Expression of the Xhox3 Homeobox Protein in Xenopus Embryos: Blocking Its Early Function Suggests the Requirement of Xhox3 for Normal Posterior Development

Antibodies directed against the product of the Xenopus homeobox gene Xhox3 were raised and used to localize the expression of Xhox3 in the embryo at different stages of development. These studies suggest that endogenous Xhox3 protein is distributed in a graded fashion in the nuclei of mesodermal cells along the anterior-posterior (A-P) and dorso-ventral (D-V) axes in the postgastrula embryo with low levels in anterior and ventral regions and higher levels in posterior and dorsal regions. Xhox3 protein is also detected at different times in the midbrain, spinal cord and hindbrain. In the hindbrain, Xhox3 displays different metameric expression patterns in dorsal and ventral regions during early embryogenesis and metamorphosis. We have tested for the early function of Xhox3 by injecting antibodies against the Xhox3 protein into the cytoplasm of developing embryos. A significant number of embryos injected with Xhox3 antibodies show posterior (trunk and tail) deficiencies. This posterior deficient phenotype constitutes the opposite of the anterior (head) deficient phenotype obtained after overexpresson of Xhox3 reported previously. These results suggest that expression of Xhox3 in the posterior mesoderm is necessary for posterior development and that the graded distribution of Xhox3 in the embryonic mesoderm is required for the development of normal embryonic axial pattern.     

Involvement of the Xenopus homeobox gene Xhox3 in pattern formation along the anterior-posterior axis

The Xenopus homeobox gene Xhox3 shows a graded expression in the axial mesoderm, with the highest concentration in the posterior end of frog gastrula and neurula embryos. To investigate the function of the Xhox3 gene, synthetic Xhox3 mRNA was injected into different regions of developing embryos. In particular, Xhox3 was supplied in excess to anterior cells, which normally have the lowest levels of Xhox3 RNA. The results show that injection of Xhox3, but not control, mRNA into prospective anterior regions of developing embryos produces a series of graded axial defects. The injected embryos gastrulate normally but fail to form anterior (head) structures. Our findings suggest that Xhox3 is involved in establishing anterior-posterior cell identities during pattern formation of the axial mesoderm in early embryonic development.     

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.     

Different spatial distribution of mRNAs for activin receptors (type IIA and IIB) and follistatin in developing embryos of Xenopus laevis

Spatial distribution of mRNAs for activin receptors and follistatin was studied by Northern blot hybridization using RNAs from different parts of dissected Xenopus embryos. mRNAs of two activin receptors (type IIA and IIB) occurred uniformly in pre-gastrular embryos, but occurred in larger amounts in ectoderm (in gastrulae), neural plate (in neurulae) and anterior (head) regions (in tailbud embryos) than in other embryonic regions. By contrast, follistatin mRNA appeared almost exclusively in the dorsal mesoderm including invaginating organizer region at the gastrula stage, in notochord and in dorsal ectoderm at the neurula stage, then in anterior part at the tailbud stage. The localized patterns of the distribution of these mRNAs may be due to the regionally different zygotic expression of genes in embryos at later stages. From the relatively widespread pattern of distribution of their mRNAs, we assume that both type IIA and type IIB activin receptors have broad functions in ectodermal and neural differentiation. On the other hand, follistatin mRNA showed quite a restricted pattern of expression, and therefore, we assume that follistatin may have functions more specifically related to the sites of expression of its mRNA. Thus, follistatin may be involved in the differentiation of notochord itself and/or directly be responsible for organizer functions such as neural induction and subsequent differentiation of induced neural tissues at the gastrula and later stages.     

Programming neural Hoxd10: in vivo evidence that early node-associated signals predominate over paraxial mesoderm signals at posterior spinal levels

Studies of the programming of Hox patterns at anterior spinal levels suggest that these events are accomplished through an integration of Hensen's node-derived and paraxial mesoderm signaling. We have used in vivo tissue manipulation in the avian embryo to examine the respective roles of node- derived and other local signals in the programming of a Hox pattern at posterior spinal levels. Hoxd10 is highly expressed in the lumbosacral (LS) spinal cord and adjacent paraxial mesoderm. At stages of LS neural tube formation (stages 12-14), the tailbud contains the remnants of Hensen's node and the primitive streak. Hoxd10 expression was analyzed after transposition of LS neural segments with and without the tailbud, after isolation of normally positioned LS segments from the stage 13 tailbud, and after axial displacement of posterior paraxial mesoderm. Data suggest that inductive signals from the tailbud are primarily responsible for the programming of Hoxd10 at neural plate and the earliest neural tube stages. After these stages, the LS neural tube appears to differ from more anterior neural segments in its lack of dependence on Hox-inductive signals from local tissues, including paraxial mesoderm. Our data also suggest that a graded system of repressive signals for posterior Hox genes is present at cervical and thoracic levels and likely to originate from paraxial mesoderm.     

Expression of estrogen induced gene 121-like (EIG121L) during early Xenopus development

Estrogen induced gene 121 (EIG121) and EIG121-like (EIG121L) are evolutionarily conserved genes. But, their function is still unknown. Here, we report the expression pattern of Xenopus EIG121-like (xEIG121L) during early development. Its expression was first detected at stage 9 after mid-blastula transition, attained its maximal level at the gastrula stage, and remained constant until the tadpole stage. Whole-mount in situ hybridization revealed that xEIG121L was expressed strongly in the ventral ectoderm at the gastrula stage, and in the anterior ectoderm surrounding the neural plate at the neurula stage. xEIG121L expression was especially high in the presumptive hatching gland and cement gland regions in the neurula. At the tailbud stage, xEIG121L expression was limited to the hatching gland; an inverted Y type staining, characteristic of the hatching gland, was observed. However, at the tadpole stage, xEIG121L was expressed broadly in the head, heart and fin.     

Identification and characterization of Xenopus kctd15, an ectodermal gene repressed by the FGF pathway

The FGF pathway regulates a variety of developmental processes in animals through activation and/or repression of numerous target genes. Here we have identified a Xenopus homolog of potassium channel tetramerization domain containing 15 (KCTD15) as an FGF-repressed gene. Kctd15 expression is first detected at the gastrula stage and gradually increases until the tadpole stage. Whole-mount in situ hybridization reveals that the spatial expression of kctd15 is tightly regulated during early embryogenesis. While kctd15 is uniformly expressed throughout the presumptive ectoderm at the early gastrula stage, its expression becomes restricted to the non-neural ectoderm and is excluded from the neural plate at the early neurula stage. At the mid-neurula stage, kctd15 shows a more restricted distribution pattern in regions that are located at the anterior, lateral or medial edge of the neural fold, including the preplacodal ectoderm, the craniofacial neural crest and the prospective roof plate. At the tailbud stage, kctd15 expression is mainly detected in neural crest- or placode-derived tissues that are located around the eye, including the mandibular arch, trigeminal ganglia and the olfactory placode. FGF represses kctd15 expression in ectodermal explants, and the inhibition of FGF receptor with a chemical compound dramatically expands the region expressing kctd15 in whole embryos. Dorsal depletion of kctd15 in Xenopus embryos leads to bent axes with reduced head structures, defective eyes and abnormal somites, while ventral depletion causes defects in ventral and caudal morphologies. These results suggest that kctd15 is an FGF-repressed ectodermal gene required for both dorsal and ventral development.     

Developmental expression of the Xenopus int-2 (FGF-3) gene: activation by mesodermal and neural induction.

We have used a probe specific for the Xenopus homologue of the mammalian proto-oncogene int-2 (FGF-3) to examine the temporal and spatial expression pattern of the gene during Xenopus development. int-2 is expressed from just before the onset of gastrulation through to prelarval stages. In the early gastrula, it is expressed around the blastopore lip. This is maintained in the posterior third of the prospective mesoderm and neuroectoderm in the neurula. A second expression domain in the anterior third of the neuroectoderm alone appears in the late gastrula, which later resolves into the optic vesicles, hypothalamus and midbrain-hindbrain junction region. Further domains of expression arise in tailbud to prelarval embryos, including the stomodeal mesenchyme, the endoderm of the pharyngeal pouches and the cranial ganglia flanking the otocyst. It is shown, by treatment of blastula ectoderm with bFGF and activin, that int-2 can be expressed in response to mesoderm induction. By heterotypic grafting of gastrula ectoderm into axolotl neural plate, we have also demonstrated that int-2 can be expressed in response to neural induction. These results suggest that int-2 has multiple functions in development, including an early role in patterning of the anteroposterior body axis and a later role in the development of the tail, brain-derived structures and other epithelia.     

Selective expression of angiopoietin 1 and 2 in mesenchymal cells surrounding veins and arteries of the avian embryo

We have isolated a new Wnt receptor frizzled family member from Xenopus laevis, Xenopus frizzled-5 (Xfz5), a likely ortholog of human frizzled-5. Based on Northern and whole-mount in situ hybridization data, Xfz5 is first detected at the late neurula stage in retinal primordia. Throughout the tailbud stage Xfz5 is expressed exclusively in the neural retina within the optic vesicles. During tadpole stage Xfz5 expression becomes restricted to the ciliary marginal zone. This highly restrictive expression pattern makes Xfz5 an excellent marker for neural retinal tissue.     

Planar and vertical induction of anteroposterior pattern during the development of the amphibian central nervous system

In amphibians and other vertebrates, neural development is induced in the ectoderm by signals coming from the dorsal mesoderm during gastrulation. Classical embryological results indicated that these signals follow a “vertical” path, from the involuted dorsal mesoderm to the overlying ectoderm. Recent work with the frog Xenopus laevis, however, has revealed the existence of “planar” neural-inducing signals, which pass within the continuous sheet or plane of tissue formed by the dorsal mesoderm and presumptive neurectoderm. Much of this work has made use of Keller explants, in which dorsal mesoderm and ectoderm are cultured in a planar configuration with contact along only a single edge, and vertical contact is prevented. Planar signals can induce the full anteroposterior (A-P) extent of neural pattern, as evidenced in Keller explants by the expression of genes that mark specific positions along the A-P axis. In this review, classical and modern molecular work on vertical and planar inductionwill be discussed. This will be followed by a discussion of various models for vertical induction and planar induction. It has been proposed that the A-P pattern in the nervous system is derived from a parallel pattern of inducers in the dorsal mesoderm which is “imprinted” vertically onto the overlying ectoderm. Since it is now known that planar signals can also induce A-P neural pattern, this kind of model must be reassessed. The study of planar induction of A-P pattern in Xenopus embryos provides a simple, manipulable, two-dimensional system in which to investigate pattern formation. © 1993 John Wiley & Sons, Inc.