Unique functions of Sonic hedgehog signaling during external genitalia development.

Coordinated growth and differentiation of external genitalia generates a proximodistally elongated structure suitable for copulation and efficient fertilization. The differentiation of external genitalia incorporates a unique process, i.e. the formation of the urethral plate and the urethral tube. Despite significant progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes for external genitalia. The sonic hedgehog (Shh) gene and its signaling genes have been found to be dynamically expressed during murine external genitalia development. Functional analysis by organ culture revealed that Shh could regulate mesenchymally expressed genes, patched 1 (Ptch1), bone morphogenetic protein 4 (Bmp4), Hoxd13 and fibroblast growth factor 10 (Fgf10), in the anlage: the genital tubercle (GT). Activities of Shh for both GT outgrowth and differentiation were also demonstrated. Shh(-/-) mice displayed complete GT agenesis, which is compatible with such observations. Furthermore, the regulation of apoptosis during GT formation was revealed for the first time. Increased cell death and reduced cell proliferation of the Shh(-/-) mice GT were shown. A search for alterations of Shh downstream gene expression identified a dramatic shift of Bmp4 gene expression from the mesenchyme to the epithelium of the Shh mutant before GT outgrowth. Regulation of mesenchymal Fgf10 gene expression by the epithelial Shh was indicated during late GT development. These results suggest a dual mode of Shh function, first by the regulation of initiating GT outgrowth, and second, by subsequent GT differentiation.

Shh and Ptc are associated with taste bud maintenance in the adult mouse.

In mammals, taste receptor cells are organized into taste buds on tongue. Taste buds are trophically maintained by taste neurons and under continuous renewal, even in adults. We found that the receptor for Sonic hedgehog (Shh), Patched1 (Ptc), was expressed around taste buds where cells were proliferating, and that Shh was expressed within basal cells of taste buds. Denervation caused the loss of Shh and Ptc expression before the degeneration of taste buds.

Developmentally regulated expression of the transcriptional cofactors/histone acetyltransferases CBP and p300 during mouse embryogenesis.

CBP (CREBBP/CREB-binding protein) and p300 are related signal-dependent transcriptional cofactors and histone acetyltransferases. They are both implicated in tumorigenesis and mutations in the human CBP gene have been found in Rubinstein-Taybi syndrome (RTS), which is characterized by multiple developmental defects and mental retardation. Studies with CBP and p300 mouse mutants indicate that both proteins are required for normal development, and that there is an essential gene dosage-sensitive role for these transcriptional cofactors in embryogenesis, cell differentiation and proliferation. Although it is generally believed that the expression of CBP and p300 is ubiquitous, we report here that they are developmentally regulated during mouse embryogenesis. In the developing CNS, CBP and p300 proteins were found throughout the newly formed neural plate, but their expression was later restricted to the dorsal parts of the developing neural tube. Later in neural development, CBP and p300 proteins could also be found in subsets of ventral neurons, including motor neurons and oligodendrocytes. During organogenesis, CBP and p300 proteins were expressed in specific cell types of the developing heart, vasculature, skin, lung and liver. Many of these tissues and organs are known to be affected in mutant mice lacking CBP and/or p300, and in RTS patients. Interestingly, while CBP and p300 proteins show extensive overlapping expression during mouse embryogenesis, we observed that their subcellular localization is developmentally regulated in several cell types. Taken together, our results suggest that there are common, as well as distinct, biochemical functions of CBP and p300 during mouse development.

Cloning and characterization of two cytoplasmic dynein intermediate chain genes in mouse and human.

Cytoplasmic dynein is a large multisubunit microtubule-based motor protein, which mediates movement of numerous intracellular organelles. We report here the identification of the human homologue of cytoplasmic dynein intermediate chain 1 gene (DNCI1) located on human chromosome 7q21.3-q22.1. The mouse orthologue (Dnci1) was identified along with another highly related gene, Dnci2, and their RNA in situ expression patterns were examined during mouse embryogenesis. Dnci1 was found to have a highly restricted expression domain in the developing forebrain as well as the peripheral nervous system (PNS), while Dnci2 displayed a broad expression profile throughout the entire central nervous system and most of the PNS. A dynamic expression profile was also found for Dnci2 in the developing mouse limb bud. The data presented here provide a framework for the further analysis of the functional role of Dnci1 and Dnci2 in mouse and DNCI1 in human.

Overlapping and non-overlapping Ptch2 expression with Shh during mouse embryogenesis.

In Drosophila, patched encodes a negative regulator of Hedgehog signaling. Biochemical experiments have demonstrated that vertebrate patched homologues might function as a Sonic hedgehog (Shh) receptor. In mice, two patched homologues, Ptch and Ptch2, have been identified. Sequence comparison have suggested that they might possess distinct properties in Shh signaling. In the developing tooth, hair and whisker, Shh and Ptch2 are co-expressed in the epithelium while Ptch is strongly expressed in the mesenchymal cells. We report here the chromosomal localization of Ptch2 and further analysis of Ptch2 expression. Throughout mouse development, the level of Ptch2 expression is significantly lower than that of Ptch. In early mouse embryos, Ptch and Ptch2 were found to be co-expressed in regions adjacent to Shh-expressing cells in the developing CNS. Similar to other epidermal structures, Shh and Ptch2 also show overlapping expression in the developing nasal gland and eyelids. Thus, during mouse development, Ptch2 is expressed in both Shh-producing and -nonproducing cells.

Essential function of Gli2 and Gli3 in the formation of lung, trachea and oesophagus.

Foregut malformations (oesophageal atresia, tracheo-oesophageal fistula, lung anomalies and congenital stenosis of the oesophagus and trachea) are relatively common anomalies occurring in 1 in 2,000-5,000 live births, although their aetiology is poorly understood. The secreted glycoprotein Sonic hedgehog (Shh) has been suggested to act as an endodermal signal that controls hindgut patterning and lung growth. In mice, three zinc-finger transcription factors, Gli1, Gli2 and Gli3, have been implicated in the transduction of Shh signal. We report here that mutant mice lacking Gli2 function exhibit foregut defects, including stenosis of the oesophagus and trachea, as well as hypoplasia and lobulation defects of the lung. A reduction of 50% in the gene dosage of Gli3 in a Gli2-/- background resulted in oesophageal atresia with tracheo-oesophageal fistula and a severe lung phenotype. Mutant mice lacking both Gli2 and Gli3 function did not form oesophagus, trachea and lung. These results indicate that Gli2 and Gli3 possess specific and overlapping functions in Shh signalling during foregut development, and suggest that mutations in GLI genes may be involved in human foregut malformations.

Defect in the maintenance of the apical ectodermal ridge in the Dactylaplasia mouse.

During vertebrate limb development the distal apex of the limb bud ectoderm is induced to form the apical ectodermal ridge (AER). The presence of the AER is required for the continued outgrowth of the limb bud. Classical embryological studies have led to the hypothesis that a secreted mesenchymal factor is required to maintain the AER. We have undertaken a detailed analysis of Dactylaplasia (Dac) mice, a semidominant mutant which displays missing central digits in the fore- and hindlimbs of heterozygous animals and monodactyly in homozygous animals. Our data show that Dac mice have a defect in the maintenance of the AER. At E10.5, the mutant AER is found to be morphologically normal. However, by E11.5 the central aspect of the AER degenerates leaving the anterior and posterior AER intact. In homozygous mice both the central and anterior AER degenerate, while the posterior extremity of the AER is unaffected. Analysis of BrdU incorporation reveals that degeneration of the AER is due to a lack of cell proliferation in the mutant AER. The loss of the AER leads to a reduction in cell proliferation in the subridge mesenchyme at E11.5. The data represent direct genetic evidence for the existence of an AER maintenance activity that is distinct from AER induction and differentiation. Moreover, the data suggest that the role of the AER maintenance factor is to promote cell proliferation in the ridge. Based on our findings, we propose a model for AER maintenance in the vertebrate limb.

Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice.

Floor plate cells at the midline of the neural tube are specified by high-level activity of Sonic hedgehog (Shh) secreted by notochord, whereas motor neurons are thought to be specified by a lower level activity of Shh secreted in turn by floor plate cells. In Drosophila, the Gli zinc finger protein Cubitus interruptus functions as a transcription factor activating Hedgehog-responsive genes. We report that the expression of known Shh-responsive genes such as Ptc and Gli1 is downregulated in mutant mice lacking Gli2 function. Gli2 mutants fail to develop a floor plate yet still develop motor neurons, which occupy the ventral midline of the neural tube. Our results imply that Gli2 is required to mediate high level but not low level Shh activity and show that the development of motor neurons can occur in the absence of floor plate induction.

Organogenesis of the liver, thymus and spleen is affected in jumonji mutant mice.

The recessive mutant mouse jumonji (jmj), obtained by a gene trap strategy, shows neural tube defects in approximately half of homozygotes with a Balb/cA and 129/Ola mixed background. Here, we show that no neural tube defects are observed with a Balb/cA background. We also found hypoplasia of the liver, thymus and spleen with full penetrance with a Balb/cA background. In the livers of homozygous embryos we found excessive cell death in the peripheral region. In both the thymus and spleen, the accumulation of hematopoietic cells is affected in mutant embryos. These phenotypes were also observed with C57BL/6J and DBA/2J backgrounds, suggesting that the jmj gene plays an essential role in the organogenesis of these tissues.

The presence of a magnocellular defect depends on the type of dyslexia.

Previous studies have identified a magnocellular pathway defect in approximately 75% of dyslexics. Since these experiments have not classified dyslexia into subtypes, the purpose of this experiment was to determine if adult dyseidetic dyslexics or dysphoneidetic dyslexics suffer from a defect in the magnocellular pathway. Nine dyseidetic dyslexics, eight dysphoneidetic dyslexics, and nine normal readers participated in the experiment. Contrast sensitivity functions (CSF) were determined with vertically oriented sine wave gratings (0.5, 1.0, 2.0, 4.0, 8.0, 12.0 c/deg drifting at 1 and 10 Hz) by employing a two-alternative, forced-choice technique. The results of the experiment indicated that dysphoneidetic dyslexics had reduced sensitivity to low spatial frequencies at 10 Hz, whereas dyseidetic dyslexics did not have reduced sensitivity at either 1 or 10 Hz. These results suggest that the type of dyslexia influences whether losses in perception are found which are consistent with a magnocellular deficit.

Gene trap capture of a novel mouse gene, jumonji, required for neural tube formation.

A mouse mutation, termed jumonji (jmj), was generated by a gene trap strategy. Expression of the trapped gene (jmj gene), as monitored by X-gal staining, was detected predominantly at the midbrain-hindbrain boundary and in the cerebellum, depending on the stage of development. All embryos homozygous for the jmj mutation died before embryonic day 15.5. Some, but not all, of the homozygotes developed an abnormal groove in a region just anterior to the midbrain-hindbrain boundary on the neural plate at embryonic day 8-8.5 and showed a defect in neural tube closure in the midbrain region. Analyses of jmj cDNA revealed that the jmj gene is novel, conserved among vertebrates, and disrupted by vector insertion in the jmj homozygotes. The amino acid sequence deduced from the cDNA shared a portion of significant homology with human retinoblastoma-binding protein RBP-2 and with a putative protein encoded by human gene XE169 that escapes X-chromosome inactivation. These results suggest that jmj gene is essential for normal morphogenesis of the neural tube.

Antisense retinoic acid receptor gamma-1 oligonucleotide enhances chondrogenesis of mouse limb mesenchymal cells in vitro.

Retinoic acid receptor (RAR) gamma gene is expressed in the precartilaginous cells during chondrogenesis in mouse embryos, but the role of the gene products is still unclear. To examine the role during chondrogenesis, we isolated mesenchymal cells from the limb bud of mouse embryos and exposed them to antisense RAR gamma-1 oligodeoxynucleotide in micromass culture. The antisense oligodeoxynucleotide inhibited RAR gamma-1 protein expression and enhanced chondrogenesis in the exposed cells. These results suggest that the complex of RAR gamma-1 protein and its ligand RA acts as a suppressor of the chondrogenesis in the limb development.

Antisense c-myc oligonucleotide promotes chondrogenesis and enhances RA responsiveness of mouse limb mesenchymal cells in vitro.

To examine the role of c-myc protein during chondrogenesis, we exposed 11 day p.c. mouse limb mesenchymal cells to the antisense c-myc oligonucleotide in micromass culture. The antisense oligonucleotide inhibited the c-myc protein expression, and intensely promoted chondrogenesis in the exposed cells. Most of the cells differentiated into cartilaginous cells, whereas they differentiated into cartilaginous and fibrous cells under the control conditions. The antisense oligonucleotide increased the inhibitory efficiency of all-trans retinoic acid (RA) to the chondrogenesis. These results suggest that the c-myc protein suppress the chondrogenesis and reduces RA responsiveness in the limb mesenchymal cells.