skittles

DEVELOPMENTAL BIOLOGY

Embryonic

In situ hybridization shows that during embryogenesis sktl is expressed at all stages, but there is a very dynamic pattern of regulation in various developing tissues. At all stages there is a basal level of expression in all cells. At stage 5, strong expression is seen in the procephalic neuroectoderm. During gastrulation, expression is elevated in the invaginating cells of the ventral and cephalic furrows. At stage 11 all central nervous system and peripheral nervous system precursor cells express high levels of sktl. At stage 13 most developing tissues (heart, gut, muscles, CNS, and PNS) express high levels of skittles. By the end of embryogenesis (stage 17) expression is prominent in a few CNS cells and the gut. This expression pattern, particularly in the nervous system, is remarkably similar, if not identical, to that of insc (Kraut, 1996a and Knirr, 1997b). This suggests that the two genes may share common regulatory elements and raises the question of whether they interact during nervous system development. Alternatively, sktl may be under the control of insc enhancers and may serve an unrelated function (Hassan, 1998).

Larval

During third instar larval development sktl is expressed widely in all imaginal discs. In the leg disc expression is ubiquitous and uniform. In the wing disc, expression is elevated in the precursors of the anterior wing margin sensory organs and along the anterior-posterior axis. Expression is very low or absent along the dorso-ventral axis. In the eye disc, expression is elevated in the row of cells anterior to the morphogenetic furrow from which the R8 photoreceptors will differentiate. In the third instar larval brain, sktl is expressed widely but not ubiquitously. Areas of expression include the outer proliferation center of the optic lobes, several patches of cells in the midbrain, and subsets of cells in the ventral ganglion (Hassan, 1998).

Adult

During oogenesis, sktl expression is first observed in region 2 of the germarium. At this stage, sktl appears to be expressed in a subset of cells building the different cysts. In egg chambers of stagee S1-S14 strong expression is observed in the oocyte. Only weak expression is detected in the nurse cells from stage S1 to S8. At stage S8, expression in the nurse cells becomes more profound and increases during subsequent stages. No expression is observed in the follicle cells surrounding the egg chambers. Sktl transcripts are uniformly distributed in the unfertilized egg. skit is also expressed in the male germline. Sktl transcripts first occur in primary spermatocytes and later during meiotic prophase, whereas in early germ cells, such as the stem cells and the spermatogonia, as well as in postmeiotic stages, Sktl transcripts are absent (Knirr, 1997a).

Effects of Mutation or Deletion

sktl mutants are lethal in early first instar larvae. Because sktl is expressed at high levels in most if not all nervous system precursors, the consequences of the loss of sktl on nervous system development were investigated. sktl^Delta20 mutant embryos were used to determine if sktl is required for nervous system development. Nervous system development was examined using anti-ELAV and Mab 22C10 antibodies to detect neurons and anti-PROS to detect neuronal precursors during early neurogenesis and glial cells during late neurogenesis. No detectable defects are seen by stage 16 with these markers. The absence of an insc phenotype in sktl mutants demonstrates that the phenotypes reported are indeed caused by the absence of insc and not by the loss of function of either sktl alone or both sktl and insc, as for example, the loss of nervous system cells due to mislocalization of Numb and Prospero during neuronal lineage development (Hassan, 1998).

PIP5KI is required for Ca²⁺-dependent neuropeptide secretion from PC12 cells (Hay, 1995). In addition, several lines of evidence suggest that PIP5Ks play a crucial role in regulating membrane trafficking. Furthermore, sktl is expressed in many cells in the ventral ganglion of third instar larvae. Some of these cells may correspond to motor neurons innervating the larval body wall muscles. The Drosophila third instar larval neuromuscular junction is an excellent system for measuring neurotransmitter release from motor neurons. To test the requirement of sktl for neurotransmitter release, third instar larvae transheterozygous for sktl alleles were used. One combination results in late second instar lethality with a few third instar escapers. Electrophysiological recordings at the neuromuscular junction were done to examine spontaneous release as well as evoked release. The size, shape, and frequency of evoked and spontaneous responses were examined in control larvae (heterozygous for either allele alone) and in transheterozygous mutant larvae. Evoked response was measured after either single or repetitive stimulation. Tests were perfomed for nerve fatigue by repetitive stimulation. No detectable defects were observed in any of the above measurements, suggesting that sktl is not required for glutamate neurotransmitter release, at least at the larval neuromuscular junction. It is therefore unlikely to play a role in regulating vesicular trafficking at that junction. The lack of a vesicular secretion phenotype may be due to the activity of other PIP5Ks in Drosophila. Drosophila has a PIP5K type II that maps to the tip of chromosome 4 and that appears, from preliminary expression analysis, to be expressed specifically in the late embryonic CNS (B. Hassan, unpublished results cited in Hassan, 1998). It remains to be established whether this form of PIP5K functions in neuronal secretion (Hassan, 1998).

Zhong (1995) showed in Drosophila that repetitive stimulation at the neuromuscular junction at 20 Hz or higher results not only in a fast evoked response but also in a slow, neuropeptide-dependent, depolarization. Neuropeptides are released by dense core vesicles from the nerve terminal. Hay (1995) showed that PIP5KI is required for dense core vesicle secretion from PC12 cells. To test the requirement of sktl for dense core vesicle secretion, motor nerves were stimulated at 30 and 50 Hz. The resulting slow depolarization profile in sktl mutants is indistinguishable from that of heterozygous controls or wild-type larvae. Therefore sktl does not appear to play a role in regulating dense core vesicle secretion, at least at the larval neuromuscular junction. It is not possible to exclude the possibility that the transheterozygous combination used in these experiments, while being strong enough to cause larval lethality, is not strong enough to have an effect on peptide release. However, this is unlikely because even viable hypomorphic mutations of numerous proteins involved in neurotransmission, such as synaptotagmin and RAS opposite (ROP), show severe electrophysiological defects (Hassan, 1998).

To characterize the function of sktl, overexpression studies were carried out. Flies with a UAS-sktl construct were used to overexpress sktl using a variety of Gal4 drivers. The neuronal-specific elav-Gal4 driver resulted in no detectable phenotypes and gave rise to fertile adults. Overexpression using the ubiquitous daughterless-Gal4 driver and the heat shock-Gal4 driver resulted in no obvious phenotypes during embryogenesis but caused early larval lethality. Expression with the ubiquitous imaginal disc driver T80-Gal4 resulted in third instar larval lethality. The lethality associated with the ubiquitous expression of sktl, which is itself very widely expressed, precluded the use of the UAS-sktl construct to rescue sktl mutants. Overexpression with the dpp-Gal4 driver, expressed in the morphogenetic furrow of the eye disc and along the anterior-posterior boundary in the wing disc, showed no detectable phenotypes in the eye (Hassan, 1998),

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date revised: 26 August 1999

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