Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis.

A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. We have explored the regulation of myosin activity by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, we screened for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. We determined that a single missense mutation in the zipper(Ebr) allele gives rise to its sensitivity to second-site noncomplementation. We then identify the Rho signal transduction pathway as necessary for proper myosin function. First we show that a lethal P-element insertion interacts genetically with zipper. Subsequently we show that this second-site noncomplementing mutation disrupts the RhoGEF2 locus. Next, we show that two EMS-induced mutations, previously shown to interact genetically with zipper(Ebr), disrupt the RhoA locus. Further, we have identified their molecular lesions and determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, we show that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. Our studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan.

Joint action of two RNA degradation pathways controls the timing of maternal transcript elimination at the midblastula transition in Drosophila melanogaster.

Maternally synthesized RNAs program early embryonic development in many animals. These RNAs are degraded rapidly by the midblastula transition (MBT), allowing genetic control of development to pass to zygotically synthesized transcripts. Here we show that in the early embryo of Drosophila melanogaster, there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is targeted to specific classes of mRNAs through cis-acting elements in the 3'-untranslated region and is conserved in Xenopus laevis. The second pathway is activated 2 h after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT.

Second-site noncomplementation identifies genomic regions required for Drosophila nonmuscle myosin function during morphogenesis.

Drosophila is an ideal metazoan model system for analyzing the role of nonmuscle myosin-II (henceforth, myosin) during development. In Drosophila, myosin function is required for cytokinesis and morphogenesis driven by cell migration and/or cell shape changes during oogenesis, embryogenesis, larval development and pupal metamorphosis. The mechanisms that regulate myosin function and the supramolecular structures into which myosin incorporates have not been systematically characterized. The genetic screens described here identify genomic regions that uncover loci that facilitate myosin function. The nonmuscle myosin heavy chain is encoded by a single locus, zipper. Contiguous chromosomal deficiencies that represent approximately 70% of the euchromatic genome were screened for genetic interactions with two recessive lethal alleles of zipper in a second-site noncomplementation assay for the malformed phenotype. Malformation in the adult leg reflects aberrations in cell shape changes driven by myosin-based contraction during leg morphogenesis. Of the 158 deficiencies tested, 47 behaved as second-site noncomplementors of zipper. Two of the deficiencies are strong interactors, 17 are intermediate and 28 are weak. Finer genetic mapping reveals that mutations in cytoplasmic tropomyosin and viking (collagen IV) behave as second-site noncomplementors of zipper during leg morphogenesis and that zipper function requires a previously uncharacterized locus, E3.10/J3.8, for leg morphogenesis and viability.

Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis.

Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. We show that maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 RNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity. In contrast, Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules--cappuccino, oskar, spire, staufen, tudor, valois, and vasa. Mislocalization of oskar RNA to the anterior pole, which has been shown to result in induction of germ cells at the anterior, leads to anterior protection of maternal Hsp83 RNA. These results suggest that Hsp83 RNA is a component of the posterior polar plasm that might be associated with polar granules. In addition, we show that zygotic expression of Hsp83 commences in the anterior third of the embryo at the syncytial blastoderm stage and is regulated by the anterior morphogen, bicoid. We consider the possible developmental significance of this complex control of Hsp83 transcript distribution.

Reciprocal effects of hyper- and hypoactivity mutations in the Drosophila pattern gene torso.

In Drosophila, five "terminal" polarity genes must be active in females in order for them to produce embryos with normal anterior and posterior ends. Hypoactivity mutations in one such gene, torso, result in the loss of the most posterior domain of fushi tarazu expression and the terminal cuticular structures. In contrast, a torso hyperactivity mutation causes the loss of central fushi tarazu expression and central cuticular structures. Cytoplasmic leakage, transplantation, and temperature-shift experiments suggest that the latter effect is caused by abnormal persistence of the torso product in the central region of the embryo during early development. Thus, the amount and timing of torso activity is key to distinguishing the central and terminal regions of the embryo. Mutations in the tailless terminal gene act as dominant maternal suppressors of the hyperactive torso allele, indicating that the torso product acts through, or in concert with, the tailless product.

Differential expression of early and late embryonic histone genes in adult tissues of the sea urchin Strongylocentrotus purpuratus.

The sea urchin synthesizes distinct classes of histone mRNAs at different stages of development. "Early" embryonic histone mRNAs are synthesized in large amount in cleavage and blastula stage embryos. "Late" embryonic histone mRNAs are the predominant forms in postblastula embryos. To learn more about how early and late histone genes are regulated during the life cycle of the sea urchin and to search for additional classes of developmentally regulated histone mRNAs, we examined histone mRNAs in sea urchin adult tissues. Using methods of primer extension and S1 nuclease protection, we found that tube foot, intestine, testis, and ovary contain a subset of the several H2b mRNA species synthesized by the embryo. We detected early H2b mRNA in ovary, but not in other tissues. Three late H2b mRNA species were present in all tissues tested, while a fourth late H2b was not detected. Using a probe that hybridized specifically with transcripts of a single-copy late H2b gene, we found that this gene was transcribed in both embryos and adults. Interestingly, its level of expression relative to other late H2b genes varied among tissues. Finally, we identified two H2b mRNA species that were distinct from early and late embryonic forms and were synthesized only in adult tissues.

Role of mRNA 5'-terminal caps in translational dormancy of Physarum polycephalum.

Translational regulation of protein synthesis accompanies sclerotization in Physarum polycephalum. Plasmodial and sclerotial poly(A)+ RNA were translated in a message-dependent wheat germ lysate in the presence of the cap analogue 7-methylguanosine-triphosphate to determine whether 5' structural alterations in mRNA accompany translational repression. The translation of plasmodial and sclerotial poly(A)+ RNA was reduced to identical levels suggesting that both RNA populations are capped. The 5'-termini of plasmodial and sclerotial poly(A)+ RNA were identified as m7G5'ppp5'Cm. Alterations in the 5'-cap of mRNA during sclerotization do not appear to be responsible for translational dormancy.