Correction to: Manipulation of Developmental Function in Turtles with Notes on Alligators.

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Manipulation of Developmental Function in Turtles with Notes on Alligators.

Reptiles have great taxonomic diversity that is reflected in their morphology, ecology, physiology, modes of reproduction, and development. Interest in comparative and evolutionary developmental biology makes protocols for the study of reptile embryos invaluable resources. The relatively large size, seasonal breeding, and long gestation times of turtles epitomize the challenges faced by the developmental biologist. We describe protocols for the preparation of turtle embryos for ex ovo culture, electroporation, in situ hybridization, and microcomputed tomography. Because these protocols have been adapted and optimized from methods used for frog, chick, and mouse embryos, it is likely that they could be used for other reptilian species. Notes are included for alligator embryos where appropriate.

Late-emigrating trunk neural crest cells in turtle embryos generate an osteogenic ectomesenchyme in the plastron.

BACKGROUND: The turtle plastron is composed of a keratinized epidermis overlying nine dermal bones. Its developmental origin has been controversial; recent evidence suggests that the plastral bones derive from trunk neural crest cells (NCCs). RESULTS: This study extends the observations that there is a turtle-specific, second wave of trunk NCC delamination and migration, after the original NCCs have reached their destination and differentiated. This second wave was confirmed by immunohistochemistry in whole-mounts and serial sections, by injecting DiI (1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate) into the lumen of the neural tube and tracing labeled cells into the plastron, and by isolating neural tubes from older turtle embryos and observing delaminating NCCs. This later migration gives rise to a plastral ectomesenchyme that expresses NCC markers and can be induced to initiate bone formation. CONCLUSIONS: The NCCs of this second migration have properties similar to those of the earlier NCCs, but also express markers characteristic of cranial NCCs. The majority of the cells of the plastron mesenchyme express neural crest markers, and have osteogenic differentiation capabilities that are similar or identical to craniofacial ectomesenchyme. Our evidence supports the contention that turtle plastron bones are derived from a late emigrating population of cells derived from the trunk neural crest.

Evidence that a late-emerging population of trunk neural crest cells forms the plastron bones in the turtle Trachemys scripta.

The origin of the turtle plastron is not known, but these nine bones have been homologized to the exoskeletal components of the clavicles, the interclavicular bone, and gastralia. Earlier evidence from our laboratory showed that the bone-forming cells of the plastron were positive for HNK-1 and PDGFRalpha, two markers of the skeletogenic neural crest. This study looks at the embryonic origin of these plastron-forming cells. We show that the HNK-1+ cells are also positive for p75 and FoxD3, confirming their neural crest identity, and that they originate from the dorsal neural tube of stage 17 turtle embryos, several days after the original wave of neural crest cells have migrated and differentiated. DiI studies show that these are migratory cells, and they can be observed in the lateral regions of the embryo and can be seen forming intramembranous bone in the ventral (plastron) regions. Before migrating ventrally, these late-emerging neural crest cells reside for over a week in a carapacial staging area above the neural tube and vertebrae. It is speculated that this staging area is where they lose the inability to form skeletal cells.

The contribution of neural crest cells to the nuchal bone and plastron of the turtle shell.

The origin of the turtle plastron is not well understood, and these nine bones have been homologized to the exoskeletal components of the clavicles, the interclavicular bone, and gastralia. Earlier data from our laboratory showed that the plastral bone-forming cells stained positively for HNK-1 and PDGFRα, two markers of skeletogenic neural crest cells. We have now shown that the HNK-1(+) cells are also positive for p75 and FoxD3, affirming their neural crest identity. These cells originate from the dorsal neural tube of stage-17 turtle embryos, several days after the original wave of neural crest cells have migrated and differentiated. Moreover, we have demonstrated the existence of a staging area, above the neural tube and vertebrae, where these late-emigrating neural crest cells collect. After residing in the carapacial staging area, these cells migrate to form the plastral bones. We also demonstrate that one bone of the carapace, the nuchal bone, also stains with HNK-1 and with antibodies to PDGFRα. The nuchal bone shares several other properties with the plastral bones, suggesting that it, too, is derived from neural crest cells. Alligator gastralia stain for HNK-1, while their ribs do not, thus suggesting that the gastralial precursor may also be derived from neural crest cells.

Epithelial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development.

Bone morphogenetic protein (BMP) signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of hair follicles. However, definitive genetic evidence for these roles has been lacking. Here, we show that Cre-mediated mutation of the gene encoding BMP receptor 1A in the surface epithelium and its derivatives causes arrest of tooth morphogenesis and lack of external hair. The hair shaft and hair follicle inner root sheath (IRS) fail to differentiate, and expression of the known transcriptional regulators of follicular differentiation Msx1, Msx2, Foxn1 and Gata3 is markedly downregulated or absent in mutant follicles. Lef1 expression is maintained, but nuclear beta-catenin is absent from the epithelium of severely affected mutant follicles, indicating that activation of the WNT pathway lies downstream of BMPR1A signaling in postnatal follicles. Mutant hair follicles fail to undergo programmed regression, and instead continue to proliferate, producing follicular cysts and matricomas. These results provide definitive genetic evidence that epithelial Bmpr1a is required for completion of tooth morphogenesis, and regulates terminal differentiation and proliferation in postnatal hair follicles.

T-box gene products are required for mesenchymal induction of epithelial branching in the embryonic mouse lung.

The regulation of signaling pathways is a prerequisite for coordinating the induction between mesenchymal and epithelial tissues during morphogenesis. Mesenchymal FGF10 is known to be an important paracrine factor regulating the branching morphogenesis of the bronchial epithelium. By using antisense oligonucleotides (AS ODNs) and in vitro culture of embryonic lungs, we demonstrate that the transcription factors Tbx4 and Tbx5 are critical for the expression of mesenchymal FGF10. Treatment of embryonic lung cultures with AS ODNs to Tbx4 and Tbx5 reduces the level of these transcripts, suppresses Fgf10 expression in the mesenchyme, and completely eliminates the formation of new lung branches. If FGF10 is locally replaced in these AS ODN-treated lungs, epithelial branching is restored. These studies provide evidence that the production of branching signals by the lung mesenchyme is mediated by T-box genes.