Three putative murine Teashirt orthologues specify trunk structures in Drosophila in the same way as the Drosophila teashirt gene

Drosophila teashirt (tsh) functions as a region-specific homeotic gene that specifies trunk identity during embryogenesis. Based on sequence homology, three tsh-like (Tsh) genes have been identified in the mouse. Their expression patterns in specific regions of the trunk, limbs and gut raise the possibility that they may play similar roles to tsh in flies. By expressing the putative mouse Tsh genes in flies, we provide evidence that they behave in a very similar way to the fly tsh gene. First, ectopic expression of any of the three mouse Tsh genes, like that of tsh, induces head to trunk homeotic transformation. Second, mouse Tsh proteins can rescue both the homeotic and the segment polarity phenotypes of a tsh null mutant. Third, following ectopic expression, the three mouse Tsh genes affect the expression of the same target genes as tsh in the Drosophila embryo. Fourth, mouse Tsh genes, like tsh, are able to induce ectopic eyes in adult flies. Finally, all Tsh proteins contain a motif that recruits the C-terminal binding protein and contributes to their repression function. As no other vertebrate or fly protein has been shown to induce such effects upon ectopic expression, these results are consistent with the idea that the three mouse Tsh genes are functionally equivalent to the Drosophila tsh gene when expressed in developing Drosophila embryos.     

Functional conservation of atonal and Math1 in the CNS and PNS

To determine the extent to which atonal and its mouse homolog Math1 exhibit functional conservation, we inserted (beta)-galactosidase (lacZ) into the Math1 locus and analyzed its expression, evaluated consequences of loss of Math1 function, and expressed Math1 in atonal mutant flies. lacZ under the control of Math1 regulatory elements duplicated the previously known expression pattern of Math1 in the CNS (i.e., the neural tube, dorsal spinal cord, brainstem, and cerebellar external granule neurons) but also revealed new sites of expression: PNS mechanoreceptors (inner ear hair cells and Merkel cells) and articular chondrocytes. Expressing Math1 induced ectopic chordotonal organs (CHOs) in wild-type flies and partially rescued CHO loss in atonal mutant embryos. These data demonstrate that both the mouse and fly homologs encode lineage identity information and, more interestingly, that some of the cells dependent on this information serve similar mechanoreceptor functions.     

Conserved and divergent functions of Drosophila atonal,amphibian, and mammalian Ath5 genes

Insect and vertebrate eyes differ in their formation, cellular composition, neural connectivity, and visual function. Despite this diversity, Drosophila atona and its vertebrate Ortholog in the eye, Ath5, each regulate determination of the first retinal neuron class-R8 photo-receptors and retinal ganglion cells (RGCs)-in their respective organisms. We have performed a cross-species functional comparison of these genes. In ato mutant Drosophila, ectopic Xenopus Ath5 (Xath5) rescues photoreceptor cell development comparably with atonaI. In contrast, mouse Ath5 (Math5) induces formation of very few ommatidia, and most of these lack R8 cells. In the developing frog eye, ectopic atonal, like Xath5, promotes the differentiation RGCs. Despite strong conservation of atonaI, Xath5, and Math5 structure and shared function, other factors must contribute to the species specificity of retinal neuron determination. These observations suggest that the atonaI family may occupy a position in a gene hierarchy where differences in gene regulation or function can be correlated with evolutionary diversity of eye development.     

Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase

Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. We have performed ple rescue experiments with modified DTH transgenes. Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila.     

The eukaryotic DNMT2 genes encode a new class of cytosine-5 DNA methyltransferases

DNMT2 is a subgroup of the eukaryotic cytosine-5 DNA methyltransferase gene family. Unlike the other family members, proteins encoded by DNMT2 genes were not known before to possess DNA methyltransferase activities. Most recently, we have shown that the genome of Drosophila S2 cells stably expressing an exogenous Drosophila dDNMT2 cDNA became anomalously methylated at the 5'-positions of cytosines (Reddy, M. N., Tang, L. Y., Lee, T. L., and Shen, C.-K. J. (2003) Oncogene, in press). We present evidence here that the genomes of transgenic flies overexpressing the dDnmt2 protein also became hypermethylated at specific regions. Furthermore, transient transfection studies in combination with sodium bisulfite sequencing demonstrated that dDnmt2 as well as its mouse ortholog, mDnmt2, are capable of methylating a cotransfected plasmid DNA. These data provide solid evidence that the fly and mouse DNMT2 gene products are genuine cytosine-5 DNA methyltransferases.     

The appendage role of insect disco genes and possible implications on the evolution of the maggot larval form

Though initially identified as necessary for neural migration, Disconnected and its partially redundant paralog, Disco-related, are required for proper head segment identity during Drosophila embryogenesis. Here, we present evidence that these genes are also required for proper ventral appendage development during development of the adult fly, where they specify medial to distal appendage development. Cells lacking the disco genes cannot contribute to the medial and distal portions of ventral appendages. Further, ectopic disco transforms dorsal appendages toward ventral fates; in wing discs, the medial and distal leg development pathways are activated. Interestingly, this appendage role is conserved in the red flour beetle, Tribolium (where legs develop during embryogenesis), yet in the beetle we found no evidence for a head segmentation role. The lack of an embryonic head specification role in Tribolium could be interpreted as a loss of the head segmentation function in Tribolium or gain of this function during evolution of flies. However, we suggest an alternative explanation. We propose that the disco genes always function as appendage factors, but their appendage nature is masked during Drosophila embryogenesis due to the reduction of limb fields in the maggot style Drosophila larva.     

The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal.

The peripheral nervous system (PNS) of Drosophila offers a powerful system to precisely identify individual cells and dissect their genetic pathways of development. The mode of specification of a subset of larval PNS cells, the multiple dendritic (md) neurons (or type II neurons), is complex and still poorly understood. Within the dorsal thoracic and abdominal segments, two md neurons, dbd and dda1, apparently require the proneural gene amos but not atonal (ato) or Achaete-Scute-Complex (ASC) genes. ASC normally acts via the neural selector gene cut to specify appropriate sensory organ identities. Here, we show that dbd- and dda1-type differentiation is suppressed by cut in dorsal ASC-dependent md neurons. Thus, cut is not only required to promote an ASC-dependent mode of differentiation, but also represses an ASC- and ato-independent fate that leads to dbd and dda1 differentiation.     

Mammalian achaete-scute and atonal homologs regulate neuronal versus glial fate determination in the central nervous system

Whereas vertebrate achaete-scute complex (as-c) and atonal (ato) homologs are required for neurogenesis, their neuronal determination activities in the central nervous system (CNS) are not yet supported by loss-of-function studies, probably because of genetic redundancy. Here, to address this problem, we generated mice double mutant for the as-c homolog Mash1 and the ato homolog Math3. Whereas in Mash1 or Math3 single mutants neurogenesis is only weakly affected, in the double mutants tectal neurons, two longitudinal columns of hindbrain neurons and retinal bipolar cells were missing and, instead, those cells that normally differentiate into neurons adopted the glial fate. These results indicated that Mash1 and Math3 direct neuronal versus glial fate determination in the CNS and raised the possibility that downregulation of these bHLH genes is one of the mechanisms to initiate gliogenesis.     

Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3

Deciphering the expression pattern of K+ channel encoding genes during development can help in the understanding of the establishment of cellular excitability and unravel the molecular mechanisms of neuromuscular diseases. We focused our attention on genes belonging to the erg family, which is deeply involved in the control of neuromuscular excitability in Drosophila flies and possibly other organisms. Both in situ hybridisation and RNase Protection Assay experiments were used to study the expression pattern of mouse (m)erg1, m-erg2 and m-erg3 genes during mouse embryo development, to allow the pattern to be compared with their expression in the adult. M-erg1 is first expressed in the heart and in the central nervous system (CNS) of embryonic day 9.5 (E9.5) embryos; the gene appears in ganglia of the peripheral nervous system (PNS) (dorsal root (DRG) and sympathetic (SCG) ganglia, mioenteric plexus), in the neural layer of retina, skeletal muscles, gonads and gut at E13.5. In the adult m-erg1 is expressed in the heart, various structures of the CNS, DRG and retina. M-erg2 is first expressed at E9.5 in the CNS, thereafter (E13.5) in the neural layer of retina, DRG, SCG, and in the atrium. In the adult the gene is present in some restricted areas of the CNS, retina and DRG. M-erg3 displayed an expression pattern partially overlapping that of m-erg1, with a transitory expression in the developing heart as well. A detailed study of the mouse adult brain showed a peculiar expression pattern of the three genes, sometimes overlapping in different encephalic areas.     

The embryonic expression patterns of zfh-1 and zfh-2, two Drosophila genes encoding novel zinc-finger homeodomain proteins.

The zfh-1 and zfh-2 genes of D. melanogaster encode novel proteins containing both homeodomain and C2-H2 zinc-finger DNA-binding motifs. Antisera against these proteins were used to investigate their expression patterns during embryonic development. The zfh-1 gene is expressed in the mesoderm of early embryos and in a number of mesodermally-derived structures of late embryos, including the dorsal vessel, support cells of the gonads, and segment-specific arrays of adult muscle precursors. In addition, zfh-1 is expressed in the majority of identified motor neurons of the developing CNS. The mesodermal zfh-1 expression requires the products of the twist and snail genes. The zfh-2 gene displays a more limited expression pattern, largely restricted to the CNS of late embryos. Ubiquitous zfh-1 expression in transgenic flies bearing an hsp70-zfh-1 construct has specific developmental consequences, including embryonic CNS defects as well as adult eye and bristle abnormalities. The expression patterns of zfh-1 and zfh-2 suggest that both genes may be involved in Drosophila neurogenesis and that zfh-1 may have additional functions in mesoderm development.     

Cooperation between Drosophila flies in searching behavior

In Drosophila melanogaster food search behaviour, groups of flies swarm around and aggregate on patches of food. We wondered whether flies explore their environment in a cooperative way as interactions between individual flies within a population might influence the flies' ability to locate food sources. We have shown that the food search behavior in the fruit fly Drosophila is a two-step process. Firstly, 'primer' flies search the environment and randomly land on different food patches. Secondly, the remaining group of flies move to the most favorable food source and aggregate there. We call this a 'search–aggregation' cycle. Our data demonstrate that flies do not individually assess all available food resources. Rather, social interactions between flies appear to affect their choice of a specific food patch. A genetic analysis of this 'search–aggregation' behavior shows that flies carrying mutations in specific genes (for example, the dunce ( dnc ) gene which codes for a phosphodiesterase) were defective in this search–aggregation behavior when compared to normal flies. Future investigations of the neuronal signaling involved in this behavior will help us to understand the complexities of this aspect of Drosophila social behaviour.     

Diverged and conserved aspects of heart formation in a spider

Heart development exhibits some striking similarities between vertebrates and arthropods, for example in both cases the heart develops as a linear tube from mesodermal cells. Furthermore, the underlying molecular pathways exhibit a significant number of similarities between vertebrates and the fruit fly Drosophila, suggesting a common origin of heart development in the last common ancestor of flies and vertebrates. However, there is hardly any molecular data from other animals. Here we show that many of the key genes are also active in heart development in the spider Cupiennius salei. Spiders belong to the chelicerates and are distantly related to insects with respect to the other arthropods. The tinman/Nkx2.5 ortholog is the first gene to be specifically expressed in the presumptive spider heart, like in flies and vertebrates. We also show that tinman is expressed in a similar way in the beetle Tribolium castaneum. Taken together this demonstrates that tinman has a conserved role in the specification of the arthropod heart. In addition, we analyzed the expression of other heart genes (decapentaplegic, Wnt5, H15, even-skipped, and Mef2 ) in Cupiennius. The expression of these genes suggests that the genetic pathway of heart development may be largely conserved among arthropods. However, a major difference is seen in the earlier expression of the even-skipped gene in the developing spider heart compared with Drosophila, implying that the role of even-skipped in heart formation might have changed during arthropod evolution. The most striking finding, however, is that in addition to the dorsal tissue of the fourth walking leg segment and the opisthosomal segments, we discovered tinman-expressing cells that arise from a position dorsal to the cephalic lobe and that contribute to the anterior dorsal vessel. In contrast to the posterior heart tissue, these cells do not express the other heart genes. The spider heart thus is composed of two distinct populations of cells.     

Identification of a mouse germ cell-less homologue with conserved activity in Drosophila

Drosophila Germ cell-less (Gcl) has previously been shown to be important in early events during the formation of pole cells, which are the germ cell precursors in the fly. We have isolated a 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homologue of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed.     

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.     

Rough eye is a gain-of-function allele of amos that disrupts regulation of the proneural gene atonal during Drosophila retinal differentiation

The regular organization of the ommatidial lattice in the Drosophila eye originates in the precise regulation of the proneural gene atonal (ato), which is responsible for the specification of the ommatidial founder cells R8. Here we show that Rough eye (Roi), a dominant mutation manifested by severe roughening of the adult eye surface, causes defects in ommatidial assembly and ommatidial spacing. The ommatidial spacing defect can be ascribed to the irregular distribution of R8 cells caused by a disruption of the patterning of ato expression. Disruptions in the recruitment of other photoreceptors and excess Hedgehog production in differentiating cells may further contribute to the defects in ommatidial assembly. Our molecular characterization of the Roi locus demonstrates that it is a gain-of-function mutation of the bHLH gene amos that results from a chromosomal inversion. We show that Roi can rescue the retinal developmental defect of ato1 mutants and speculate that amos substitutes for some of ato's function in the eye or activates a residual function of the ato1 allele.