Drosophila couch potato mutants exhibit complex neurological abnormalities including epilepsy phenotypes

RNA-binding proteins play critical roles in regulation of gene expression, and impairment can have severe phenotypic consequences on nervous system function. We report here the discovery of several complex neurological phenotypes associated with mutations of couch potato (cpo), which encodes a Drosophila RNA-binding protein. We show that mutation of cpo leads to bang-sensitive paralysis, seizure susceptibility, and synaptic transmission defects. A new cpo allele called cpo(EG1) was identified on the basis of a bang-sensitive paralytic mutant phenotype in a sensitized genetic background (sda/+). In heteroallelic combinations with other cpo alleles, cpo(EG1) shows an incompletely penetrant bang-sensitive phenotype with approximately 30% of flies becoming paralyzed. In response to electroconvulsive shock, heteroallelic combinations with cpo(EG1) exhibit seizure thresholds less than half that of wild-type flies. Finally, cpo flies display several neurocircuit abnormalities in the giant fiber (GF) system. The TTM muscles of cpo mutants exhibit long latency responses coupled with decreased following frequency. DLM muscles in cpo mutants show drastic reductions in following frequency despite exhibiting normal latency relationships. The labile sites appear to be the electrochemical GF-TTMn synapse and the chemical PSI-DLMn synapses. These complex neurological phenotypes of cpo mutants support an important role for cpo in regulating proper nervous system function, including seizure susceptibility.     

Analysis of Gene Expression Following Sciatic Nerve Crush and Spinal Cord Hemisection in the Mouse by Microarray Expression Profiling

1. The responses of periphery (PNS) and central nervous systems (CNS) towards nerve injury are different: while injured mammalian periphery nerons can successfully undergo regeneration, axons in the central nervous system are usually not able to regenerate.2. In the present study, the genes which were differentially expressed in the PNS and CNS following nerve injury were identified and compared by microarray profiling techniques.3. Sciatic nerve crush and hemisection of the spinal cord of adult mice were used as the models for nerve injury in PNS and CNS respectively.4. It was found that of all the genes examined, 14% (80/588) showed changes in expression following either PNS or CNS injury, and only 3% (18/588) showed changes in both types of injuries.5. Among all the differentially expressed genes, only 8% (6/80) exhibited similar changes in gene expression (either up- or down-regulation) following injury in both PNS and CNS nerve injuries.6. Our results indicated that microarray expression profiling is an efficient and useful method to identify genes that are involved in the regeneration process following nerve injuries, and several genes which are differentially expressed in the PNS and/or CNS following nerve injuries were identified in the present study.     

THE INTENSITY OF SELECTION ACTING ON THE COUCH POTATO GENE—SPATIAL–TEMPORAL VARIATION IN A DIAPAUSE CLINE

Cosmopolitan populations of Drosophila melanogaster have co‐opted a form of reproductive diapause to overwinter in northern populations. Polymorphism in the couch potato gene has been implicated in genetic variation for this diapause trait. Using a collection of 20 populations from Florida to Canada and 11 collections from 3 years in a Pennsylvania orchard, we estimated the allele frequencies for 15 single nucleotide polymorphisms (SNPs) in the couch potato gene. These include the specific polymorphism associated with diapause inducability. We find that the SNP polymorphism, 48034(A/T), is correlated with latitude and its frequencies are predicted by the incidence of diapause trait. We find that the clinal patterns for cpo SNPs sampled in 1997 are similar to the same SNPs sampled in 2009–2010. SNPs that show apparent associations with cpo expression are also clinal with the low‐expression allele increasing in frequency, as would be predicted from functional knockout studies of cpo. Finally, we see a significant pattern where the frequency of the diapause‐causing allele drops in frequency during the summer season, consistent with the drop in the incidence of the diapause trait. The selection required to drive this response is large, roughly 24% to 59% per generation depending on the degree of dominance.     

Polymorphism in the couch potato gene clines in eastern Australia but is not associated with ovarian dormancy in Drosophila melanogaster

Natural selection can generate parallel latitudinal clines in traits and gene frequencies across continents, but these have rarely been linked. An amino acid (isoleucine to lysine, or I462K) polymorphism of the couch potato (cpo) gene in Drosophila melanogaster is thought to control female reproductive diapause cline in North America (Schmidt et al. 2008, Proc Natl Acad Sci USA, 105, 16207-16211). Here, we show that under standard diapause-inducing conditions (12 °C and short photoperiod) (Saunders et al. 1989, Proc Natl Acad Sci USA, 86, 3748-3752), egg maturation in Australian flies is delayed, but not arrested at previtellogenic stages. At 12 °C, the phenotypic distribution in egg development was bimodal at stages 8 and 14 and showed a strong nonlinear pattern on the east coast of Australia, with incidence of egg maturation delay (ovarian dormancy) increasing both toward tropical and temperate climates. Furthermore, we found no evidence for an association between the cpo I462K polymorphism and ovarian dormancy at either 12 or 10 °C (when egg maturation was often delayed at stage 7). Owing to strong linkage disequilibrium, the latitudinal cline in cpo allele frequencies was no longer evident once variation in the In(3R)P inversion polymorphism was taken into account. Our results suggest that the standard diapause-inducing conditions (12 °C and short photoperiod) were not sufficient to cause the typical previtellogenic developmental arrest in Australian flies and that the cpo I462K polymorphism does not explain the observed delay in egg development. In conclusion, ovarian dormancy does not show a simple latitudinal cline, and the lack of cpo-dormancy association suggests a different genetic basis to reproductive dormancy in North America and Australia.     

Expression level of sarah, a homolog of DSCR1, is critical for ovulation and female courtship behavior in Drosophila melanogaster

To better understand the genetic bases of postmating responses in Drosophila melanogaster females, we screened a collection of P{GS} insertion lines and identified two insertions in sarah (sra), whose misexpression in the nervous system induced high levels of ovulation in virgins. The gene sra encodes a protein similar to human Down syndrome critical region 1 (DSCR1). The ovulation phenotype was reproduced in transgenic virgins expressing UAS-sra in the nervous system. The flies also extruded the ovipositor toward courting males as seen in wild-type mated females, supporting the notion that ovulation and behavioral patterns are physiologically coupled. The sra insertions were found to be hypomorphic alleles with reduced expression levels. Females homozygous for these alleles show: (1) spontaneous ovulation in virgins, (2) sterility with impaired meiotic progression, and (3) compromised postmating responses with lower ovulation level, higher remating rate, and shorter period for restoration of receptivity. No obvious defects were observed in the homozygous males. The gene sra is predominantly expressed in oocytes, nurse cells, and the nervous system. Taken together, these results indicate that the expression level of sra is critical for ovulation and female courtship behavior, including their postmating changes.     

赤链蛇不同组织Sox基因表达的RT-PCR分析

采用RT-PCR技术,研究了赤链蛇不同组织Sox基因的表达。通过PCR产物直接克隆法和SSCP技术筛选阳性克隆,分析了雄性睾丸和雌性卵巢组织中的Sox基因序列。结果显示,在赤链蛇雌雄成体组织中,Sox基因在睾丸、卵巢、脑和脾组织中均有不同程度的表达,而在雌雄成体肌肉组织中均无表达,显示该基因表达有一定的组织特异性。序列分析显示睾丸组织中表达的是DRSox3,卵巢组织中表达的是DRSox22。在Sox家族中,Sox3表达于中枢神经系统和尿生殖嵴的发育过程中;Sox22则表达于多种组织和神经系统中,可横跨CNS和PNS的整个过程。此结果表明Sox基因不仅在性别决定中起作用,还可能在胚胎发育过程中担负重要功能。     

Mutations in neuromusculin,a gene encoding a cell adhesion molecule,cause nervous system defects

The Drosophila neuromusculin (nrm) gene encodes an immunoglobulin-like (Ig-like) cell adhesion molecule expressed in the precursors of the embryonic peripheral nervous system (PNS), in the midline precursors of the central nervous system (CNS), and in muscles. During the initial phases of CNS axonogenesis, nrm is expressed in cells involved in the development of commissures and longitudinal tracts. Mutations which alter expression of nrm mRNAs cause aberrant development of commissures and longitudinal axon pathways. Defects in the PNS and muscles of nrm mutants are also observed. In most nrm embryos, abnormal development can be detected in a subset of abdominal segments; however, in approximately 1 of 10 nrm embryos, the defects extend to all segments. Herein, we present evidence that nrm plays an important role in early morphogenesis, possibly by mediating or facilitating inductive cell contacts and movements.     

Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila

The senseless (sens) gene is required for proper development of most cell types of the embryonic and adult peripheral nervous system (PNS) of Drosophila. Sens is a nuclear protein with four Zn fingers that is expressed and required in the sensory organ precursors (SOP) for proper proneural gene expression. Ectopic expression of Sens in many ectodermal cells causes induction of PNS external sensory organ formation and is able to recreate an ectopic proneural field. Hence, sens is both necessary and sufficient for PNS development. Our data indicate that proneural genes activate sens expression. Sens is then in turn required to further activate and maintain proneural gene expression. This feedback mechanism is essential for selective enhancement and maintenance of proneural gene expression in the SOPs.     

Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system

Targeted disruption of the retinoblastoma gene in mice leads to embryonic lethality in midgestation accompanied by defective erythropoiesis. Rb(-/-) embryos also exhibit inappropriate cell cycle activity and apoptosis in the central nervous system (CNS), peripheral nervous system (PNS), and ocular lens. Loss of p53 can prevent the apoptosis in the CNS and lens; however, the specific signals leading to p53 activation have not been determined. Here we test the hypothesis that hypoxia caused by defective erythropoiesis in Rb-null embryos contributes to p53-dependent apoptosis. We show evidence of hypoxia in CNS tissue from Rb(-/-) embryos. The Cre-loxP system was then used to generate embryos in which Rb was deleted in the CNS, PNS and lens, in the presence of normal erythropoiesis. In contrast to the massive CNS apoptosis in Rb-null embryos at embryonic day 13.5 (E13.5), conditional mutants did not have elevated apoptosis in this tissue. There was still significant apoptosis in the PNS and lens, however. Rb(-/-) cells in the CNS, PNS, and lens underwent inappropriate S-phase entry in the conditional mutants at E13.5. By E18.5, conditional mutants had increased brain size and weight as well as defects in skeletal muscle development. These data support a model in which hypoxia is a necessary cofactor in the death of CNS neurons in the developing Rb mutant embryo.     

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.     

P-Element Mutations Affecting Embryonic Peripheral Nervous System Development in Drosophila Melanogaster

The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study the molecular mechanisms governing neural development. To identify genes controlling PNS development, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the P-element insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS.     

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.     

Satellite cells of dorsal root ganglia are multipotential glial precursors

The evolutionary origin of myelinating cells in the vertebrate nervous system remains a mystery. A clear delineation of the developmental potentialities of neuronal support cells in the CNS and PNS might aid in formulating a hypothesis about the origins of myelinating cells. Although a glial-precursor cell in the CNS can differentiate into oligodendrocytes (OLs), Schwann cells (SCs) and astrocytes, a homologous multipotential cell has not yet been found in the PNS. Here, we identify a cell type of embryonic dorsal root ganglia (DRG) of the PNS - the satellite cell - that develops into OLs, SCs and astrocytes. Interestingly,satellite-cell-derived OL precursors were found in cultures prepared from embryonic day 17 (E17) to postnatal day 8 (P8) ganglia,but not from adult DRGs, revealing a narrow developmental window for multipotentiality. We suggest that compromising the organization of the ganglia triggers a differentiation pathway in a subpopulation of satellite cells, inducing them to become myelinating cells with either a CNS or PNS phenotype. Our data provide an additional, novel piece in the myelinating cell-precursor puzzle, and lead to the concept that cells in the CNS and PNS that function to ensheath neuronal cell bodies and axons can differentiate into OLs, SCs and astrocytes. In sum, it appears that glial fate might be determined over and above the CNS/PNS dichotomy. Last, we suggest that primordial ensheathing cells form the original cell population in which the myelination program first evolved.     

Restricted neural epidermal growth factor-like like 2 (NELL2) expression during muscle and neuronal differentiation

We have identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a screen designed to isolate molecules regulating sensory neuron genesis and differentiation in the dorsal root ganglia (DRG). In investigating NELL2 expression during embryogenesis, we demonstrate here that NELL2 is highly regulated spatially and temporally, being only transiently expressed in discrete regions of the central (CNS) and peripheral nervous systems (PNS) and in a subset of mesoderm derived structures during their peak periods of development. In the CNS and PNS, NELL2 is maximally expressed as motor and sensory neurons differentiate. Interestingly, its expression is restricted to sublineages of the neural crest, being strongly expressed throughout the immature DRG, but excluded from sympathetic ganglia. Similarly during muscle development, NELL2 is specifically expressed by hypaxial muscle precursor cells in the differentiating somite and derivatives in the forelimbs and body wall, but not by epaxial muscle precursors. Furthermore, NELL2 is differentially regulated in the CNS and PNS; in the CNS, NELL2 is only expressed by nascent, post-mitotic neurons as they commence their differentiation, yet in the PNS, NELL2 is expressed by subsets of progenitor cells in addition to nascent neurons. Based on this restricted spatial and temporal expression pattern, functional studies are in progress to determine NELL2's role during neuronal differentiation in both the PNS and CNS.     

Restricted neural epidermal growth factor-like like 2 (NELL2) expression during muscle and neuronal differentiation

We have identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a screen designed to isolate molecules regulating sensory neuron genesis and differentiation in the dorsal root ganglia (DRG). In investigating NELL2 expression during embryogenesis, we demonstrate here that NELL2 is highly regulated spatially and temporally, being only transiently expressed in discrete regions of the central (CNS) and peripheral nervous systems (PNS) and in a subset of mesoderm derived structures during their peak periods of development. In the CNS and PNS, NELL2 is maximally expressed as motor and sensory neurons differentiate. Interestingly, its expression is restricted to sublineages of the neural crest, being strongly expressed throughout the immature DRG, but excluded from sympathetic ganglia. Similarly during muscle development, NELL2 is specifically expressed by hypaxial muscle precursor cells in the differentiating somite and derivatives in the forelimbs and body wall, but not by epaxial muscle precursors. Furthermore, NELL2 is differentially regulated in the CNS and PNS; in the CNS, NELL2 is only expressed by nascent, post-mitotic neurons as they commence their differentiation, yet in the PNS, NELL2 is expressed by subsets of progenitor cells in addition to nascent neurons. Based on this restricted spatial and temporal expression pattern, functional studies are in progress to determine NELL2's role during neuronal differentiation in both the PNS and CNS.     

Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.