Le facteur de transcription Egr1 dans le développement du cartilage chez le zebrafish

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 regulators 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

RUNX3, EGR1 and SOX9B form a regulatory cascade required to modulate BMP-signaling during cranial cartilage development in zebrafish

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.status: publishe

Fgf receptors Fgfr1a and Fgfr2 control the function of pharyngeal endoderm in late cranial cartilage development.

Cranial cartilage derives mainly from cranial neural crest cells and its formation requires fibroblast growth factor (Fgf) signaling for early differentiation and survival of developing chondrocytes as well as patterning of the endodermal pouches. Here, we investigate the role of Fgf receptors in chondrocyte maturation at later stages, beyond 24hpf. Using inducible expression of a dominant-negative Fgf receptor, we show that Fgf signaling is required around 30hpf for correct cartilage formation. The receptor genes fgfr1a and fgr2 are expressed in pharyngeal endodermal pouches after 24hpf or 26hpf, respectively. Depletion of any of these two receptors by microinjection of antisense morpholinos results in severe defects in cartilage formation at 4dpf and a decrease in expression of the late chondrocyte markers barx1 and runx2b. Although endodermal pouches are correctly formed and patterned, receptor knock down leads to decreased expression of runx3, egr1 and sox9b in this tissue, while expression of fsta, coding for a secreted BMP/Tgfss inhibitor, is clearly increased. Rescue experiments revealed that each Fgfr1a or Fgfr2 receptor is able to compensate for the loss of the other. Thus, we show that minimal amounts of Fgfr1a or Fgfr2 are required to initiate a regulatory cascade in pharyngeal endoderm reducing expression of fsta, thereby allowing correct BMP signaling to the maturing chondrocytes of the head cartilage

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

<div><p>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 <em>fsta</em> 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 <em>runx2b</em>, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that e<em>gr1</em> depletion leads to increased expression of <em>fsta</em> 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.</p> </div

Only late chondrogenic and osteogenic marker genes display decreased expression in <i>egr1</i> morphants between 24 and 48 hpf.

<p><i>In situ</i> hybridization was performed at the indicated stages for various cartilage markers, lateral views, anterior to the left. Scale bars 100 µm. (A–E) 4 ng MOcon treated control embryos, (F,G,H,I,J) 4ng splicing MOegr1 injected embryos and (K) rescue. (A,F) At 24 hpf, <i>ap2α3</i> expression in cranial neural crest cells (cNCC) is not altered in morphants. (B,C,G,H) cNCC marker <i>dlx2a</i> is normally expressed in e<i>gr1</i> morphants (G,H) compared to control embryos (B,C) at 24 and 48 hpf. (D,I) Expression of the essential chondrogenic gene <i>sox9a</i> is not changed at 48 hpf by <i>egr1</i> knock-down. (E,J,K) At 48 hpf, <i>runx2b</i> transcripts are absent in pharyngeal cartilage precursor cells in 4 ng MOegr1 spl embryos. Expression of <i>runx2b</i> is maintained in the cleithrum (cl) and ethmoid plate (ep). (K) Rescue by injection of 80 pg mRNA <i>egr1</i> restores all <i>runx2b</i> expression domains at 48 hpf. Otic vesicle (ov), mandible (m), ceratohyal (ch), hyosymplectic (hs), ceratobranchial pairs 1 to 5 (cb1-5), cleithrum (cl), ethmoid plate (ep), stream of cNCCs (S1–S3).</p

Runx3, Egr1 and Sox9b form a regulatory cascade required to modulate Bmp-signaling during cranial cartilage development in zebrafish.

<p>Signaling model in wild-type embryos (A) and in embryos lacking of endodermal regulatory cascade (B). (A) In wild-type embryos, pharyngeal endoderm expresses a regulatory cascade composed of three transcription factors, Runx3, Egr1 and Sox9b, which down-regulates <i>fsta</i> expression that codes for a Bmp antagonist. This down-regulation of <i>fsta</i> enables Bmp ligands to bind to their heterodimeric receptor (BmpRI and BmpRII) and induce <i>runx2b</i> expression in cranial neural crest cells (cNCC). (B) Embryos lacking of any member of Runx3-Egr1-Sox9b cascade have an over-expression of <i>fsta</i>, which its coding protein is secreted from the endoderm. Antagonist Fsta binds to Bmp ligands and inhibit them to bind to their receptor, having for consequence no Bmp-signaling towards the cNCC and no <i>runx2b</i> expression.</p