Drosophila single-minded gene and the molecular genetics of CNS midline development.

Our goal is to understand the molecular mechanisms that govern the formation of the central nervous system. In particular, we have focused on the development of a small group of neurons and glia that lie along the midline of the Drosophila CNS. These midline cells possess a number of unique attributes which make them particularly amenable to molecular, cellular, and genetic examinations of nervous system formation and function. In addition, the midline cells exhibit distinctive ontogeny, morphology, anatomical position, and patterns of gene expression which suggest that they may provide unique functions to the developing CNS. The single-minded gene encodes a nuclear protein which is specifically expressed in the midline cells and has been shown to play a crucial role in midline cell development and CNS formation. Genetic experiments reveal that sim is required for the expression of many CNS midline genes which are thought to be involved in the proper differentiation of these cells. In order to identify additional genes which are expressed in some or all of the midline cells at different developmental stages, a technique known as enhancer trap screening was employed. This screen led to the identification of a large number of potential genes which exhibit various midline expression patterns and may be involved in discrete aspects of midline cell development. Further molecular, genetic, and biochemical analyses of sim and several of the enhancer trap lines are being pursued. This should permit elucidation of the genetic hierarchy which acts in the specification, differentiation, and function of these CNS midline cells.     

The jing and ras1 pathways are functionally related during CNS midline and tracheal development

The Drosophila jing gene encodes a zinc finger protein required for the differentiation and survival of embryonic CNS midline and tracheal cells. We show that there is a functional relationship between jing and the Egfr pathway in the developing CNS midline and trachea. jing function is required for Egfr pathway gene expression and MAPK activity in both the CNS midline and trachea. jing over-expression effects phenocopy those of the Egfr pathway and require Egfr pathway function. Activation of the Egfr pathway in loss-of-function jing mutants partially rescues midline cell loss. Egfr pathway genes and jing show dominant genetic interactions in the trachea and CNS midline. Together, these results show that jing regulates signal transduction in developing midline and tracheal cells.     

The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells

The single-minded (sim) gene of Drosophila encodes a nuclear protein that plays a critical role in the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the embryonic CNS. Using distinct cell fate markers, we observe that in sim mutant embryos the midline cells fail to differentiate properly into their mature CNS cell types and do not take their appropriate positions within the developing CNS. We further present evidence that sim is required for midline expression of a group of genes including slit, Toll, rhomboid, engrailed, and a gene at 91F; that the sim mutant CNS defect may be largely due to loss of midline slit expression; and that the snail gene is required to repress sim and other midline genes in the presumptive mesoderm.     

The CNS midline cells coordinate proper cell cycle progression and identity determination of the Drosophila ventral neuroectoderm

The CNS midline cells, specified by the single-minded (sim) gene, are required for the proper patterning of the ventral CNS and epidermis, which are derived from the Drosophila ventral neuroectoderm. Defects in the sim mutant are characterized by the loss of the gene expression, which is required for the proper formation of the ventral neurons and epidermis, and by a decrease in the spacing of longitudinal and commissural axon tracks. Molecular and cellular mechanisms for these defects were analyzed to elucidate the precise role of the CNS midline cells in proper patterning of the ventral neuroectoderm during embryonic neurogenesis. These analyses showed that the ventral neuroectoderm in the sim mutant fails to carry out its proper formation and characteristic cell division cycle. This resulted in the loss of the dividing neuroectodermal cells that are located ventral to the CNS midline. The CNS midline cells are also required for the cell cycle-independent expression of the neural and epidermal markers. This indicates that the CNS midline cells are essential for the establishment and maintenance of the ventral epidermal and neuronal cell lineage by cell-cell interaction. On the other hand, the CNS midline cells do not cause extensive cell death in the ventral neuroectoderm. This study indicates that the CNS midline cells play important roles in the coordination of the proper cell cycle progression and the correct identity determination of the adjacent ventral neuroectoderm along the dorsoventral axis.     

MidExDB: A database of Drosophila CNS midline cell gene expression

Background  

The Drosophila CNS midline cells are an excellent model system to study neuronal and glial development because of their diversity of cell types and the relative ease in identifying and studying the function of midline-expressed genes. In situ hybridization experiments generated a large dataset of midline gene expression patterns. To help synthesize these data and make them available to the scientific community, we developed a web-accessible database.     

CNS midline cells are required for establishment and differentiation of Drosophila MP2 interneurons

The Drosophila CNS develops from the ventral neuroectoderm (VNE) on both sides of the midline along the dorsoventral axis. During early neurogenesis, three homeodomain and Egfr signaling genes are required for the dorsoventral patterning of the VNE. However, the roles of CNS midline cells in patterning of the specific neural lineages are not well understood. Their roles in identity determination and differentiation of the well-established MP2 lineage were studied using several molecular markers. We showed that these cells are essential for identity determination of the MP2 lineage that originates from the VNE. The midline cells and the Egfr signaling genes were also required for the proper maintenance of MP2 and the correct formation of MP2 axonal pathways. Overexpression of sim in the midline cells activated ectopic expression of MP2 markers in the VNE. This analysis suggests that CNS midline cells and Egfr signaling genes play essential roles in the proper establishment and differentiation of the MP2 lineage.     

Effects of IKAP/hELP1 deficiency on gene expression in differentiating neuroblastoma cells: implications for familial dysautonomia

Familial dysautonomia (FD) is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype.     

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.     

Interaction of notochord-derived fibrinogen-like protein with Notch regulates the patterning of the central nervous system of Ciona intestinalis embryos

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.     

The phosphoinositide phosphatase Sac1 is required for midline axon guidance

Sac1 phosphoinositide (PI) phosphatases are important regulators of PtdIns(4)P turnover at the ER, Golgi, and plasma membrane (PM) and are involved in diverse cellular processes including cytoskeletal organization and vesicular trafficking. Here, we present evidence that Sac1 regulates axon guidance in the embryonic CNS of Drosophila. Sac1 is expressed on three longitudinal axon tracts that are defined by the cell adhesion molecule Fasciclin II (Fas II). Mutations in the sac1 gene cause ectopic midline crossing of Fas II-positive axon tracts. This phenotype is rescued by neuronal expression of wild-type Sac1 but not by a catalytically-inactive mutant. Finally, sac1 displays dosage-sensitive genetic interactions with mutations in the genes that encode the midline repellent Slit and its axonal receptor Robo. Taken together, our results suggest that Sac1-mediated regulation of PIs is critical for Slit/Robo-dependent axon repulsion at the CNS midline.     

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.     

Segregation of postembryonic neuronal and glial lineages inferred from a mosaic analysis of the Drosophila larval brain

Due to its intermediate complexity and its sophisticated genetic tools, the larval brain of Drosophila is a useful experimental system to study the mechanisms that control the generation of cell diversity in the CNS. In order to gain insight into the neuronal and glial lineage specificity of neural progenitor cells during postembryonic brain development, we have carried an extensive mosaic analysis throughout larval brain development. In contrast to embryonic CNS development, we have found that most postembryonic neurons and glial cells of the optic lobe and central brain originate from segregated progenitors. Our analysis also provides relevant information about the origin and proliferation patterns of several postembryonic lineages such as the superficial glia and the medial-anterior Medulla neuropile glia. Additionally, we have studied the spatio-temporal relationship between gcm expression and gliogenesis. We found that gcm expression is restricted to the post-mitotic cells of a few neuronal and glial lineages and it is mostly absent from postembryonic progenitors. Thus, in contrast to its major gliogenic role in the embryo, the function of gcm during postembryonic brain development seems to have evolved to the specification and differentiation of certain neuronal and glial lineages.