Early limb patterning in the direct‐developing salamander Plethodon cinereus revealed by sox9 and col2a1

Abstract

Direct-developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct-developing frogs, but little attention has been given to direct-developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct-developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior-to-posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote-like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct-developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct-developing salamander species.

Fgf-signaling is compartmentalized within the mesenchyme and controls proliferation during salamander limb development

“…These findings support independent molecular control of digit specification and differentiation and hint at the wide diversity in ontogenetic and heterochronic shifts that have occurred in the limb during urodele evolution (Blanco and Alberch, 1992; Franssen et al, 2005). Similarly, Sox9 expression shows spatiotemporal variability across urodeles (Kerney et al, 2018) and this underscores the need for more comparative limb studies to better understand the ancestral condition. While preaxial vs. postaxial dominance of skeletal formation clearly separates urodele limb development from anurans and amniotes, it is likely this difference points to alterations in the upstream genetic control of digit specification involving Fgfs , Bmps , and retinoic acid (Montero et al, 2017; ten Berge et al, 2008).…”

Whispers from vestigial nubbins: Arrested development provokes trait loss in toads

“…In amphibians, developmental mechanisms underlying the loss or reduction of traits may be inherently tied to metamorphosis (Eigenmann & Denny, 1900); it has been suggested that pre‐metamorphic stages are more fixed than late labile stages, and that postmetamorphic changes in gene expression or timing serve to compartmentalize trait evolution and loss (Moran, 1994; Wilkens, 2007). Given that toad development is prolonged overall (Fabrezi & Goldberg, 2009; Hetherington, 1987; Smirnov, 1989; Womack, Stynoski, et al, 2018), postmetamorphic truncation of the columella could have resulted from desynchronization of gene regulatory networks for endochondral chondrification and ossification such as runx2, col2a1 , and sox9 (Chapman, 2011; Gómez‐Picos & Eames, 2015; Kerney et al, 2018; Sienknecht, 2013), and/or release of thyroid hormone, which stimulates both metamorphosis and skeletogenesis in anurans (Bassett & Williams, 2016; Hanken et al, 1989). The reversal of postmetamorphic degeneration via resynchronization could explain the inferred regain of tympanic middle ear structures in at least two bufonid lineages, including within Atelopus (Lande, 1978; Pereyra et al, 2016).…”

Genetic basis for an evolutionary shift from ancestral preaxial to postaxial limb polarity in non-urodele vertebrates

“…Apparently Bmp activity, even when down-modulated, is in excess during limb skeletal element formation and suffices for mesenchymal condensation following cell-cycle exit, but the timing of cell-cycle slowing in anterior relative to posterior limb bud (Figure 5B) determines when cells become permissive to Bmp response and sets the polarity of limb axis formation. Notably, even direct-developing salamanders with a ''paddle'' handplate stage, have a comparatively narrow shaped limb bud, 32 suggesting a lower degree of proliferative expansion. Bmps have recently been implicated in directly regulating cell cycle in the limb, 33 but our results indicate a more direct effect of Gli3 on cell-cycle gene expression.…”