Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis.

We describe the cloning, expression pattern and functional overexpression analysis of Xotx5b, a new member of the Otx gene family in Xenopus laevis. Early expression of Xotx5b resembles that of Xotx2, being detected in the organizer region at early gastrula stage, and, shortly after, also in anterior neuroectoderm. During neurula stages Xotx5b exhibits a changing and dynamic pattern of expression. After neural tube closure, Xotx5b is expressed in the eye and pineal gland, both involved in photoreception. Overexpression of Xotx5b has a similar effect to that of Xotx2, producing posterior truncations and inducing ectopic cement gland and neural tissue in whole embryos. In animal cap assays, Xotx5b and Xotx2 are both able to activate XAG, to strongly suppress the expression of the epidermal marker XK81, and to reciprocally activate each other. Finally, in einsteck transplantation assays, Xotx5b is able to respecify a tail/trunk organizer to a head organizer.

HNF1(beta) is required for mesoderm induction in the Xenopus embryo.

XHNF1(&bgr;) is a homeobox-containing gene initially expressed at the blastula stage in the vegetal part of the Xenopus embryo. We investigated its early role by functional ablation, through mRNA injection of an XHNF1(beta)/engrailed repressor fusion construct (XHNF1(beta)/EngR). Dorsal injections of XHNF1(beta)/EngR mRNA abolish dorsal mesoderm formation, leading to axial deficiencies; ventral injections disrupt ventral mesoderm formation without affecting axial development. XHNF1(beta)/EngR phenotypic effects specifically depend on the DNA-binding activity of its homeodomain and are fully rescued by coinjection of XHNF1(beta) mRNA. Vegetal injection of XHNF1(beta)/EngR mRNA blocks the mesoderm-inducing ability of vegetal explants. Both B-Vg1 and VegT maternal determinants trigger XHNF1(beta) expression in animal caps. XHNF1(beta)/EngR mRNA blocks B-Vg1-mediated, but not by eFGF-mediated, mesoderm induction in animals caps. However, wild-type XHNF1(beta) mRNA does not trigger Xbra expression in animal caps. We conclude that XHNF1(beta) function is essential, though not sufficient, for mesoderm induction in the Xenopus embryo.

The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals.

We have isolated and studied the expression pattern of Xemx1 and Xemx2 genes in Xenopus laevis. Xemx genes are the homologues of mouse Emx genes, related to Drosophila empty spiracles. They are expressed in selected regions of the developing brain, particularly in the telencephalon, and, outside the brain, in the otic vesicles, olfactory placodes, visceral arches and the developing excretory system. We also report on experiments concerning the tissue and molecular signals responsible for their activation in competent ectoderm. Xemx genes are activated in ectoderm conjugated with head organizer tissue, but not with tail organizer tissue. Furthermore, they are not activated in animal cap either by noggin or by Xnr3, thus suggesting that a different inducer or the integration of several signals may be responsible for their activation.

Unusual features of the urodele genome: do they have a role in evolution and development?

Urodeles are amongst the organisms with the highest C-values. They provide a useful system for studies of genome organization at both the chromosomal and the molecular level. In this contribution we discuss the general features of "excess" DNA in urodeles and emphasize that the urodele genome is in a state of plasticity. That fluidity is due to various molecular mechanisms which are involved in its continuous turnover. The implications of this fluid, "excess DNA" for evolution and/or development are considered.

Xotx genes in the developing brain of Xenopus laevis.

The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. We previously reported on the expression of Xotx2 gene, homologous to the murine Otx2 gene, during early Xenopus development. In the present paper we report an extensive analysis of the expression pattern of Xotx2 during later stages of development and also the cloning and developmental expression of two additional Otx Xenopus genes, Xotx1 and Xotx4. These latter two genes bear a good degree of homology to murine Otx1, higher for Xotx1 than for Xotx4. Both these genes are expressed in the forebrain and midbrain regions and their developmental patterns of expression are very similar, although not perfectly superimposable. Spatial and temporal expression patterns of the three Xotx genes suggest that they may be involved in the early subdivision of the rostral brain, providing antero-posterior positional information within the most anterior districts of the neuraxis. The three Xotx genes are expressed in all the developing sense organs of the head, eyes, olfactory system and otic vesicles. By in situ hybridization the earliest detectable expression is found in anterior mesendoderm for Xotx2, and in presumptive anterior neuroectoderm for Xotx1 and Xotx4. In addition, we examined whether Xotx1 is expressed in exogastrulae, finding that Xotx1 expression can be activated in the apparent absence of vertical signals of neural induction.

The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions.

In this paper we study Xotx2, a Xenopus homeobox gene related to orthodenticle, a gene expressed in the developing head of Drosophila. The murine cognate, Otx2, is first expressed in the entire epiblast of prestreak embryos and later in very anterior regions of late-gastrulae, including the neuroectoderm of presumptive fore- and mid-brain. In Xenopus, RNase protection experiments reveal that Xotx2 is expressed at low levels throughout early development from unfertilized egg to late blastula, when its expression level significantly increases. Whole-mount in situ hybridization shows a localized expression in the dorsal region of the marginal zone at stage 9.5. At stage 10.25 Xotx2 is expressed in dorsal bottle cells and in cells of the dorsal deep zone fated to give rise to prechordal mesendoderm, suggesting a role in the specification of very anterior structures. In stage 10.5 gastrulae, Xotx2 transcripts start to be detectable also in presumptive anterior neuroectoderm, where they persist in subsequent stages. Various treatments of early embryos cause a general reorganization of Xotx2 expression. In particular, retinoic acid treatment essentially abolishes Xotx2 expression in neuroectoderm. Microinjection of Xotx2 mRNA in 1-, 2- and 4-cell stage embryos causes the appearance of secondary cement glands and partial secondary axes in embryos with reduced trunk and tail structures. The presence of the Xotx2 homeodomain is required to produce these effects. In particular, this homeodomain contains a specific lysine residue at position 9 of the recognition helix. Microinjected transcripts of Xotx2 constructs containing a homeodomain where this lysine is substituted by a glutamine or a glutamic acid residue fail to cause these effects.

Cloning and developmental expression of LFB3/HNF1 beta transcription factor in Xenopus laevis.

We have cloned the Xenopus laevis homologue of the LFB3/HNF1 beta transcription factor. RNase protection and in situ hybridisation experiments show that XLFB3 transcription starts in the gastrulating endoderm at stage 10.5 (mid-gastrula). At later stages, XLFB3 transcripts within the endoderm are restricted to mid- and hindgut and to their derivative organs and tissues. XLFB3 is also expressed in the neuroectoderm and in the pronephros anlage. XLFB3 is not expressed in the rostral part of all three germ layers, with coincident anterior borders that are shifted anteriorly by treatment of developing embryos with retinoic acid. XLFB3 is a useful marker of early endoderm differentiation and its expression pattern along the antero-posterior axis, as well as the response to retinoic acid treatment, suggests a role in early morphogenesis.

Genetic control of development in Xenopus laevis.

In this paper we address the question of how genes can control development by using Xenopus as a model system, since it combines the classical advantages of amphibian embryology with advanced molecular techniques. Several developmental regulator genes have been shown to encode for transcription factors which trigger the activation of downstream genes, thus resulting in a cascade of regulatory events. In the first two examples, we deal with regulatory events that underlie early body patterning in vertebrates, and with the role of homeobox transcription factors in deciphering positional information along the body axis. In the third example, we address the question of the role of post-transcriptional regulation in development by studying the possible regulatory role of a cytoplasmic zinc finger protein, presumably acting through RNA-protein interactions. The general idea is that understanding how genes can control development will hopefully lead to understanding the construction of a shape, and eventually of an organism.

Two dispersed highly repeated DNA families of Triturus vulgaris meridionalis (Amphibia, Urodela) are widely conserved among Salamandridae.

Two BamHI families of repeated sequences were characterized from the genome of the Italian smooth newt, Triturus vulgaris meridionalis (Amphibia, Urodela). The first family, which is divided into subfamilies, consists of tandemly arranged arrays whose basic repeat is around 398 bp long; these arrays are dispersed throughout the entire chromosome sets of the various species of Triturus tested. Moreover the family is widely conserved among Salamandridae, being detected by genomic DNA blotting of Notophthalmus viridescens, Taricha granulosa, Salamandrina terdigitata and Euproctus platycephalus. The second BamHI family is represented by a cloned sequence of 419 bp, which is dispersed in the chromosome set of several species of Triturus. The sequence is also conserved in S. terdigitata and in E. platycephalus but is not detectable in N. viridescens or T. granulosa. The cloned sequence is most probably only part of a longer unit interspersed within the Triturus genome.

An autoradiographic analysis of vitellogenin synthesis and secretion in the fat body of the stick insect Bacillus rossius.

The fat body of the stick insect Bacillus rossius was studied with a view to clarifying the metabolic pathway leading to secretion of vitellogenin (VG). Electrophoretic analysis of ovarian follicles and hemolymph from egg-laying females showed that the two tissues shared a common polypeptide composition consisting of five major polypeptides with molecular weights ranging from 60-180 Kd. Following in vivo exposure to [(35)S]-methionine for up to 24 h, these polypeptides were labeled in a stage- and time-dependent manner, suggesting that they were transferred from the hemolymph to the oocyte during vitellogenesis. Fat body pulse-labeled with [(35)S]-methionine for up to 240 min and immunoprecipitated with an anti-yolk serum was labeled only in a fraction containing high molecular weight polypeptides. We presume these polypeptides to be VG precursors bearing a precursor-product relationship with the five major polypeptides of the hemolymph and developing ovarian follicles. Fat body exposed in vivo to [(3)H]-leucine for time intervals ranging from 20-240 min were processed for EM autoradiography. The results of this analysis showed that incorporated radioactivity was progressively transferred from the endoplasmic reticulum to the Golgi apparatus and from there to the composite granules. The data provided in this study are consonant with previous findings by which composite granules were shown to contain two compartments differing both in content and origin. In addition, the autoradiographical data of in vivo labeled fat body demonstrate that only the material partitioned into the electron-dense compartment of these granules is exocytosed.

Structure and expression of a Xenopus gene encoding an snRNP protein (U1 70K).

A cDNA and two genes for the Xenopus laevis U snRNP 70K protein have been cloned and partially sequenced. The cDNA encodes a protein whose predicted mol. wt is 57 kd but which migrates as a 70 kd protein in SDS-PAGE when translated in vitro from a cDNA transcript. The predicted protein sequences of the human and Xenopus U1 70K are shown to be very similar. Analysis of several genomic clones suggests that there are at least two, and possibly more, different genes coding for the 70K protein in the Xenopus genome. The two genes analysed in detail cover approximately 16 kb and are divided into 10 exons of which the last exon covers more than half of the protein coding sequence. During Xenopus development several different stage-specific RNAs hybridizing to the U1 70K cDNA are detected. The promoter region of one of the cloned genes is demonstrated to be functionally active, and to show apparent differences from other pol II promoters.

Heterochromatic DNA in Triturus (Amphibia, Urodela) II. A centromeric satellite DNA.

The MspI family of highly repeated sequences is a centromeric satellite DNA representing about 1% of the genome of the Italian smooth newt, Triturus vulgaris meridionalis. We have studied the structure, genomic organization, chromosomal localization and conservation across species of this family. MspI sequences are around 197 bp long, as shown by sequencing of three cloned units. The family is organized in large clusters of tandemly arrayed units, present at almost all the centromeres of T.v. meridionalis, and is well conserved in the T.v. vulgaris subspecies. Conserved MspI sequences are also present in the related species T. helveticus, where they appear to be clustered at the centromeres of only a few chromosomes. MspI sequences are not found in other Triturus species analysed. The correlation of these sequences with the overall distribution pattern of heterochromatin and the extent of their conservation within the genus Triturus, are discussed.

Evolution of highly repeated DNA within the genusTriturus (Amphibia, Urodela).

Highly repeated DNA is a main feature of urodele amphibian genomes. InTriturus this class of DNA consists of several sequence families differently arranged at both the molecular and the chromosomal level, showing varying degrees of conservation across species. Present data on highly repeated DNA inTriturus are here summarized and discussed with regard to the evolution and possible functional role of these sequences.