Normal levels of p27Xic1 are necessary for somite segmentation and determining pronephric organ size

The Xenopus laevis cyclin dependent kinase inhibitor p27^Xic1 has been shown to be involved in exit from the cell cycle and differentiation of cells into a quiescent state in the nervous system, muscle tissue, heart and retina. We show that p27^Xic1 is expressed in the developing kidney in the nephrostomal regions. Using overexpression and morpholino oligonucleotide (MO) knock-down approaches we show normal levels of p27^Xic1 regulate pronephros organ size by regulating cell cycle exit. Knock-down of p27^Xic1 expression using a MO prevented myogenesis, as previously reported; an effect that subsequently inhibits pronephrogenesis. Furthermore, we show that normal levels of p27^Xic1 are required for somite segmentation also through its cell cycle control function. Finally, we provide evidence to suggest correct paraxial mesoderm segmentation is not necessary for pronephric induction in the intermediate mesoderm. These results indicate novel developmental roles for p27^Xic1, and reveal its differentiation function is not universally utilised in all developing tissues.     

Normal levels of p27Xic1 are necessary for somite segmentation and determining pronephric organ size

The Xenopus laevis cyclin dependent kinase inhibitor p27^Xic1 has been shown to be involved in exit from the cell cycle and differentiation of cells into a quiescent state in the nervous system, muscle tissue, heart and retina. We show that p27^Xic1 is expressed in the developing kidney in the nephrostomal regions. Using over-expression and morpholino oligonucleotide (MO) knock-down approaches we show normal levels of p27^Xic1 regulate pronephros organ size by regulating cell cycle exit. Knock-down of p27^Xic1 expression using a MO prevented myogenesis, as previously reported; an effect that subsequently inhibits pronephrogenesis. Furthermore, we show that normal levels of p27^Xic1 are required for somite segmentation also through its cell cycle control function. Finally, we provide evidence to suggest correct paraxial mesoderm segmentation is not necessary for pronephric induction in the intermediate mesoderm. These results indicate novel developmental roles for p27^Xic1, and reveal its differentiation function is not universally utilised in all developing tissues.     

Regulated expression of the X. tropicalis connexin43 promoter

The spatio-temporal expression pattern of the connexin43 gene during Xenopus development has been described (Van der Heyden et al. 2001). To further investigate the regulation and function of connexin43 (Cx43) in amphibians, we have isolated the gene from Xenopus tropicalis (Xt) and determined its structure. The X. tropicalis Cx43 gene displays the typical two exon-one intron connexin configuration, where the first exon is non-coding. The predicted amino acid sequence of the XtCx43 protein is highly homologous to that of X. laevis, chicken and mammals. Expression of XtCx43 cDNA in N2A cells results in gap-junction plaque formation. Promoter activity of a 3.5 kb upstream region of the X. tropicalis Cx43 gene, including exon 1, mimics endogenous timing of expression after injection of reporter constructs in X. laevis embryos.     

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.     

Identification of Xenopus cyclin-dependent kinase inhibitors, p16Xic2 and p17Xic3

The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.     

Regulation of nuclear transport and degradation of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1

The regulation of the vertebrate cell cycle is controlled by the function of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. The Xenopus laevis kinase inhibitor, p27(Xic1) (Xic1) is a member of the p21(Cip1)/p27(Kip1)/p57(Kip2) CDK inhibitor family and inhibits CDK2-cyclin E in vitro as well as DNA replication in Xenopus egg extracts. Xic1 is targeted for degradation in interphase extracts in a manner dependent on both the ubiquitin conjugating enzyme, Cdc34, and nuclei. Here we show that ubiquitination of Xic1 occurs exclusively in the nucleus and that nuclear localization of Xic1 is necessary for its degradation. We find that Xic1 nuclear localization is independently mediated by binding to CDK2-cyclin E and by nuclear localization sequences within the C terminus of Xic1. Our results also indicate that binding of Xic1 to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation. Moreover, we show that amino acids 180-183 of Xic1 are critical determinants of Xic1 degradation. This region of Xic1 may define a motif of Xic1 essential for recognition by the ubiquitin conjugation machinery or for binding an alternate protein required for degradation.     

The cdk inhibitor p27Xic1 is required for differentiation of primary neurones in Xenopus

We have investigated the role of the cyclin-dependent kinase inhibitor, p27(Xic1), in the coordination of cell cycle exit and differentiation during early neurogenesis. We demonstrate that p27(Xic1) is highly expressed in cells destined to become primary neurones and is essential for an early stage of neurogenesis. Ablation of p27(Xic1) protein prevents differentiation of primary neurones, while overexpressing p27(Xic1) promotes their formation. p27(Xic1) may enhance neurogenesis by stabilising the bHLH protein, neurogenin. Moreover, the ability of p27(Xic1) to stabilise neurogenin and enhance neurogenesis localises to an N-terminal domain of the molecule and is separable from its ability to inhibit the cell cycle.     

One of the duplicated matrix metalloproteinase-9 genes is expressed in regressing tail during anuran metamorphosis

The drastic morphological changes of the tadpole are induced during the climax of anuran metamorphosis, when the concentration of endogenous thyroid hormone is maximal. The tadpole tail, which is twice as long as the body, shortens rapidly and disappears completely in several days. We isolated a cDNA clone, designated as Xl MMP-9TH, similar to the previously reported Xenopus laevis MMP-9 gene, and showed that their Xenopus tropicalis counterparts are located tandemly about 9 kb apart from each other in the genome. The Xenopus MMP-9TH gene was expressed in the regressing tail and gills and the remodeling intestine and central nervous system, and induced in thyroid hormone-treated tail-derived myoblastic cultured cells, while MMP-9 mRNA was detected in embryos. Three thyroid hormone response elements in the distal promoter and the first intron were involved in the upregulation of the Xl MMP-9TH gene by thyroid hormone in transient expression assays, and their relative positions are conserved between X. laevis and X. tropicalis promoters. These data strongly suggest that the MMP-9 gene was duplicated, and differentiated into two genes, one of which was specialized in a common ancestor of X. laevis and X. tropicalis to be expressed in degenerating and remodeling organs as a response to thyroid hormone during metamorphosis.     

Localization of the single copy gene Mdh2 on Xenopus tropicalis chromosomes by FISH-TSA

A single copy gene, mitochondrial malate dehydrogenase 2 (Mdh2), was localized on Xenopus tropicalis chromosomes by fluorescence in situ hybridization coupled with tyramide signal amplification (FISH-TSA). The respective cDNA was cloned and sequenced. The labeled probe hybridized with a subcentromeric region of the long arms of homologous chromosomes 3. Results of comparison of the gene localization with previously mapped X. laevis paralogs strongly suggest a common evolutionary origin of chromosomes 3 and 8 in X. laevis and chromosome 3 in X. tropicalis. This is the first time that a single copy gene has been visualized on X. tropicalis chromosomes. The FISH-TSA method gives a strong signal with a 1-kb labeled probe.     

Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries

Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.     

Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence

Voltage-sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization.