Insight into the genetic control of the identity of specific regions along the body axis of vertebrates has resulted primarily from the study of vertebrate homologues of regulatory genes operating in the Drosophila trunk, but little is known about the development of most anterior regions of the body either in flies or vertebrates. Three Drosophila genes have been identified that are important in controlling the development of the head, two of which, empty spiracles and orthodenticle, have been cloned and shown to contain a homeobox. We previously cloned and characterized Emx1 and Emx2, two mouse genes related to empty spiracles that are expressed in restricted regions of the developing forebrain, including the presumptive cerebral cortex and olfactory bulbs. Here we report the identification of Otx1 and Otx2, which are related to orthodenticle. We have compared the expression domains of the four genes in the developing rostral brain of mouse embryos at a developmental stage, day 10 post coitum, when they are all expressed. 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 the time when regional specification of major brain regions takes place, the expression domains of the four genes seem to be continuous regions contained within each other in the sequence Emx1 less than Emx2 less than Otx1 less than Otx2.

We cloned two homeobox genes, Emx1 and Emx2, related to empty spiracles, a gene expressed in very anterior body regions during early Drosophila embryogenesis, and studied their expression in mouse embryos. Emx1 expression is detectable from day 9.5 of gestation whereas Emx2 appears to be already expressed in 8.5 day embryos. Both genes are expressed in the presumptive cerebral cortex and olfactory bulbs. Emx1 is expressed exclusively there, whereas Emx2 is also expressed in some neuroectodermal areas in embryonic head including olfactory placodes in earlier stages and olfactory epithelia later in development.

The branchial region of the vertebrate head forms through complex interactions involving rhombomeric segments, neural crest and branchial arches. It is though that aspects of their patterning mechanisms are linked and involve Hox-2 genes, whose overlapping and spatially restricted expression domains represent a combinatorial code for generating regional diversity. Vertebrates possess four Hox clusters of Antennapedia class homeobox genes, related to each other by duplication and divergence from a common ancestral complex. In consequence, at equivalent positions in different clusters there are highly related genes known as subfamilies or paralogous groups. As Hox-2 genes cannot fully account for patterning individual rhombomeres, we investigated whether offsets in expression limits of paralogous genes could account for the generation of regional diversity. We report here that, with the exception of the labial subfamily, paralogues show identical expression limits in rhombomeres, cranial ganglia and branchial arches, providing a combinatorial Hox code for the branchial region that seems to be different in organization to that of the trunk.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes uGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

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...