit identity through the interdigits.

Rethinking positional information and digit identity: The role of late interdigit signaling.

Seminal work from John Fallon's lab has illuminated how digit identity determination involves ongoing late regulation and occurs progressively during phalanx formation. Complementary genetic analyses in mice and several papers in this special issue have begun to flesh out how interdigit signaling accomplishes this, but major questions remain unaddressed, including how uncommitted progenitors from which phalanges arise are maintained, and what factors set limits on digit extension and phalanx number, particularly in mammals. This review summarizes what has been learned in the two decades since control of digit identity by late interdigit signals was first identified and what remains poorly understood, and will hopefully spark renewed interest in a process that is critical to evolutionary limb adaptations but nevertheless remains enigmatic.

Downregulation of Grem1 expression in the distal limb mesoderm is a necessary precondition for phalanx development.

BACKGROUND: The phalanges are the final skeletal elements to form in the vertebrate limb and their identity is regulated by signaling at the phalanx forming region (PFR) located at the tip of the developing digit ray. Here, we seek to explore the relationship between PFR activity and phalanx morphogenesis, which define the most distal limb skeletal elements, and signals associated with termination of limb outgrowth. RESULTS: As Grem1 is extinguished in the distal chick limb mesoderm, the chondrogenesis marker Aggrecan is up-regulated in the metatarsals and phalanges. Fate mapping confirms that subridge mesoderm cells contribute to the metatarsal and phalanges when subridge Grem1 is down-regulated. Grem1 overexpression specifically blocks chick phalanx development by inhibiting PFR activity. PFR activity and digit development are also disrupted following overexpression of a Gli3 repressor, which results in Grem1 expression in the distal limb and downregulation of Bmpr1b. CONCLUSIONS: Based on expression and fate mapping studies, we propose that downregulation of Grem1 in the distal limb marks the transition from metatarsal to phalanx development. This suggests that downregulation of Grem1 in the distal limb mesoderm is necessary for phalanx development. Grem1 downregulation allows for full PFR activity and phalanx progenitor cell commitment to digit fate.

Digit ratio in the common toad Bufo bufo: the effects of reduced fingers and of age dependency.

INTRODUCTION: Despite the growing number of studies describing digit ratio patterns in tetrapods, knowledge concerning certain basic issues is still scarce. In lower vertebrates such as tailless amphibians (Anura), the numbering of individual fingers on the forelimbs and their homology with the fingers of other vertebrates pose an unsolved problem. Based on reviewed data on anuran limb development, we argue that the correct finger numbering scheme should be based on the assumption that the first finger, not the fifth finger, was reduced on the forelimbs. We analyzed the digit ratio in the common toad (Bufo bufo, Bufonidae), a species characterized by well-developed sexual dimorphism whereby females are larger than males, using both numbering schemes present in the literature. RESULTS: We found that the digit ratio on hindlimbs differed significantly between the sexes only in the cases of left 2D:3D, with lower digit ratios in females, and of left 3D:4D, with lower digit ratios in males. We found that sex was the only significant variable for forelimbs, differentiating 2D:3D on the left forelimb, with lower digit ratios in females; 2D:4D on the right forelimb, with lower digit ratios in males; and 3D:4D on both forelimbs, with lower digit ratios in males. These results relate to variant II reflecting the hypothesis that the first digit was reduced during phylogenesis. There was no relationship between the body size (SVL) of individuals and any digit ratio, excluding 2D:4D on the right forelimbs in models with age variables. Additionally, for a subset of data where individual age was known, the models indicated that age was linked to significant differences in 2D:4D and 3D:4D on the left hindlimbs, while age, SVL, and sex influenced 2D:4D on the right forelimbs. CONCLUSION: We emphasize the importance of the problem of the correct numbering of forelimb digits in Anura and, under the assumption that it was the fifth digit that was reduced, argue that earlier results on digit ratio in this group should be interpreted with caution. The detected relationship between digit ratio and age in amphibians expands our knowledge, indicating that the age of individuals should be included in future digit ratio studies. This relationship may also apply to studies using digit ratio as a noninvasive indicator of endocrine disruption in amphibians.

The dynamic spatial and temporal relationships between the phalanx-forming region and the interdigits determine digit identity in the chick limb autopod.

BACKGROUND: Interdigits (IDs) determine digit identity in chick limbs. They are located between the digital rays and act as secondary signaling centers downstream of sonic hedgehog to provide positional information for determining digit identity in the phalanx-forming region (PFR). We examined the dynamic developmental mechanism by which PFR cells obtain positional information from IDs to determine the identity of individual digits in the chick hindlimb. RESULTS: We identified the specific region of the IDs responsible for determining digit identity and showed that PFR cells actively receive positional information only from the posteriorly, and not the anteriorly, located IDs. We also demonstrated that digits 1, 2, and 3 are interchangeable with each other, but not with digit 4. Finally, we found that both ID4 and digital ray 4 are necessary for determining digit 4 identity. CONCLUSIONS: The digital rays are naïve during the initial stages of their development, at which time digit identity is not determined. To determine digit identity, each PFR cell shows a unidirectional response to obtain positional information specifically from the IDs located posterior to the PFR, regardless of the signal strength from the anteriorly located IDs.

Gremlin1 induces anterior-posterior limb bifurcations in developing Xenopus limbs but does not enhance limb regeneration.

Gremlin1 (grem1) has been previously identified as being significantly up-regulated during regeneration of Xenopus laevis limbs. Grem1 is an antagonist of bone morphogenetic proteins (BMPs) with a known role in limb development in amniotes. It forms part of a self-regulating feedback loop linking epithelial (FGF) and mesenchymal (shh) signalling centres, thereby controlling outgrowth, anterior posterior and proximal distal patterning. Spatiotemporal regulation of the same genes in developing and regenerating Xenopus limb buds supports conservation of this mechanism. Using a heat shock inducible grem1 (G) transgene to created temperature regulated stable lines, we have shown that despite being upregulated in regeneration, grem1 overexpression does not enhance regeneration of tadpole hindlimbs. However, both the regenerating and contralateral, developing limb of G transgenics developed skeletal defects, suggesting that overexpressing grem1 negatively affects limb patterning. When grem1 expression was targeted earlier in limb bud development, we saw dramatic bifurcations of the limbs resulting in duplication of anterior posterior (AP) pattern, forming a phenotypic continuum ranging from duplications arising at the level of the femoral head to digit bifurcations, but never involving the pelvis. Intriguingly, the original limbs have AP pattern inversion due to de-restricted Shh signalling. We discuss a possible role for Grem1 regulation of limb BMPs in regulation of branching pattern in the limbs.