Lallier TE and DeSimone DW (2000), Separation of neural induction and neurulation ...

XB-ART-10421

Dev Biol 2000 Sep 01;2251:135-50. doi: 10.1006/dbio.2000.9833.

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Separation of neural induction and neurulation in Xenopus.

Lallier TE , DeSimone DW .

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Cellular interactions with laminin are important for numerous morphogenetic events. In Xenopus, the first of these is neurulation. The integrin alpha6 subunit mediates an attachment of the cells of the neural plate to the underlying basal lamina. A disruption of this interaction results in embryos that fail to neurulate (T. E. Lallier et al., 1996, Development 122, 2539-2554). Here we provide evidence supporting the specificity of this phenomenon and characterize developmental events as either disrupted or unaffected by a perturbation of alpha6 integrin expression. First, reduction of alpha6 integrin expression does not halt mitotic division throughout the embryo, indicating that the neural defects observed are not simply a global perturbation of all developmental processes. Second, a gene associated with dorsal mesoderm formation, brachyury, is expressed normally in alpha6 integrin-perturbed embryos. Third, the expression of BMP4, noggin, chordin, and follistatin, all of which are critical for neural induction, are at near normal levels. In addition, several genes expressed shortly after neural induction (N-CAM, nrp1, and Xanf1) are not perturbed in nonneurulating embryos. Interestingly, expression of one neural-specific gene (synaptobrevin), which is normally detectable late in neurulation, is abolished in these alpha6 integrin-perturbed embryos. Furthermore, the spatial expression of several transcripts is expanded in alpha6 integrin-perturbed embryos (orthodenticle and engrailed). Taken together, these data indicate that while alpha6 integrin-mediated interactions with laminin are required for neurulation, they are not required for the initial processes of neural induction. However, these cell-extracellular matrix interactions appear to be important in later inductive events and rostrocaudal patterning of the neural tube.

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Species referenced: Xenopus
Genes referenced: bmp4 chrd en2 fst gsc hesx1 itga6 neurod1 nog nrp1 otx2 snai2 tbx2 tbxt vamp2

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	FIG. 1. Control embryos that are injected with a construct lacking the integrin a6 insert develop normally (A and C), as do embryos injected with constructs containing a6 in the sense orientation (Lallier et al., 1996). Xenopus embryos that have been microinjected with antisense integrin a6 constructs display a distinct morphology of perturbed neurulation (B and D–F). (C) An example of a control embryo at higher magnification, displaying a closed neural tube on its dorsal surface. (D) A dorsal view of a neurulation-perturbed sibling embryo displaying a flattened neural plate. (E) A rostral view, while (F) is a caudal view of a similarly perturbed embryo. Scale bar represents 200 mm.
	FIG. 2. Embryos displaying neurulation defects resulting from reduced a6 integrin expression possess cell numbers and DNA content comparable to that of sibling embryos of tail-bud stage. (A) The numbers of cells present in individual Xenopus embryos were compared by direct cell counts for control (black bars) neural plate (stage 12 and 15), neurula (stage 25), and tail-bud (stage 35) embryos, as well as antisense a6 integrin-treated embryos (gray bars) grown until siblings reached stages 15, 25, and 35. (B) The DNA content of individual embryos was quantified fluorimetrically. Control embryo (dark diamonds) and antisense a6 integrin-perturbed embryo (gray squares) were compared. Error bars represent the standard deviations of the mean.
	FIG. 3. The temporal expression of early mesodermal genes, neural genes, and genes that mediate neural induction are unaffected in antisense a6 integrin-treated embryos. Control embryos and embryos injected with antisense a6 integrin constructs that displayed neurulation arrest were evaluated semiquantitatively by RT-PCR. Equivalent RNA samples isolated from neural plate (12 and 13), neurula (15, 18, 20, and 25), and tail bud (30, 32, and 35), as well as antisense a6 integrin-treated embryos (stages 13, 20, and 32) were used as templates for cDNAs. Each sample was subjected to RT-PCR using primers specific for (A) a6 integrin, (B) the mesodermally expressed gene Xbra, and (C) the neurally expressed genes N-CAM and neuroD and genes involved in neural induction: BMP4, noggin, follistatin, and chordin. Parallel RT-PCRs were performed using primers specific for b1 integrin to normalize for RNA content.
	FIG. 4. The temporal expression of neural-specific genes is altered in antisense a6 integrin-treated embryos. Control and antisense a6 neurulation-arrested embryos were evaluated semiquantitatively by RT-PCR. Equivalent RNA samples isolated from neural plate (13), neurula (20 and 25), and tail-bud (32 and 35) stage embryos, as well as antisense a6 integrin-treated embryos (stages 13, 20, and 32), were use as templates for cDNAs. Each sample was subjected to RT-PCR using primers specific for Xanf1, sybII, and nrp1. Parallel RT-PCR reactions were performed using primers specific for b1 integrin to control for RNA content.
	FIG. 5. Spatial expression of neural-specific genes is unaffected in antisense a6 integrin-treated embryos. Controls and embryos with repressed a6 integrin expression were examined by in situ hybridization for several neural-selective transcripts: nrp1 (A–F), Xanf1 (G–L), and sybII (M–R). In uninjected, control embryos the expression of these transcripts was examined in embryos of neural plate (stage 13; A, G, and M), neurula (stage 25; B, H, and N) and tail-bud (stage 35; C, I, and O) stages. The expression of these genes was also examined in embryos injected with antisense a6 integrin constructs and that displayed severe perturbation of neurulation at sibling stages of neural plate (stage 15; D, J, and P), neurula (stage 25; E, K, and Q), and tail-bud (stage 35; F, L, and R) stages. Scale bar represents 200 mm.
	FIG. 6. Spatial expression of neural-specific genes is altered in antisense a6 integrin-treated embryos. Controls and embryos with repressed a6 integrin expression were examined by in situ hybridization for several neural-selective transcripts: otx (A–F), engrailed (G–L), and slug (M–R). In uninjected control embryos the expression of these transcripts was examined at neural plate (stage 13; A, G, and M), neurula (stage 25; B, H, and N) and tail-bud (stage 35; C, I, and O) stages. The expression of these genes was also examined in embryos injected with antisense a6 integrin constructs and that displayed severe perturbation of neurulation at sibling stages of neural plate (stage 15; D, J, and P), neurula (stage 25; E, K, and Q), and tail-bud (stage 35; F, L, and R) stages. Scale bar represents 200 mm.
	FIG. 7. The temporal expression of neural-specific genes unaffected in antisense a6 integrin-treated embryos. Controls and embryos injected with antisense a6 integrin constructs that displayed neurulation-arrest were evaluated semiquantitatively by RT-PCR. Equivalent RNA samples isolated from neural plate (13), neurula (20 and 25), and tail-bud (32 and 35) stage embryos, as well as antisense a6 integrin-treated embryos (stages 13, 20, and 32), were use as templates for cDNAs. Each sample was subjected to RT-PCR using primers specific for otx, engrailed, and slug. All three transcripts were detected in quantities comparable to those of late neurula (stage 25) through tail-bud (stage 35) embryos. Parallel RT-PCRs were performed using primers specific for b1 integrin to control for RNA content.
	FIG. 8. The spatial restriction of several neural-specific transcripts was perturbed in antisense a6-treated embryos. (A) CSKA vector control-injected and a6 integrin antisense-injected, neurulation-perturbed embryos were quartered along their rostrocaudal axis. Lanes 1–4 represent control embryo quarters arranged rostrocaudally, and lanes 5–8 represent chronologically equivalent perturbed embryos quartered along the equivalent rostrocaudal axis. (B) Equivalent RNA samples from control and perturbed stage 23 embryo quarters were used as templates for cDNAs. Each sample was subjected to RT-PCR using primers for the injected CSKA construct, a6 integrin, nrp1, Xanf1, otx, engrailed, slug, and HoxA7. Parallel RT-PCRs were performed using primers specific for b1 integrin to control for RNA content.