IQGAP2 is required for the cadherin-mediated cell-to-cell adhesion in Xenopus laevis embryos

We have previously identified two Xenopus homologues of mammalian IQGAP, XIQGAP1 and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. In order to clarify function of the IQGAPs during development, we performed knock-down experiments on the XIQGAPs in Xenopus laevis embryos by microinjecting morpholino antisense oligonucleotides into blastomeres at the two-cell stage. Suppression of XIQGAP2 expression caused ectodermal lesions in the neurula stage embryos. While suppression of XIQGAP1 expression alone did not show any obvious defect in subsequent developmental processes, simultaneous knock-down of both XIQGAPs caused the ectodermal lesions during the gastrula stage. Histological studies suggested that a loss of cell adhesion in the ectodermal and mesodermal layers of the embryos caused the defect. The suppression of XIQGAP2 expression resulted in loss of actin filaments, beta-catenin, and XIQGAP1 from cell borders in the ectoderm, although it did not affect the expression levels of these proteins. Furthermore, it inhibited Ca(2+)-induced reaggregation of embryonic cells which had been dissociated in a Ca(2+)/Mg(2+)-free medium. These results strongly suggest that XIQGAP2 is crucial for cell adhesion during early development in Xenopus.     

The Notch-regulated ankyrin repeat protein is required for proper anterior-posterior somite patterning in mice

The Notch-regulated ankyrin repeat protein (Nrarp) is a component of a negative feedback system that attenuates Notch pathway-mediated signaling. In vertebrates, the timing and spacing of formation of the mesodermal somites are controlled by a molecular oscillator termed the segmentation clock. Somites are also patterned along the rostral-caudal axis of the embryo. Here, we demonstrate that Nrarp-deficient embryos and mice exhibit genetic background-dependent defects of the axial skeleton. While progression of the segmentation clock occurred in Nrarp-deficient embryos, they exhibited altered rostrocaudal patterning of the somites. In Nrarp mutant embryos, the posterior somite compartment was expanded. These studies confirm an anticipated, but previously undocumented role for the Nrarp gene in vertebrate somite patterning and provide an example of the strong influence that genetic background plays on the phenotypes exhibited by mutant mice.     

Zygotic Wnt activity is required for Brachyury expression in the early Xenopus laevis embryo

The canonical, beta-catenin-dependent Wnt pathway is a crucial player in the early events of Xenopus development. Dorsal axis formation and mesoderm patterning are accepted effects of this pathway, but the regulation of expression of genes involved in mesoderm specification is not. This conclusion is based largely on the inability of the Wnt pathway to induce mesoderm in animal cap explants. Using injections of inhibitors of canonical Wnt signaling, we demonstrate that expression of the general mesodermal marker Brachyury (Xbra) requires a zygotic, ligand-dependent Wnt activity throughout the marginal zone. Analysis of the Xbra promoter reveals that putative TCF-binding sites mediate Wnt activation, the first sites in this well-studied promoter to which an activation role can be ascribed. However, established mesoderm inducers like eFGF and activin can bypass the Wnt requirement for Xbra expression. Another mesoderm promoting factor, VegT, activates Xbra in a Wnt-dependent manner. We also show that the activin/nodal signaling is necessary for ectopic Xbra induction by the Wnt pathway, but not by VegT. Our data significantly change the understanding of Brachyury regulation in Xenopus, implying the existence of an unknown zygotic Wnt ligand in Spemann's organizer. Since Brachyury is considered to have a major role in mesoderm formation, it is possible that Wnts might play a role in mesoderm specification, in addition to patterning.     

Cyclin E2 is required for embryogenesis in Xenopus laevis

In mammalian cells, E-type cyclins (E1 and E2) are generally believed to be required for entry into S phase. However, in mice, cyclin E is largely dispensable for normal embryogenesis. Moreover, Drosophila cyclin E plays a critical role in cell fate determination in neural lineages independently of proliferation. Thus, the functions of cyclin E, particularly during early development, remain elusive. Here, we investigated the requirement for E-type cyclins during Xenopus embryogenesis. Although cyclin E1 has been reported as a maternal cyclin, inhibition of its translation in the embryo caused no serious defects. We isolated a Xenopus homologue of human cyclin E2, which was zygotically expressed. Sufficient inhibition of its expression led to death at late gastrula, while partial inhibition allowed survival. These observations indicate distinct roles for Xenopus cyclins E1 and E2, and an absolute requirement of cyclin E2 for Xenopus embryogenesis.     

The Xenopus laevis Hox 2.1 homeodomain protein is expressed in a narrow band of the hindbrain

The expression pattern of the Xenopus homeodomain protein Hox 2.1 during development was determined using an affinity-purified antibody directed against a carboxyterminal peptide. Nuclear staining was detected in a very narrow band of the hindbrain. This pattern was compared to that of the previously described Xenopus gene XIHbox 1 in serial sections and found to be more anterior than the XIHbox 1 long protein expression but overlapping with that of the short protein. Xenopus Hox 2.1 protein expression is restricted to a much narrower antero-posterior band than that reported for mouse Hox 2.1 RNA expression by in situ hybridization.     

Xmab21l3 mediates dorsoventral patterning in Xenopus laevis

Specification of the dorsoventral (DV) axis is critical for the subsequent differentiation of regional fate in the primary germ layers of the vertebrate embryo. We have identified a novel factor that is essential for dorsal development in embryos of the frog Xenopus laevis. Misexpression of Xenopus mab21-like 3 (Xmab21l3) dorsalizes gastrula-stage mesoderm and neurula-stage ectoderm, while morpholino-mediated knockdown of Xmab21l3 inhibits dorsal differentiation of these embryonic germ layers. Xmab21l3 is a member of a chordate-specific subclass of a recently characterized gene family, all members of which contain a conserved, but as yet ill-defined, Mab21 domain. Our studies suggest that Xmab21l3 functions to repress ventralizing activity in the early vertebrate embryo, via regulation of BMP/Smad and Ras/ERK signaling.     

Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos.

Retinoic acid is a very potent teratogen and has also been implicated as an endogenous developmental signalling molecule in vertebrate embryos. One of the regions of the embryo reliably affected by exogenously applied RA is the hindbrain. In this paper, we describe in detail the hindbrain of Xenopus laevis embryos briefly treated with various levels of RA at gastrula stages. Such treatments lead to development of embryos with loss of anterior structures. In addition, RA has a general effect on rhombomere morphology and specific effects on the development of the anterior rhombomeres. This effect is demonstrated using neurofilament antibodies, HRP staining and in situ hybridisation using a probe for expression of the Xenopus Krox-20 gene. Anatomically it is evident that the development of the hindbrain normally anterior to the otocyst (rhombomeres 1-4) is abnormal following RA treatment. Sensory and motor axons of cranial nerves V and VII form a single root and the peripheral paths of V and VII and IX and X are also abnormal, as is the more anterior location of the otocyst. These anatomical changes are accompanied by changes in the pattern of expression for the gene XKrox-20, which normally expresses in rhombomeres 3 and 5, but is found in a single band in the anterior hindbrain of treated embryos which standardly fail to generate the normal external segmental appearance. The results are discussed in terms of both the teratogenic and possible endogenous roles of RA during normal development of the central nervous system. We conclude that low doses of RA applied during gastrulation have specific effects on the anterior Xenopus hindbrain which appear to be evolutionarily conserved in the light of similar recent findings in zebrafish.     

Expression of XMyoD protein in early Xenopus laevis embryos.

A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anterior somitic mesoderm, and its concentration then declines in anterior somites from the tailbud stage onwards.     

Inhibition of BMPs by follistatin is required for FGF3 expression and segmental patterning of the hindbrain

A network of molecular interactions is required in the developing vertebrate hindbrain for the formation and anterior-posterior patterning of the rhombomeres. FGF signaling is required in this network to upregulate the expression of the Krox20 and Kreisler segmentation genes, but little is known of how FGF gene expression is regulated in the hindbrain. We show that the dynamic expression of FGF3 in chick hindbrain segments and boundaries is similar to that of the BMP antagonist, follistatin. Consistent with a regulatory relationship between BMP signaling and FGF3 expression, we find that an increase in BMP activity due to blocking of follistatin translation by morpholino antisense oligonucleotides or overexpression of BMP results in strong inhibition of FGF3 expression. Conversely, addition of follistatin leads to an increase in the level of FGF3 expression. Furthermore, the segmental inhibition of BMP activity by follistatin is required for the expression of Krox20, Hoxb1 and EphA4 in the hindbrain. In addition, we show that the maintenance of FGF3 gene expression requires FGF activity, suggestive of an autoregulatory loop. These results reveal an antagonistic relationship between BMP activity and FGF3 expression that is required for correct segmental gene expression in the chick hindbrain, in which follistatin enables FGF3 expression by inhibiting BMP activity.     

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning.     

Fission yeast tmsl protein abrogates normal development in Xenopus laevis embryos

Recently we cloned tms1 (a putative dehydrogenase) by complementation of a human tumour-derived mutant p53 induced growth arrest in fission yeast. Microinjection of purified tmsl protein into Xenopus laevis embryos abrogated normal embryo development by causing cleavage retardation or cleavage arrest of injected blastomeres in a concentration dependant manner, whereas injection of specific affinity purified tms1 antiserum showed no significant morphological defects. Microinjection of tms1 protein together with affinity purified tms1 antibody resulted in a significantly reduced number of cleavage arrested embryos.     

Xenopus glucose transporter 1 (xGLUT1) is required for gastrulation movement in Xenopus laevis

Glucose transporters (GLUTs) are transmembrane proteins that play an essential role in sugar uptake and energy supply. Thirteen GLUT genes have been described and GLUT1 is the most abundantly expressed member of the family in animal tissues. Deficiencies in human GLUT1 are associated with many diseases, such as metabolic abnormalities, congenital brain defects and oncogenesis. It was suggested recently that Xenopus GLUT1 (xGLUT1) is upregulated by Activin/Nodal signaling, although the developmental role of xGLUT1 remains unclear. Here, we investigated the expression pattern and function of xGLUT1 during Xenopus development. Whole-mount in situ hybridization analysis showed expression of xGLUT1 in the mesodermal region of Xenopus embryos, especially in the dorsal blastopore lip at the gastrula stage. From the neurula stage, it was expressed in the neural plate, eye field, cement gland and somites. Loss-of-function analyses using morpholino antisense oligonucleotides against xGLUT1 (xGLUT1MO) caused microcephaly and axis elongation error. This elongation defect of activin-treated animal caps occurred without downregulation of early mesodermal markers. Moreover, dorsal-marginal explant analysis revealed that cell movement was suppressed in dorsal marginal zones injected with xGLUT1MO. These findings implicate xGLUT1 as an important player during gastrulation cell movement in Xenopus.