Multiple spatially specific enhancers are required to reconstruct the pattern of Hox-2.6 gene expression.

Murine Hox genes are organized into four clusters that share many features with the homeotic clusters of Drosophila. This evolutionary conservation and the clear relationships between the position of a gene within a cluster and its expression pattern have led to the suggestion that the structure of the cluster is essential for proper regulation. Using a Hox-2.6-lacZ reporter gene in transgenic mice we have shown that the overall expression pattern of the endogenous Hox-2.6 gene can be reconstructed when it is isolated from the complex. The transgene was expressed in the proper tissues, with the correct spatial distribution and temporal pattern. Furthermore, direct comparison by in situ hybridization revealed that the levels of transgene expression are similar to those of the endogenous gene. This has allowed us to define three elements that regulate particular aspects of the Hox-2.6 pattern, two of which act as spatially specific enhancers. One enhancer, region A, directed expression only in the neural tube, whereas the other, region C, specified the majority of the Hox-2.6 pattern. Both were also capable of imposing the correct boundaries of expression on heterologous promoters. The definition of such elements will allow the characterization of the trans-acting factors that mediate spatial regulation in the mammalian embryo.     

Complementary homeo protein gradients in developing limb buds

A new human homeo box-containing gene designated Hox-5.2 was cloned and mapped to human chromosome 2. This homeo box is related in sequence to Abdominal-B, a Drosophila homeotic gene that specifies identity of posterior segments. An antibody probe was made using a human Hox-5.2 fusion protein and was found to stain posterior regions of mouse, chicken, and Xenopus embryos. Unexpectedly, when the distribution of Hox-5.2 antigen was compared with that of X1Hbox 1 antigen, a non-overlapping and mutually exclusive pattern was detected (e.g., in developing limb buds, intestine, and somites). Regions expressing Hox-5.2 do not express X1Hbox 1 protein, and vice versa. Hox-5.2 antigen is detected strongly in developing fore- and hindlimb buds, where it forms a gradient of nuclear protein throughout most of the mesenchyme. This gradient is maximal in distal and posterior regions. Hox-5.2 expression is activated in Xenopus limb regeneration blastemas, as expected for any gene involved in pattern formation. As described previously, a gradient of X1Hbox 1 protein can be detected in the forelimb. The latter gradient has the opposite polarity to that of Hox-5.2. i.e., maximal in anterior and proximal mesoderm. These two opposing gradients (and possibly others) could be involved in determining positional values in developing limb buds.     

Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development

During development, several genes that specify neuronal subtype identity are expressed in distinct dorsoventral domains of the spinal cord and hindbrain. Cadherin-7 (Cad7), a member of the cadherin family of adhesion molecules, is expressed by radial glia in a dorsal domain of the spinal cord basal plate in chicken. To study the regulation of the Cad7 gene, we ectopically expressed two known dorsoventral patterning genes, Shh and Pax7, in the caudal neural tube and in two brain regions at different stages of development by in vivo electroporation. Results showed that Shh regulated the expression of Cad7 by radial glia in a concentration-dependent manner. Shh induced or repressed the expression of Cad7, at low and high concentrations, respectively. Furthermore, Pax7 inhibited the expression of Cad7. These results are compatible with a role of Shh and Pax7 in regulating endogenous Cad7 expression during spinal cord and hindbrain development. Our data show, for the first time, that Shh can regulate the expression not only of other gene regulatory factors, but also of Cad7, a morphoregulatory molecule that plays a role in axon elongation and neural circuit formation.     

Expression of zTlxA, a Hox11-like gene, in early differentiating embryonic neurons and cranial sensory ganglia of the zebrafish embryo.

We have isolated a cDNA encoding a member of the Tlx/Hox11 family of homeodomain factors from the zebrafish, most closely related to the vertebrate Tlx-1/Hox11 and Tlx-3/Hox11L2 proteins. The gene is expressed in a set of early differentiating neurons that project to a common tract, the lateral longitudinal fascicle. We show that the gene is specifically expressed in spinal cord Rohon Beard neurons, in nucleus of the posterior commissure neurons of the midbrain, in a set of hindbrain neurons that include RoL3 reticulospinal interneurons, and in the trigeminal, statoacoustic, anterior lateral line, glossopharyngeal, and vagal cranial sensory ganglia. Timing of expression of the gene in these neurons correlates with the phase of axonal outgrowth and target innervation. Expression of the gene is also observed in several non-neural tissues, including the pharyngeal arches, budding gill filaments, outgrowing semicircular protrusions in the otic vesicle, and in the pectoral fin buds.     

Novel murine homeo box gene on chromosome 1 expressed in specific hematopoietic lineages and during embryogenesis.

We describe here a new murine homeo box gene, denoted Hlx, which is expressed within specific hematopoietic lineages. The cDNA sequence indicates that Hlx differs markedly from known vertebrate homeo box genes, and linkage analysis of an interspecific murine backcross showed that it resides at a novel homeo box locus on the distal portion of mouse chromosome 1. The Hlx homeo domain is most similar to that of the Drosophila H2.0 gene, but outside this region the two polypeptides are related only within a few short segments, the most notable being a motif (denoted Hep) also partially conserved in the engrailed and invected homeo proteins and possibly related to an octapeptide in certain paired box proteins. The presence of an intron within the Hlx homeo box at the same position as in several divergent Drosophila genes (H2.0, labial, Distal-less, proboscipedia, Abdominal-B, NK-1) suggests an ancient evolutionary relationship between these genes. RNA analysis of 67 murine hematopoietic cell lines and normal hematopoietic cells revealed Hlx expression throughout the myeloid/macrophage lineage and at early stages of B lymphocyte development but not in T lymphocytes, erythroid cells, or mast cells. Hence, Hlx is a candidate regulator of hematopoietic lineage commitment and maturation. It probably also functions outside the hematopoietic system, however, because Hlx mRNA could be detected in diverse adult tissues and in embryos from as early as day 8 of development.     

Survival motor neuron SMN1 and SMN2 gene promoters: identical sequences and differential expression in neurons and non-neuronal cells

Spinal muscular atrophy (SMA) is a recessive disorder involving the loss of motor neurons from the spinal cord. Homozygous absence of the survival of motor neuron 1 gene (SMN1) is the main cause of SMA, but disease severity depends primarily on the number of SMN2 gene copies. SMN protein levels are high in normal spinal cord and much lower in the spinal cord of SMA patients, suggesting neuron-specific regulation for this ubiquitously expressed gene. We isolated genomic DNA from individuals with SMN1 or SMN2 deletions and sequenced 4.6 kb of the 5' upstream regions of the these. We found that these upstream regions, one of which is telomeric and the other centromeric, were identical. We investigated the early regulation of SMN expression by transiently transfecting mouse embryonic spinal cord and fibroblast primary cultures with three transgenes containing 1.8, 3.2 and 4.6, respectively, of the SMN promoter driving beta-galactosidase gene expression. The 4.6 kb construct gave reporter gene expression levels five times higher in neurons than in fibroblasts, due to the combined effects of a general enhancer and a non-neuronal cell silencer. The differential expression observed in neurons and fibroblasts suggests that the SMN genes play a neuron-specific role during development. An understanding of the mechanisms regulating SMN promoter activity may provide new avenues for the treatment of SMA.     

Zebrafish wnt3 is expressed in developing neural tissue

Wnt signaling regulates embryonic patterning and controls stem cell homeostasis, while aberrant Wnt activity is associated with disease. One Wnt family member, Wnt3, is required in mouse for specification of mesoderm, and later regulates neural patterning, apical ectodermal ridge formation, and hair growth. We have identified and performed preliminary characterization of the zebrafish wnt3 gene. wnt3 is expressed in the developing tailbud and neural tissue including the zona limitans intrathalamica (ZLI), optic tectum, midbrain‐hindbrain boundary, and dorsal hindbrain and spinal cord. Expression in these regions suggests that Wnt3 participates in processes such as forebrain compartmentalization and regulation of tectal wiring topography by retinal ganglia axons. Surprisingly, wnt3 expression is not detectable during mesoderm specification, making it unlikely that Wnt3 regulates this process in zebrafish. This lack of early expression should make it possible to study later Wnt3‐regulated patterning events, such as neural patterning, by knockdown studies in zebrafish. Developmental Dynamics 238:1768–1795, 2009. © 2009 Wiley‐Liss, Inc.     

Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell migration.

We report the cloning and expression patterns of three novel zebrafish Roundabout homologs. The Roundabout (robo) gene encodes a transmembrane receptor that is essential for axon guidance in Drosophila and Robo family members have been implicated in cell migration. Analysis of extracellular domains and conserved cytoplasmic motifs shows that zebrafish Robo1 and Robo2 are orthologs of mammalian Robo1 and Robo2, respectively, while zebrafish Robo3 is likely to be an ortholog of mouse Rig-1. The three zebrafish robos are expressed in distinct but overlapping patterns during embryogenesis. They are highly expressed in the developing nervous system, including the olfactory system, visual system, hindbrain, cranial ganglia, spinal cord, and posterior lateral line primordium. They are also expressed in several nonneuronal tissues, including somites and fin buds. The timing and patterns of expression suggest roles for zebrafish robos in axon guidance and cell migration. Wiley-Liss, Inc.     

Spectrum of malformations of the hindbrain (cerebellum, pons, and medulla) in a cohort of children with high rate of parental consanguinity

We review 25 patients with a spectrum of hindbrain (cerebellum, pons, and medulla) malformations from a cohort of children with high parental consanguinity rate. Twenty-three of the 25 patients were born to consanguineous parents. The patients were classified in four groups. Eleven patients of 6 families had malformation of the hindbrain and midbrain with molar tooth sign (10 patients of 5 families with typical Joubert syndrome), 5 patients showed severe supratentorial anomalies in addition to the hindbrain malformations, 5 patients had pontocerebellar or cerebellar hypoplasia with anterior horn cell disease in the spinal cord (spinal muscular atrophy), and 4 patients showed malformations affecting predominantly the hindbrain without substantial involvement of other systems. A locus for Joubert syndrome was previously identified on chromosome 9q34.3 in two families, and a second locus on chromosome 11p12-q13.3 in another family. A third Joubert syndrome locus has been mapped at 6q23 and a mutation in the AHI1 gene at this site has been found recently in a further family from this cohort. Delineation of homogeneous subgroups of patients with hindbrain malformations and molecular genetic analysis of these groups may lead to identification of further loci, genes and mutations responsible for the malformations.