Bradley RS (2018), Neural crest development in Xenopus requires Pr...

XB-ART-54581

Mech Dev 2018 Feb 01;149:41-52. doi: 10.1016/j.mod.2018.01.002.

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Neural crest development in Xenopus requires Protocadherin 7 at the lateral neural crest border.

Bradley RS .

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In vertebrates, the neural crest is a unique population of pluripotent cells whose development is dependent on signaling from neighboring tissues. Cadherin family members, including protocadherins, are emerging as major players in neural crest development, largely through their roles in cell adhesion and sorting in embryonic tissues. Here, we show that Protocadherin 7 (Pcdh7), previously shown to function in sensorial layer integrity and neural tube closure in Xenopus, is also involved in neural crest specification and survival. Pcdh7 expression partly overlaps the neural crest domain at the lateral neural crest border. Pcdh7 knockdown in embryos does not alter neural crest induction; however, neural crest specification markers, including Snail2 and Sox9, are lost, due to apoptosis of the neural crest starting after stage 13. Pcdh7 knockdown also results in downregulation of Wnt11b; both of which are co-expressed in the sensorial layer lateral to the neural crest, suggestive of a role for Wnt11b in the neural crest apoptosis. Confirming this role, apoptosis, Snail2 expression and the developmental fate of the neural crest can be partially rescued by ectopic expression of Wnt11b. These results indicate that Pcdh7 plays an important role in maintaining the sensorial layer at the lateral neural crest border, which is necessary for the secretion of survival factors, including Wnt11b.

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Species referenced: Xenopus laevis
Genes referenced: bcl2l1 casp3.2 ctnnb1 krt12.4 myc pax3 pcdh7 six1 snai1 snai2 sox15 sox2 sox9 tp63 twist1 wnt11b zic1
GO keywords: apoptotic process [+]
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	Fig. 1. Pcdh7 is expressed in a subset of neural crest cells. Embryos at stage 17/18were subjected to in situ hybridization for Pcdh7 (A, F), or the neural crestmarker Snail2 (B, G) or double in situ for Pcdh7 and Snail2 (C, D, H) or the neural crestmarker Sox9 (E, I). Embryos are viewed inwholemount (Aâ€“E) or in cross-section (F\\I). Pcdh7 is expressed in the sensorial layer of the ectodermand at the tips of the neural folds (A, arrows in F), with highest expression along the lateral border of the neural crest. Snail2 is expressed in the neural crest, which derives fromthe sensorial layer (B, G). Embryos subjected to double in situ for Pcdh7 and Snail 2 or Sox9 (C, D, E) reveal overlap along the lateral border of the neural crest (arrowheads),which is confirmed in cross section (H, I). Anterior is the right in Aâ€“E. Sections F\\I are anterior, through the prospective hindbrain region. A summary diagram of the domains of the neural plate, neural crest and Pcdh7 is shown (J). Abbreviations: np, neural plate; nc, notochord; psm, presomitic mesoderm.
	Fig. 2. Pcdh7 knockdown disrupts expression of neural crest specification markers, but not neural crest induction or neural platemarkers. Embryoswere injected with Pcdh7MO, Pcdh7Î´E orCMO, together with LacZmRNA, fixed at stage 15â€“17, stained for Î²-galactosidase, and subjected to in situ hybridization for neuralmarkers. Neither Pcdh7MO nor Pcdh7Î´E has an effect on expression of the inductionmarkers Pax3 (A, B), Zic1 (C,D) or c-myc (E, F), nor on the neural plate markers Sox15 and Sox2 (G, H). In contrast, Pcdh7MO- or Pcdh7Î´E-injectedembryos exhibit reduced expression of the specification markers Sox9 (I, J), Snail2 (K, L) and Twist (M, N). Control (CMO)-injected embryos show no change in specificationmarkers Snail2 (O) or Twist (P). Anterior is towards the top of the photos and the injected side of the embryos is to the right.
	Fig. 3. Disrupting Pcdh7 results in cranial neural crest apoptosis. Embryoswere injectedwith Pcdh7MOor the dominant-negative Pcdh7Î´EmRNA, togetherwith LacZmRNA, fixed at stage 17, stained for Î²-galactosidase, and immunostained for activated Caspase 3 to detect dying cells. Both Pcdh7MO (panel A) and Pcdh7Î´E (panel C) result in apoptosis of cranial neural crest cells (arrowheads), as compared to CMO-injected embryos (panel D). The specificity of the Pcdh7MO-induced neural crest is demonstrated by rescuing apoptosis with co-injected Pcdh7 mRNA (panel B). A, B, C, D show caspase immunofluorescence; Aâ€²Bâ€²Câ€²Dâ€² show the Î²-galactosidase staining; while Aâ€², Bâ€², Câ€², Dâ€² are the corresponding overlays. The injected side of the embryo is to the right. Results of at least 6 embryos are quantified in E. * indicates a statistical significant difference (p b 0.001).
	Fig. 4. Pcdh7MO-induced neural crest apoptosis begins at stage 13 and is evident by stage 15. Embryos were injected with Pcdh7MO, together with LacZ mRNA, fixed at stages 11â€“17, stained for Î²-galactosidase, and immunostained for activated Caspase 3. At stage 11 (panel A), mainly background staining is observed. By stage 13 (panel B), embryos exhibit staining for activated Caspase 3, that includes both nuclei and cell bodies (arrowheads). At stage 15 (panel C) and continuing through stage 17 (panel D), the neural crest exhibits strong Caspase 3 staining throughout the cells on the Pcdh7MO-injected side, indicating that the cell death caused by Pcdh7MO coincides with the timing of neural crest specification. A, B, C, D show caspase immunofluorescence; Aâ€²Bâ€²Câ€²Dâ€² show the Î²-galactosidase staining; while Aâ€², Bâ€², Câ€², Dâ€² are the corresponding overlays. The injected side of the embryo is to the right. Results of at least 6 embryos are quantified in F. * indicates a statistical significant difference between the injected and uninjected sides at each stage (p b 0.001). Abbreviations: np, neural plate.
	Fig. 5. Pcdh7 knockdown disrupts the sensorial layer. Embryos were injected with Pcdh7MO, together with LacZ mRNA (A, Aâ€²), or the dominant-negative Pcdh7Î´E, together with GFP mRNA (B, Bâ€²), then fixed, sectioned and immunostained for the sensorial layer marker p63. While p63 is observed in sensorial layer nuclei on the uninjected side of the embryos (arrowheads in A and B), disrupting Pcdh7 via either morpholino or dominant-negative results in loss of sensorial layer cells on the injected side. In C and D, embryos were injected with Pcdh7Î´E and LacZ mRNA, then fixed, sectioned and immunostained for Î²-catenin, which localizes to the cell membranes on the uninjected side of the embryo (arrowheads in C); in contrast, the injected side exhibits reduced Î²-catenin staining (D), and an increase in the pigmented outer layer (arrows in Dâ€²). To confirm that the ectoderm is disrupted, Pcdh7Î´Einjected embryos were subjected to in situ hybridization for epidermal keratin at stage 20 (E, F). The injected side of the embryos reveals a thicker, disorganized ectoderm (arrows in E) as compared to the uninjected side (F). Abbreviations: np, neural plate; nc, notochord; psm, presomitic mesoderm; nt, neural tube.
	Fig. 6.Wnt11b is disrupted in Pcdh7MO-injected embryos. Embryos were analyzed by in situ hybridization for Wnt11b (A) or double in situ hybridization for Pcdh7 and Wnt11b (B), demonstrating that Wnt11b and Pcdh7 expression in the sensorial layer overlap at the neural crest border (arrows in B). To examine the effect of Pcdh7 knockdown on Wnt11b expression, embryos were injected with Pcdh7MO, together with LacZ mRNA, fixed at stage 17/18, stained for Î²-galactosidase, and subjected to in situ hybridization for Wnt11b (C). Results demonstrate that Pcdh7MO-injected embryos exhibit reducedWnt11b expression on the injected side (top of photo in C), as compared to CMO-injected embryos (D).
	Fig. 7. Ectopic expression ofWnt11b, Snail2 or Bcl2l1 rescues Pcdh7MO-induced neural crest apoptosis. Embryos were co-injectedwith Pcdh7MOandmRNA encodingWnt11b, Snail2 or Bcl2l1, together with LacZ mRNA, then fixed at stage 17, stained for Î²-galactosidase, and immunostained for activated Caspase 3. The mean number of caspase positive cells was determined for at least 6 embryos for each injection, demonstrating that Pcdh7-induced apoptosis is rescuable by co-injecting Wnt11b, Snail2 or Bcl2l1 mRNAs. Injecting Wnt11b, Snail2 or Bcl2l1 mRNA alone results in few apoptotic neural crest cells. * indicates statistical significant difference (p b 0.001) as compared to Pcdh7MO-injected embryos.
	Fig. 8. Ectopic expression ofWnt11b or Bcl2l1 rescues Snail2 expression and cranial cartilage in Pcdh7MO-injected embryos. Aâ€“C) Embryos were co-injected with Pcdh7MO and mRNA encoding Wnt11b or Bcl2l1, togetherwith LacZ mRNA, fixed at stage 17, stained for Î²-galactosidase, and analyzed by in situ hybridization for Snail2. Pcdh7MO (A) causes reduced Snail2 expression,which is rescued by eitherWnt11b (B) or Bcl2l1 (C). The injected side of the embryos is up and anterior is to the right in Aâ€“C. D) Pcdh7 knockdown results in cranial cartilage defects. Embryos were injected with Pcdh7MO, together with GFP mRNA, sorted for GFP expression in the head, fixed at stage 46 and stained for cranial cartilage with Alcian Blue. Pcdh7MO-injected embryos exhibit defects in the branchial basket (bb) cartilage that range mild (arrow, left panel), to moderate (arrow, middle panel). Similarly, Pcdh7Î´E -injected embryos (arrow, right panel), exhibit bb defects, confirming the specificity of Pcdh7 knockdown. E) The Pcdh7MO-induced cranial cartilage defects are rescued by ectopic expression of Wnt11b (left panel) or Bcl2l1 (middle panel). In comparison, CMO-injected embryos exhibit no cranial cartilage defects (right panel). The injected side of the embryo is to the right in D and E. Abbreviations: bb, branchial basket; bh, basihyobranchial cartilage; ch, ceratohyal cartilage; m, Meckel's cartilage.

References [+] :

Abbruzzese, ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site. 2016, Pubmed, Xenbase