Li C et al. (2018), Alteration of the Retinoid Acid-CBP Signaling P...

XB-ART-55553

Front Pediatr 2018 Dec 03;6:382. doi: 10.3389/fped.2018.00382.

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Alteration of the Retinoid Acid-CBP Signaling Pathway in Neural Crest Induction Contributes to Enteric Nervous System Disorder.

Li C , Hu R , Hou N , Wang Y , Wang Z , Yang T , Gu Y , He M , Shi Y , Chen J , Song W , Li T .

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Hirschsprung Disease (HSCR) and/or hypoganglionosis are common pediatric disorders that arise from developmental deficiencies of enteric neural crest cells (ENCCs). Retinoid acid (RA) signaling has been shown to affect neural crest (NC) development. However, the mechanisms underlying RA deficiency-induced HSCR or hypoganglionosis are not well-defined. In this report, we found that in HSCR patient bowels, the RA nuclear receptor RARα and its interacting coregulator CREB-binding protein (CBP) were expressed in enteric neural plexuses in the normal ganglionic segment. However, the expression of these two genes was significantly inhibited in the pathological aganglionic segment. In a Xenopus laevis animal model, endogenous RARα interacted with CBP and was expressed in NC territory. Morpholino-mediated knockdown of RARα blocked expression of the NC marker genes Sox10 and FoxD3 and inhibited NC induction. The morphant embryos exhibited reduced nervous cells in the gastrointestinal anlage, a typical enteric nervous deficiency-associated phenotype. Injection of CBP mRNA rescued NC induction and reduced enteric nervous deficiency-associated phenotypes. Our work demonstrates that RARα regulates Sox10 expression via CBP during NC induction, and alteration of the RA-CBP signaling pathway may contribute to the development of enteric nervous system disorders.

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Species referenced: Xenopus laevis
Genes referenced: creb1 crebbp foxd3 msx1 pax3 rab40b rara rbfox3 snai2 sox10 tubb2b zic1
???displayArticle.antibodies??? Crebbp Ab1 Crebbp Ab2 Rara Ab2
???displayArticle.morpholinos??? Crebbp MO1 hbg1 cMO1 rara MO8

???displayArticle.omims??? HIRSCHSPRUNG DISEASE, SUSCEPTIBILITY TO, 1; HSCR1

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	Figure 1. RARα and CBP expression patterns in the enteric nervous system of HSCR patients. (A–D) In normal ganglionic segments, β-tubulin III (green)-labeled nerve fibers (A) and NeuN protein (red) indicate ganglion cells (B). DAPI-stained cell nuclei (C) β-tubulin III and NeuN displayed partially overlapping expression patterns in the neural plexus, as shown in the merged panel (D). (E–H) In normal ganglionic segments, CBP protein (green) (F)-labeled ganglion cells are detected by colocalization (H) with NeuN (red) (E). (I–L) In aganglionic segments, NeuN (I) and CBP (J) are absent within the neural plexus, and CBP is expressed faintly in the muscularis propria of the colon, as shown in the merged panel (L). (M–P) In normal ganglionic segments, RARα (M) colocalizes with β-tubulin III (N), as shown in the merged panel (P). (Q–T) RARα (Q) is absent and β-tubulin III (R) is weakly expressed in aganglionic segments. (U–X) In normal ganglionic segments, RARα (U) labels nerve fibers and CBP (V) indicates ganglion cells. (X) Both proteins display partially overlapping patterns within the neural plexus. (Y) RARα (0.459 ± 0.273) and CBP (0.569 ± 0.196) transcripts were significantly lower in aganglionic segments than in normal regions. NS, normal ganglionic segments; AS, aganglionic segments. Numbers in brackets indicate the relative expression ratio of pathogenic aganglionic segments to normal ganglionic segments. Dashed lines encircle the neural plexus. **P < 0.01, with T-test. Scale bar: 100 μm.
	Figure 2. RARα and CBP [crebbp] expression in presumptive neural crest in Xenopus Laevis. (A) Developmental expression of the RARα and CBP proteins in Xenopus laevis. β-actin was used as an internal control. Statistics revealed the ratio of protein levels relative to stage 2. RARα (B) and CBP (G) are maternally expressed at the animal hemisphere, and the expression intensity increases at stage 7 (C,H). At the neural plate stage (stage 15), RARα (D) and CBP (I) appear in the central neural plate (* white asterisk means neural plate) and anterior neural plate border (black arrowhead). At stage 25 (E) and stage 30 (F), RARα is in the neural tube and branchial arch (red arrowhead). CBP has a similar expression pattern in the branchial arch (red arrowhead) and neural tube at stage 25 (J) and stage 30 (K). np, neural plate; b, neural plate border region; ba, branchial arch. Scale bar: 100 μm.
	Figure 3. RARα and CBP are cooperatively required for enteric nervous cell development in Xenopus laevis. Morpholino-mediated knockdown of RARα (A) or CBP (B) is efficient at embryonic stage 15. (C–J) RARα (D,H) or CBP (E,I) knockdown decreases enteric nervous cells (labeled with N-tubulin) and enteric neural crest cells (labeled with Sox10) compared to control guts (C,G). CBP mRNA coinjection rescues RARα morpholino effects on the gut (F,J). Gut anlage was harvested at stage 41. Enteric nervous cells are detected as dispersed blue-spots by N-tubulin in situ hybridization, whereas enteric neural crest are marked by Sox10. (K) Statistical analysis of enteric nervous cells among different groups. Nervous cells were quantified by counting the number of blue spots. In each group, at least three guts were analyzed and presented as the mean ± sd. (L) RARα coimmunoprecipitates with CBP in stage 15 Xenopus laevis embryos. (M–R) RARα morpholino injection at the 2- or 4-cell stage into the dorsal blastomere leads to dose-dependent microcephaly (red arrowhead) and pigment cell abnormalities (black arrowhead) at stage 37. (Q) CBP morpholino injection has similar phenotypes to RARα knockdown. (R) CBP mRNA largely rescues the RARα morphant small head and pigment deformity phenotypes. The numbers of embryos showing similar phenotypes and total injected embryos in each group are indicated. Scale bar: 100 μm. *P < 0.05.
	Figure 4. CBP functions downstream of RARα in regulating neural crest induction. The effects of the control morpholino (A–D), RARα knockdown (E–H) and CBP knockdown (I–L) on the expression of indicated neural border and neural crest markers. (M,N) The RAR and CBP morpholino-induced reduction in Sox10 is rescued by coinjection of morpholino-resistant RAR and CBP mRNAs, respectively. (O,P) The RARα knockdown-induced inhibition of FoxD3 and Sox10 is rescued by CBP mRNA coinjection. All of the embryos were collected at stage 15–16. (Q) Schematics of neural crest from the dorsal view along the anterior-posterior axis at stage 15. IS means injected side, and the left and right embryo halves are denoted with the dashed line. The numbers of embryos showing similar changes of gene expression and the total injected embryos in each group are indicated. Scale bar: 100 μm.
	rara (retinoic acid receptor alpha) gene gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 7.
	rara (retinoic acid receptor alpha ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, anterior view, dorsal up.
	rara (retinoic acid receptor alpha ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, dorsal view, anterior upper left.
	rara (retinoic acid receptor alpha ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28-30, lateral view, anterior left, dorsal up.
	crebbp (CREB binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 7.
	crebbp (CREB binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage15, anterior view, dorsal up.
	crebbp (CREB binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, dorsal view, anterior upper left.
	crebbp (CREB binding protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28-30, lateral view, anterior left, dorsal up.

References [+] :

Aoki, Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. 2003, Pubmed, Xenbase