RUNX3, EGR1 and SOX9B Form a Regulatory Cascade Required to Modulate BMP-Signaling during Cranial Cartilage Development in Zebrafish

Abstract

The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the fsta gene, encoding a BMP antagonist, in pharyngeal endoderm. Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of runx2b, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that egr1 depletion leads to increased expression of fsta and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish.

Transcriptional regulation of gilthead seabream bone morphogenetic protein (BMP) 2 gene by bone- and cartilage-related transcription factors

“…Both MEF2C and SOX9 factors have been implicated in the regulation of bone and cartilage formation (Dy et al, 2012;Mackie et al, 2008) and are co-expressed with BMP2 in several cell lines including the human osteosarcoma U-2 OS and HEK-293 cells (see the Human Protein Atlas database at www.proteinatlas.org). There are also evidences of Mef2c and Sox9 being regulators of BMP signaling pathway mediators (Dalcq et al, 2012;Wu et al, 2010) and vice versa; the expression of both transcription factors was shown to be regulated by Bmp2 (Kawakami et al, 2006;Zheng et al, 2013). In agreement with data presented here, Liao and co-workers recently demonstrated that Bmp2 expression is lowered in Sox9-enhanced chondrogenesis in mouse cells (Liao et al, 2014).…”

Pregnancy-associated plasma protein-A2 modulates development of cranial cartilage and angiogenesis in zebrafish embryos

“…Accordingly, the expression of papp-a2 in the lower jaw at 48 hpf suggests that Papp-a2 might be required for normal jaw development at such later stages. Precise regulation of BMP signaling is crucial for cranial neural crest development (Schumacher et al, 2011), and, interestingly, BMP signaling is required during the second day post fertilization for the normal development of cranial cartilages (Dalcq et al, 2012). In addition, knockdown of runx3, an upstream transcriptional repressor of the BMP antagonist Follistatin A, results in loss of cranial cartilages similar to papp-a2 knockdown (Dalcq et al, 2012;Flores et al, 2006).…”

“…Precise regulation of BMP signaling is crucial for cranial neural crest development (Schumacher et al, 2011), and, interestingly, BMP signaling is required during the second day post fertilization for the normal development of cranial cartilages (Dalcq et al, 2012). In addition, knockdown of runx3, an upstream transcriptional repressor of the BMP antagonist Follistatin A, results in loss of cranial cartilages similar to papp-a2 knockdown (Dalcq et al, 2012;Flores et al, 2006). Taken together with our biochemical analysis of Papp-a2, these data support a role of Papp-a2 as a modulator of Bmp signaling, and Papp-a2 thus presents itself as a plausible candidate for modulating the development of cartilages of the cranium through its proteolytic activity.…”

Identification of cis -regulatory elements in the upstream regions of zebrafish runx3 through in silico analysis: implications for function

“…RUNX2 and RUNX3 have also been shown to regulate chondrocyte differentiation and maturation (Yoshida et al., ). In zebrafish, loss of function of runx3 was shown to lead to severe reduction of head cartilage at 4 days post‐fertilization (dpf) (Flores et al., ; Dalcq et al., ). Furthermore, it was shown that a regulatory cascade formed by Runx3‐Egr1‐Sox9b controls late chondrogenesis by reducing expression of Follistatin A, a BMP inhibitor (Dalcq et al., ).…”

“…In zebrafish, loss of function of runx3 was shown to lead to severe reduction of head cartilage at 4 days post‐fertilization (dpf) (Flores et al., ; Dalcq et al., ). Furthermore, it was shown that a regulatory cascade formed by Runx3‐Egr1‐Sox9b controls late chondrogenesis by reducing expression of Follistatin A, a BMP inhibitor (Dalcq et al., ). This down‐regulation allows the correct activation of BMP signalling required for expression of runx2b in developing chondrocytes (Dalcq et al., ).…”