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.

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.

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.

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.

EMX2 protein in the developing mouse brain and olfactory area.

The distribution of EMX2, the protein product of the homeobox gene Emx2, was analyzed in the developing mouse CNS by means of a polyclonal antibody we raised against it. The protein is present in the rostral brain, the olfactory area and a set of scattered cells lying between the nasal pits and the telencephalon. In the cortical neuroepithelium EMX2 is expressed all along the rostro-caudal axis in a graded distribution with a caudal-medial maximum and a rostral-lateral minimum. Anti-EMX2 immunoreactivity is also detectable in Cajal-Retzius cells as well as in apical dendrites of marginal neurons of the cortical plate. We also observe that the EMX2 and EMX1 homeoproteins display complementary expression patterns in olfactory bulbs and amygdaloid complex. Here, they demarcate different neuronal populations, involved in processing olfactory information coming from the vomero-nasal organ and from the main olfactory epithelium, respectively. EMX2 is also detectable in mesencephalic structures, such as the optic tectum and tegmentum. The graded distribution of EMX2 along antero-posterior and medial-lateral axes of the primitive cortex prefigures a role of this protein in the subdivision of the cortex in cytoarchitectonic regions and possibly functional areas, whereas its presence in Cajal-Retzius cells suggests a role in the process of cortical lamination.

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.

Implication of OTX2 in pigment epithelium determination and neural retina differentiation.

The expression pattern of Otx2, a homeobox-containing gene, was analyzed from the beginning of eye morphogenesis until neural retina differentiation in chick embryos. Early on, Otx2 expression was diffuse throughout the optic vesicles but became restricted to their dorsal part when the vesicles contacted the surface ectoderm. As the optic cup forms, Otx2 was expressed only in the outer layer, which gives rise to the pigment epithelium. This early Otx2 expression pattern was complementary to that of PAX2, which localizes to the ventral half of the developing eye and optic stalk. Otx2 expression was always observed in the pigment epithelium at all stages analyzed but was extended to scattered cells located in the central portion of the neural retina around stage 22. The number of cells expressing Otx2 transcripts increased with time, following a central to peripheral gradient. Bromodeoxyuridine labeling in combination with immunohistochemistry with anti-OTX2 antiserum and different cell-specific markers were used to determine that OTX2-positive cells are postmitotic neuroblasts undergoing differentiation into several, if not all, of the distinct cell types present in the chick retina. These data indicate that Otx2 might have a double role in eye development. First, it might be necessary for the early specification and subsequent functioning of the pigment epithelium. Later, OTX2 expression might be involved in retina neurogenesis, defining a differentiation feature common to the distinct retinal cell classes.

OTX2 homeoprotein in the developing central nervous system and migratory cells of the olfactory area.

We analyzed the distribution of OTX2 during mouse development. OTX2 is a homeoprotein encoded by Otx2, a vertebrate homeobox gene expressed in the developing brain and anterior head regions. The protein is already detectable in pre-streak embryos, in nuclei of embryonic ectoderm or epiblast and primitive endoderm or hypoblast. Its distribution is uniform along the entire epiblast, while showing an antero-posterior gradient along the hypoblast at the time when primitive streak first forms. Between embryonic day 7 (E7) and E7.5 there is a progressive confinement of the protein to the anterior ectoderm corresponding to the forming headfold. At E7.5-E7.8, the protein is mainly confined in this region but is still present, though at lower level, in more posterior ectoderm. Starting from day 8 of development it is essentially confined to anterior neuroectoderm corresponding to presumptive fore- and midbrain. Its subsequent distribution in forebrain, midbrain, developing isthmo-cerebellum and posterior central nervous system is analyzed in detail. Of particular interest is the presence of OTX2 in nuclei of cells of the olfactory system starting from its origin in the olfactory placode. OTX2 protein is present in some cells of the olfactory epithelium, in both the major olfactory epithelium and the vomero-nasal organ, and in scattered migratory cells present in the mesenchyme outside it. These cells surround the axon bundles of the olfactory nerve along its path from the olfactory epithelium in the nasal cavities to the olfactory bulb in rostral telencephalon and include both ensheathing glial cells and luteinizing hormone-releasing hormone (LHRH)-positive cells.

EMX1 homeoprotein is expressed in cell nuclei of the developing cerebral cortex and in the axons of the olfactory sensory neurons.

We analyzed the distribution of EMX1 during mouse development. EMX1 is a homeoprotein encoded by Emx1, a regulatory homeobox gene expressed in the developing forebrain. Its distribution essentially overlaps the expression domains of Emx1 transcripts. The EMX1 protein is present in the developing dorsa telencephalon, that is in the cerebral cortex, olfactory bulb and hippocampus. In the cerebral cortex EMX1 is present in nuclei of proliferating, differentiating and most mature neurons belonging to all cortical layers. In the olfactory bulb it is present in all proliferating cells during development, whereas postnatally it is faintly expressed in some mitral cells. Non-cerebral localizations include a transient expression in branchial pouches, in the apical ectodermal ridge of the developing limbs and in the developing kidney. Of particular interest is the presence of EMX1 in the olfactory nerve from its first appearance during embryogenesis to birth. The protein is present in axons of olfactory sensory neurons along their entire length, including their terminals in spherical regions of neuropil in the olfactory bulb called glomeruli.

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.

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.

Vertebrate homeobox genes.

In the former part of the review the principal available data about Hox genes, their molecular organisation and their expression in vertebrate embryos, with particular emphasis for mammals, are briefly summarized. In the latter part 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 ems, and Otx1 and Otx2, related to otd.

A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo.

We studied the expression of two vertebrate homeobox genes, Otx1 and Otx2, related to orthodenticle, a gene expressed in the developing head of Drosophila. Both genes are expressed in restricted regions of the developing rostral brain including the presumptive cerebral cortex and olfactory bulbs. The expression patterns of the two genes in diencephalon suggest that they both have a role in establishing the boundary between presumptive dorsal and ventral thalamus. They are also expressed in regions of the developing olfactory, auricolar and ocular system, including the covering of the optic nerve. Otx1 expression is detectable from day 8 of gestation in telencephalic, diencephalic and mesencephalic regions. From day 10.5 of gestation its expression extends to some metencephalic areas. Otx2 appears to be already expressed in the epiblast of prestreak embryos. It persists in the entire embryonic ectoderm for some time after the onset of gastrulation. In midstreak embryos its expression appears progressively restricted to the anterior embryonic ectoderm corresponding to presumptive fore- and mid-brain. In early midgestation embryos it is expressed in telencephalic, diencephalic and mesencephalic regions but from day 11.75 of gestation its expression disappears from dorsal telencephalon and is confined to diencephalic and mesencephalic regions. Otx2 is one of the earliest genes expressed in the epiblast and immediately afterwards is expressed in anterior neuroectoderm, demarcating rostral brain regions even before headfold formation. Its gene product contains a homeodomain of the bicoid class and is able to recognize and transactivate a bicoid target sequence.

Isolation and mapping of EVX1, a human homeobox gene homologous to even-skipped, localized at the 5' end of HOX1 locus on chromosome 7.

We isolated and mapped the human homeobox gene EVX1. This gene encodes a protein of 407 amino acid residues containing a homeodomain closely related to the Drosophila even-skipped (eve) segmentation gene of the pair-rule class. EVX1 belongs to a small family of vertebrate eve-related homeobox genes including human EVX1 and EVX2 genes, their murine homologs, Evx 1 and Evx 2, and the frog Xhox-3 gene. We previously reported that EVX2 is localized at the 5' end of the HOX4 locus on chromosome 2. We show here that EVX1 is localized at the 5' end of the HOX1 locus on chromosome 7, 48 kb upstream from the most 5' of the eleven HOX1 genes, namely HOX1J. Both EVX genes are transcribed in an opposite orientation as compared to that of adjacent HOX genes. Human HOX1 and HOX4 complex loci appear to be both closely linked to a homeobox gene of the EVX family.