Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling

Abstract

Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination.in ovo electroporation | basement membrane N eural crest cells (NCCs) belong to a transient population of embryonic multipotent stem cells that are induced at the interface between the neuroectoderm and the prospective epidermis. These cells delaminate from the epithelium and adopt a characteristic migratory pathway into the periphery, where they contribute to several tissues, notably cranial facial structures and peripheral nervous system (1). During delamination, NCCs undergo an epithelial-mesenchyme transition (EMT), characterized by loss of cell-cell contacts and polarity, as well as acquisition of migratory capabilities (2). Successful EMT and delamination are essential steps for the subsequent migration and differentiation of NCCs. Consequently, mutations in genes involved in NCCs EMT are often associated with congenital defects (3).Previous studies showed that Sry-related HMG box-9 (Sox9), a member of the SoxE family of transcription factors, plays several roles in NC formation, survival, EMT, and differentiation (4-6). In mice harboring a null mutation in Sox9, NCCs were still generated but died by apoptosis before or shortly after NC delamination, suggesting that Sox9 is required for NC survival (4). In chick, misexpression of Sox9 in the neural tube was sufficient to induce NC identity at the expense of neural fate but only inefficiently induced delamination and EMT (5). By contrast, coexpression of Sox9 with the zinc finger-type transcription factor, Snail2, promoted systematic ectopic EMT and NC delamination along the dorsal-ventral extent of the neural tube (4). In addition, in vitro studies showed that Sox9 physically interacts with Snail2 (6). Altogether, these data suggest that the partnership between Sox9 and Snail2 may underlie the ability of Sox9 to induce NC delamination and EMT. However, how the combination of Sox9 and Snail2 achieves this is unclear.The ability of Sox9 to initiate NC delamination appears to be regulated by protein kinase A (PKA)-mediated phosphorylation at two serine residues (S64 a...

Controlled levels of canonical Wnt signaling are required for neural crest migration

“…Interestingly Snail2 has been also shown to transcriptionally repress cadherin-6B, which belongs to type II classical cadherins in chick cranial and trunk NC cells during delamination . The action of Snail2 is supported by interaction with Sox9, another transcription factor, which upon phosphorylation, induced by BMP and canonical Wnt signaling, binds directly Snail2 and promotes EMT of chick trunk NC (Cheung and Briscoe, 2003;Liu et al, 2013). Furthermore, canonical Wnt signaling is also thought to be involved in EMT by transcriptional and post-transcriptional regulation of Snail2 (Vallin et al, 2001;Yook et al, 2006).…”

Sox proteins: regulators of cell fate specification and differentiation

“…Sry and Sox9 are phosphorylated by protein kinase A (PKA), which enhances their DNA-binding activity (Malki et al, 2010). This phosphorylation of Sox9 also leads to its translocation into the nucleus in cells of the testis (Malki et al, 2010), and is essential in neural crest (NC) cells for the Sox9-Snail interaction that promotes NC delamination (Liu et al, 2013). The functional relevance of sumoylation has been best demonstrated for SoxE proteins in NC development.…”

“…Sry and Sox9 are phosphorylated by protein kinase A (PKA), which enhances their DNA-binding activity (Malki et al, 2010). This phosphorylation of Sox9 also leads to its translocation into the nucleus in cells of the testis (Malki et al, 2010), and is essential in neural crest (NC) cells for the Sox9-Snail interaction that promotes NC delamination (Liu et al, 2013 miR-145 expression is highly upregulated during the differentiation of human embryonic stem cells (ESCs). miR-145 directly targets 3Ј UTRs (untranslated regions) of the mRNAs of Sox2 and other 'core pluripotency factors', and promotes differentiation into mesoderm and ectoderm lineages (Xu et al, 2009b).…”

Establishing neural crest identity: a gene regulatory recipe

“…This is accomplished through direct interaction with a pair of E-box sites located in the vicinity of the Kozak consensus sequence. An important partner of Snai1/2 during the process of EMT is the transcription factor Sox9, which when phosphorylated physically interacts with Snai1/2 to drive EMT (Cheung and Briscoe, 2003;Liu et al, 2013b).…”