Mayor R et al. (1995), Induction of the prospective neural crest of Xe...

XB-ART-20029

Development 1995 Mar 01;1213:767-77.

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Induction of the prospective neural crest of Xenopus.

Mayor R , Morgan R , Sargent MG .

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The earliest sign of the prospective neural crest of Xenopus is the expression of the ectodermal component of Xsna (the Xenopus homologue of snail) in a low arc on the dorsal aspect of stage 11 embryos, which subsequently assumes the horseshoe shape characteristic of the neural folds as the convergence-extension movements shape the neural plate. A related zinc-finger gene called Slug (Xslu) is expressed specifically in this tissue (i.e. the prospective crest) when the convergence extension movements are completed. Subsequently, Xslu is found in pre- and post-migratory cranial and trunk neural crest and also in lateral plate mesoderm after stage 17. Both Xslu and Xsna are induced by mesoderm from the dorsal or lateral marginal zone but not from the ventral marginal zone. From stage 10.5, explants of the prospective neural crest, which is underlain with tissue, are able to express Xslu. However expression of Xsna is not apparently specified until stage 12 and further contact with the inducer is required to raise the level of expression to that seen later in development. Xslu is specified at a later time. Embryos injected with noggin mRNA at the 1-cell stage or with plasmids driving noggin expression after the start of zygotic transcription express Xslu in a ring surrounding the embryo on the ventroposterior side. We suggest this indicates (a) that noggin interacts with another signal that is present throughout the ventral side of the embryo and (b) that Xslu is unable to express in the neural plate either because of the absence of a co-inducer or by a positive prohibition of expression. The ventral co-inducer, in the presence of overexpressed noggin, seems to generate an anterior/posterior pattern in the ventral part of the embryo comparable to that seen in neural crest of normal embryos. We suggest that the prospective neural crest is induced in normal embryos in the ectoderm that overlies the junction of the domains that express noggin and Xwnt-8. In support of this, we show animal cap explants from blastulae and gastrulae, treated with bFGF and noggin express Xslu but not NCAM although the mesoderm marker Xbra is also expressed. Explants treated with noggin alone express NCAM only. An indication that induction of the neural plate border is regulated independently of the neural plate is obtained from experiments using ultraviolet irradiation in the precleavage period. At certain doses, the cranial crest domains are not separated into lateral masses and there is a reduction in the size of the neural plate.

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Species referenced: Xenopus
Genes referenced: eef1a2 fgf2 ncam1 nog snai1 snai2 tbxt wnt8a

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	Fig. 1. Sequence alignment of Xslu and Xsna. Start of the five zinc fingers shown by bold numbers.
	Fig. 2. Expression pattern of Xslu in Xenopus embryos obtained using whole mount in situ hybridisation. (A) Stage 12, Xslu on lateral aspect of embryo (c), (b) blastopore. (B) Stage 14, Xslu in prospective cranial neural crest (d, deep layer of ectoderm; s, superficial). Weak expression in trunk crest. (C) Stage 16, Xslu in cranial premigratory crest (Sadaghiani and Thiebaud, 1987). d, deep layer of ectoderm; s, superficial layer Xslu expression extending over the neural plate. (D) Stage 18. Neural tube now closed with cranial crest masses condensed (m, h and b, mandibular, hyoid and branchial aggregates. Xslu expression over neural plate demarcating rhombomeres. t, trunk neural crest. (E) Stage 22, Xslu in trunk crest (t), branchial arches and, in head surrounding the eyes (e) and forebrain, Xslu seems to demarcate rhombomeres. (F) Stage 25, Xslu in branchial arches (m, mandibular; h, hyoid and b, branchial), in lateral plate mesoderm (lp) with pronephros not expressing (p). (G) Transverse section through cephalic crest of stage 18 embryo. Xslu expression in deep layer (d) and superficial layer (s). np, neural plate; n, notochord; so, somites; a, archenteron. (H) Transverse section through trunk of stage 18 embryo. Xslu expression on top of neural folds in deep layer (d); nt, neural tube. (I) Parasagittal section of stage 20 embryo showing Xslu in three premigratory masses (m, mandibular; h, hyoid and b, branchial).
	Fig. 3. Fate map of stage 10.25 embryos and expression of Xslu in dorsal marginal zone explants. (A) Fate map of stage 10.25 embryos. Each spot represents dye marks applied to embryo at this stage. Fate of each spot is d, neural folds; s, epidermis; (, eye lens; x, neural plate. a, V, D and b = Animal pole, vegetal pole, dorsal and blastopore respectively. (114 embryos analysed). (B) Dissection of UDMZ (A) and LDMZ (B). Both explants were cut as strips extending round 180° of the embryo as shown; m indicates height of the floor of the blastocoel. C-F were hybridised with Xslu (C) UDMZ (A) (D) LDMZ (B) (E) Complete DMZ (A)+(B) (F) Remainder of embryo (C).
	Fig. 4. Time of commitment to Xslu and Xsna expression. Animal cap explants containing the prospective neural crest, dissected at stages shown and cultured until the equivalent of stage 16. A-D stained with Xsna (122 caps analysed), E-H stained with Xslu (245 caps analysed). (Arrows show expression). (A-C) Prospective folds dissected at stages 10, 11 and 12, cultured to equivalent of stage 16. (D) The prospective folds dissected at stage 12 and fixed immediately. (E-G) Prospective folds dissected at stages 10, 11 and 12, cultured to equivalent of stage 16. (H) Prospective folds dissected at stage 12 with a small amount of mesoderm (m) attached cultured to equivalent of stage 16.
	Fig. 5. Induction of Xslu in stage 10.25 animal caps by mesoderm measured by in situ hybridisation. (A) Animal caps alone (43 caps analysed). (B) Organiser in conjugate with stage 10.25 ectoderm (32 conjugates analysed). (C) Organiser alone (12). (D-F) Dorsal, lateral and ventral marginal zone respectively in conjugate with stage 10.25 ectoderm (41 conjugates analysed). (Arrows show expression.)
	Fig. 6. Effect of overexpression of Xwnt-8 and noggin on Xslu. (A) Embryo viewed from ventral aspect at equivalent of stage 13 injected with noggin RNA (100 pg) at 1 cell stage. (52 embryos). (B) Embryo viewed from posterior side, at equivalent of stage 17 injected with noggin RNA (5 ng) at 1 cell stage. Normal cephalic Xslu expression on lateral aspect extended in three banded pattern to ventral side (v). Microscopic examination of these embryos indicates a three-banded structure unambiguously (64 embryos). (C) Embryo viewed from lateral aspect at equivalent of stage 16 injected with pCSKA-noggin DNA (100 pg) at 1 cell stage (16 embryos). (D) Group of embryos at equivalent of stage 13 injected with Xwnt-8 RNA at 1 cell stage. Note greatly reduced expression (32 embryos).
	Fig. 7. RNAse protection study of the effect of soluble noggin and bFGF on ectodermal explants. Animal caps of stage 8 (Fig. A) and stage 10.5 (Fig. B) embryos were cultured 1´ NAM containing (a-c) noggin 100, 30 and 10 u/ml, respectively; (d-f) bFGF 90, 30 10 u/ml respectively; (g) noggin (30 u/ml) plus bFGF 10 u/ml; (h) noggin (30 u/ml) plus bFGF (30 u/ml); (i) noggin (10 u/ml) plus bFGF (10 u/ml); (j) no additions. At the equivalent of stage 17, the relative proportions of Xslu, Xsna, NCAM, Xbra and EF1a in the RNA prepared from the explants was determined by RNAse protection. Each track contains RNA from 10 explants. Individual probes hybridised with whole embryo RNA are shown in Fig. 7C.
	Fig. 8. Effect of UV irradiation on Xslu and Xsna. (A) Effect of UV on Xslu distribution at stage 12 Embryos are viewed from dorsal side. (a) Control; (b) regions of cephalic crest slightly closer; (c) cephalic crest regions joined; (d) cephalic fold fused in single dorsal spot; (e) no expression perfectly radially symmetrical embryo. (B) Effect of UV on Xsna distribution at stage 12.5. Note presence of labelled mesoderm underlying ectodermal pattern. Embryos are viewed from dorsal side, arrows indicate ectodermal expression. (a) Control; (b) Xsna in cephalic crest with slightly reduced transverse fold; (c) transverse fold diminished further; (d) cephalic fold fused in single dorsal spot; (e) no expression perfectly radially symmetrical embryo. c, cranial crest; m, mesoderm; y, yolk. (C) Effect of UV on NCAM distribution at stage 17 in a population of selected slow developing embryos. N indicates a normal control. All of this group have shortened axes at the equivalent stage. Most extreme NCAM patterns (x) are extremely short and extend around the blastopore.
	Fig. 9. Model of induction of the neural plate border. (A) The neural plate border is induced in ectoderm when it receives two signals from the underlying mesoderm one from the dorsal mesoderm region (expressing noggin; //////) and another from the ventral mesoderm region (expressing Xwnt-8; \\\\\\). M,H,S, mandibular, hyoid and branchial aggregates of the cranial crest in ectoderm. Note expression of the neural plate border occurs above the region of mesoderm expression. (B) Effect of noggin on distribution of cranial folds. M,H and S in three-banded Xslu expressing region encircling the ventral part of embryo. (C) Distribution of the three influences that determine expression in the neural plate border. (a) noggin expression (//////); (b) Ubiquitous ‘ventral’ signal (\\\\\\); (c) prohibitory signal (<<). Expression occurs only in the presence of a and b but without c.