Drosophila LAR regulates R1-R6 and R7 target specificity in the visual system.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye connect to specific targets in the optic lobe. Using a behavioral screen, we identified LAR, a receptor tyrosine phosphatase, as being required for R cell target specificity. In LAR mutant mosaic eyes, R1-R6 cells target to the lamina correctly, but fail to choose the correct pattern of target neurons. Although mutant R7 axons initially project to the correct layer of the medulla, they retract into inappropriate layers. Using single cell mosaics, we demonstrate that LAR controls targeting of R1-R6 and R7 in a cell-autonomous fashion. The phenotypes of LAR mutant R cells are strikingly similar to those seen in N-cadherin mutants.

N-cadherin regulates target specificity in the Drosophila visual system.

Using visual behavioral screens in Drosophila, we identified multiple alleles of N-cadherin. Removal of N-cadherin selectively from photoreceptor neurons (R cells) causes deficits in specific visual behaviors that correlate with disruptions in R cell connectivity. These defects include disruptions in the pattern of neuronal connections made by all three classes of R cells (R1-R6, R7, and R8). N-cadherin is expressed in both R cell axons and their targets. By inducing mitotic recombination in a subclass of eye progenitors, we generated mutant R7 axons surrounded by largely wild-type R cell axons and a wild-type target. R7 axons lacking N-cadherin mistarget to the R8 recipient layer. We consider the implications of these findings in the context of the proposed role for cadherins in target specificity.

Afferent growth cone interactions control synaptic specificity in the Drosophila visual system.

In the Drosophila compound eye, photoreceptors (R cells) that respond to light from the same point in space are distributed across the retina and connect to the same target neurons. This complex connectivity pattern reconstructs visual space in the first optic ganglion, the lamina. We have used mutations that delete specific R cell subtypes or alter their retinal organization to define the cellular mechanisms that generate this pattern. R cell axons are programmed to search for targets within a local region in the lamina but their selection of appropriate postsynaptic targets requires specific interactions among R cell growth cones. The orientation of the projections is controlled both by the spatial arrangement of R cells in the retina and by cues in the target.

Inositol trisphosphate mediates a RAS-independent response to LET-23 receptor tyrosine kinase activation in C. elegans.

Activity of LET-23, the C. elegans homolog of the epidermal growth factor receptor, is required in multiple tissues. RAS activation is necessary and sufficient for certain LET-23 functions. We show that an inositol trisphosphate receptor can act as a RAS-independent, tissue-specific positive effector of LET-23. Moreover, an inositol trisphosphate kinase negatively regulates this transduction pathway. Signals transduced by LET-23 control ovulation through changes in spermathecal dilation, possibly dependent upon calcium release regulated by both IP3 and IP4. Our results demonstrate that one mechanism by which receptor tyrosine kinases can evoke tissue-specific responses is through activation of distinct signal transduction cascades in different tissues.

Caenorhabditis elegans HOM-C genes regulate the response of vulval precursor cells to inductive signal.

Factors that determine the competence of cells to respond to extracellular cues are not well understood. We demonstrate that two HOM-C transcription factors have antagonistic roles in determining the ability of Caenorhabditis elegans vulval precursor cells (VPCs) to respond to the inductive signal from the anchor cell of the somatic gonad. The vulva develops from a subset of ectodermal vulval precursor cells distributed along the anteroposterior axis. Vulval patterning depends on both a localized inductive signal, the LIN-3 growth factor, and lateral signaling between induced VPCs. One HOM-C gene, the Antp homolog mab-5, is expressed in the posterior two VPCs. By examining the response of single VPCs to controlled doses of inductive signal in wild-type and in mab-5 mutant animals, we demonstrate that mab-5 reduces the competence of these two cells. Moreover, a gain-of-function allele of mab-5 that causes ectopic expression of MAB-5 in all VPCs reduces the sensitivity of all VPCs to inductive signal. Additional experiments suggest that another HOM-C gene, the Scr homolog lin-39, is required for VPCs in wild-type animals to respond to activation of inductive signal. Genetic epistasis tests are consistent with models in which lin-39 acts downstream of the RAS pathway to regulate response to inductive signal. We propose that the spatial pattern of HOM-C gene expression may enhance the precision of vulval fate patterning.

A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates.

LET-23-mediated signal transduction during Caenorhabditis elegans development.

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming alpha-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the "vulvaless" phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the "multivulva" phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development--larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways.

Different levels of the C. elegans growth factor LIN-3 promote distinct vulval precursor fates.

An invariant spatial pattern of three cell fates (3 degrees-3 degrees-2 degrees-1 degree-2 degrees-3 degrees) is generated from a field of multipotent precursor cells during C. elegans vulval development. We demonstrate that the epidermal growth factor-like domain of the LIN-3 protein can induce either of two distinct vulval cell fates: a high dose of LIN-3 induces a 1 degree fate; a lower dose of LIN-3 induces a 2 degrees fate. A high dose of LIN-3 can also induce adjacent vulval precursor cells to assume 1 degree fates; thus, high levels of LIN-3 can override the lateral signaling that normally inhibits formation of adjacent 1 degree fates. We propose that the invariant pattern of vulval cell fates is generated by a graded distribution of LIN-3 that promotes different vulval fates according to local concentration and by a lateral signal that reinforces this initial bias.

sli-1, a negative regulator of let-23-mediated signaling in C. elegans.

By screening for suppressors of hypomorphic mutations of let-23, a receptor tyrosine kinase necessary for vulval induction in Caenorhabditis elegans, we recovered > or = 12 mutations defining the sli-1 (suppressor of lineage defect) locus. sli-1 mutations suppress four of five phenotypes associated with hypomorphic alleles of let-23 but do not suppress let-23 null alleles. Thus, a sli-1 mutation does not bypass the requirement for functional let-23 but rather allows more potent LET-23-dependent signaling. Mutations at the sli-1 locus are otherwise silent with respect to vulval differentiation and cause only a low-penetrance abnormal head phenotype. Mutations at sli-1 also suppress the vulval defects but not other defects associated with mutations of sem-5, whose product likely interacts with LET-23 protein during vulval induction. Mutations at sli-1 suppress lin-2, lin-7 and lin-10 mutations but only partially suppress lin-3 and let-60 mutations and do not suppress a lin-45 mutation. The sli-1 locus displays dosage sensitivity: severe reduction of function alleles of sli-1 are semidominant suppressors; a duplication of the sli-1(+) region enhances the vulvaless phenotype of hypomorphic mutations of let-23. We propose that sli-1 is a negative regulator that acts at or near the LET-23-mediated step of the vulval induction pathway. Our analysis suggests that let-23 can activate distinct signaling pathways in different tissues: one pathway is required for vulval induction; another pathway is involved in hermaphrodite fertility and is not regulated by sli-1.

Genetic studies of mei-1 gene activity during the transition from meiosis to mitosis in Caenorhabditis elegans.

Genetic evidence suggests that the mei-1 locus of Caenorhabditis elegans encodes a maternal product required for female meiosis. However, a dominant gain-of-function allele, mei-1(ct46), can support normal meiosis but causes defects in subsequent mitotic spindles. Previously identified intragenic suppressors of ct46 lack functional mei-1 activity; null alleles suppress only in cis but other alleles arise frequently and suppress both in cis and in trans. Using a different screen for suppressors of the dominant ct46 defect, the present study describes another type of intragenic mutation that also arises at high frequency. These latter alleles appear to have reduced meiotic activity and retain a weakened dominant effect. Characterization of these alleles in trans-heterozygous combinations with previously identified mei-1 alleles has enabled us to define more clearly the role of the mei-1 gene product during normal embryogenesis. We propose that a certain level of mei-1 activity is required for meiosis but must be eliminated prior to mitosis. The dominant mutation causes mei-1 activity to function at mitosis; intragenic trans-suppressors act in an antimorphic manner to inactivate multimeric mei-1 complexes. We propose that inactivation of meiosis-specific functions may be an essential precondition of mitosis; failure to eliminate such functions may allow ectopic meiotic activity during mitosis and cause embryonic lethality.

Dietary fat: exogenous determination of membrane structure and cell function.

Evidence indicates that principal features of the membrane involve structural organization of lipids in the form of a bilayer with functional proteins either bound to the bilayer surface or inserted into the bilayer and interacting within specific domains in the lipid milieux. In homeotherms, intrinsic and extrinsic factors apparently form the basis for determination of membrane lipid composition and thus membrane physicochemical properties. Moreover, many intrinsic metabolic controls, such as fatty acid desaturation and phospholipid biosynthesis, may be attenuated by change in the nature of the extrinsic or dietary influence. This review will focus on the role of dietary fat as a determinant of subcellular structural constituents to illustrate that feeding nutritionally adequate diets differing in fatty acid composition can induce physiological transitions in membrane function involving the activity of enzymes responsible for synthesis of membrane constituents, hormone-activated functions and expression of activity in the cell nucleus.