Bmp signaling is down-regulated in <i>egr1</i> morphants.

<p>Pharyngeal cartilage precursor cells were visualized by immunohistochemistry using anti-GFP antibodies (green) in <i>fli-</i>GFP embryos. Activity of the BMP signaling pathway was assessed using antibodies against phospho-Smad1/5/8 (red) in 32 hpf embryos. Ventral view of pharyngeal arches, scale bar 40 µm. (A–F) Pharyngeal cartilage precursor cells were visualized by immunohistochemistry using anti-GFP antibodies (green) in <i>fli1-</i>GFP embryos. Activity of the BMP signaling pathway was assessed using antibodies against phospho-Smad1/5/8 (red) in 32 hpf embryos. Ventral view of pharyngeal arches, scale bar 40 µm. (A,B,C) 4 ng MOcon injected embryos, (D, E, F) 4 ng MOegr1 spl injected embryos. <i>fli1-</i>GFP embryos express the GFP transgene in cartilage precursors and endothelial cells in control (A) and in e<i>gr1</i> morphants (D). In contrast, phospho-Smad1/5/8 is is clearly down regulated in e<i>gr1</i> morphants (E) compared to control embryos (B). (C,F) Overlay images of the two anti-body signals clearly show that phospho-Smad1/5/8 is present in GFP-epressing cartilage precursor cells in control embryos (C), while no colocalization is observed in e<i>gr1</i> morphants (F). (a1) first arch, (a2) second arch, (a3) third arch, (a4) fourth arch, (bv) blood vessel.</p

Expression of <i>fsta</i> is increased in <i>runx3</i> and <i>egr1</i> morphants and <i>sox9b</i> mutants.

<p>Lateral views of <i>in situ</i> hybridizations, anterior to the left. Scale bars 100 µm. (A–F) Compared to controls or wild-type embryos, expression of <i>fsta</i> is up-regulated in e<i>gr1</i> morphants (A,B), <i>runx3</i> morphants (C,D), and homozygous <i>sox9b</i> mutants (E,F) at 48 hpf. pharyngeal endoderm (pe). (G,H) 4 dpf Alcian Blue stained larvae injected with MOcon 6 ng (K) and MOfsta 6ng (L). Knock-down of <i>fsta</i> causes a hyperplasia of the viscerocranium. Meckel’s cartilage (m), ceratohyal (ch), ceratobranchials 1 to 5 (cb1-5).</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

Runx3 is required for pharyngeal <i>egr1</i> and <i>sox9b</i> expression at 48 hpf.

<p>Lateral views of <i>in situ</i> hybridizations (A,B,E–L) with indicated markers and ventral views of Alcian Blue stained embryos (C,D), anterior to the left. Scale bars 100 µm. (A,B) Endodermal <i>runx3</i> expression in the pharyngeal region is not altered in 4 ng MOegr1 spl morphants. (C,D) <i>runx3</i> knock-down using 2 ng MOrunx3 tr leads to total absence of viscerocranium and the anterior neurocranium (D) compared to control (C) embryos. (E,F) <i>runx3</i> morphants do not express r<i>unx2b</i> in pharyngeal cartilage precursor cells. (G,H) <i>runx3</i> morphants do not express e<i>gr1</i> transcripts in pharyngeal endoderm. (I,J) The endodermal marker s<i>ox9b</i> is absent in pharyngeal endoderm when <i>runx3</i> expression is blocked. (K,L) <i>runx3</i> knock-down does not affect expression of pharyngeal endodermal marker <i>nkx2.3 a</i>t 48 hpf. Trigeminal ganglia (tg), pharyngeal endoderm (pe), cleithrum (cl), Meckel’s cartilage (m), palatoquadrate (pq), hyosymplectic (hs), ceratohyal (hs), ceratobranchials 1 to 5 (cb1-5), ethmoid plate (ep), otic vesivle (ov).</p

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

Expression of <i>egr1</i> in the pharyngeal region between 30 hpf to 5 dpf is restricted to endoderm and epithelium.

<p>Lateral (A–G,I) and ventral (H,J) views, anterior to the left. Scale bars 100 µm. Images of double <i>in situ</i> hybridizations were taken by confocal microscopy and pictures of individual Z-sections are shown. (A) e<i>gr1</i> transcripts are observed in the pharyngeal region starting at 30 hpf in endoderm. (B,C) At 48 hpf, double <i>in situ</i> hybridization for <i>egr1</i> (green) and <i>fli1</i> (red); <i>egr1</i> transcripts are localized in pharyngeal endoderm and do not colocalize with <i>fli1</i> mRNA in pharyngeal cartilage precursor cells. (D) <i>egr1</i> is expressed in pharyngeal endoderm. (E–G) At 3 dpf, e<i>gr1</i> (green) does not colocalize with <i>runx2b</i> (red) (E) or s<i>ox9a</i> (red) (F) in cartilage, while (G) e<i>gr1</i> (green) mRNAs colocalize with those for the pharyngeal endoderm marker <i>sox9b</i> (red). (H) At 4 dpf, e<i>gr1</i> (green) is never expressed in cells in pharyngeal cartilage precursor cells expressing <i>fli1</i> (red). (I) Expression of <i>egr1</i> at 4 dpf in pharyngeal endoderm. (J) At 5 dpf, e<i>gr1</i> is still expressed in pharyngeal endoderm (stars) and not in pharyngeal cartilage. Pharyngeal endoderm (pe), cranial neural crest cells (cNCC).</p

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

<i>egr1</i> and <i>fsta</i> knock-down do not affect ventralisation of cranial neural crest cells.

<p><i>In situ</i> hybridization was performed at 24 hpf, lateral views, anterior to the left. Scale bars 100 µm. (A,D,G) MOcon 4 ng, (B,E,H) MOegr1 4 ng and (C,F) MOfsta 6 ng. No modification in the expression of markers <i>hand2</i>, <i>edn1</i> and <i>fsta</i> was observed in MOegr1 4 ng or MOfsta 6 ng injected embryos compared to control.</p

Runx3 depleted embryos can be rescued by <i>runx3</i> and <i>egr1</i> mRNA.

<p>(A–F) Head cartilages were stained with Alcian Blue in morpholino treated larvae at 4 dpf; ventral views are shown. (A) MOcon 2 ng injected larvae. (B) MOrunx3 2 ng injected larvae do not develop viscerocranium. (C) 100 pg of <i>runx3</i> mRNA do not affect 4 dpf old larvae cartilage morphology. (D) Injection of 100 pg <i>runx3</i> mRNA rescues 89% of MOrunx3 2 ng injected eggs. (E) 80 pg of <i>egr1</i> mRNA. (F) Injection of 80 pg <i>egr1</i> mRNA rescues 62% of MOrunx3 2 ng injected eggs.</p

Egr1 is required for expression of <i>sox9b</i> in pharyngeal endoderm.

<p>Endodermal gene expression by <i>in situ</i> hybridization (A,C,D,F,G,H,I) or in living transgenic embryos (B,E) in control embryos (A–C,H), e<i>gr1</i> morphants (D–F), rescued embryos (G) and <i>sox9b</i> mutants (I) at 48 hpf. Lateral views, anterior to the left. Scale bars 100 µm. (A,D) <i>nkx2.3</i> expression is not altered in 4 ng MOegr1 spl injected embryos. (B,E) In living <i>sox17:GFP</i> transgenic embryos, the transgene is correctly expressed in <i>egr1</i> morphants. (D,F,G) The endodermal marker s<i>ox9b</i> is not expressed in the pharyngeal endoderm in 4 ng MOegr1 spl injected embryos, but its expression is rescued upon co-injection of 80 pg e<i>gr1</i> mRNA and spl 4 ng MOegr1. (H, I) In homozygous <i>sox9b^−/−</i> embryos, <i>egr1</i> transcripts are still observed in the pharyngeal endoderm like in the wild-type or heterozygous <i>sox9b^+/−</i> embryos. Pharyngeal endoderm (pe), otic vesicle (ov).</p