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.

Homeobox genes in normal and malignant cells.

Homeobox genes are transcription factors primarily involved in embryonic development. Several homeobox gene families have so far been identified: Hox, EMX, PAX, MSX as well as many isolated divergent homeobox genes. Among these, Hox genes are most intriguing for having a regulatory network structure organization. Recent indications suggest the involvement of homeobox genes in (i) crucial adult eukariotic cell functions and (ii) human diseases, spanning from diabetes to cancer. In this review we will discuss the mechanisms through which homeobox genes act, and will propose a model for the function of the Hox gene network as decoding system for achieving specific genetic programs. New technologies for whole-genome RNA expression will be crucial to evaluate the clinical relevance of homeobox genes in structural and metabolic diseases.

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.

Otx and Emx homeobox genes in brain development.

Over the last few years great progress has been made in the understanding of the formation and regionalisation of the mouse brain. In this review we will focus our attention on two families of homeobox-containing genes essentially coding for four transcription factors involved in brain and forebrain development: the two Emx and the two Otx genes. Here we describe the expression pattern of these genes in the developing mouse, as well as the characterisation of the corresponding knockout mice with special emphasis on Emx2. Whereas Otx genes are clearly involved in the formation and regionalisation of the whole rostral brain, comprised of forebrain and midbrain, our data suggest a role for Emx2 in the specification of the cytoarchitecture of the cerebral cortex, achieved through the control of proliferation of neuronal precursors and of migration of newly-formed neurons to their final destination.

Genetic control of regional identity in the developing vertebrate forebrain.

In the past we isolated and characterized a number of vertebrate homeobox genes expressed in the developing brain. In particular, Emx1 and Emx2 are expressed in the developing forebrain of mouse embryos, in a region including the presumptive cerebral cortex. In the developing cerebral cortex, Emx1 is expressed in most neuroblasts and neurons at all stages of development, whereas Exm2 expression is restricted to proliferating neuroblasts of the so-called ventricular zone and to Cajal-Retzius cells, but is undetectable in most postmitotic cortical neurons. It is conceivable to hypothesize that Emx2 plays a role in the control of proliferation of cortical neuroblasts and in the regulation of their subsequent migration. This latter process has been recently analysed in some detail in null mutant mice. The expression of these and other genes has also been analysed in the developing brain of different species of vertebrates. Homologies between forebrain subdivisions have been proposed based on the conservation and divergence of gene expression patterns.

The Otx2 homeoprotein regulates expression from the gonadotropin-releasing hormone proximal promoter.

The GnRH gene is expressed exclusively in a highly restricted population of approximately 800 neurons in the mediobasal hypothalamus in the mouse. The Otx2 homeoprotein has been shown to colocalize with GnRH in embryonic mouse brain. We have identified a highly conserved bicoid-related Otx target sequence within the proximal promoter region of the GnRH gene from several species. This element from the rat GnRH promoter binds baculovirus-expressed Otx2 protein and Otx2 protein in nuclear extracts of a hypothalamic GnRH-expressing neuronal cell line, GT1-7. Transient transfection assays indicate that the GnRH promoter Otx/bicoid site is required for specific expression of the GnRH gene in GT1-7 cells and that it can confer specificity to a neutral Rous sarcoma virus (RSV) promoter in GT1-7 cells but not in NIH3T3 cells. Overexpression of mouse Otx2 in GT1-7 cells induces expression of a GnRH promoter plasmid, an effect that is dependent upon the Otx binding site. Thus, the GnRH proximal promoter is regulated by the Otx2 homeoprotein. Finally, we have now demonstrated the presence of Otx2 protein in the GnRH neurons of the adult mouse hypothalamus. These data suggest that Otx2 is important in the development of the GnRH neuron and/or in the maintenance of GnRH expression in the adult mouse hypothalamus.

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.

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.

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.

A mouse model for valproate teratogenicity: parental effects, homeotic transformations, and altered HOX expression.

Valproate (VPA) is one of several effective anti-epileptic and mood-stabilizing drugs, many of which are also potent teratogens in humans and several other mammalian species. Variable teratogenicity among inbred strains of laboratory mice suggests that genetic factors influence susceptibility. While studying the genetic basis for VPA teratogenicity in mice, we discovered that parental factors influence fetal susceptibility to induced malformations. Detailed examination of these malformations revealed that many were homeotic transformations. To test whether VPA, like retinoic acid (RA), alters HOX expression, pluripotent human embryonal carcinoma cells were treated with VPA or RA and Hox expression assessed. Altered expression of specific Hox genes may thus account for the homeotic transformations and other malformations found in VPA-treated fetuses.

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.

Mouse forebrain development. The role of Emx2 homeobox gene.

Over the last few years great progress has been made in the understanding of the formation of the mouse forebrain. Among the genes involved in this process, the mouse Emx homeobox genes Emx1 and particularly Emx2 play a primary role. Here we describe the mRNA and protein expression related to Emx2 in the developing mouse telencephalon, as well as the results obtained studying the corresponding knock-out mice. Our findings indicate a role for this gene in the specification of the forebrain via the control of cell proliferation, as well as in guiding neuronal migration during development through the cortical plate. These studies will hopefully enable us to better understand the molecular mechanisms underlying the formation of the mouse cerebral cortex as well as to establish relevant interactions between the various proteins present in this region of the brain.

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.

The caudal limit of Otx2 expression positions the isthmic organizer.

The homeobox gene Otx2 is expressed in the anterior neural tube with a sharp limit at the midbrain/hindbrain junction (the isthmic organizer). Otx2 inactivation experiments have shown that this gene is essential for the development of its expression domain. Here we investigate whether the caudal limit of Otx2 expression is instrumental in positioning the isthmic organizer and in specifying midbrain versus hindbrain fate, by ectopically expressing Otx2 in the presumptive anterior hindbrain using a knock-in strategy into the En1 locus. Transgenic offspring display a cerebellar ataxia. Morphological and histological studies of adult transgenic brains reveal that most of the anterior cerebellar vermis is missing, whereas the inferior colliculus is complementarily enlarged. During early neural pattern formation expression of the midbrain markers Wnt1 and Ephrin-A5, the isthmic organizer markers Pax2 and Fgf-8 and the hindbrain marker Gbx2 are shifted caudally in the presumptive hindbrain territory. These findings show that the caudal limit of Otx2 expression is sufficient for positioning the isthmic organizer and encoding caudal midbrain fate within the mid/hindbrain domain.

Homeobox genes and cancer.

Homeobox-containing genes are a family of regulatory genes encoding transcription factors that primarily play a crucial role during development. Several indications suggest their involvement in the control of cell growth and, when dysregulated, in oncogenesis. We will describe the implications, in tumor origin and evolution, of members of the homeobox gene families HOX, EMX, PAX, and MSX as well as of other divergent homeobox genes. We will also propose a model for the function of the HOX gene network in controlling cell identity to account for the involvement of some HOX genes in both normal development and oncogenesis.

TargetFinder: searching annotated sequence databases for target genes of transcription factors.

UNLABELLED: TargetFinder is a new software tool to search a database of annotated sequences for transcription factor binding sites located in context with other important transcription regulatory signals and regions, like the TATA element, the promoter, and so on, thereby greatly reducing the background usually associated with this kind of search. AVAILABILITY: The TargetFinder Web service is available at http://hercules.tigem.it/TargetFinder.html CONTACT: giovanni.lavorgna@hsr.it

Laminin receptor alpha6beta4 integrin is highly expressed in ENU-induced glioma in rat.

Laminins and their receptors influence neoplastic growth and invasiveness. We recently reported the abnormal expression of a laminin receptor, alpha6beta4 integrin, in human astrocytomas. To further investigate the role of alpha6beta4 in gliomas, we produced an experimental model of glioma in rat by transplacental ethylnitrosourea (ENU) administration. This animal model allowed us to study the timing of alpha6beta4 expression during tumor development and the topography of expression in the tumor and the surrounding tissue. Immunohistochemistry, in situ hybridization, and immunoprecipitation studies demonstrated that alpha6beta4 heterodimer forms in experimental gliomas, and confirmed that alpha6beta4 is expressed diffusely in neoplastic cells and reactive astrocytes, but not in normal glia surrounding the tumors. Interestingly, alpha6beta4 was expressed from the early phases of tumor development, and more highly expressed by cells in the proliferative centers of the tumors. Both neoplastic cells and reactive astrocytes also expressed the glial growth factor (neuregulin) receptors, Erb-B2 and Erb-B3. Finally, alpha6beta4 expression was reduced in a subset of tumor blood vessels. Thus, this study suggests a potential role for alpha6beta4 in the pathogenesis of gliomas. Furthermore, this is the first description of altered integrin expression in experimental gliomas; transplacental ENU-induced gliomas in rat will provide a useful model to study the role of altered adhesion in the pathogenesis of human gliomas.