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.

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 amaed DlxS and Dax6, in human and mouse. The two human genes, DLXS and DLX6, are closely linked in an inverted convergent configuration in a region of chromosome 7, at 7q22. Similarly, the two human genes DLXI and DLK2 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 DlxS and Dlx6 mRNA in restricted regions of ventral diencephalon and basal teencephalon, with a distribution very similar to that reported forDMl and Dx2 mRNA. A surprising feature of DlxS and Dlx6 is that they are also expressed in all skeletal sutures of dtion embryos after the first cartilae formation. The expreion pattern of these genes, together with their chromosome calation, may provide useftl cues for the study of congenital disorders in which there is a combination of cranlofacial and limb defects.Many vertebrate genes have been identified by virtue of their nucleotide sequence similarity with Drosophila developmental genes. Many homeobox-containing genes (1) have been identified on this basis. The Dlx gene family (2-7) has been identified because these genes contain a homeobox related to that of Distalless (Dli, also known as Ba) a gene expressed in the head and limbs of the developing fruit fly (8-9).Cloned Dlx sequences in the mouse (2-4), frog (6, 7), and zebrafish (5) have been shown to correspond to at least four different genes, Dlxi-Dlx4. A detailed expression analysis has been carried out for murine Dlxi (2, 10, 11) and Dlx2 (3, 4, 12) genes. They appear to be expressed within the central nervous system of midgestation mouse embryos in specific regions ofthe forebrain, but not in more posterior parts of the neural tube. In early embryos they are also expressed in branchial arches, in the otic vesicle, and in facial and limb primordia. Expression in the developing inner ear has been also reported (5) for the zebrafish cognate of Dlx3. With the notable exception of Xdli2 (7), several frog genes (Xenopus) of the Dlx family have been identified (6, 7) that are similarly expressed in the anterior portion of the embryonic neural tube. In many instances, a correlation of their expression domain with forebrain regionalization (13,14) has been suggested (2)(3)(4)(5)(6)(7)(10)(11)(12) MATERIALS AND METHODSExpression Analysis. A cDNA library prepared from 8-week human embryos (15) was screened at low-stringency conditions with a short Dli genomic sequence including the homeobox (8). Four classes of homologous cDNA clones, corresponding to DLX), DLX2, DLX5, and DLX6, were found. Using these cDNA clones as probes, we screened in turn a human genomic library constructed in cosmids (15) to study the transcrip...

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.

It has recently been demonstrated that the transcription factor genes Emx2 and Pax6, expressed in the developing cerebral cortex along two complementary tangential gradients, are essential for the shaping of the cortical areal profile at late developmental ages, when cortical neuronogenesis is almost completed. In this study we addressed the question of whether cortical regionalization is already affected in Emx2 and Pax6 loss of function mutants at the beginning of neuronogenesis. By comparing expression patterns of selected molecular markers in these mutants at this age, we found that: (i) Emx2 and Pax6 are necessary for the establishment of their own specific expression profiles and are able to down-regulate each other; and (ii) absence of functional EMX2 or PAX6 proteins results in reduction of caudal-medial and rostral-lateral cortical regions, respectively, as well as in impairment of the WNT signalling center at the medial-caudal edge of the cortical field, crucial for cortical growth. These results suggest that pre-neuronogenic cortical regionalization may rely on mutual interactions between these two transcription factors and that the late areal phenotype of Emx2(-/-) and Pax6(-/-) mutants may possibly arise from both misconfiguration of the cortical molecular protomap and distortion of the cortical growth profile.

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.