Functional equivalence of Hox gene products in the specification of the tritocerebrum during embryonic brain development of Drosophila.

Hox genes encode evolutionarily conserved transcription factors involved in the specification of segmental identity during embryonic development. This specification of identity is thought to be directed by differential Hox gene action, based on differential spatiotemporal expression patterns, protein sequence differences, interactions with co-factors and regulation of specific downstream genes. During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere; in the absence of labial, the cells in this brain region do not acquire a neuronal identity and major axonal pathfinding deficits result. We have used genetic rescue experiments to investigate the functional equivalence of the Drosophila Hox gene products in the specification of the tritocerebral neuromere. Using the Gal4-UAS system, we first demonstrate that the labial mutant brain phenotype can be rescued by targeted expression of the Labial protein under the control of CNS-specific labial regulatory elements. We then show that under the control of these CNS-specific regulatory elements, all other Drosophila Hox gene products, except Abdominal-B, are able to efficiently replace Labial in the specification of the tritocerebral neuromere. We also observe a correlation between the rescue efficiency of the Hox proteins and the chromosomal arrangement of their encoding loci. Our results indicate that, despite considerably diverged sequences, most Hox proteins are functionally equivalent in their ability to replace Labial in the specification of neuronal identity. This suggests that in embryonic brain development, differences in Hox gene action rely mainly on cis-acting regulatory elements and not on Hox protein specificity.

Identification of candidate downstream genes for the homeodomain transcription factor Labial in Drosophila through oligonucleotide-array transcript imaging.

BACKGROUND: Homeotic genes are key developmental regulators that are highly conserved throughout evolution. Their encoded homeoproteins function as transcription factors to control a wide range of developmental processes. Although much is known about homeodomain-DNA interactions, only a small number of genes acting downstream of homeoproteins have been identified. Here we use a functional genomic approach to identify candidate target genes of the Drosophila homeodomain transcription factor Labial. RESULTS: High-density oligonucleotide arrays with probe sets representing 1,513 identified and sequenced genes were used to analyze differential gene expression following labial overexpression in Drosophila embryos. We find significant expression level changes for 96 genes belonging to all functional classes represented on the array. In accordance with our experimental procedure, we expect that these genes are either direct or indirect targets of labial gene action. Among these genes, 48 were upregulated and 48 were downregulated following labial overexpression. This corresponds to 6.3% of the genes represented on the array. For a selection of these genes, we show that the data obtained with the oligonucleotide arrays are consistent with data obtained using quantitative RT-PCR. CONCLUSIONS: Our results identify a number of novel candidate downstream target genes for Labial, suggesting that this homeoprotein differentially regulates a limited and distinct set of embryonically expressed Drosophila genes.

Differential expression and function of the Drosophila Pax6 genes eyeless and twin of eyeless in embryonic central nervous system development.

We analyzed the expression and function of eyeless (ey) and twin of eyeless (toy) in the embryonic central nervous system (CNS) of Drosophila. Both genes are differentially expressed in specific neuronal subsets (but not in glia) in every CNS neuromere, and in the brain, specific cell populations co-expressing both proteins define a longitudinal domain which is intercalated between broad exclusive expression domains of ey and toy. Studies of genetic null alleles and dsRNA interference did not reveal any gross neuroanatomical effects of ey, toy, or ey/toy elimination in the embryonic CNS. In contrast, targeted misexpression of ey, but not of toy, resulted in profound axonal abnormalities in the embryonic ventral nerve cord and brain.

Drosophila transcription factor AP-2 in proboscis, leg and brain central complex development.

We report loss- and gain-of-function analyses that identify essential roles in development for Drosophila transcription factor AP-2. A mutagenesis screen yielded 16 lethal point mutant alleles of dAP-2. Null mutants die as adults or late pupae with a reduced proboscis, severely shortened legs (approximately 30% of normal length) lacking tarsal joints, and disruptions in the protocerebral central complex, a brain region critical for locomotion. Seven hypomorphic alleles constitute a phenotypic series yielding hemizygous adults with legs ranging from 40-95% of normal length. Hypomorphic alleles show additive effects with respect to leg length and viability; and several heteroallelic lines were established. Heteroallelic adults have moderately penetrant defects that include necrotic leg joints and ectopic growths (sometimes supernumerary antennae) invading medial eye territory. Several dAP-2 alleles with DNA binding domain missense mutations are null in hemizygotes but have dominant negative effects when paired with hypomorphic alleles. In wild-type leg primordia, dAP-2 is restricted to presumptive joints. Ectopic dAP-2 in leg discs can inhibit but not enhance leg elongation indicating that functions of dAP-2 in leg outgrowth are region restricted. In wing discs, ectopic dAP-2 cell autonomously transforms presumptive wing vein epithelium to ectopic sensory bristles, consistent with an instructive role in sensory organ development. These findings reveal multiple functions for dAP-2 during morphogenesis of feeding and locomotor appendages and their neural circuitry, and provide a new paradigm for understanding AP-2 family transcription factors.

Expression and function of the LIM homeodomain protein Apterous during embryonic brain development of Drosophila.

We analyzed the expression and function of the LIM-homeodomain transcription factor Apterous (Ap ) in embryonic brain development of Drosophila. Expression of Ap in the embryonic brain begins at early stage 12 and is subsequently found in approximately 200 protocerebral neurons and in 4 deutocerebral neurons. Brain glia do not express Ap. Most of the Ap-expressing neurons are interneurons and project their axons across the midline to the contralateral hemisphere; a smaller subset projects their axons into the ventral nerve cord. A few Ap-expressing neurons project to the ring gland, suggesting that they are neurosecretory cells. In ap loss-of-function mutants, some of the protocerebral and deutocerebral interneurons that express Ap in the wild type show axon pathfinding errors and fasciculation defects in the brain, notably in the fascicles of the brain commissure. In contrast, the interneurons that project to the ring gland do not appear to be affected in ap mutants. Thus, in brain development, Ap is required for correct axon guidance and fasciculation of interneurons, and Ap-expressing cells may also be involved in the brain neuroendocrine system.

Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays.

Embryonic development in Drosophila is characterized by an early phase during which a cellular blastoderm is formed and gastrulation takes place, and by a later postgastrulation phase in which key morphogenetic processes such as segmentation and organogenesis occur. We have focused on this later phase in embryogenesis with the goal of obtaining a comprehensive analysis of the zygotic gene expression that occurs during development under normal and altered environmental conditions. For this, a functional genomic approach to embryogenesis has been developed that uses high-density oligonucleotide arrays for large-scale detection and quantification of gene expression. These oligonucleotide arrays were used for quantitative transcript imaging of embryonically expressed genes under standard conditions and in response to heat shock. In embryos raised under standard conditions, transcripts were detected for 37% of the 1,519 identified genes represented on the arrays, and highly reproducible quantification of gene expression was achieved in all cases. Analysis of differential gene expression after heat shock revealed substantial expression level changes for known heat-shock genes and identified numerous heat shock-inducible genes. These results demonstrate that high-density oligonucleotide arrays are sensitive, efficient, and quantitative instruments for the analysis of large scale gene expression in Drosophila embryos.

Expression, regulation and function of the homeobox gene empty spiracles in brain and ventral nerve cord development of Drosophila.

We analyse the role of the empty spiracles (ems) gene in embryonic brain and ventral nerve cord development. ems is differentially expressed in the neurectoderm of the anterior head versus the trunk region of early embryos. A distal enhancer region drives expression in the deutocerebral brain anlage and a proximal enhancer region drives expression in the VNC and tritocerebral brain anlage. Mutant analysis indicates that in the anterior brain ems is necessary for regionalized neurogenesis in the deutocerebral and tritocerebral anlagen. In the posterior brain and VNC ems is necessary for correct axonal pathfinding of specific interneurons. Rescue experiments indicate that the murine Emx2 gene can partially replace the fly ems gene in CNS development.

Conserved genetic programs in insect and mammalian brain development.

In recent years it has become evident that the developmental regulatory genes involved in patterning the embryonic body plan are conserved throughout the animal kingdom. Striking examples are the orthodenticle (otd/Otx) gene family and the Hox gene family, both of which act in the specification of anteroposterior polarity along the embryonic body axis. Studies carried out in Drosophila and mouse now demonstrate that these genes are also involved in the formation of the insect and mammalian brain; the otd/Otx genes are involved in rostral brain development and the Hox genes are involved in caudal brain development. These studies also show that the genes of the otd/Otx family can functionally replace each other in cross-phylum rescue experiments and indicate that the genetic mechanisms underlying pattern formation in insect and mammalian brain development are evolutionarily conserved.

Homeotic gene action in embryonic brain development of Drosophila.

Studies in vertebrates show that homeotic genes are involved in axial patterning and in specifying segmental identity of the embryonic hindbrain and spinal cord. To gain further insights into homeotic gene action during CNS development, we here characterize the role of the homeotic genes in embryonic brain development of Drosophila. We first use neuroanatomical techniques to map the entire anteroposterior order of homeotic gene expression in the Drosophila CNS, and demonstrate that this order is virtually identical in the CNS of Drosophila and mammals. We then carry out a genetic analysis of the labial gene in embryonic brain development. Our analysis shows that loss-of-function mutation and ubiquitous overexpression of labial results in ectopic expression of neighboring regulatory genes. Furthermore, this analysis demonstrates that mutational inactivation of labial results in regionalized axonal patterning defects which are due to both cell-autonomous and cell-nonautonomous effects. Thus, in the absence of labial, mutant cells are generated and positioned correctly in the brain, but these cells do not extend axons. Additionally, extending axons of neighboring wild-type neurons stop at the mutant domains or project ectopically, and defective commissural and longitudinal pathways result. Immunocytochemical analysis demonstrates that cells in the mutant domains do not express neuronal markers, indicating a complete lack of neuronal identity. An alternative glial identity is not adopted by these mutant cells. Comparable effects are seen in Deformed mutants but not in other homeotic gene mutants. Our findings demonstrate that the action of the homeotic genes labial and Deformed are required for neuronal differentiation in the developing brain of Drosophila.

Equivalence of the fly orthodenticle gene and the human OTX genes in embryonic brain development of Drosophila.

Members of the orthodenticle gene family are essential for embryonic brain development in animals as diverse as insects and mammals. In Drosophila, mutational inactivation of the orthodenticle gene results in deletions in anterior parts of the embryonic brain and in defects in the ventral nerve cord. In the mouse, targeted elimination of the homologous Otx2 or Otx1 genes causes defects in forebrain and/or midbrain development. To determine the morphogenetic properties and the extent of evolutionary conservation of the orthodenticle gene family in embryonic brain development, genetic rescue experiments were carried out in Drosophila. Ubiquitous overexpression of the orthodenticle gene rescues both the brain defects and the ventral nerve cord defects in orthodenticle mutant embryos; morphology and nervous system-specific gene expression are restored. Two different time windows exist for the rescue of the brain versus the ventral nerve cord. Ubiquitous overexpression of the human OTX1 or OTX2 genes also rescues the brain and ventral nerve cord phenotypes in orthodenticle mutant embryos; in the brain, the efficiency of morphological rescue is lower than that obtained with overexpression of orthodenticle. Overexpression of either orthodenticle or the human OTX gene homologs in the wild-type embryo results in ectopic neural structures. The rescue of highly complex brain structures in Drosophila by either fly or human orthodenticle gene homologs indicates that these genes are interchangeable between vertebrates and invertebrates and provides further evidence for an evolutionarily conserved role of the orthodenticle gene family in brain development.

Embryonic development of the Drosophila brain: formation of commissural and descending pathways.

The establishment of initial axonal pathways in the embryonic brain of Drosophila melanogaster was investigated at the cellular and molecular level using antibody probes, enhancer detector strains and axonal pathfinding mutants. During embryogenesis, two bilaterally symmetrical cephalic neurogenic regions form, which are initially separated from each other and from the ventral nerve cord. The brain commissure that interconnects the two brain hemispheres is pioneered by axons that project towards the midline in close association with an interhemispheric cellular bridge. The descending longitudinal pathways that interconnect the brain to the ventral nerve cord are prefigured by a chain of longitudinal glial cells and a cellular bridge between brain and subesophageal ganglion; pioneering descending and ascending neurons grow in close association with these structures. The formation of the embryonic commissural and longitudinal pathways is dependent on cells of the CNS midline. Mutations in the commissureless gene, which affects growth cone guidance towards the midline, result in a marked reduction of the brain commissure. Mutations in the single-minded gene and in other spitz group genes, which affect the differentiation of CNS midline cells, result in the absence or aberrant projection of longitudinal pathways. The analysis of axon pathway formation presented here reveals remarkable similarities as well as distinct differences in the embryonic development of the brain and the segmental ganglia, and forms the basis for a comprehensive genetic and molecular genetic dissection of axonal pathfinding processes in the developing brain.

Developmental defects in brain segmentation caused by mutations of the homeobox genes orthodenticle and empty spiracles in Drosophila.

We have studied the roles of the homeobox genes orthodenticle (otd) and empty spiracles (ems) in embryonic brain development of Drosophila. The embryonic brain is composed of three segmental neuromeres. The otd gene is expressed predominantly in the anterior neuromere; expression of ems is restricted to the two posterior neuromeres. Mutation of otd eliminates the first (protocerebral) brain neuromere. Mutation of ems eliminates the second (deutocerebral) and third (tritocerebral) neuromeres. otd is also necessary for development of the dorsal protocerebrum of the adult brain. We conclude that these homeobox genes are required for the development of specific brain segments in Drosophila, and that the regionalized expression of their homologs in vertebrate brains suggests an evolutionarily conserved program for brain development.