Spatially restricted patterns of expression of the homeobox-containing gene Hox 2.1. during mouse embryogenesis

The mouse Hox 2.1 gene contains a homeobox sequence and is therefore a candidate for a vertebrate gene involved in the control of embryonic patterning or positional specification. To investigate this possibility, we have used in situ hybridization to determine the pattern of Hox 2.1 expression during mouse embryogenesis. At 8.5 days post coitum, Hox 2.1 is expressed at a low level in the posterior neuroectoderm and mesoderm, and in the neuroectoderm of the presumptive hindbrain. At 12.5 days p.c., Hox 2.1 is expressed in an anteroposterior restricted domain extending from the hindbrain throughout the length of the spinal cord, predominantly in the dorsal region. Between 12.5 and 13.5 days p.c. the domain becomes localized to the occipital and cervical regions. We also detect Hox 2.1 RNA in the embryonic lung, stomach, mesonephros and metanephros, as well as in myenteric plexus, dorsal root ganglia and the nodose ganglion, and in mature granulocytes. The embryonic expression of Hox 2.1 in neural tissue is compared with that of Hox 3.1, which also shows anteroposterior restricted domains of gene expression. These patterns of expression are not clearly consistent with Hox 2.1 or Hox 3.1 having roles in segmental patterning. However, the data are consistent with these genes having regulatory roles in anteroposterior positional specification in the neuroectoderm and mesoderm, and suggest that Hox 2.1 may also have functions during organogenesis.     

Cloning and developmental expression of WSTF during Xenopus laevis embryogenesis

The gene WSTF is deleted in the autosomal dominant hereditary disorder Williams-Beuren syndrome. This disorder is caused by a 1.3 megabase deletion in human chromosome 7, encompassing at least 17 genes. The WSTF protein contains a bromodomain, found predominantly in chromatin-associated proteins. Reported association of WSTF with chromatin remodeling factors and functional data support a role for WSTF during chromatin remodeling. Here, we report the cloning and developmental expression pattern of Xenopus laevis WSTF. Xenopus laevis WSTF is a protein with a predicted amino acid sequence of 1441 amino acids. Three discrete domains can be identified in the Xenopus laevis WSTF protein, a PHD finger, a DDT domain and a bromodomain. Alignment of Xenopus WSTF with the corresponding orthologues from Homo sapiens, Gallus gallus, Mus musculus and Danio rerio demonstrates an evolutionary conservation of WSTF amino acid sequence and domain organization. In situ hybridization reveals a dynamic expression profile during embryonic development. WSTF is expressed differentially in neural tissue, especially during neurulae stages in the eye, in neural crest cells and the brain.     

Tunicamycin-inducible polypeptide synthesis during Xenopus laevis embryogenesis

Tunicamycin treatment of Xenopus laevis embryos enhanced the synthesis of a specific set of polypeptides with molecular masses of 98, 78, 59 and 58 kDa. The 78-kDa polypeptide was tentatively identified as glucose-regulated protein (GRP) 78 on the basis of molecular mass, pl (5.2), and tunicamycin inducibility, which took place upon treating embryos after the midblastula transition (MBT). The synthesis of a polypeptide with this electrophoretic mobility was detected but was not tunicamycin-inducible at stages prior to the MBT. GRP78 mRNA was detectable before the MBT but was not inducible by tunicamycin until the tailbud stage. A comparison of tunicamycin-induced polypeptide synthesis in Xenopus embryos, A6 cell line, and white blood cells by 2D-PAGE and fluorography revealed three spots in the GRP78 region of the gel. One was observed in both embryos and adult cells; another was adult-specific; and the third one was possibly an embryo-specific form. These results suggest that GRP78 synthesis might undergo a switch from an embryonic to an adult pattern during Xenopus development.     

An atlas of differential gene expression during early Xenopus embryogenesis

We have carried out a large-scale, semi-automated whole-mount in situ hybridization screen of 8369 cDNA clones in Xenopus laevis embryos. We confirm that differential gene expression is prevalent during embryogenesis since 24% of the clones are expressed non-ubiquitously and 8% are organ or cell type specific marker genes. Sequence analysis and clustering yielded 723 unique genes displaying a differential expression pattern. Of these, 18% were already described in Xenopus, 47% have homologs and 35% are lacking significant sequence similarity in databases. Many of them encode known developmental regulators. We classified 363 of the 723 genes for which a Gene Ontology annotation for molecular function could be attributed and found 'DNA binding' and 'enzyme' the most represented terms. The most common protein domains encoded in these embryonic, differentially expressed genes are the homeobox and RNA Recognition Motif (RRM). Fifty-nine putative orthologs of human disease genes, and 254 organ or cell specific marker genes were identified. Markers were found for nasal placode and archenteron roof, organs for which a specific marker was previously unavailable. Markers were also found for novel subdomains of various other organs. The tissues for which most markers were found are muscle and epidermis. Expression of cell cycle regulators fell in two classes, containing proliferation-promoting and anti-proliferative genes, respectively. We identified 66 new members of the BMP4, chromatin, endoplasmic reticulum, and karyopherin synexpression groups, thus providing a first glimpse of their probable cellular roles. Cluster analysis of tissues to measure tissue relatedness yielded some unorthodox affinities besides expectable lineage relationships. In conclusion, this study represents an atlas of gene expression patterns, which reveals embryonic regionalization, provides novel marker genes, and makes predictions about the functional role of unknown genes.     

Regulated gene expression of hyaluronan synthases during Xenopus laevis development

Here reported is the developmental gene expression pattern of the three known vertebrate hyaluronan synthases (XHas1, XHas2 and XHas3) and a comparative analysis of their mRNAs spatio-temporal distribution during Xenopus laevis development. We found that while XHas2 shows a steady-state expression from gastrula to late tailbud stage, XHas1 is mainly present in the early phases of development while XHas3 is predominantly transcribed in tailbud embryos. XHas1, XHas2 and XHas3 show distinct tissue expression patterns. In particular, XHas1 is localized in ectodermal derivatives and in cranial neural crest cells, whereas XHas2 is mainly found in mesoderm-derived structures and in trunk neural crest cells. Moreover, the expression pattern of XHas2 overlaps that of MyoD in cells committed to a muscle fate. Unlike the other hyaluronan synthases, XHas3 mRNA distribution is very restricted. In particular, XHas3 is expressed in the otic vesicles and closely follows the inner ear development. In conclusion, XHas1, XHas2 and XHas3 mRNAs have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis.     

Microtubule protein synthesis during oogenesis and early embryogenesis in Xenopus laevis.

A method is described which permits the preparation of descrete classes of oocytes of different sizes from all stages of oogenesis in Xenopus laevis. This technique is used in the determination of the content of microtubule protein in oocytes during the course of oogenesis. These experiments show that microtubule protein is present in oocytes of all sizes assayed and that the amount is simply related to the volume of the oocyte. In the largest oocytes microtubule protein constitutes 1% of the soluble protein and this amount does not change on maturation and fertilization. These results show that the changes occurring in the oocyte on maturation which allow the cytoplasm to support microtubule polymerization occur as a result of a modification of the pre-existing microtubule protein, not from protein synthesis de novo. These experiments also indicate that the synthesis of microtubule protein either form 'masked' mRNA or from newly synthesized mRNA plays an insignificant role in microtubule protein synthesis at maturation, ovulation and immediately post-fertilization.     

Role of TSC-22 during early embryogenesis in Xenopus laevis

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.     

Tetracycline-regulated gene expression switch in Xenopus laevis

Xenopus is a well-characterized model system for the investigation of biological processes at the molecular, cellular, and developmental level. The successful application of a rapid and reliable method for transgenic approaches in Xenopus has led to renewed interest in this system. We have explored the applicability of tetracycline-regulated gene expression, first described by Gossen and Bujard in 1992, to the Xenopus system. By optimizing conditions, tetracycline repressor induced expression of a luciferase reporter gene was readily and reproducibly achieved in both the Xenopus oocyte and developing embryo. This high level of expression was effectively abrogated by addition of low levels of tetracycline. The significance of this newly defined system for studies of chromatin dynamics and developmental processes is discussed.