Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew.

The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associated in vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function.

Daughter of sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during sevenless signaling.

The SH2 domain-containing phosphotyrosine phosphatase Corkscrew (CSW) is an essential component of the signaling pathway initiated by the activation of the sevenless receptor tyrosine kinase (SEV) during Drosophila eye development. We have used genetic and biochemical approaches to identify a substrate for CSW. Expression of a catalytically inactive CSW was used to trap CSW in a complex with a 115 kDa tyrosine-phosphorylated substrate. This substrate was purified and identified as the product of the daughter of sevenless (dos) gene. Mutations of dos were identified in a screen for dominant mutations which enhance the phenotype caused by overexpression of inactive CSW during photoreceptor development. Analysis of dos mutations indicates that DOS is a positive component of the SEV signaling pathway and suggests that DOS dephosphorylation by CSW may be a key event during signaling by SEV.

The mouse tissue plasminogen activator gene 5' flanking region directs appropriate expression in development and a seizure-enhanced response in the CNS.

Tissue plasminogen activator (t-PA) is a secreted serine protease implicated in multiple aspects of development. In the adult rat brain, transcription of t-PA is an immediate-early response in the hippocampus following treatments that induce neuronal plasticity. To study the sequence elements that govern transcription of this gene, in situ analysis was used to define t-PA's temporal and spatial expression pattern in midgestation embryos. Transgenic mice were then generated carrying t-PA 5' flanking sequences linked to the E. coli lacZ gene. Constructs containing 4 kb of the flanking sequences (4.0TAMGAL) confer beta-galactosidase activity mostly to the same tissues that exhibit high levels of t-PA mRNA by in situ analysis. In 4.0TAMGAL embryos from embryonic day 8.5 (E8.5) to 13.5 (E13.5), the majority of expression observed is localized to neural ectoderm-derived tissues. beta-galactosidase activity is first detected in restricted neuromeres in the midbrain and diencephalon, at E8.5 and E9.5 respectively. At E10.5, transgene expression is observed in neural crest-derived cranial nerves and dorsal root ganglia, but not placode-derived cranial nerves. From E10.5 to E13.5, beta-galactosidase activity is observed in postmitotic neurons of the midbrain, spinal cord, neural retina and the developing olfactory system. beta-galactosidase activity is also detected in areas undergoing tissue remodeling such as the pinna of the ear, whisker follicles and the limbs. In adult mice, lacZ is expressed in the hippocampus and this expression was found to be enhanced upon seizure in the giant pyramidal neurons of CA3. These results reinforce the concept that t-PA plays a role in neurogenesis and morphogenesis, and identifies the promoter region that directs its transcriptional regulation both in development and in the CNS.

Creating maternal effect mutations in transgenic mice: antisense inhibition of an oocyte gene product.

Gene products present in mouse oocytes direct development until the two-cell stage and may be important in later development. Here, we demonstrate that expression of a specific maternal protein can be disrupted in mouse oocytes using transgenic antisense RNA technology. An oocyte-specific promoter (mZP3) was utilized to express antisense RNA directed against maternal mRNA encoding tissue-type plasminogen activator (tPA). Antisense expression results in reduced levels of tPA mRNA and enzyme activity in mouse oocytes. We also provide evidence for a novel mechanism of antisense-mediated translational inhibition, whereby the cytoplasmic polyadenylation of maternal tPA mRNA is altered. This strategy should prove applicable to functional studies of other murine maternal mRNAs in an in vivo environment.

Preimplantation mouse embryos express a cell surface receptor for tissue-plasminogen activator.

The serine protease tissue-plasminogen activator (t-PA) has previously been shown to be intracellular in mouse secondary oocytes and extracellular in fertilized eggs. Here we demonstrate that extracellular t-PA activity is bound to the surface of the fertilized egg. The level of t-PA activity associated with preimplantation mouse embryos decreases in the 2-cell stage embryo, then increases in 4-cell and morula stage embryos. However, morulae grown in culture from fertilized eggs lack t-PA activity but are able to bind exogenously added mouse t-PA. Additionally, northern analysis indicates that preimplantation embryos do not contain detectable levels of t-PA mRNA. Therefore, the enzyme activity associated with 4-cell and morula stage embryos in vivo is derived from t-PA present in the oviduct lumen that binds the embryo, and not from protein produced from translation of embryonic mRNA. The binding activity is species and protein specific in that neither mouse urokinase-type plasminogen activator (u-PA) nor human t-PA bind to cultured morulae. Furthermore, binding activity is dose-dependent and saturable, and does not require the active site of t-PA. These data indicate that a cell surface-specific t-PA-binding activity exists in the preimplantation mouse embryo and may localize function and concentrate the proteolytic activity of t-PA in early mouse development.

Abrogation of IL-3 dependent growth requires a functional v-src gene product: evidence for an autocrine growth cycle.

We have investigated the ability of the v-src oncogene to abrogate growth factor dependent growth in the interleukin-3 dependent myeloid progenitor cell line 32D c13. Growth factor independent clones were isolated following infection of 32D c13 cells with murine retroviruses containing the v-src oncogene. v-src was demonstrated to be directly responsible for growth factor independence in experiments utilizing temperature-sensitive v-src mutants. The v-src infected cells released a growth factor capable of stimulating the proliferation of normal 32D c13 cells. Analysis of the mRNA from v-src infected 32D c13 did not identify the putative autocrine growth factor as one of the currently identified murine or human hematopoietic growth factors.

The effect of hypertonic solution on the wet weight and contractions of rat uterus and vas deferens.

The role of propagated activity in the responses to agonist drugs was studied for the rat uterus and vas deferens. Hypertonic solutions were used to inhibit propagation of activity by shrinking cells. Tissue weight was used to indicate cell volume. Hypertonic solutions after 10 min caused weight loss and reduced the size of contractions in response to submaximal doses of drugs, to KCl, and to external electrical stimulation. Contractions in response to KCl and drugs were diminished to a similar degree in the vas deferens, but in the uterus, drug contractions were depressed much more. Prolonged action of hypertonic solution also differed for the two tissues. In the uterus, weight changes correlated with changes in size of the drug-induced contractions. Uterine contractions reduced in hypertonic solution could be increased by using supramaximal doses of drug. When stimulation was applied to one end of the uterus in a three compartment bath, propagation of spontaneous drug- and KCl-induced contraction occurred, but it was prevented by placing hypertonic solution in the center compartment. An increase of the KCl to 44 mM in the hypertonic solution restored propagation. These experiments yielded no evidence of propagated responses in the rat vas deferens. It was concluded that propagated activity plays a role in drug-induced contractions in the rat uterus but not in the rat vas deferens. Hyperpolarization of shrunken cells might be involved in inhibition of propagation by hypertonic solutions.