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.

Homeobox genes and disease.

To date, not many disorders have been associated with homeobox genes, especially with those belonging to the HOX family. This is particularly surprising, considering the body of evidence accumulated for a role of these genes in the control of mammalian development. Recently, this situation has changed and some congenital or somatic defects have been demonstrated to involve mutations in homeobox genes of the HOX, EMX, PAX, and MSX families, as well as in other novel genes containing either a paired- or bicoid-type homeobox.

Homeobox genes in vertebrate gastrulation.

The formation and anteroposterior patterning of the three definitive germ layers, ectoderm, or epiblast, is the common theme of vertebrate gastrulation. What changes from system to system is the geometry of these events and the nature of the non-epiblast transient structures implicated. A number of molecular markers, including a few homeobox genes and in particular goosecoid and Otx2, are now available that will hopefully allow us to explore the underlying molecular mechanisms and to establish biologically relevant homologies between the various systems.

Area identity shifts in the early cerebral cortex of Emx2-/- mutant mice.

The specification of area identities in the cerebral cortex is a complex process, primed by intrinsic cortical cues and refined after the arrival of afferent fibers from the thalamus. Little is known about the genetic control of the early steps of this process, but the distinctive expression pattern of the homeogene Emx2 in the developing cortex has prompted suggestions that it is critical in this context. We tested this hypothesis using Emx2 -/- mice. We found that the normal spectrum of cortical areal identities was encoded in these mutants, but areas with caudal-medial identities were reduced and those with anterior-lateral identities were relatively expanded in the cortex.

From fly head to mammalian forebrain: the story of otd and Otx.

Many of the genes responsible for axial patterning have been conserved throughout evolution. Recent studies of invertebrates and vertebrates are extending our understanding of this molecular conservation into the anterior region of the animal embryo, including the developing brain. These studies suggest that this domain is specified according to a genetic paradigm that is different from that governing trunk development, and are also beginning to provide insights into the structures that underlie the rostral brain.

Downstream of Otx2, or how to get a head.

Otx2 is a member of a highly conserved family of homeodomain-containing transcription factors that function in early brain development. Recent studies have identified a significant number of target genes downstream of Otx2, allowing us to address the question of how it fulfils its diverse developmental roles. Interestingly, many of these target genes are not transcription factors or signaling molecules, and they probably have no direct affect on gene expression. Furthermore, there is evidence that Otx2 coordinates the activity of unrelated genes that have overlapping functions, and that it does so directly without intermediate transcriptional or signaling activity.

Were protein internal repeats formed by "bricolage"?

Is evolution an engineer, or is it a tinkerer--a "bricoleur"--building up complex molecules in organisms by increasing and adapting the materials at hand? An analysis of completely sequenced genomes suggests the latter, showing that increasing repetition of modules within the proteins encoded by these genomes is correlated with increasing complexity of the organism.

HOX gene activation by retinoic acid.

Vertebrate homeobox genes of the Hox family are, like Drosophila homeotic genes, organized in gene clusters and show a strict correspondence, or collinearity, between the order of the genes (3' to 5') within the chromosomal cluster and that of their expression domains (anterior to posterior) in the embryo. Recent data obtained from embryonal carcinoma cells induced to differentiate by retinoic acid cast some light on the molecular mechanisms underlying the collinear expression of the Hox genes.

Alteration of homeobox gene expression by N-ras transformation of PA-1 human teratocarcinoma cells.

We used a series of cell clones from a human teratocarcinoma cell line, PA-1, to study the effect of transformation by an activated N-ras oncogene on the expression of genes involved in retinoic acid (RA)-induced differentiation and growth regulation. Recently, it has been shown that expression of human HOX 2 genes is sequentially activated by RA beginning from Hox 2.9 at the 3' end of the HOX 2 cluster (A. Simeone, D. Acampora, L. Arcioni, P. W. Andrews, E. Boncinelli, and F. Mavilio, Nature [London] 346:763-766, 1990). We now report that six different genes of the cluster HOX 1 are sequentially induced by RA in a similar temporal pattern, beginning with genes at the 3' end of the cluster. However, in N-ras-transformed cell clones, RA-induced expression of these homeobox genes is delayed. Hox 1.4 and Hox 1.3, genes abundantly induced in nontransformed clones after 3 days of RA treatment, are expressed in N-ras-transformed cells only after 10 days of RA treatment. At this time, the cells' growth is arrested at very high density, and no differentiated morphologic characteristics are observed. Constitutive expression of a transfected Hox 1.4 gene under the control of a simian virus 40 promotor leads to differentiated cell morphology similar to that of the RA-induced phenotype and restores the growth-inhibitory effects of RA in N-ras-transformed cells. These observations provide evidence that enhanced proliferation in N-ras-transformed cells compromises teratocarcinoma cell differentiation by a mechanism that transiently suppresses homeobox gene induction and implies a central role for homeobox genes in RA-induced cell differentiation. We conclude that stimulation of a putative growth factor signal pathway, associated with ras-induced proliferation, transiently suppresses the induction of transcription factors functionally involved in cell growth and differentiation.

Emx homeogenes and mouse brain development.

Mammalian homeogenes of the Emx family, Emx1 and Emx2, are expressed in the developing cerebral cortex and are involved in the patterning of the rostral brain. Although very little is known about the role of Emx1, details of the function of EMX2 are emerging from the observation of cortical phenotypes in normal and mutant mice. Emx2 is expressed in proliferating neuroblasts and in the so-called postmitotic Cajal-Retzius cells, known to control migration of cortical neurons. The graded distribution of EMX2 homeoprotein suggests a potential role for Emx2 in the subdivision of the cortex into territories and possibly areas.

Early CNS development: distal-less related genes and forebrain development.

The ever increasing number of molecular markers available for the study of developing structures is revolutionizing developmental neurobiology. It is now possible to identify entirely new groups of cells and trace their lineages among all other cell populations. Recent studies on the expression domains of a variety of genes specifically expressed in the developing CNS are beginning to give us a better understanding of the early regionalization of the CNS and, in particular, of the forebrain.

The relationship of modulation of major histocompatibility complex class I antigens to retrovirus transformation in rat cell lines.

The expression of major histocompatibility complex (MHC) Class I antigens has been studied, by means of monoclonal antibodies directed against nonpolymorphic determinants of MHC Class I molecules, in two epithelial differentiated cell lines (FRTL-5 clone 2 and PC clone 3) and in one fibroblast cell line (FRT Fibro) of Fischer rat thyroid origin, before and after infection with various acute retroviruses carrying the v-ras-Ha, v-mos, v-src, polyoma middle T, and c-myc oncogenes. The results obtained indicate that a single virus does not produce identical changes in MHC Class I molecule expression in all tested lines, but a general increase occurs in lines derived from FRTL-5 clone 2 and a decrease occurs in lines derived from PC clone 3 and from FRT Fibro. Thus the modulation of expression seems to proceed always in the same direction in each cell line regardless of the infecting retrovirus and appears to involve posttranscriptional mechanisms, since no modification of expression of mRNA levels has been observed between normal and transformed cells. Only one line of PC clone 3 origin, transformed by the cooperation of two oncogenes (human c-myc and middle T), almost completely lost MHC Class I antigens on the cell surface and presented a significantly reduced synthesis of Class I mRNA.

The lack of Emx2 causes impairment of Reelin signaling and defects of neuronal migration in the developing cerebral cortex.

Neocorticogenesis in mice homozygous for an Emx2 null allele is the topic of this article. The development of both main components of neocortex, primordial plexiform layer derivatives and cortical plate, was analyzed, paying special attention to radial migration of neurons forming the cortical plate. The products of the Reelin gene, normally playing a key role in orchestrating radial migration of these neurons, display normal distribution at the beginning of the cortical neuronogenesis but are absent in the neocortical marginal zone of the mutant mice at the time when the cortical plate is laid down. As a consequence, the development of radial glia is impaired, and neurons making up the cortical plate display abnormal migration patterns. In addition, restricted defects along the rostrocaudal and the mediolateral axes are present in the subplate, suggesting an Emx2-specific role in priming the proper development of this layer.

Molecular analysis of the heterogeneity region of the human ribosomal spacer.

The human ribosomal non-transcribed spacers are 30 X 10(3) base-pairs (or 30 kb) in length with a limited length heterogeneity localized in a specific region downstream from the 3' end of the transcribed region. Total DNA digested with EcoRI and BamHI and hybridized with a probe containing the 3' end of the 28 S ribosomal RNA coding region shows four major bands of 3.9 kb, 4.6 kb, 5.4 kb and 6.2 kb. The 5.4 kb band is the most abundant in every individual, followed by the 4.6 kb band. The longest and the shortest size classes are less well-represented and may even be absent. Every individual shows his own pattern of relative abundance of non-transcribed spacer length classes that can be followed through generations. We decided to investigate the molecular structure of the heterogeneity region, in order to cast light onto the mechanisms underlying the origin and maintenance of this length heterogeneity. Pertinent spacer regions of eight ribosomal clones from two human genomic libraries were subcloned and analyzed by restriction mapping and nucleotide sequencing. In the minimal length class, there is a sequence of 700 base-pairs that appears to be tandemly duplicated once, twice or three times in the other length classes. This repeated DNA module contains a region consisting of repetitions of simple pyrimidine groups like C-T, C-T-T-T or C-C-C-T. DNA module repeats may differ by the length of this pyrimidine-rich region. However, these length variations are not continuous, as revealed by Southern transfer analysis of several individuals and different cloned gene units: instead, the repeated modules fall into two discrete length classes of about 700 base-pairs and 800 base-pairs. An imperfect duplication of a short sequence of 86/89 base-pairs is present at the boundary between the heterogeneity region and the upstream flanking region, representing a very ancient duplication event.

Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors.

We describe the expression pattern of cSix3, a chick homologue of the murine Six3. cSix3 transcripts are expressed from presomitic stages in the most anterior portion of the neural plate. As the neural tube folds and the optic vesicles evaginate, cSix3 is expressed in the optic vesicle and the rostroventral forebrain. At later stages, cSix3 is found in most of the structures derived from the anterior neural plate, i.e. olfactory epithelium, septum, adenohypophysis, hypothalamus and preoptic areas. During eye development, cSix3 expression is first found in the entire optic vesicle and the overlying ectoderm but soon becomes restricted to the prospective neural retina and to the lens placode. In the developing neural retina, cSix3 is expressed in the entire undifferentiated neuroepithelium but is rapidly downregulated, first in the postmitotic photoreceptors and later in the majority of retinal ganglion cells.

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.

Changes in the prevalence of an homeobox gene product during muscle differentiation.

We have studied by immunohistofluorescence and confocal microscopy the localization of the XlHbox-1 protein, the product of a Xenopus class 1 homeobox gene corresponding to the human HOX 3C, during the development of Xenopus laevis mesodermal derivatives. The protein, not present at early stages of embryonic development, can first be detected in the neurula where it is weakly expressed in the rostral part of the spinal cord and in the nuclei of the corresponding somites. At later stages of mesodermal development, very high levels of the molecule are present in the nuclei of a small group of myogenic cells in the most dorsal aspect of the myotome, while the nuclei of differentiated muscle fibers within the myotome are either stained weakly or completely negative. A similar transient expression of XlHbox-1 gene product during myogenesis occurs during muscle differentiation in the limb bud and during differentiation of visceral smooth muscles from the lateral plate mesoderm. In both cases the nuclei of precursor cells contain high level of this protein which is rapidly down regulated during further muscle differentiation. In myogenic areas the modulation of XlHbox-1 expression invariably parallels that of the neural cell adhesion molecule N-CAM. These data are the first evidence that a homeobox gene belonging to the Antennapedia-Bithorax complex is transiently expressed in early phases of muscle differentiation. The transient expression of homeobox genes in early phases of embryonic development could act synergistically with the expression of other myogenic transcriptional factors to specify a fine level of differentiation of the muscle cells along the body axis.

The Hox-4.8 gene is localized at the 5' extremity of the Hox-4 complex and is expressed in the most posterior parts of the body during development.

We report the isolation and expression pattern of a novel mouse homeobox gene, Hox-4.8. Hox-4.8 is the most 5'-located homeobox gene in the HOX-4 complex. Sequence analysis confirmed that Hox-4.8 is a member of the subfamily of AbdominalB-related Hox-4 genes and revealed strong interspecies conservation. As for the human locus, Hox-4.8 is probably the last Hox gene in this part of the HOX-4 complex. During development, Hox-4.8 transcripts are restricted to the extremities of the embryonic anteroposterior axis and limbs as well as in the developing tail bud and to the most posterior segment of the gut (the rectum). Within the limb mesenchyme, Hox-4.8 is expressed in more posterodistal regions than those of its neighbour Hox-4.7. Hence, Hox-4.8 expression appears to be related to the last significant phenotypic changes towards the extremities of the embryonic body and limb axes.

Inactivation of the zebrafish homologue of Chx10 by antisense oligonucleotides causes eye malformations similar to the ocular retardation phenotype.

We report the cloning of a zebrafish paired-type homeobox gene, Alx, closely related to the murine Chx10 and the gold fish Vsx-I homeodomain proteins. Alx is first expressed at about 12 h post-fertilization (hpf) when optic vesicles appear. Its expression is restricted to the early retinal neuroepithelium, whereas no signal can be detected in the optic placode. Later, Alx expression follows the differentiation of the neural retina. Inhibition experiments with antisense oligonucleotides resulted in specific eye malformations which are reminiscent of the phenotype of ocular retardation (or) mice, caused by a spontaneous Chx10 mutation. The expression of other developmentally relevant genes such as pax(zf-a), pax(zf-b) and krx-20 was not affected in the antisense treated embryos.