Abstract
We have identified a novel EMS-induced maternal effect mutation called ?N12". Loss of function mutation of 4N12 leads to dorsalization of the embryo within a wildtype eggshell. However, the phenotypes of 4N12 germ-line clone embryos range from moderately dorsalized with no ventral denticle band left to near wild type. Since mutation in 4N12 did not perturb the polarity of the surrounding eggshell, 4N12 is apparently a novel dorsal group gene and is required, after fertilization, in the spatzle-Toll pathway to generate a nuclear gradient of the transcription factor Dorsal within the embryo.In addition to the cuticular phenotype, we performed twist, snail mRNA in situ hybridization, and clearly demonstrated the dorsalized phenotypes of 4N12 at the molecular level. To determine where 4N12 acts in the spatzle-Toll pathway, we examined the 4N12,cactus double mutant embryos and found that 4N12 is upstream of cactus. Furthermore, by microinjection of in vitro transcribed Toll[10B] mRNA into 4N12 germline clone embryos, we demonstrate that 4N12 is downstream of Toll. We can not separate the dorsalized and the lethal phenotypes from which we map 4N12 at 35D1-35E1. By performing complementation tests between 4N12 and all known mutations (including P elements) at 35D1-35E1, we identify that 4N12 is allelic to l(2)35Ea. So far, we have 4 different alleles, including a P element l(2)5271. Since P element l(2)5271 is allelic to 4N12, we perform both inverse PCR and plasmid rescue to isolate genomic DNA flanking the P line and get 379 and 282 bp genomic DNA fragments, respectively. From these genomic DNA sequence, we identify two EST and one P1 clones. Sequence analysis of the EST clones revealed that it encodes a zinc finger protein. To prove that these EST clones are indeed 4N12, we carry out double strand RNA interference. However, only low fraction of embryos exhibit dorsalized phenotype. We are repeating this experiment with higher concentration of RNA.