McGrew LL et al. (1992), Analysis of Xwnt-4 in embryos of Xenopus laevis...

XB-ART-23685

Development 1992 Jun 01;1152:463-73.

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Analysis of Xwnt-4 in embryos of Xenopus laevis: a Wnt family member expressed in the brain and floor plate.

McGrew LL , Otte AP , Moon RT .

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This study characterizes the temporal and spatial expression during early Xenopus development of Xwnt-4, a member of the Wnt gene family. The Xwnt-4 protein contains all of the sequence motifs that are hallmarks of the Wnt gene family and is 84% identical to the mouse homolog, Wnt-4. The highest level of Xwnt-4 expression occurs during the early neurula stage of development although its expression persists throughout embryogenesis and can be found in the adult testis, brain and epithelium. Consistent with its localization to head and dorsal regions of microdissected embryos, the expression of Xwnt-4 is enhanced in anterodorsalized embryos resulting from treatment with LiCl, and the expression of Xwnt-4 is suppressed in UV-ventralized embryos that lack anterior neural tissue. These results suggested that expression of Xwnt-4 is dependent on the induction of neural tissue. This idea was tested using induction experiments with dorsal or ventral ectoderm from a stage 10 embryo, recombined with dorsal marginal zone mesoderm from the same embryo. Recombinant tissue and ectoderm alone were cultured until stage 14, when Xwnt-4 expression was assayed using Northern analysis. In the recombinant assay, Xwnt-4 expression does not occur in the uninduced ectoderm but is expressed in both the dorsal and ventral recombinants. Xwnt-4 expression in neural ectoderm was confirmed in isolated, induced neural ectoderm, dissected away from the dorsal mesoderm, in a stage 12.5 embryo. Whole-mount in situ hybridization confirmed the dissection studies and demonstrated that Xwnt-4 transcripts are expressed in the dorsal midline of the midbrain, hindbrain and the floor plate of the neural tube. Collectively, the data indicate that Xwnt-4 is a unique member of the Wnt family whose expression is dependent on neural induction. The specific pattern of expression following neural induction suggests that Xwnt-4 plays a role in the early patterning events responsible in the formation of the nervous system in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: krt18.1 prph wnt1 wnt4

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	Fig. 1. Comparison of the predicted XwM-4, Xwflt-1 and Wnt-4 protein Sequences. GapS introduced to align the sequences are shown as dashes and identical residues are marked with asterisks. A putative hydrophobic leader sequence
	Fig. 2. Expression of Xwnt-4 in adult tissues and during early Xenopus development. (A) Northern analysis of 20 lig of total RNA isolated from each of the adult tissues indicated and hybridized with ^P-labelled antisense Xwnt-4 RNA probe. The filters were rehybridized with an EFl-ar cDNA clone to determine the integrity of RNA in each lane. (B) Temporal expression of Xwnt-4 mRNA during early development. Northern analysis of RNA from different developmental stages (Nieuwkoop and Faber, 1967). 20 ug aliquots of total RNA were analyzed at each stage. Equivalent loading of RNA was determined by rehybridization of the filters in the presence of a Xenopus 5S cDNA probe. The 28S and 18S ribosomal RNA markers are noted.
	Fig. 3. Xwnt-4 expression in UV-irradiated or lithiumtreated embryos. Northern blot containing 20 ng of total RNA from tailbud embryos (stage 25) that either received no treatment (—UV, —Li), or were treated with UV (+UV) or lithium (+Li) as described. The blot was rehybridized with a ^P-labeled X3F3 antisense RNA probe to determine the amount of anterior neural tissue in each sample, followed by EFl-a cDNA probe to determine the relative loading of RNA in each sample.
	Fig. 4. Xwnt-4 expression in neural recombinant tissues cultured in vitro. (A) Dissected tissues were assembled from a stage 10 embryo as diagrammed. (B) Recombinant tissue and isolated ectodermal tissue were cultured until control embryos reached stage 14, total RNA was isolated and 20 /ig aliquots were examined using Northern analysis. The integrity of the RNA in the sample was verified by rehybridization of the filter with the 5S cDNA probe. Control embryo, C.
	Fig. 5. Xwnt-4 expression in neural ectoderm as compared to the expression of XIF3, a marker of anterior neural tissue and XK endo B, a marker of dorsal mesoderm tissue. Tissues were dissected as described and cultured until control embryos reached stage 23. 20 jig of total RNA from each sample, including control embryos (lanel), were hybridized to a Xwnt-4 antisense RNA probe followed by successive rehybridizations to XIF3 antisense RNA probe, XK endo B cDNA probe and the 5S ribosomal cDNA probe.
	Fig. 6. Localization of Xwnt-4 transcripts during early development using whole-mount in situ hybridization. (A) Antisense Xwnt-4 probe hybridized to a late gastrula stage embryo (stage 12; on the left), the blastopore is pointing down (arrowhead), and hybridized to a slightly later stage embryo (stage 13; on the right). Xwnt-4 transcripts are localized to a thin band of cells above the archenteron (arrow) in the embryo on the right. (B) Antisense Xwnt-4 probe hybridized to a neurula stage embryo (stage 14/15), shown from the side. Xwnt-4 expression occurs along the entire anteroposterior axis where it is localized to the inner layer of the neural ectoderm (arrow). A distinct anterior patch of staining is denoted by the arrowhead. (C) Antisense Xwnt-4 probe hybridized to a neurula stage embryo (stage 14/15), shown from the top. Two anterior patches of Xwnt-4 expression are clearly seen (arrowhead) on the medial region of the neural plate. At this stage, there is an absence of Xwnt-4 expression along the midline of the neural groove (arrow). (D) Antisense XwntA probe hybridized to an early tailbud stage embryo (stage 23). Xwnt-4 expression in the neural tube changes by this stage, becoming concentrated along the length of the floor plate and restricted to the posterior end of the embryo. Three overlapping regions of expression are seen in the border of the prosencephalon/mesencephalon (arrow 1), the anterior rhombencephalon (arrow 2) and the posterior rhombencephalon (arrow 3). (E) Antisense Xwnt-4 probe hybridized to a late tailbud stage embryo (stage 27). The same regions of expression in the brain are denoted as in D. (F) Antisense Xwnt-4 probe hybridized to a tadpole stage embryo (stage 38). (G) Sense Xwnt-4 probe hybridized to early and late tailbud stage embryos. All embryos were rendered transparent with Murray's clear prior to photography. The intensity of the stain varies with respect to the length of time the embryos are incubated in the chromogenic reaction and should not be taken as a measure of transcript abundance when compared between different embryos.
	Fig. 7. Comparison of the expression of Xwnt-4 and Xwnt-1 in the neural tube of a late tadpole stage embryo (stage 28). (A) Antisense Xwnt-4 and Xwnt-1 probes hybridized to late tadpole stage embryos and processed for whole-mount in situ hybridization. Embryos were embedded in paraffin and sectioned. Representative sections are denoted by the lettered arrows. (B) A transverse section through an Xwnt-4 hybridized embryo taken at the level of the optic vesicle (ov). Xwnt-4 expression occurs on the dorsal midline of the mesencephalon (me). (C) A transverse section through an Xwnt-4 hybridized embryo taken at the level of the auditory vesicle (av). At this position in the anteroposterior axis Xwnt-4 expression is seen in the dorsolateral region of the rhombencephalon (rh) and in the ventral midline directly above the notochord (nc). The ventral portion of the auditory vesicle is also positive for Xwnt-4 expression. (D) A transverse section through an Xwnt-4 hybridized embryo, taken at the posterior edge of the rhombencephalon. Xwnt-4 expression is localized to the floor plate of the neural tube (nt). (E) A transverse section taken at the posterior edge of the optic vesicle (ov), through an embryo hybridized with Xwnt-1. Xwnt-1 expression occurs in both the dorsal midline of the mesencephalon (me) and in the ventral-lateral margin of the mesencephalon. (F) A transverse section taken through an Xwnt-1 hybridized embryo in the rhombencephalon (rh). All of the Xwnt-1 expressing cells at this point in the rhombencephalon are localized to the dorsal midline. There is no Xwnt-1 expression in the ventral midline overlying the notochord (nc).