Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis

Abstract

The vertebrate transcription factor protein Runx2 is regarded as a "master regulator" of bone formation due to the dramatic loss of the osseous skeleton in the mouse homozygous knockout. However, Runx2 mRNA also is expressed in the pre-hypertrophic cartilaginous skeleton of the mouse and chicken, where its developmental function is largely unknown. Several tiers of Runx2 regulation exist in the mouse, any of which may account for its seeming biological inactivity during early stages of skeletogenesis. Unlike mouse and chicken, zebrafish require Runx2 function in early cartilage differentiation. The present study reveals that the earlier functional role of Runx2 in cartilage differentiation is shared between zebrafish and Xenopus. A combination of morpholino oligonucleotide injections and neural crest transplants indicate that Runx2 is involved in differentiation of the cartilaginous hyobranchial skeleton in the frog, Xenopus laevis. Additionally, in situ hybridizations show runx2 mRNA expression in mesenchymal precursors of the cartilaginous skull, which reveals the earliest pre-patterning of these cartilages described to date. The early distribution of runx2 resolves the homology of the larval suprarostral plate, which is one of the oldest controversies of anuran skull development. Together these data reveal a shift in Runx2 protein function during vertebrate evolution towards its exclusive roles in cartilage hypertrophy and bone differentiation within the amniote lineage. Developmental Dynamics 236:1650 -1662, 2007.

Regulatory elements of Xenopus col2a1 drive cartilaginous gene expression in transgenic frogs

“…Whereas several studies examine the early distribution of col2a1 mRNA in Xenopus laevis (Su et al, 1991;Bieker and Yazdani-Buicky, 1992;Larrain et al, 2000;Kerney et al, 2007), this study is the first to investigate its expression through metamorphosis (Fig. 1).…”

“…These constructs can be used to drive dominant-negative protein expression or to over-express various genes in the cartilaginous skeleton. Whereas several studies investigate the skeletal morphology of Xenopus laevis during development (Bernasconi, 1951;Sedra and Michael, 1957;Sokol, 1977;Trueb and Hanken, 1992), few examine the underlying transcriptional regulators involved in skeletogenesis (Pasqualetti et al, 2000;Spokony et al, 2002;Moriishi et al, 2005;Kerney et al, 2007). Despite ongoing use of Xenopus as a model system of organogenesis (Thomsen, 2006) and the quickly growing field of skeletal developmental biology (Karsenty, 2003;Zelzer and Olsen, 2003;Hall, 2005;Goldring et al, 2006) few studies research skeletal development in Xenopus.…”

“…The second, metamorphic phase of skeleton formation results in both new cartilage and bone formation in the post-metamorphic froglet (reviewed in Hanken, 1992). Previous investigations have concentrated on the cellular origins of the tadpole skull (Stone, 1929;Sadaghiani and Thièbaud, 1987;Olsson and Hanken, 1996) and the differentiation of tadpole chondrocytes (Spokony et al, 2002;Baltzinger et al, 2005;Kerney et al, 2007). Few studies, however, have investigated the cellular origins and differentiation of chondrocytes during the second-metamorphic-phase of anuran skull development (e.g., Gross and Hanken, 2008).…”

The protein kinase MLTK regulates chondrogenesis by inducing the transcription factor Sox6

“…2A), a migrating neural crest marker xTwist (Hopwood et al, 1989) (Fig. 2B) and a cranial neural crest marker xSox9 (Spokony et al, 2002;Kerney et al, 2007) at stage 35/36, when neural crest cells reside in the branchial arches after migration (Fig. 2C).…”

“…S4A-C). However, xSox9 was expressed at stage 35/36 in the branchial arch and also in cartilages at stage 41 (Kerney et al, 2007) (supplementary material Fig. S4D).…”