Sox neuro, a new Drosophila Sox gene expressed in the developing central nervous system

We describe the identification and detailed expression pattern of a second Drosophila Sox gene, SoxNeuro (SoxN), highly related to mammalian group B Sox1, 2, 3 genes. SoxN is expressed in a highly dynamic pattern during embyogenesis, being associated with the development of the central nervous system (CNS), from the early steps onwards. We present strong evidence that the early SoxN neuroectoderm expression is controlled by the zygotic dorso-ventral patterning genes (dpp, sog, brk, twi).     

A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus

Retinoic acid (RA) mediates both anterior/posterior patterning and neuronal specification in the vertebrate central nervous system (CNS). However, the molecular mechanisms downstream of RA are not well understood. To investigate these mechanisms, we used the invertebrate chordate amphioxus, in which the CNS, although containing only about 20,000 neurons in adults, like the vertebrate CNS, has a forebrain, midbrain, hindbrain, and spinal cord and is regionalized by RA-signaling. Here we show, first, that domains of genes with expression normally limited to diencephalon and midbrain are generally not affected by altered RA-signaling, second, that contrary to previous reports, not only Hox1, 3, and 4, but also Hox2 and Hox6 are collinearly expressed in the amphioxus CNS, and third, that collinear expression of all these Hox genes is controlled by RA-signaling. Finally, we show that Hox1 is involved in mediating both the role of RA-signaling in regionalization of the hindbrain and in specification of hindbrain motor neurons. Thus, morpholino knock-down of the single amphioxus Hox1 mimics the effects of treatments with an RA-antagonist. This analysis establishes RA-dependent regulation of collinear Hox expression as a feature common to the chordate CNS and indicates that the RA-Hox hierarchy functions both in proper anterior/posterior patterning of the developing CNS and in specification of neuronal identity.     

Mammalian DSCAMs: roles in the development of the spinal cord,cortex, and cerebellum?

Central nervous system (CNS) development involves neural patterning, neuronal and axonal migrations, and synapse formation. DSCAM, a chromosome 21 axon guidance molecule, is expressed by CNS neurons during development and throughout adult life. We now report that DSCAM and its chromosome 11 paralog DSCAML1 exhibit inverse ventral-dorsal expression patterns in the developing spinal cord and distinct, partly inverse, expression patterns in the developing cortex, beginning in the Cajal-Retzius cells. In the adult cortex, DSCAM predominates in layer 3/5 pyramidal cells and DSCAML1 predominates in layer 2 granule cells. In the cerebellum, DSCAM is stronger in the Purkinje cells and DSCAML1 in the granule cells. Finally, we find that the predicted DSCAML1 protein contains 60 additional N-terminal amino acids which may contribute to its distinct expression pattern and putative function. We propose that the DSCAMs comprise novel elements of the pathways mediating dorsal-ventral patterning and cell-fate specification in the developing CNS.     

The CNS midline cells and spitz class genes are required for proper patterning of Drosophila ventral neuroectoderm.

The Drosophila embryonic central nervous system (CNS) develops from sets of neuroblasts (NBs) which segregate from the ventral neuroectoderm during early embryogenesis. It is not well established how each individual NB in the neuroectoderm acquires its characteristic identity along the dorsal-ventral axis. Since it is known that CNS midline cells and spitz class genes (pointed, rhomboid, single-minded, spitz and Star) are required for the proper patterning of ventral CNS and epidermis originated from the ventral neuroectoderm, this study was carried out to determine the functional roles of the CNS midline cells and spitz class genes in the fate determination of ventral NBs and formation of mature neurons and their axon pathways. Several molecular markers for the identified NBs, neurons, and axon pathways were employed to examine marker gene expression profile, cell lineage and axon pathway formation in the spitz class mutants. This analysis showed that the CNS midline cells specified by single-minded gene as well as spitz class genes are required for identity determination of a subset of ventral NBs and for formation of mature neurons and their axon pathways. This study suggests that the CNS midline cells and spitz class genes are necessary for proper patterning of the ventral neuroectoderm along the dorsal-ventral axis.     

Hremx, the ascidian homologue of ems/emx, is expressed in the anterior and posterior-lateral epidermis but not in the central nervous system during embryogenesis

Recent comparative studies on expression patterns of homeobox genes in the development between ascidians and vertebrates have come to suggest a possibility that a common basic mechanism may exist in the patterning of the central nervous system (CNS). The ems/emx genes have been demonstrated to be involved in the formation and patterning of the anterior CNS in Drosophila and vertebrate embryos. In the present study, we have isolated and analyzed expression of Hremx, the ascidian homologue of ems/emx with particular attention to whether it is expressed in the larval ascidian CNS. Expression of Hremx was detected in the anterior trunk and lateral tail epidermis but not in the anterior CNS. The two expression domains of the epidermis responded in different ways upon treatment with retinoic acid: the anterior expression domain was unaltered, while the posterior expression domain extended to the anterior. The present result suggests that Hremx may have a function in anterior patterning but not in the patterning of the CNS in the ascidian embryo. We suggest the possibility that the function of ems/emx genes in the patterning of the anterior CNS in Drosophila and vertebrate embryos might have been acquired independently in the lineages to Drosophila and vertebrates.     

3.6 kb genomic sequence from Takifugu capable of promoting axon growth-associated gene expression in developing and regenerating zebrafish neurons

Unlike mammals, fish have the capacity for functional adult CNS regeneration, which is due, in part, to their ability to express axon growth-related genes in response to nerve injury. One such axon growth-associated gene is gap43, which is expressed during periods of developmental and regenerative axon growth, but is not expressed in CNS neurons that do not regenerate in adult mammals. We previously demonstrated that cis-regulatory elements of gap43 that are sufficient for developmental expression are not sufficient for regenerative expression in the zebrafish. Here we have identified a 3.6kb genomic sequence from Fugu rubripes that can promote reporter gene expression in the nervous system during both development and regeneration in zebrafish. This compact sequence is advantageous for functional dissection of regions important for axon growth-associated gene expression during development and/or regeneration. In addition, this sequence will also be useful for targeting gene expression to neurons during periods of growth and plasticity.     

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.     

Tissue-specific requirements for a phosphorylation site in the Fushi tarazu homeodomain

 The homeodomain protein Fushi tarazu (Ftz) is required for several embryonic patterning processes including segmentation and neurogenesis. During the stages that these processes are regulated the protein is differentially phosphorylated, suggesting that phosphorylation plays a role in helping the protein to regulate different functions in different tissues. We showed in a recent study that one of the Ftz phosphorylation sites, a protein kinase A-type site in the N-terminal arm of the homeodomain, is required for normal Ftz-dependent segmentation. Here we test whether phosphorylation of this site (Thr-263) is also required in the developing central nervous system (CNS). A well-established role for Ftz in the CNS is for the differentiation of neurons referred to as RP2 neurons. Absence of Ftz expression in these cells causes a failure of certain target genes to be expressed and subsequent defects in RP2 differentiation. In contrast to its effect on segmentation, we find that mutation of Thr-263 to Ala (or Asp) has no effect on these CNS functions. This suggests that the phosphorylation state of this site is irrelevant for Ftz function in the CNS, and that there are tissue-specific differences in the requirements for Ftz phosphorylation. Received: 28 February 1999 / Accepted: 10 March 1999     

Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology

Neurons establish diverse dendritic morphologies during development, and a major challenge is to understand how these distinct developmental programs might relate to, and influence, neuronal function. Drosophila dendritic arborization (da) sensory neurons display class-specific dendritic morphology with extensive coverage of the body wall. To begin to build a basis for linking dendrite structure and function in this genetic system, we analyzed da neuron axon projections in embryonic and larval stages. We found that multiple parameters of axon morphology, including dorsoventral position, midline crossing and collateral branching, correlate with dendritic morphological class. We have identified a class-specific medial-lateral layering of axons in the central nervous system formed during embryonic development, which could allow different classes of da neurons to develop differential connectivity to second-order neurons. We have examined the effect of Robo family members on class-specific axon lamination, and have also taken a forward genetic approach to identify new genes involved in axon and dendrite development. For the latter, we screened the third chromosome at high resolution in vivo for mutations that affect class IV da neuron morphology. Several known loci, as well as putative novel mutations, were identified that contribute to sensory dendrite and/or axon patterning. This collection of mutants, together with anatomical data on dendrites and axons, should begin to permit studies of dendrite diversity in a combined developmental and functional context, and also provide a foundation for understanding shared and distinct mechanisms that control axon and dendrite morphology.     

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.     

Distinct rac activation pathways control Caenorhabditis elegans cell migration and axon outgrowth

Rac GTPases act as molecular switch in various morphogenic events. However, the regulation of their activities during the development of multicellular organisms is not well understood. Caenorhabditis elegans rac genes ced-10 and mig-2 have been shown to act redundantly to control P cell migration and the axon outgrowth of D type motoneurons. We showed that ced-10 and mig-2 also control amphid axon outgrowth and amphid dendrite fasciculation in a redundant fashion. Our biochemical and genetic data indicate that unc-73, which encodes a protein related to Trio-like guanine nucleotide exchange factor, acts as a direct activator of ced-10 and mig-2 during P cell migration and axon outgrowth of D type motoneurons and amphid sensory neurons. Furthermore, rac regulators ced-2/crkII and ced-5/dock180 function genetically upstream of ced-10 and mig-2 during axon outgrowth of D type motoneurons and act upstream of mig-2 but not ced-10 during P cell migration. However, neither ced-2/crkII nor ced-5/dock180 is involved in amphid axon outgrowth. Therefore, distinct rac regulators control ced-10 and mig-2 differentially in various cellular processes.     

Evidence for differential and redundant function of the Sox genes Dichaete and SoxN during CNS development in Drosophila

Group B Sox-domain proteins encompass a class of conserved DNA-binding proteins expressed from the earliest stages of metazoan CNS development. In all higher organisms studied to date, related Group B Sox proteins are co-expressed in the developing CNS; in vertebrates there are three (Sox1, Sox2 and Sox3) and in Drosophila there are two (SoxNeuro and Dichaete). It has been suggested there may be a degree of functional redundancy in Sox function during CNS development. We describe the CNS phenotype of a null mutation in the Drosophila SoxNeuro gene and provide the first direct evidence for both redundant and differential Sox function during CNS development in DROSOPHILA: In the lateral neuroectoderm, where SoxNeuro is uniquely expressed, SoxNeuro mutants show a loss or reduction of achaete expression as well as a loss of many correctly specified lateral neuroblasts. By contrast, in the medial neuroectoderm, where the expression of SoxNeuro and Dichaete overlaps, the phenotypes of both single mutants are mild. In accordance with an at least partially redundant function in that region, SoxNeuro/Dichaete double mutant embryos show a severe neural hypoplasia throughout the central nervous system, as well as a dramatic loss of achaete expressing proneural clusters and medially derived neuroblasts. However, the finding that Dichaete and SoxN exhibit opposite effects on achaete expression within the intermediate neuroectoderm demonstrates that each protein also has region-specific unique functions during early CNS development in the Drosophila embryo.