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.     

Regulatory elements involved in Drosophila Adh gene expression are conserved in divergent species and separate elements mediate expression in different tissues.

Alcohol dehydrogenase (ADH) is expressed in a complex temporal and spatial pattern from tandem promoters (proximal and distal) in Drosophila melanogaster, and from two closely linked genes (Adh-1 and Adh-2) in D. mulleri. The expression patterns of Adh-1 and the proximal promoter, and Adh-2 and the distal promoter are similar, but not identical. We show that the mulleri Adh genes are appropriately expressed when introduced into the melanogaster genome, indicating that the cis- and trans-acting elements which regulate the corresponding promoters are functionally equivalent in the two species. By analyzing the expression of in vitro generated mutants of the mulleri Adh locus, we define at least three regulatory regions of the mulleri Adh genes and show that different control elements mediate the expression of Adh in different tissues.     

Maternal expression of genes that regulate the bithorax complex of Drosophila melanogaster

A relatively large number of genes have been described that are required for the normal spatial expression of the genes of the bithorax complex. Most of these regulators appear to act negatively and are required to prevent indiscriminate expression of bithorax complex (BX-C) functions. In this report we examine five negative BX-C regulators to determine whether these are maternally expressed in germ-line derived cells. The genes studied include Additional sex combs (Asx), Polycomblike (Pcl), Sex comb extra (Sce), Sex comb on midleg (Scm), and lethal(4)29 [l(4)29]. The maternal germ-line dependent expression of each of these genes is assessed by comparison of zygotes from mothers whose functional germ cells carry no wild-type alleles to zygotes from mothers whose germ cells contain one wild-type allele. Because mutant alleles of each of the genes studied are recessive lethals, mosaic females with homozygous or hemizygous mutant germ lines were produced by pole cell transplantation. The results demonstrate that all of the negative regulators tested are expressed in the maternal germ line and all play important roles in the regulation of BX-C activities during embryogenesis. The absence of maternally supplied products from all of the genes studied except l(4)29 can be largely or completely compensated for by the activity in the zygote of a paternally contributed wild-type allele. It is argued that, with the exception of l(4)29, the genes studied in this report are qualitatively similar in function to the previously described BX-C regulators Pc, esc, and sxc. The available evidence indicates that genes within this group have functions that are not restricted to the regulation of genes that control segmental identity.     

Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.

Four consensus sequences are conserved with the same linear arrangement in RNA-dependent DNA polymerases encoded by retroid elements and in RNA-dependent RNA polymerases encoded by plus-, minus- and double-strand RNA viruses. One of these motifs corresponds to the YGDD span previously described by Kamer and Argos (1984). These consensus sequences altogether lead to 4 strictly and 18 conservatively maintained amino acids embedded in a large domain of 120 to 210 amino acids. As judged from secondary structure predictions, each of the 4 motifs, which may cooperate to form a well-ordered domain, places one invariant amino acid in or proximal to turn structures that may be crucial for their correct positioning in a catalytic process. We suggest that this domain may constitute a prerequisite 'polymerase module' implicated in template seating and polymerase activity. At the evolutionary level, the sequence similarities, gap distribution and distances between each motif strongly suggest that the ancestral polymerase module was encoded by an individual genetic element which was most closely related to the plus-strand RNA viruses and the non-viral retroposons. This polymerase module gene may have subsequently propagated in the viral kingdom by distinct gene set recombination events leading to the wide viral variety observed today.     

CNS midline cells influence the division and survival of lateral glia in the Drosophila nervous system

Central nervous system (CNS) midline cells are essential for identity determination and differentiation of neurons in the Drosophila nervous system. It is not clear, however, whether CNS midline cells are also involved in the development of lateral glial cells. The roles of CNS midline cells in lateral glia development were elucidated using general markers for lateral glia, such as glial cell missing and reverse polarity, and specific enhancer trap lines labeling the longitudinal, A, B, medial cell body, peripheral, and exit glia. We found that CNS midline cells were necessary for the proper expression of glial cell missing, reverse polarity, and other lateral glia markers only during the later stages of development, suggesting that they are not required for initial identity determination. Instead, CNS midline cells appear to be necessary for proper division and survival of lateral glia. CNS midline cells were also required for proper positioning of three exit glia at the junction of segmental and intersegmental nerves, as well as some peripheral glia along motor and sensory axon pathways. This study demonstrated that CNS midline cells are extrinsically required for the proper division, migration, and survival of various classes of lateral glia from the ventral neuroectoderm.     

Fingerprinting genomes by use of PCR with primers that encode protein motifs or contain sequences that regulate gene expression

PCR primers of arbitrary nucleotide sequence have identified DNA polymorphisms useful for genetic mapping in a large variety of organisms. Although technically very powerful, the use of arbitrary primers for genome mapping has the disadvantage of characterizing DNA sequences of unknown function. Thus, there is no reason to anticipate that DNA fragments amplified by use of arbitrary primers will be enriched for either transcribed or promoter sequences that may be conserved in evolution. For these reasons, we modified the arbitrarily primed PCR method by using oligonucleotide primers derived from conserved promoter elements and protein motifs. Twenty-nine of these primers were tested individually and in pairwise combinations for their ability to amplify genomic DNA from a variety of species including various inbred strains of laboratory mice and Mus spretus. Using recombinant inbred strains of mice, we determined the chromosomal location of 27 polymorphic fragments in the mouse genome. The results demonstrated that motif sequence-tagged PCR products are reliable markers for mapping the mouse genome and that motif primers can also be used for genomic fingerprinting of many divergent species.     

Notch and Numb are required for normal migration of peripheral glia in Drosophila

A prominent feature of glial cells is their ability to migrate along axons to finally wrap and insulate them. In the embryonic Drosophila PNS, most glial cells are born in the CNS and have to migrate to reach their final destinations. To understand how migration of the peripheral glia is regulated, we have conducted a genetic screen looking for mutants that disrupt the normal glial pattern. Here we present an analysis of two of these mutants: Notch and numb. Complete loss of Notch function leads to an increase in the number of glial cells. Embryos hemizygous for the weak Notch(B-8X) allele display an irregular migration phenotype and mutant glial cells show an increased formation of filopodia-like structures. A similar phenotype occurs in embryos carrying the Notch(ts1) allele when shifted to the restrictive temperature during the glial cell migration phase, suggesting that Notch must be activated during glial migration. This is corroborated by the fact that cell-specific reduction of Notch activity in glial cells by directed numb expression also results in similar migration phenotypes. Since the glial migration phenotypes of Notch and numb mutants resemble each other, our data support a model where the precise temporal and quantitative regulation of Numb and Notch activity is not only required during fate decisions but also later during glial differentiation and migration.     

Transient expression of Bis protein in midline radial glia in developing rat brainstem and spinal cord

Bis (Bcl-2 interacting death suppressor) has been reported to contribute to the differentiation and maturation of specific neuronal populations in the developing rat forebrain, in addition to its well-established functions as a stress or survival-related protein. In the present study, we have analyzed the expression of Bis in the rat brainstem and cervical spinal cord during development by using immunohistochemistry. Bis immunoreactivity was detected in radial glial cells flanking the midline from embryonic day 14. During embryonic and early postnatal development, Bis expression persisted in differentiating radial glia at the midline but disappeared first in the spinal cord by postnatal day 7 (P7) and later also in the brainstem by P14. Bis expression was restricted to a subpopulation of the midline radial glia, i.e., the dorsal midline of the midbrain and spinal cord and the ventral midline of the hindbrain, which were double- or triple-labeled with vimentin and nestin, markers for radial glia, and S100B. However, these markers also labeled all radial glia including the ventral midline glia in the midbrain and spinal cord, with Bis being absent from these structures. In addition, the dorsal midline glia in the midbrain and spinal cord expressed Bis prior to the timing of expression for radial glial markers. Therefore, our results demonstrate the early and transient expression of Bis in the subpopulation of midline glia in the developing brainstem and spinal cord, suggesting that Bis has a unique role in association with the radial glial cells in the developing central nervous system. This research was supported by a grant (10029970) from the Ministry of Knowledge Economy, The Republic of Korea and by a grant (M103KV010010-08 K2201-01010) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, The Republic of Korea.     

Elimination of oncogenic cells that regulate epithelial homeostasis in Drosophila

Normal epithelial tissues often put anti-tumorigenic pressure on newly emerged oncogenic cells through cell–cell communications. In Drosophila epithelium, clones of oncogenic cells mutant for evolutionarily conserved apico-basal polarity genes such as scribble (scrib) and discs large (dlg) are actively eliminated when surrounded by normal cells. It has been reported that c-Jun N-terminal kinase (JNK) signaling in polarity-deficient cells is crucial for their cell death. However, the mechanism by which normal epithelial tissues exert anti-tumorigenic effects on polarity-deficient cells had been elusive. Here, I describe our genetic studies in Drosophila epithelium especially focused on the role of surrounding normal epithelial cells in response to the emergence of polarity-deficient cells. Furthermore, I also describe recent studies regarding the mechanism by which polarity-deficient cells are extruded from the tissue, and discuss future perspectives on the study of cell–cell communications in epithelial homeostasis.     

The functions of the multiproduct and rapidly evolving dec-1 eggshell gene are conserved between evolutionarily distant species of Drosophila.

The Drosophila dec-1 gene encodes multiple proteins that are required for female fertility and proper eggshell morphogenesis. Genetic and immunolocalization data suggest that the different DEC-1 proteins are functionally distinct. To identify regions within the proteins with potential biological significance, we cloned and sequenced the D. yakuba and D. virilis dec-1 homologs. Interspecies comparisons of the predicted translation products revealed rapidly evolving sequences punctuated by blocks of conserved amino acids. Despite extensive amino acid variability, the proteins produced by the different dec-1 homologs were functionally interchangeable. The introduction of transgenes containing either the D. yakuba or the D. virilis dec-1 open reading frames into a D. melanogaster DEC-1 protein null mutant was sufficient to restore female fertility and wild-type eggshell morphology. Normal expression and extracellular processing of the DEC-1 proteins was correlated with the phenotypic rescue. The nature of the conserved features highlighted by the evolutionary comparison and the molecular resemblance of some of these features to those found in other extracellular proteins suggests functional correlates for some of the multiple DEC-1 derivatives.     

Calmodulin and son of sevenless dependent signaling pathways regulate midline crossing of axons in the Drosophila CNS

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues which prevent axons crossing the midline.