A hierarchy of Runx transcription factors modulate the onset of chondrogenesis in craniofacial endochondral bones in zebrafish

Abstract

The Runx (runt-related) family of transcription factors are important regulators of cell fate decisions in early embryonic development, and in differentiation of tissues including blood, neurons, and bone. During skeletal development in mammals, while only Runx2 is essential for osteoblast differentiation, all family members seem to be involved in chondrogenesis. Runx2 and Runx3 control chondrocyte maturation. Both Runx1 and Runx2 are expressed early in mesenchymal condensations, but how they contribute to the initial stages of chondrocyte differentiation is unclear. Here we show that a hierarchy of Runx transcriptional regulation promotes the early program of chondrocyte differentiation from pre-cartilage mesenchyme in the zebrafish head skeleton. We have previously characterized the zebrafish orthologs for all Runx genes. Zebrafish runx2 is duplicated, but not runx1 or runx3. In the work presented here, we determined the early expression pattern of the runx genes in the craniofacial region. The earliest expression detected was that of runx3 in the pharyngeal endoderm, then runx2a and b in mesenchymal condensations, and later runx1 in the epithelium. Using antisense morpholino knockdown analysis, we examined their respective activities in early chondrogenesis. Depletion of runx2b (but not runx2a) and runx3 severely compromised craniofacial cartilage formation. Because runx2b expression was abolished in Runx3 morphants, we propose that endodermal Runx3 has a role in influencing signaling activities from the endoderm to promote chondrocyte differentiation. We also show that, in contrast to data from mouse studies, zebrafish Runx1 is not required in the initial steps of chondrogenesis leading to endochondral bone formation. Developmental Dynamics 235:3166 -3176, 2006.

Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis

“…Several stage-46 clearedand-stained tadpoles injected with both morpholinos lack cranial trabeculae and otic capsules (Fig. 4B), as has been demonstrated for zebrafish runx2b-II (Flores et al, 2006). However, many hatchlings recover from the effect of morpholinos by this stage.…”

“…5). This stronger effect is shared with the zebrafish runx2b-II morpholino in comparison with runx2b-I (Flores et al, 2006). Mouse cell culture studies also have found the RUNX2-II protein to have a greater effect on regulating the osteocalcin gene than RUNX2-I (Harada et al, 1999), which indicates a stronger role for this isoform as a transcription factor during bone formation.…”

“…Early suppression of runx2a function does not affect early cartilage formation. However, the zebrafish runx2b paralog, which is strongly expressed in precursors of the pharyngeal arch cartilages, is essential for pharyngeal cartilage formation (Flores et al, 2006). This developmental role of runx2b differs from the amniote model, and may indicate a replacement of runx1 function in zebrafish cartilage formation.…”

“…Three vertebrate paralogs, derived from a triplication of an ancestral runx (Levanon et al, 2003;Glusman et al, 2004), share the highly conserved DNA-binding "runt" domain. While Runx2 is of primary importance in bone development (Banerjee et al, 1997;Ducy et al, 1997;Komori et al, 1997;Otto et al, 1997), Runx1 and 3 are also expressed in the developing skeleton (Yamashiro et al, 2002;Lian et al, 2003;Smith et al, 2005) suggesting some functional redundancy between these family members (Cameron et al, 2003;Flores et al, 2006). Overexpression and RNA interference of mouse Runx1 has revealed its role in early cartilage formation (Wang et al, 2005).…”

“…Overexpression and RNA interference of mouse Runx1 has revealed its role in early cartilage formation (Wang et al, 2005). However, Runx1 is not involved in zebrafish cartilage formation, where Runx2 appears to have taken over the functional role in chondrogenesis (Flores et al, 2006). Runx family members interact with core binding factor beta (also known as Cbfb, Pea2, Pebp2, Pebp2b, Pebpb2), which increases their transcriptional activity (Speck and Dzierzak, 2001;Kundu et al, 2002).…”