Highly conserved during evolution, the enzyme Ubc9 activates the small ubiquitin-like modifier (SUMO) prior to its covalent ligation to target proteins. We have used mutations in the Drosophila Ubc9 (dUbc9) gene to understand Ubc9 functions in vivo. Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. Some blood cells are polyploid and multinucleate, exhibiting signs of genomic instability. We also observe an overabundance of highly differentiated blood cells (lamellocytes), normally not found in healthy larvae. Lamellocytes in mutants are either free in circulation or recruited to form tumorous masses. Hematopoietic defects of dUbc9 mutants are strongly suppressed in the absence of the Rel/NF-kappaB-family transcription factors Dorsal and Dif or in the presence of a non-signaling allele of Cactus, the IkappaB protein in Drosophila. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Together, our results support a role for dUbc9 in the negative regulation of the Drosophila NF-kappaB signaling pathways in larval hematopoiesis and humoral immunity.

The hermaphroditic Mangrove Killifish, Kryptolebias marmoratus, is the world's only vertebrate that routinely self-fertilizes. As such, highly inbred and presumably isogenic “clonal” lineages of this androdioecious species have long been maintained in several laboratories and used in a wide variety of experiments that require genetically uniform vertebrate specimens. Here we conduct a genetic inventory of essentially all laboratory stocks of the Mangrove Killifish held worldwide. At 32 microsatellite loci, these stocks proved to show extensive interline differentiation as well as some intraline variation, much of which can be attributed to post-origin de novo mutations and/or to the segregation of polymorphisms from wild progenitors. Our genetic findings also document that many of the surveyed laboratory strains are not what they have been labeled, apparently due to the rather frequent mishandling or unintended mixing of various laboratory stocks over the years. Our genetic inventory should help to clarify much of this confusion about the clonal identities and genetic relationships of laboratory lines, and thereby help to rejuvenate interest in K. marmoratus as a reliable vertebrate model for experimental research that requires or can capitalize upon “clonal” replicate specimens.

Kryptolebias marmoratus is a synchronous hermaphroditic vertebrate that utilizes an ovotestis for reproduction. This fish develops externally, is easy to maintain, and has about a 100-day life cycle, making it a desirable developmental genetic model organism. Here, we present a pilot zygotic mutant screen utilizing the common chemical mutagen, N-ethyl-N-nitrosourea (ENU) to establish genetics in this model species. Selection of clonal stocks and optimal conditions for mutagenizing this fish are presented and the types and frequencies of zygotic mutants are documented in comparison to other fish models. Kryptolebias marmoratus is an exemplar model organism that will complement future developmental genetic screens in vertebrates.

The mangrove killifish, Kryptolebias marmoratus, is unique among vertebrates due to its self-fertilizing mode of reproduction involving an ovotestis. As a result, it constitutes a simplistic and desirable vertebrate model for developmental genetics as it is easily maintained, reaches sexual maturity in about 100 days, and provides a manageable number of relatively clear embryos. After the establishment and characterization of an initial mutagenesis pilot screen using N-ethyl-N-nitrosourea, a three-generation genetic screen was performed to confirm zygotic mutant allele heritability and simultaneously score for homozygous recessive mutant sterile F2 fish. From a total of 307 F2 fish screened, 10 were found to be 1° males, 16 were sterile, 92 wild-type, and the remaining 189, carriers of zygotic recessive alleles. These carriers produced 25% progeny exhibiting several zygotic phenotypes similar to those previously described in zebrafish and in the aforementioned pilot screen, as expected. Interestingly, new phenotypes such as golden yolk, no trunk, and short tail were observed. The siblings of sterile F2 mutants were used to produce an F3 generation in order to confirm familial sterility. Out of the 284 F3 fish belonging to 10 previously identified sterile families, 12 were found to be 1° males, 69 were wild-type, 83 sterile, and 120 were classified as */+ (either wild-type or carriers) with undefined genotypes. This screen provides proof of principle that K. marmoratus is a powerful vertebrate model for developmental genetics and can be used to identify mutations affecting fertility.

At the onset of metamorphosis in Drosophila melanogaster, the steroid hormone 20-OH ecdysone induces a small number of early and early-late puffs in the polytene chromosomes of the third-instar larval salivary gland whose activity is required for regulating the activity of a larger set of late puffs. Most of the corresponding early and early-late genes have been found to encode transcription factors that regulate a much larger set of late genes. In contrast, we describe here the identification of an ecdysone-regulated gene in the 62E early-late puff, denoted D-spinophilin, that encodes a protein similar to the mammalian protein spinophilin/neurabin II. The D-spinophilin protein is predicted to contain a highly conserved PP1-binding domain and adjacent PDZ domain, as well as a coiled-coil domain and SAM domain, and belongs to a family of related proteins from diverse organisms. Transcription of D-spinophilin is correlated with 62E puff activity during the early stages of metamorphosis and is ecdysone-dependent, making this the first member of this gene family shown to be regulated by a steroid hormone. Examination of the dynamic patterns of D-spinophilin expression during the early stages of metamorphosis are consistent with a role in central nervous system metamorphosis as well as a more general role in other tissues. As D-spinophilin appears to be the only member of this gene family in Drosophila, its study provides an excellent opportunity to elucidate the role of an important adaptor protein in a genetic model organism.