Development of fore‐ and hindlimb muscles in GFP‐transgenic axolotls: Morphogenesis, the tetrapod bauplan, and new insights on the Forelimb‐Hindlimb Enigma

Abstract

The axolotl is becoming one of the most used model organisms in developmental and regenerative studies but no publication has described in detail the development of its forelimb and hindlimb muscles. We describe and illustrate the ontogeny of these muscles in transgenic axolotls that express GFP in muscle fibers and discuss our results and data previously obtained by us and by other authors about limb regeneration in axolotls and ontogeny in frogs and other tetrapods. Our observations and comparisons: (1) demonstrate radio-ulnar and ventro-dorsal morphogenetic gradients in the order of axolotl forelimb muscle formation and differentiation, while in axolotl hindlimb ontogeny there are only proximo-distal and tibio-fibular gradients; some of the axolotl gradients are therefore different from the ulno-radial/fibulo-tibial fore- and hindlimb and the dorso-ventral hindimb ontogenetic morphogenetic gradients seen in frogs and amniotes such as chickens; (2) provide a potential explanation for the usual presence, in both limbs of taxa from all major tetrapod groups, of more radial/tibial muscles than ulnar/fibular muscles; (3) support the "in-out" developmental mechanism of appendicular muscle formation; (4) offer new insights about the ancestral Bauplan of tetrapod limbs, including the striking similarity of the zeugopodial (forearm/leg) and autopodial (hand/foot) muscles of the two limbs and the ventro-dorsal symmetry of the zeugopodial muscles of a same limb; and (5) provide further evidence to corroborate the hypothesis that these similarities are due to derived homoplastic events that occurred during the fins-limbs transition and not due to forelimb-hindlimb serial homology.

Characteristic tetrapod musculoskeletal limb phenotype emerged more than 400 MYA in basal lobe-finned fishes

“…Images were resized in ImageJ (NIH) and 3D reconstruction of fin skeleton and muscles was performed with Amira 5.2.1 (Visage Imaging) with manual segmentation of structures. Information about Ambystoma is from our previous works on the development, regeneration, and adult anatomy of the limb muscles of salamanders23505152. No experiments on live vertebrates were performed for this study.…”

“…Regarding the muscular anatomy of lobe-finned fishes, the major novel contributions of this work are 1) description of new muscles; 2) re-appraisal of evolutionary origin (e.g., from ventral/abductor vs. dorsal/adductor masses) and identity of previously described muscles; and 3) first comprehensive comparisons of pelvic and pectoral appendages (PELA, PECA) among these and other fish and in tetrapods, leading to proposal of new names, evolutionary origins and one-to-one homology hypotheses for all muscles of these taxa (see SI for more details, in particular between the differences between our work and previous studies). We discuss our results in the context of the anatomical, developmental, and paleontological literature, including recent papers on the soft tissues of adult dipnoans1920 and coelacanths14, of phylogenetically basal extant bony fishes such as Polypterus 21, and works on appendicular muscle development in most gnathostome clades111222232425262728293031323334.…”

Reconstructing pectoral appendicular muscle anatomy in fossil fish and tetrapods over the fins‐to‐limbs transition

“…Diogo et al (2016) included a new analysis of fin muscles in Latimeria based on dissections and computerized tomography (CT) scans (see also Miyake et al, 2016), and compared the resulting data with similar data from Neoceratodus and Ambystoma. The study proposed homology hypotheses based upon: (i) embryonic primordia and sequence of development [from Neoceratodus and Ambystoma (Boisvert et al, 2013); from Ambystoma (Diogo & Tanaka, 2014)]; (ii) comparative anatomy including architecture, innervation, topology, and attachments; and (iii) distribution of muscles within the phylogeny of each group (see Diogo et al, 2016, for detailed methods). Based on this evidence, the study concluded that the common ancestor of extant sarcopterygians probably had both superficial and deep dorsal and ventral muscle masses that extended from the girdles to the distal region of the fin, a series of even deeper dorsal and ventral muscles that spanned only a few axial segments (similar to the pronators and supinators of Latimeria), and pre-and postaxial muscles that spanned more than one joint.…”

“…We include both a terrestrial (S. salamandra) and an aquatic (A. mexicanum) salamander to represent extant lissamphibians because salamanders are hypothesized to display a more plesiomorphic limb musculoskeletal configuration among tetrapods (Miner, 1925;Diogo & Tanaka, 2014;Diogo et al, 2015). Among amniotes, we include Sphenodon as a representative of Rhynchocephalia, the extant sister group of all other lepidosaurs (i.e.…”

Carpus and tarsus of Temnospondyli

“…As in Acheloma, the fibular articulation occupies only the medial portion of its medial edge. The remaining free lateral projection of the fibulare bears a concavity that probably served as the attachment site for one or more of the flexor accessorius lateralis and medialis, extensor cruris et tarsi fibularis, and abductor digit minimi (Diogo and Tanaka 2014). Although the intermedium in specimen MCZ 4169 of Dissorophus is damaged (Fig.…”