A mutually stimulating loop involving emx2 and canonical wnt signalling specifically promotes expansion of occipital cortex and hippocampus.

The correct size of the different areas composing the mature cerebral cortex depends on the proper early allocation of cortical progenitors to their distinctive areal fates, as well as on appropriate subsequent tuning of their area-specific proliferation-differentiation profiles. Whereas much is known about the genetics of the former process, the molecular mechanisms regulating proliferation and differentiation rates within distinctive cortical proto-areas are still largely obscure. Here we show that a mutual stimulating loop, involving Emx2 and canonical Wnt signalling, specifically promotes expansion of the occipito-hippocampal anlage. Collapse of this loop occurring in Emx2-/- mutants leads progenitors within this region to slow down DNA synthesis and exit prematurely from the cell cycle, due to misregulation of cell cycle-, proneural- and lateral inhibition-molecular machineries, and eventually results in dramatic and selective size-reduction of occipital cortex and hippocampus. Reactivation of canonical Wnt signalling in the same mutants rescues a subset of molecular abnormalities and corrects differentiation rates of occipito-hippocampal progenitors.

Xotx1 maternal transcripts are vegetally localized in Xenopus laevis oocytes.

Xotx1 is a Xenopus homeobox gene related to the Drosophila gene orthodenticle (otd). We previously reported that Xotx1 transcripts are already present in unfertilized egg. Here we report that maternal Xotx1 mRNA is vegetally localized during oogenesis. In stage II oocytes Xotx1 transcripts are localized within the mitochondrial cloud, in a perinuclear position; later on, they are translocated to the vegetal cortex within the mitochondrial cloud. We also observed that in stage III oocytes the expression domain of Wnt11 is contained within the one of Xotx1 while, at stage IV, the Xotx1 expression domain is contained within the one of Vg1.

Calponin modulates the exclusion of Otx-expressing cells from convergence extension movements.

Otx2, a vertebrate homologue of the Drosophila orthodenticle gene, coordinates two processes in early embryonic development. Not only does it specify cell fate in the anterior regions of the embryo, it also prevents the cells that express it from participating in the convergence extension movements that shape the rest of the body axis. Here we show that, in Xenopus, this latter function is mediated by XclpH3, transcription of which is directly stimulated by Xotx2. XclpH3 is a Xenopus homologue of the mammalian calponin gene, the product of which binds both actin and myosin and prevents the generation of contractile force by actin filaments.

Novel interactions between vertebrate Hox genes.

Understanding why metazoan Hox/HOM-C genes are expressed in spatiotemporal sequences showing colinearity with their genomic sequence is a central challenge in developmental biology. Here, we studied the consequences of ectopically expressing Hox genes to investigate whether Hox-Hox interactions might help to order gene expression during very early vertebrate embryogenesis. Our study revealed conserved autoregulatory loops for the Hox4 and Hox7 paralogue groups, detected following ectopic expression Hoxb-4 or HOXD4, and Hoxa-7, respectively. We also detected specific induction of 5' posterior Hox genes; Hoxb-5 to Hoxb-9, following ectopic expression of Hoxb-4/HOXD4; Hoxb-8 and Hoxb-9 following ectopic expression of Hoxa-7. Additionally, we observed specific repression of 3' anterior genes, following ectopic expression of Hox4 and Hox7 paralogues. We found that induction of Hoxb-4 and Hoxb-5 by Hoxb-4 can be direct, whereas induction of Hoxb-7 is indirect, suggesting the possibility of an activating cascade. Finally, we found that activation of Hoxb-4 itself and of posterior Hox genes by Hoxb-4 can be both non-cell-autonomous, as well as direct. We believe that our findings could be important for understanding how a highly ordered Hox expression sequence is set up in the early vertebrate embryo.

[cDNA cloning of three new homeobox-containing genes of Anf class from human, chicken and newt]. / Klonirovanie kDNK trekh novykh gomeobokssoderzhashchikh genov klassa Anf iz cheloveka, kuritsy i ispanskogo tritona.

On the basis of the analysis of cDNA of three new homeobox-containing genes from human, chicken, and newt, a new class of homeobox genes Anf is characterized homologous to the Xanf-1 gene from Xenopus laevis, earlier cloned by us. These genes may be largely involved in the specification of embryonic subdivisions in the forebrain region of the embryo. The homeodomains of the proteins encoded by these genes differ greatly in the primary structure from all previously described homeobox genes. The high variability of the homeodomain sequences of the proteins of this class imply their rapid evolution.

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.

Anf: a novel class of vertebrate homeobox genes expressed at the anterior end of the main embryonic axis.

Five novel genes homologous to the homeobox-containing genes Xanf-1 and Xanf-2 of Xenopus and Hesx-1/Rpx of mouse have been identified as a result of a PCR survey of cDNA in sturgeon, zebrafish, newt, chicken and human. Comparative analysis of the homeodomain primary structure of these genes revealed that they belong to a novel class of homeobox genes, which we name Anf. All genes of this class investigated so far have similar patterns of expression during early embryogenesis, characterized by maximal transcript levels being present at the anterior extremity of the main embryonic body axis. The data obtained also suggest that, despite considerable high structural divergence between their homeodomains, all known Anf genes may be orthologues, and thus represent one of the most quickly evolving classes of vertebrate homeobox genes.

Activating and repressing signals in head development: the role of Xotx1 and Xotx2.

Xotx1 and Xotx2 are two Xenopus homologues of the Drosophila orthodenticle gene that are specifically expressed in presumptive head regions that do not undergo convergent extension movements during gastrulation. We studied the function of Xotx1 and compared it with that of Xotx2. Ectopic expression of each of the two genes has similar effects in impairing trunk and tail development. Experimental evidence suggests that posterior deficiencies observed in microinjected embryos are due to negative interference with convergent extension movements. Transplantations of putative tail-forming regions showed that, while Xotx1 overexpression inhibits tail organizer activity, Xotx2 overexpression is able to turn a tail organizer into a head organizer. Finally, Xotx1 and Xotx2 are activated by factors involved in head formation and repressed by a posteriorizing signal like retinoic acid. Taken together, these data suggest that Xotx genes are involved in head-organizing activity. They also suggest that the head organizer may act not only stimulating the formation of anterior regions, but also repressing the formation of posterior structures.

Evolution of Emx genes and brain development in vertebrates.

Emx1 and Emx2 genes are known to be involved in mammalian forebrain development. In order to investigate the evolution of the Emx gene family in vertebrates, a phylogenetic analysis was carried out on the Emx genes sequenced in man, mice, frogs, coelacanths and zebrafish. The results demonstrated the existence of two clades (Emx1 and Emx2), each grouping one of the two genes of the investigated taxa. The only exception was the zebrafish Emx1-like gene which turned out to be a sister group to both the Emx1 and Emx2 clusters. Such striking sequence divergence observed for the zebrafish Emx1-like gene could indicate that it is not orthologous to the other Emx1 genes, and therefore, in vertebrates there must be three Emx genes. Alternatively, if the zebrafish emx1 gene is orthologous to the tetrapod one, it must have undergone to strong diversifying selection.

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.

Expression patterns of Hoxb genes in the Xenopus embryo suggest roles in anteroposterior specification of the hindbrain and in dorsoventral patterning of the mesoderm.

Hox genes are thought to participate in patterning the anteroposterior (a-p) axis during vertebrate embryogenesis. In this investigation, the spatial expression of six Hoxb genes was analyzed in early embryos of Xenopus laevis by in situ hybridization. Hoxb gene expression was first detected in late gastrulae/early neurulae, by which stage, the characteristic spatially colinear Hoxb gene expression sequence was already apparent. Dissection experiments indicated that the establishment of these localized expression patterns coincides with the acquisition of anteroposterior positional information along the main body axis. The Hoxb genes continued to be expressed in similar domains along the anteroposterior axis at all developmental stages examined, although there were some changes in expression at the cellular level. Interestingly, the 3' genes, Hoxb-1, Hoxb-3, and Hoxb-4 were expressed in very restricted domains in the future hindbrain, while Hoxb-5, Hoxb-7, and Hoxb-9 transcripts were present along the entire presumptive spinal cord. It was thus notable that the 5' Hoxb genes exhibited different types of expression domain than the 3' Hoxb genes. These observations suggest that there may be different mechanisms regulating the expression of the 3' and 5' Hoxb genes. Expression of all of the Hoxb genes analyzed, except Hoxb-4, was predominantly detectable in the central nervous system or in neural crest-derived structures. Hoxb-4 mRNA was detected in the central nervous system, but interestingly, the major expression site for this gene was the somites. The other Hoxb genes tested failed to show significant expression in the somitic mesoderm, although transcripts from genes 5' from Hoxb-4 were detected in other mesodermal tissues. In the vertebrate trunk, anteroposterior patterning of the CNS is thought to be regulated by the somites. The results obtained here for Xenopus embryos did not explicitly support the idea of a Hoxb code for the somites, although we cannot rule this out. Instead, interestingly, the data were consistent with a role for Hoxb genes in dorsoventral patterning of the mesoderm.

Cloning and characterization of two members of the vertebrate Dlx gene family.

A number of vertebrate genes of the Dlx gene family have been cloned in mouse, frog, and zebrafish. These genes contain a homeobox related to that of Distalless, a gene expressed in the developing head and limbs of Drosophila embryos. We cloned and studied the expression of two members of this family, which we named Dlx5 and Dlx6, in human and mouse. The two human genes, DLX5 and DLX6, are closely linked in an inverted convergent configuration in a region of chromosome 7, at 7q22. Similarly, the two human genes DLX1 and DLX2 are closely linked in a convergent configuration at 2q32, near the HOXD (previously HOX4) locus. In situ hybridization experiments in mouse embryos revealed expression of Dlx5 and Dlx6 mRNA in restricted regions of ventral diencephalon and basal telencephalon, with a distribution very similar to that reported for Dlx1 and Dlx2 mRNA. A surprising feature of Dlx5 and Dlx6 is that they are also expressed in all skeletal structures of midgestation embryos after the first cartilage formation. The expression pattern of these genes, together with their chromosome localization, may provide useful cues for the study of congenital disorders in which there is a combination of craniofacial and limb defects.

Conserved homeobox genes in the developing brain.

We analysed the expression of four mouse homeobox genes related to two Drosophila genes expressed in the developing head of the fly: Emx1 and Emx2, related to empty spiracles and Otx1 and Otx2, related to orthodenticle. The four genes are all expressed in the developing rostral brain of E10 mouse embryos in a specific manner. Otx2 is expressed in every dorsal and most ventral regions of telencephalon, diencephalon and mesencephalon. The Otx1 expression domain is similar to that of Otx2, but contained within it. The Emx2 expression domain is comprised of dorsal telencephalon and small diencephalic regions, both dorsally and ventrally. Finally, Emx1 expression is exclusively confined to the dorsal telencephalon. Thus, at this stage the expression domains of the four genes appear to be continuous regions contained within each other in the sequence Emx1 < Emx2 < Otx1 < Otx2. The first appearance of transcripts of these genes is also sequential: Otx2 is expressed first (E5.5), followed by Otx1 and Emx2 (E8-8.5) and finally by Emx1 (E9.5). These findings suggest a role of the four genes in establishing cell fates within the limits of the various embryonic brain regions in a discrete progressive process with its center in the dorsal telencephalon. The structural organization and the regulation of these genes appear remarkably conserved in evolution.

Colinearity in the Xenopus laevis Hox-2 complex.

Here we describe experiments detailing the developmental expression, and the inducibility by all-trans retinoic acid (RA) of six members of the Xenopus Hox-2 complex of homeobox-containing genes. We first report the cloning and characterisation of two novel Xenopus Hox-2 genes (Xhox2.7 and Xhox2.9), and provide evidence that the six genes studied are indeed closely linked in the same chromosomal complex. We next show that all six genes are expressed in a spatial sequence which is colinear with their putative 3' to 5' chromosomal sequence and that five of them are also expressed in a 3' to 5' colinear temporal sequence. The sixth gene (Xhox2.9) has an exceptional spatial and temporal expression pattern. The six genes all respond to RA by showing altered spatiotemporal expression patterns, and are also hyperinduced by RA, with a sequence of magnitudes which is colinear with their 3' to 5' chromosomal sequence and with their spatial and temporal expression sequences. Our data also suggest a pre-existing anteroposterior polarity in the embryo's competence to respond to RA. These results complement and extend previous findings made using murine and avian embryos and mammalian cell lines. They suggest a mechanism whereby an endogenous retinoid could help to provide positional information in the early embryo.

Xenopus Hox-2 genes are expressed sequentially after the onset of gastrulation and are differentially inducible by retinoic acid.

In this paper, we review experiments to characterise the developmental expression and the responses to all-trans retinoic acid (RA) of six members of the Hox-2 complex of homeobox-containing genes, during the early development of Xenopus laevis. We showed that the six genes are expressed in a spatial sequence which is colinear with their putative 3' to 5' chromosomal sequence and that five of them are also expressed rapidly after the beginning of gastrulation, in a 3' to 5' colinear temporal sequence. The sixth gene (Xhox2.9) has an exceptional spatial and temporal expression pattern. The six genes all respond to RA by showing altered spatiotemporal expression patterns, and are also RA-inducible, the sequence of the magnitudes of their RA responses being colinear with their 3' to 5' chromosomal sequence, and with their spatial and temporal expression sequences. Our data also reveal that there is a pre-existing anteroposterior polarity in the embryo's competence for a response to RA. These results complement and extend previous findings made using murine and avian embryos and mammalian cell lines. They suggest that an endogenous retinoid could contribute to positional information in the early Xenopus embryo.

Expression of HOX homeogenes in human neuroblastoma cell culture lines.

Mammalian genes containing a class-I homeobox (HOX genes) are highly expressed in the embryonic nervous system. As a first step towards the molecular analysis of the role these genes play in neural cells, we studied the expression of four human HOX genes in five neuroblastoma (NB) cell lines - SK-N-BE, CHP-134, IMR-32, SK-N-SH and LAN-1 - during the process of differentiation induced by treatment with retinoic acid (RA). The four genes, HOX1D, 2F, 3E and 4B, located at corresponding positions in the four HOX loci, share a high degree of sequence similarity with the Drosophila Deformed homeotic gene and constitute a homology group, group 10. One of these genes, HOX1D, is not expressed in the cells used, whereas the other three are highly expressed in untreated and RA-induced NB cells, even though the expression pattern in the various lines is slightly different for the three genes. Our analysis reveals a complex and specific expression pattern in these lines, paving the way to an identification of different NB-cell populations by means of specific HOX gene expression schemes. On the other hand, in every line studied, morphological maturation toward a neuronal differentiated phenotype appears to be associated with increased HOX gene expression.

Human HOX genes are differentially activated by retinoic acid in embryonal carcinoma cells according to their position within the four loci.

We studied the expression of 33 human homeobox genes belonging to four complex HOX loci in embryonal carcinoma NT2/D1 cells. These cells can be induced to differentiate by culturing them in media containing retinoic acid. Northern blot analysis reveals that no expression of these genes was detectable in NT2/D1 stem cells, whereas 22 HOX genes are well expressed in NT2/D1 cells treated with 10 microM retinoic acid for 14 days. The 11 HOX genes the expression of which remained undetectable in NT2/D1 cells after this treatment are located at the 5' end of their loci: four in HOX1, five in HOX3 and two in HOX4. The boundary between induced and silent genes roughly corresponds to the HOX genes constituting the homology group 5, related to the Abdominal-B homeotic gene of Drosophila. All nine identified HOX2 genes are well expressed in fully induced NT2/D1 cells and none of them maps 5' genes of this homology group. We conclude that HOX genes are differentially activated by retinoic acid in these cells according to their physical location within the four chromosomal loci.

The three most downstream genes of the Hox-3 cluster are expressed in human extraembryonic tissues including trophoblast of androgenetic origin.

Human first trimester extraembryonic tissues of normal and androgenetic origin (molar pregnancies) were investigated for the expression of 6 homeobox genes from the chromosome 12-encoded Hox-3 cluster by non-autoradiographic in situ hybridization with biotinylated RNA probes. By comparative in situ hybridization involving the use of exon- or region-specific RNA probes, analysis included the cellular distribution of alternative Hox-3 transcripts in chorionic villous tissues. A bias in extraembryonic distribution was seen between transcripts of the three most upstream Hox-3 genes (Hox-3.7, -3.6, and -3.1) versus transcripts of the 3 most downstream genes (Hox-3.3, 3.4, and 3.5). Only genes from the latter group are transcribed in human extraembryonic tissues including extraembryonic tissues of androgenetic origin. Moreover, comparative in situ hybridization showed that distinct alternative transcripts of Hox-3.3, Hox 3.4 and Hox-3.5 are exclusively found in trophoblast cells while others are present in chorionic villous stromal cells as well. These data demonstrate the existence of tissue- and cell-specific use of transcriptional (alternative gene promoters) or post-transcriptional (alternative splicing) regulation of homeobox genes in extraembryonic tissues.

The human HOX gene family.

We report the identification of 10 new human homeobox sequences. Altogether, we have isolated and sequenced 30 human homeoboxes clustered in 4 chromosomal regions called HOX loci. HOX1 includes 8 homeoboxes in 90 kb of DNA on chromosome 7. HOX2 includes 9 homeoboxes in 180 kb on chromosome 17. HOX3 contains at least 7 homeoboxes in 160 kb on chromosome 12. Finally, HOX4 includes 6 homeoboxes in 70 kb on chromosome 2. Homeodomains obtained from the conceptual translation of the isolated homeoboxes can be attributed to 13 homology groups on the basis of their primary peptide sequence. Moreover, it is possible to align the 4 HOX loci so that corresponding homeodomains in all loci share the maximal sequence identity. The complex of these observations supports and extends an evolutionary hypothesis concerning the origin of mammalian and fly homeobox gene complexes. We also determined the coding region present in 3 HOX2 cDNA clones corresponding to HOX2G, HOX2H and HOX2I.