Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse.

During early mouse development the homeobox gene Hesx1 is expressed in prospective forebrain tissue, but later becomes restricted to Rathke's pouch, the primordium of the anterior pituitary gland. Mice lacking Hesx1 exhibit variable anterior CNS defects and pituitary dysplasia. Mutants have a reduced prosencephalon, anopthalmia or micropthalmia, defective olfactory development and bifurcations in Rathke's pouch. Neonates exhibit abnormalities in the corpus callosum, the anterior and hippocampal commissures, and the septum pellucidum. A comparable and equally variable phenotype in humans is septo-optic dysplasia (SOD). We have cloned human HESX1 and screened for mutations in affected individuals. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain which destroyed its ability to bind target DNA. These data suggest an important role for Hesx1/HESX1 in forebrain, midline and pituitary development in mouse and human.

Expression of Meis and Pbx genes and their protein products in the developing telencephalon: implications for regional differentiation.

The Meis and Pbx genes encode for homeodomain proteins of the TALE class and have been shown to act as co-factors for other homeodomain transcription factors (Mann and Affolter, 1998. Curr. Opin. Genet. Dev. 8, 423-429). We have studied the expression of these genes in the mouse telencephalon and found that Meis1 and Meis2 display region-specific patterns of expression from embryonic day (E)10.5 until birth, defining distinct subterritories in the developing telencephalon. The expression of the Meis genes and their proteins is highest in the subventricular zone (SVZ) and mantle regions of the ventral telencephalon. Compared to the Meis genes, Pbx genes show a broader expression within the telencephalon. However, as is the case in Drosophila (Rieckhof et al., 1997. Cell 91, 171-183; Kurrant et al., 1998. Development 125, 1037-1048; Pai et al., 1998. Genes Dev. 12, 435-446), nuclear localized PBX proteins were found to correlate highly with Meis expression. In addition, DLX proteins co-localize with nuclear PBX in distinct regions of the ventral telencephalon.

Cloning and expression of three members of the zebrafish Bmp family: Bmp2a, Bmp2b and Bmp4.

In vertebrates, Bmps (bone morphogenetic proteins) play critical roles in establishing the basic embryonic body plan and are involved in the development of a large variety of organs and tissues. To study the evolution of Bmps, we isolated cDNAs for three members of the zebrafish Bmp gene family: Bmp2a, Bmp2b and Bmp4. The deduced amino acid sequences of Bmp2a and Bmp4 consist of 386 and 400 aa, respectively and show high homologies to their counterparts in mouse, chick and Xenopus. The deduced Bmp2b aa sequence consists of 411 aa and the mature protein shows 88% and 86% identities to zebrafish Bmp2a and Bmp4, respectively. The expression of the mRNA of these three genes has been analyzed by whole mount in situ hybridization and RT-PCR. Areas of zebrafish Bmp2 and Bmp4 expression suggest evolutionary conserved mechanisms of Bmp2/4 dependent differentiation between lower and higher vertebrates.

Fission and fusion of the neuronal endoplasmic reticulum.

The endoplasmic reticulum (ER) is central for protein synthesis and is the largest intracellular Ca2+ store in neurons. The neuronal ER is classically described to have a continuous lumen spanning all cellular compartments. This allows neuronal ER to integrate spatially separate events in the cell. Recent in vitro as well as in vivo findings, however, demonstrate that the neuronal ER is a structurally dynamic entity, capable of rapid fragmentation, i.e., ER fission. The ER fragments can fuse back together and reinstate ER continuity. This reversible phenomenon can be induced repeatedly within the same cell, is temperature-dependent, and compatible with cell survival. The key trigger for dendritic ER fission is N-methyl D-aspartate (NMDA) receptor stimulation in the presence of extracellular Ca2+. However, the exact molecular machinery responsible for the fission and fusion of neuronal ER remains unknown. Reversible ER fission represents a new cell biological event downstream of NMDA receptor-gated Ca2+ influx and may thus influence many aspects of neuronal function in physiology and disease. Hence, it constitutes a new field for exploration in neuroscience that will benefit greatly from recent advances in light microscopy imaging techniques allowing dynamic characterization of cellular events in vitro and in vivo.

A role for Gsh1 in the developing striatum and olfactory bulb of Gsh2 mutant mice.

We have examined the role of the two closely related homeobox genes Gsh1 and Gsh2, in the development of the striatum and the olfactory bulb. These two genes are expressed in a partially overlapping pattern by ventricular zone progenitors of the ventral telencephalon. Gsh2 is expressed in both of the ganglionic eminences while Gsh1 is largely confined to the medial ganglionic eminence. Previous studies have shown that Gsh2(-/-) embryos suffer from an early misspecification of precursors in the lateral ganglionic eminence (LGE) leading to disruptions in striatal and olfactory bulb development. This molecular misspecification is present only in early precursor cells while at later stages the molecular identity of these cells appears to be normalized. Concomitant with this normalization, Gsh1 expression is notably expanded in the Gsh2(-/-) LGE. While no obvious defects in striatal or olfactory bulb development were detected in Gsh1(-/-) embryos, Gsh1/2 double homozygous mutants displayed more severe disruptions than were observed in the Gsh2 mutant alone. Accordingly, the molecular identity of LGE precursors in the double mutant is considerably more perturbed than in Gsh2 single mutants. These findings, therefore, demonstrate an important role for Gsh1 in the development of the striatum and olfactory bulb of Gsh2 mutant mice. In addition, our data indicate a role for Gsh genes in controlling the size of the LGE precursor pools, since decreasing copies of Gsh2 and Gsh1 alleles results in a notable decrease in precursor cell number, particularly in the subventricular zone.

Genetic control of dorsal-ventral identity in the telencephalon: opposing roles for Pax6 and Gsh2.

We have examined the genetic mechanisms that regulate dorsal-ventral identity in the embryonic mouse telencephalon and, in particular, the specification of progenitors in the cerebral cortex and striatum. The respective roles of Pax6 and Gsh2 in cortical and striatal development were studied in single and double loss-of-function mouse mutants. Gsh2 gene function was found to be essential to maintain the molecular identity of early striatal progenitors and in its absence the ventral telencephalic regulatory genes Mash1 and Dlx are lost from most of the striatal germinal zone. In their place, the dorsal regulators, Pax6, neurogenin 1 and neurogenin 2 are found ectopically. Conversely, Pax6 is required to maintain the correct molecular identity of cortical progenitors. In its absence, neurogenins are lost from the cortical germinal zone and Gsh2, Mash1 and Dlx genes are found ectopically. These reciprocal alterations in cortical and striatal progenitor specification lead to the abnormal development of the cortex and striatum observed in Pax6 (small eye) and Gsh2 mutants, respectively. In support of this, double homozygous mutants for Pax6 and Gsh2 exhibit significant improvements in both cortical and striatal development compared with their respective single mutants. Taken together, these results demonstrate that Pax6 and Gsh2 govern cortical and striatal development by regulating genetically opposing programs that control the expression of each other as well as the regionally expressed developmental regulators Mash1, the neurogenins and Dlx genes in telencephalic progenitors.

Conservation of BF-1 expression in amphioxus and zebrafish suggests evolutionary ancestry of anterior cell types that contribute to the vertebrate telencephalon.

The forkhead domain containing transcription factor BF-1 has been shown to play a major role in the correct development of the cerebral hemispheres in the mouse. BF-1 orthologs have been isolated from zebrafish and the cephalocordate amphioxus. In both species, BF-1 is expressed in the anterior neural tube. In zebrafish zBF-1 expression is restricted to anterior portions of the otic vesicle and to the presumptive telencephalon. In amphioxus AmphiBF-1 is transiently seen in the frontal part of the first somite and, at 3 days of development, in a small number of cells in the cerebral vesicle (cv). The anterior expression of BF-1 in chordates and vertebrates and of slp-1/2 in Drosophila suggests that BF-1 is crucial for an evolutionarily conserved specification of anterior neuronal cell types.

Retinoids are produced by glia in the lateral ganglionic eminence and regulate striatal neuron differentiation.

In order to identify molecular mechanisms involved in striatal development, we employed a subtraction cloning strategy to enrich for genes expressed in the lateral versus the medial ganglionic eminence. Using this approach, the homeobox gene Meis2 was found highly expressed in the lateral ganglionic eminence and developing striatum. Since Meis2 has recently been shown to be upregulated by retinoic acid in P19 EC cells (Oulad-Abdelghani, M., Chazaud, C., Bouillet, P., Sapin, V., Chambon, P. and Dollé, P. (1997) Dev. Dyn. 210, 173-183), we examined a potential role for retinoids in striatal development. Our results demonstrate that the lateral ganglionic eminence, unlike its medial counterpart or the adjacent cerebral cortex, is a localized source of retinoids. Interestingly, glia (likely radial glia) in the lateral ganglionic eminence appear to be a major source of retinoids. Thus, as lateral ganglionic eminence cells migrate along radial glial fibers into the developing striatum, retinoids from these glial cells could exert an effect on striatal neuron differentiation. Indeed, the treatment of lateral ganglionic eminence cells with retinoic acid or agonists for the retinoic acid receptors or retinoid X receptors, specifically enhances their striatal neuron characteristics. These findings, therefore, strongly support the notion that local retinoid signalling within the lateral ganglionic eminence regulates striatal neuron differentiation.

Expression patterns of the human sarcoma-associated genes FUS and EWS and the genomic structure of FUS.

FUS (TLS) was first identified as the 5'-part of a fusion gene with CHOP (GADD153, DDIT3) in myxoid liposarcomas with t(12; 16)(q13; p11). Homologies were found with the EWS oncogene, which is rearranged in Ewing sarcomas and other neoplasias. The genomic structure of FUS shows extensive similarities with that of EWS, but the exon/intron structures differ in the 5' parts, and overall FUS is smaller than EWS. Exon 3 of FUS corresponds to exons 3 and 4 in EWS. FUS exons 4-6 correspond to EWS exons 5-8. Exons 7 to 15 of FUS are very similar to those in EWS, although the EWS exons are larger than the corresponding FUS exons. FUS and EWS were expressed in all tissues investigated. The transcripts were stable within the 160-min half-life experiments. No or little variation in FUS or EWS expression was seen when resting lymphocytes were activated. These observations indicate that FUS and EWS belong to the housekeeping type of genes. This view is supported by the presence of the housekeeping gene type of promoter region in both genes.