Innexin Specificity in Neural Development

Research Article 3379 Gap junction proteins are not interchangeable in development of neural function in the Drosophila visual system Kathryn D. Curtin*, Zhan Zhang and Robert J. Wyman Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA *Author for correspondence (e-mail: [email protected]) Accepted 12 June 2002 Journal of Cell Science 115, 3379-3388 (2002) © The Company of Biologists Ltd Summary Gap junctions (GJs) are composed of proteins from two Specifically, we tested several innexins for their ability to distinct families. In vertebrates, GJs are composed of rescue shakB2 and ogre mutant ERGs and found that, by connexins; a connexin hexamer on one cell lines up with and large, innexins are not interchangeable. We mapped a hexamer on an apposing cell to form the intercellular the protein regions required for this specificity by making channel. In invertebrates, GJs are composed of an molecular chimeras between shakB(N) and ogre and unrelated protein family, the innexins. Different testing their ability to rescue both mutants. Each chimera connexins have distinct properties that make them largely rescued either shakB or ogre but never both. Sequences in non-interchangeable in the animal. Innexins are also a the first half of each protein are necessary for functional large family with high sequence homology, and some specificity. Potentially crucial residues include a small functional differences have been reported. The biological number in the intracellular loop as well as a short stretch implication of innexin differences, such as their ability to just N-terminal to the second transmembrane domain. substitute for one another in the animal, has not been Temporary GJs, possibly between the retina and lamina, explored. may play a role in final target selection and/or chemical Recently, we showed that GJ proteins are necessary for synapse formation in the Drosophila visual system. In that the development of normal neural transmission in the case, specificity in GJ formation or function could Drosophila visual system. Mutations in either of two contribute, directly or indirectly, to chemical synaptic Drosophila GJ genes (innexins), shakB and ogre, lead to a specificity by regulating which neurons couple and what loss of transients in the electroretinogram (ERG), which is signals they exchange. Cells may couple only if their indicative of a failure of the lamina to respond to retinal innexins can mate with each other. The partially cell depolarization. Ogre is required presynaptically and overlapping expression patterns of several innexins make shakB(N) postsynaptically. Both act during development. this ‘mix and match’ model of GJ formation a possibility. Here we ask if innexins are interchangeable in their role of promoting normal neural development in flies. Key words: Gap junctions, Innexins, Chemical synapses Introduction are sometimes interchangeable, and sometimes not, as shown Gap junctions (GJs) allow cytoplasm-to-cytoplasm in mice when one connexin gene is replaced with another via communication via ions or other small molecules (Kumar and genetic knock-in (Plum et al., 2000). Specifically, there is Gilula, 1996; Nicholson and Bruzzone, 1997). In vertebrates, tissue-specific variability in the ability of Cx43 or Cx32 GJs are composed of multimers of connexins (Swenson et al., to functionally replace Cx40. Differences in intercellular 1989), and the connexin family has at least 25 different communication requirements are suggested to be the main members (Simon and Goodenough, 1998). In invertebrates, reason for failure of one connexin to substitute for another, innexins, which have no sequence homology to connexins, are although the exact causes of such failure are generally the structural molecules for GJs (Krishnan et al., 1993; Starich unknown (Plum et al., 2000). A large number of studies on et al., 1996; Phelan et al., 1998). The innexins are a large connexin chimeras have implicated discreet portions of family with approximately 25 genes in Caenorhabditis elegans connexin proteins as responsible for the observed functional and eight genes in Drosophila. Mammalian genes with a very variability, from mating compatibility to ion selectivity (see low homology to innexins have been reported, which suggests Discussion). that this may be a universal family (Panchin et al., 2000). Systematic studies examining innexin interchangeability The reason for the large number of connexin genes have not been done as innexins are a relatively new GJ protein seems to be related to their variable permeability and family. However, different innexins behave differently in electrophysiological properties, as well as their ability to experimental situations, suggesting that they have distinct regulate cellular coupling through selective connexin mating properties. First, some innexins form GJs in a paired Xenopus (White and Bruzzone, 1996) (also see Discussion). Connexins oocyte assay, whereas others do not (Phelan et al., 1998; 3380 Journal of Cell Science 115 (17) Landesman et al., 1999; Stebbings et al., 2000) (K.D.C., T. Materials and Methods White, R.J.W. and D. Paul, unpublished). Those innexins that Transgenic Drosophila lines fail to form GJs in this assay, nevertheless apparently code for A 4.5 kB XhoI-HindIII fragment from upstream of the ogre message GJs as their mutation leads to a loss of GJs between specific was cloned into the Gal4 vector pGatB (Brand and Perrimon, 1993). cells in animals (Krishnan et al., 1993; Starich et al., 1996) The XhoI site at the 5′ end was the same site as that in the 5′ end of (Y.-A. Sun and R.J.W., unpublished). Second, pan-neural the clone used by Watanabe and Kankel for ogre rescue (Watanabe expression of GJ transgenes causes a variety of phenotypes. and Kankel, 1990). The HindIII site is within the 3′ end just inside Such expression of two different, but highly homologous, the ogre message start site. ShakBN1 and ogre cDNAs were amplified splice variant products of shakingB (shakB), shakB(L) and by PCR and cloned into pUAST (Brand and Perrimon, 1993). shakB(N) (Krishnan et al., 1995; Zhang et al., 1999) leads, Constructs were introduced into flies by P-element-mediated transformation (Spradling et al., 1982). The elav-Gal4 enhancer trap respectively, to lethality or to no obvious phenotype (Stebbings line, C155, was obtained from Corey Goodman. ShakB2 was et al., 2000; Curtin et al., 2002). Pan-neural expression of D- recombined with C155, and this recombinant was used in rescue inx-2 is lethal, whereas expression of D-inx-7 has no obvious experiments by crossing to UAS-geneX lines. Gene X refers to any phenotype (K.D.C. and R.J.W., unpublished). Pan-neural one of several innexins, connexin43 or chimeric genes used for rescue. expression of ogre (optic ganglia reduced) causes cell loss in the eye, whereas expression of D-inx-2 in the eye along with 2 ogre reduces ogre-induced cell loss (K.D.C. and R.J.W., ShakB rescue experiments unpublished). For shakB2 rescue, we genetically recombined the elav-Gal4 enhancer 2 These differences in innexin function could be due to trap (C155) with the shaking shakB mutation as both are on the X specificity in innexin-innexin interactions. Some innexins chromosome. One copy each of the driver and the UAS construct was adequate for rescue. We did the following type of cross for all rescue form heteromeric junctions: the hemi-junction is composed of experiments of shakB2: more than one innexin (Stebbings et al., 2000). Innexins may also form heterotypic junctions: hemi-junctions on apposing elav-Gal4, shakB2/FM7 × +/Y; UAS-geneX. cells are made from different innexins. The observed Male progeny of the genotype, elav-Gal4, shakB2/Y; UAS-geneX/+, overlapping innexin expression patterns in flies would allow were tested. such mixing and matching (Curtin et al., 1999; Stebbings et al., 2000; Zhang et al., 1999). Innexins could also differ in Ogrecb8 rescue experiments crucial permeability or biophysical properties. By whichever For ogrecb8 rescue experiments, we found that animals with double mechanism, innexin specificity must be mediated by protein copies of both the driver and UAS constructs showed noticeably better sequence differences. rescue (data not shown) than flies with single copies. To obtain double We examined innexins interchangeablity in the Drosophila copies, the following cross was done with appropriately constructed visual system. We have recently shown that shakB and ogre are lines: required for the development of normal neural information ogrecb8/FM7; ogre-promoter-Gal4; ogre-promoter-Gal4 × flow between retinal photoreceptors and their lamina synaptic +/Y; UAS-geneX; UAS-geneX. partners in the adult (Curtin et al., 2002). Mutations in either gene eliminate neural transmission from the retina to the We then tested male progeny of the following general genotype: lamina (Homyk et al., 1980; Lipshitz et al., 1985; Curtin et al., ogrecb8/Y; ogre-promoter-Gal4/UAS-geneX; 2002). Ogre is required in retinal neurons, whereas shakB(N) ogre-promoter-Gal4/ UAS-geneX. is required, at a minimum, in lamina neurons (Curtin et al., For two of the UAS constructs we obtained inserts on only one 2002). Expression of both proteins is required during chromosome. So for those, we made fly lines of the following general development (Curtin et al., 2002). genotype: During development, temporary GJs have frequently been cb8 observed between cells that will later form chemical synapses ogre /FM7; ogre-promoter Gal4; UAS-geneX. with each other (Fischbach, 1972; Lopresti et al., 1974; Allen Ogrecb8/Y; ogre-promoter Gal4; UAS-geneX males were tested for and Warner, 1991). However, tools have not been available to rescue. determine whether this relationship is causal or coincidental. Our work suggests that developmental GJs, possibly between Making chimeric genes pre- and post-synaptic cells, are needed for normally We made chimeric genes by making two half-genes by PCR and functioning connections to form in the Drosophila visual ligating them together.

Load more