Xenopus Bicaudal-C is required for the differentiation of the amphibian pronephros

The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which--when disrupted--results in PKD.     

Expression of the highly conserved RNA binding protein KOC in embryogenesis.

The human KOC gene which is highly expressed in cancer shows typical structural features of an RNA binding protein. We analyzed the temporal and spatial expression pattern of KOC in mouse embryos at different gestational ages. The expression of KOC seems to be ubiquitous at early stages. During advanced gestation highest KOC expression occurs in the gut, pancreas, kidney, and in the developing brain. The expression pattern of KOC was compared to its Xenopus homologue Vg1-RBP during frog development. Similar expression was found in these organs suggesting an important functional role of the homologous proteins in embryonic development.     

The mouse Cer1 (Cerberus related or homologue) gene is not required for anterior pattern formation.

Cer1 is the mouse homologue of the Xenopus Cerberus gene whose product is able to induce development of head structures during embryonic development. The Cer1 protein is a member of the cysteine knot superfamily and is expressed in anterior regions of the mouse gastrula. A segmental pattern of expression with nascent and newly formed somites is also seen. This suggests an additional role in development of the axial skeleton, musculature, or peripheral nervous system. Xenopus animal cap assays and mouse germ-layer explant recombination experiments indicate that the mouse protein can act as a patterning molecule for anterior development in Xenopus, including induction of Otx2 expression, and suggest it may have a similar role in mouse development. However, we present here genetic data that demonstrate that Cer1 is not necessary for anterior patterning, Otx2 expression, somite formation, or even normal mouse morphogenesis.     

Cloning and expression of a novel zinc finger gene, Fez, transcribed in the forebrain of Xenopus and mouse embryos

We have identified and cloned a novel zinc finger gene, Fez (forebrain embryonic zinc-finger), as a potential downstream determinant of anterior neural plate formation in Xenopus. Fez was isolated as one of several neural-specific genes that was induced by the neuralizing factor, noggin (Smith and Harland, 1992. Cell 70, 829-840), in uncommitted ectoderm. Fez has an open reading frame comprising 466 amino acids, and contains six C(2)H(2) type zinc finger domains, which are highly conserved among Drosophila, zebrafish, mouse, and human. In Xenopus, the expression of Fez begins at stage 12 in the rostral end of the neural plate, and by stage 45, it is localized to several telencephalic regions, including the olfactory bulbs, nervus terminalis, and ventricular zone. The mouse homologue of Fez is similarly expressed in the mouse forebrain by embryonic day 11.     

Isthmin is a novel secreted protein expressed as part of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain organizer

Patterning of the central nervous system is regulated by a signaling center located at the midbrain-hindbrain boundary (MHB), or isthmus organizer. Fibroblast growth factors secreted from the MHB are required and sufficient to direct the ordered growth and regionalization of the midbrain and anterior hindbrain. In an unbiased secretion cloning screen of Xenopus gastrula embryos we identified a novel gene, which we designated as Isthmin (xIsm) due to its prominent expression at the MHB. xIsm encodes a secreted protein of 449 amino acids containing one copy of the thrombospondin type 1 repeat (TSR). We also found orthologous Isthmin genes in human (hIsm) and mouse (mIsm), as well as a gene encoding an Isthmin-like human unknown protein (hIsm-l). The conservation of a unique carboxy-terminal region between hIsm and hIsm-l suggests that Isthmin is the founding member of a new family of secreted proteins. xIsm was strongly expressed maternally in the Xenopus egg and showed zygotic expression in the ventral blastopore lip, notochord, and MHB. Additional expression domains were detected in neural crest, ear vesicle, and developing blood islands. Interestingly, xIsm was co-expressed with Fibroblast growth factor-8 (xFgf-8) at multiple sites including the MHB, indicating that these two genes are part of a synexpression group which also includes sprouty and sef homologs.     

Regulation of vertebrate eye development by Rx genes

The paired-like homeobox-containing gene Rx has a critical role in the eye development of several vertebrate species including Xenopus, mouse, chicken, medaka, zebrafish and human. Rx is initially expressed in the anterior neural region of developing embryos, and later in the retina and ventral hypothalamus. Abnormal regulation or function of Rx results in severe abnormalities of eye formation. Overexpression of Rx in Xenopus and zebrafish embryos leads to overproliferation of retinal cells. A targeted elimination of Rx in mice results in a lack of eye formation. Mutations in Rx genes are the cause of the mouse mutation eyeless (ey1), the medaka temperature sensitive mutation eyeless (el) and the zebrafish mutation chokh. In humans, mutations in Rx lead to anophthalmia. All of these studies indicate that Rx genes are key factors in vertebrate eye formation. Because these results cannot be easily reconciled with the most popular dogmas of the field, we offer our interpretation of eye development and evolution.     

The cerberus-related gene, Cerr1, is not essential for mouse head formation

The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus-related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells. We found that head formation is normal in Cerr1(-/-) embryos and we detected no obvious phenotypic defects in adult Cerr1(-/-) mice. However, in embryonic tissue layer recombination assays, Cerr1(-/-) presomitic/somitic mesoderm, unlike Cerr1-expressing wild-type presomitic/somitic mesoderm, was unable to maintain expression of the anterior neural marker gene Otx2 in ectoderm explants. These findings suggest that establishment of anterior identity in the mouse may involve the action of multiple functionally redundant factors.     

Sequence and expression of a novel mouse gene PRDC (protein related to DAN and cerberus) identified by a gene trap approach

Gene trapping in embryonic stem (ES) cells was used to identify a novel gene involved in mouse development. In order to screen trapped ES cell lines for the presence of developmentally regulated genes, an in vitro differentiation test was used. One of the G418 resistant cell lines, in conjunction with the lacZ reporter gene, showed differential expression patterns under differentiated and undifferentiated conditions. The gene trap insertion in this cell line was germ-line transmitted and X-gal staining was used to assess the expression pattern of lacZ in embryos heterozygous for the trapped allele. The reporter gene's expression was detected in commissural neurons in the developing spinal cord, suggesting functions for the trapped gene in mouse neural development. Structural analysis of the cDNA revealed that this trapped gene, named PRDC (protein related to DAN and cerberus), is a novel gene that encodes a putative secretory protein consisting of 168 amino acid residues. PRDC gene product shows limited similarities to the products of DAN (differential screening-selected gene aberrative in neuroblastoma) and cerberus . (DAN is a possible tumor-suppressor for neuroblastoma in human. Cerberus can induce an ectopic head in Xenopus embryos when ectopically expressed.) These three gene products may form a novel family of signaling molecules.     

Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction.

The Brachyury (T) gene is required for mesoderm formation in the mouse. In this paper we describe the cloning and expression of a Xenopus homolog of Brachyury, Xbra. As with Brachyury in the mouse, Xbra is expressed in presumptive mesodermal cells around the blastopore, and then in the notochord. We show that expression of Xbra occurs as a result of mesoderm induction in Xenopus, both in response to the natural signal and in response to the mesoderm-inducing factors activin A and basic FGF. Expression of Xbra in response to these factors is rapid, and will occur in dispersed cells and in the presence of a protein synthesis inhibitor, indicating that this is an "immediate-early" response to mesoderm induction.     

Molecular cloning and characterization of a novel inhibitor of apoptosis protein from Xenopus laevis

A novel inhibitor of apoptosis protein family member termed SIX was identified in Xenopus containing a single baculoviral IAP repeat (BIR) domain and no COOH-terminal RING finger domain. It exhibited striking amino acid sequence similarity with human survivin, mouse TIAP, and recently found Xenopus survivin, especially a part of BIR domain was highly conserved. Interestingly, SIX interacted with RXRalpha through the AF2 domain in the absence of ligand, which was weakened when the ligand was present. Northern blot analysis demonstrated that SIX mRNA was not detectable in adult with exception of the ovary and testis, and whole-mount in situ hybridization and Northern blot analyses revealed strong and homogeneous expression of SIX in the developing oocytes. In the embryos, the expression of SIX was observed in the animal hemisphere from one-cell to yolk plug stages and high level of expression was detected in the future brain and dorsal region of the neural tube at the neurula stage and early tail-bud stage. These results strongly support the fact that survivin is evolutionarily conserved in structure and SIX is likely to be the Xenopus counterpart of human and mouse survivin.     

TRIQK, a novel family of small proteins localized to the endoplasmic reticulum membrane, is conserved across vertebrates

Here we report a novel small protein that is highly conserved across vertebrates. The protein, which we have named TRIQK, has no homology to any previously reported proteins or functional domains, but all vertebrate homologs of this protein share a characteristic triple repeat of the sequence QXXK/R, as well as a hydrophobic C-terminal region. The Xenopus triqk gene (xTriqk) was isolated in an expression screen on the basis of its ability to cause dramatic changes in cell size and nuclear size and morphology in developing embryos. The Xenopus and mouse triqk genes are broadly expressed throughout embryogenesis, and mtriqk is also generally expressed in mouse adult tissues. TRIQK proteins are localized to the endoplasmic reticulum membrane. Depletion of endogenous xTRIQK protein in Xenopus embryos causes no detectable morphological or functional changes in tadpoles.