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We have investigated the role of the ryanodine receptor in Drosophila development by using pharmacological and genetic approaches. We identified a P element insertion in the Drosophila ryanodine receptor gene, Ryanodine receptor 44F ( Ryr ), and used it to generate the hypomorphic allele Ryr 16 . An examination of hypodermal, visceral, and circulatory muscle showed that, in each case, muscle contraction was impaired in Ryr 16 larvae. Treatment with the drug ryanodine, a highly specific modulator of ryanodine receptor channel activity, also inhibited muscle function, and, at high levels, completely blocked hypodermal muscle contraction. These results suggest that the ryanodine receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval body wall muscles. Nonmuscle roles of Ryr were also investigated. Ryanodine-sensitive Ca 2+ stores had previously been implicated in phototransduction; to address this, we generated Ryr 16 mutant clones in the adult eye and performed whole-cell, patch–clamp recordings on dissociated ommatidia. Our results do not support a role for Ryr in normal light responses.

Connector enhancer of KSR (CNK) is a multidomain protein required for RAS signaling. Its C-terminal portion (CNK C-term ) directly binds to RAF. Herein, we show that the N-terminal portion of CNK (CNK N-term ) strongly cooperates with RAS, whereas CNK C-term efficiently blocks RAS- and RAF-dependent signaling when overexpressed in the Drosophila eye. Two effector loop mutants of RAS V12 , S35 and C40, which selectively activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase pathways, respectively, do not cooperate with CNK. However, a strong cooperation is observed between CNK and RAS V12G37 , an effector loop mutant known in mammals to activate specifically the RAL pathway. We have identified two domains in CNK N-term that are critical for cooperation with RAS. Our results suggest that CNK functions in more than one pathway downstream of RAS. CNK c-term seems to regulate RAF, a component of the MAPK pathway, whereas CNK N-term seems to be involved in a MAPK-independent pathway.

Secreted and transmembrane proteins play an essential role in intercellular communication during the development of multicellular organisms. Because only a small number of these genes have been characterized, we developed a screen for genes encoding extracellular proteins that are differentially expressed during Drosophila embryogenesis. Our approach utilizes a new method for screening large numbers of cDNAs by whole-embryo in situ hybridization. The cDNA library for the screen was prepared from rough endoplasmic reticulum-bound mRNA and is therefore enriched in clones encoding membrane and secreted proteins. To increase the prevalence of rare cDNAs in the library, the library was normalized using a method based on cDNA hybridization to genomic DNA-coated beads. In total, 2,518 individual cDNAs from the normalized library were screened by in situ hybridization, and 917 of these cDNAs represent genes differentially expressed during embryonic development. Sequence analysis of 1,001 cDNAs indicated that 811 represent genes not previously described in Drosophila . Expression pattern photographs and partial DNA sequences have been assembled in a database publicly available at the Berkeley Drosophila Genome Project website ( http://fruitfly.berkeley.edu ). The identification of a large number of genes encoding proteins involved in cell–cell contact and signaling will advance our knowledge of the mechanisms by which multicellular organisms and their specialized organs develop.