The lmx1b gene is pivotal in glomus development in Xenopus laevis.

We have previously shown that lmx1b, a LIM homeodomain protein, is expressed in the pronephric glomus. We now show temporal and spatial expression patterns of lmx1b and its potential binding partners in both dissected pronephric anlagen and in individual dissected components of stage 42 pronephroi. Morpholino oligonucleotide knock-down of lmx1b establishes a role for lmx1b in the development of the pronephric components. Depletion of lmx1b results in the formation of a glomus with reduced size. Pronephric tubules were also shown to be reduced in structure and/or coiling whereas more distal tubule structure was unaffected. Over-expression of lmx1b mRNA resulted in no significant phenotype. Given that lmx1b protein is known to function as a heterodimer, we have over-expressed lmx1b mRNA alone or in combination with potential interacting molecules and analysed the effects on kidney structures. Phenotypes observed by over-expression of lim1 and ldb1 are partially rescued by co-injection with lmx1b mRNA. Animal cap experiments confirm that co-injection of lmx1b with potential binding partners can up-regulate pronephric molecular markers suggesting that lmx1b lies upstream of wt1 in the gene network controlling glomus differentiation. This places lmx1b in a genetic hierarchy involved in pronephros development and suggests that it is the balance in levels of binding partners together with restricted expression domains of lmx1b and lim1 which influences differentiation into glomus or tubule derivatives in vivo.

GDNF expression during Xenopus development.

Glial cell line-derived neurotrophic factor (GDNF) has multiple roles in kidney morphogenesis, spermatogenesis, and neurogenesis during development. In this study, we report the cloning and expression pattern of Xenopus laevis GDNF. The X. laevis GDNF cDNA sequence has a complete open reading frame of 684 bases, predicting 227 amino acid residues at the protein level. Comparison of the X. laevis GDNF amino acid sequence with those of chick, human, mouse, rat and zebrafish indicates that X. laevis GDNF has 60%-52% and 75%-62% identity over the whole amino acid sequence and for the putative mature forms, respectively. All known functional motifs of GDNF were conserved in the X. laevis sequence. Temporal expression analysis by RT-PCR indicated that GDNF transcripts were first detectable at stage 12 at a low level, and gradually increased up to stage 22. From stage 24, the expression sharply increased and continued at a similar level as development progressed. Spatial expression analysis by whole-mount in situ hybridization showed that the GDNF mRNA was predominantly detected in somites, pronephros, pharyngeal arches, epibranchial placodes, digestive tract and some of the lateral line structure. These results suggest that this X. laevis gene is the orthologue for GDNF.

Characterization of a gene respondent to clinorotation in Xenopus A6 cells.

The A6 epithelial cell line, derived from the kidney of Xenopus laevis, spontaneously form domes after it has reached confluence. In a previous study, we demonstrated that formation of domes is strongly inhibited in the cells cultured using a three-dimensional clinostat. In this study, we performed staining of filamentous actin and examination using electron microscopy to investigate morphological changes of A6 cells exposured to clinorotation for 10 days. Micrographs show that A6 cells in clinorotation lose cortical actin that is characteristic of epithelial cells. Therefore, we search for genes differentially expressed in A6 cells cultured in clinorotation and identified Xenopus laevis N-myc downstream-regulated gene-1 (xNDRG1) as a clinorotation respondent gene in A6 cells. In northern blots analysis, xNDRG1 mRNA significantly increased only in A6 cells cultured in clinorotation for 10 days, and maintained at a similar level in the cells cultured for 15 days. Centrifugations of A6 cells have no effect on expression of xNDRG1. We also aimed to characterize xNDRG1 during Xenopus laevis development by examining the temporal and spatial expression patterns of xNDRG1 transcripts in embryos. Our results suggest that xNDRG1 is necessary for pronephros development in Xenopus laevis.

The lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) receptor gene families: cloning and comparative expression analysis in Xenopus laevis.

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are endogenous bioactive lipids which mediate a variety of biological cell responses such as cell proliferation, migration, differentiation and apoptosis. Their actions are mediated by binding to the G-protein-coupled endothelial differentiation gene (Edg) receptor subfamily, referred to as S1P1-5 and LPA1-5, and regulate a variety of signalling pathways involved in numerous physiological processes and pathological conditions. Their importance during embryogenesis has been demonstrated by the generation of knock-out mice and specific roles have been assigned to these receptors. However, potential functional redundancy and the lethality of some mutants have complicated functional analysis in these models. Here we report the cloning of the S1P and LPA receptors in Xenopus laevis and tropicalis. Phylogenetic analyses demonstrate the high level of conservation of these receptors between amphibian and other vertebrate species. We have conducted a comparative expression analysis of these receptors during development and in the adult frog, by both RT-PCR and whole mount in situ hybridisation. In particular, we show that S1P1, 2 and 5 display distinct embryonic specific expression patterns, suggesting potentially different developmental roles for these receptors, and therefore for their ligands, during amphibian embryogenesis.

A functional screen for genes involved in Xenopus pronephros development.

In Xenopus, the pronephros is the functional larval kidney and consists of two identifiable components; the glomus, the pronephric tubules, which can be divided into four separate segments, based on marker gene expression. The simplicity of this organ, coupled with the fact that it displays the same basic organization and function as more complex mesonephros and metanephros, makes this an attractive model to study vertebrate kidney formation. In this study, we have performed a functional screen specifically to identify genes involved in pronephros development in Xenopus. Gain-of-function screens are performed by injecting mRNA pools made from a non-redundant X. tropicalis full-length plasmid cDNA library into X. laevis eggs, followed by sib-selection to identify the single clone that caused abnormal phenotypes in the pronephros. Out of 768 egg and gastrula stage cDNA clones, 31 genes, approximately 4% of the screened clones, affected pronephric marker expression examined by whole mount in situ hybridization or antibody staining. Most of the positive clones had clear expression patterns in pronephros and predicted/established functions highly likely to be involved in developmental processes. In order to carry out a more detailed study, we selected Sox7, Cpeb3, P53csv, Mecr and Dnajc15, which had highly specific expression patterns in the pronephric region. The over-expression of these five selected clones indicated that they caused pronephric abnormalities with different temporal and spatial effects. These results suggest that our strategy to identify novel genes involved in pronephros development was highly successful, and that this strategy is effective for the identification of novel genes involved in late developmental events.

Identification and characterization of Xenopus NDRG1.

NDRG1 is a member of the N-myc downstream-regulated gene (NDRG) family and is involved in cellular differentiation, activation of p53, cell cycle arrest, metastasis, and hypoxia. Expression of NDRG1 is repressed by the proto-oncogene, N-myc during mouse development, although the exact functional role of NDRG1 in development remains unknown. Here, we report the characterization of Xenopus laevis NDRG1 (xNDRG1) during X. laevis development. Expression of xNDRG1 transcript was first detected at stage 15, and was localized to the presumptive pronephric anlagen at stage 26 and to pronephros, eye, branchial arches, and tail-bud at stage 32. Overexpression of xNDRG1 results in a reduced pronephros and disorganized somites. Depletion of xNDRG1, using morpholinos, causes failure of pronephros development. These results suggest that xNDRG1 is required for pronephros development in X. laevis.

Role of the thrombopoietin (TPO)/Mpl system: c-Mpl-like molecule/TPO signaling enhances early hematopoiesis in Xenopus laevis.

Multiple organs are induced in the primitive embryonic ectoderm excised from blastula stage Xenopus laevis embryos, under the strict control of mesoderm inducing factors. This in vitro system is useful for exploring the mechanisms of development. In this study, the function of thrombopoietin (TPO)/c-Mpl signaling in the development of hematopoietic cells was investigated. An optimal hematopoietic cell induction system was established to evaluate the influence of growth factors on hematopoiesis. It was found that exogenous TPO enhanced hematopoiesis in explants induced by activin and bone morphogenetic protein (BMP)-4 and increased the number of both erythrocytes and leukocytes in a dose-dependent manner. Addition of anti-c-Mpl antibody completely inhibited the expansion of hematopoietic cells stimulated by TPO, and the antibody specifically recognized blood-like cells. These results demonstrate that TPO acts on hematopoietic progenitors induced in explants and the c-Mpl-like molecule in Xenopus mediates the cellular function of TPO. We also found that forced expression of TPO in embryos promoted hematopoiesis in the ventral blood island and the dorsal-- lateral plate mesoderm. These results suggest that hematopoietic stem and progenitor cells are regulated by TPO/c-Mpl signaling from when they appear in their ontogeny. They also suggest that TPO/c-Mpl signaling play a crucial role in the formation of hematopoietic cells in Xenopus.