Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains.

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15 agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 ± 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 ± 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

No Wolbachia infection was detected in Drosophila elegans collected from the wild in the Ryukyu Islands, Japan.

Flower breeding, tropical and subtropical Drosophila elegans is distributed in the Ryukyu Islands and Taiwan (black morph) and in southern China, Philippines, Indonesia, and New Guinea (brown morph). Although reproductive and behavioral manipulations by Wolbachia are reported in many insect taxa, Wolbachia infection in D. elegans is unclear. There is only a report of no Wolbachia detected in a laboratory strain of brown morph. This PCR diagnosis study revealed no Wolbachia infection in D. elegans males collected from the wild in the Ryukyu Islands. We concluded that D. elegans black morph in the Ryukyu Islands is not infected with Wolbachia .

A cross-species approach for the identification of Drosophila male sterility genes.

Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. We focused on one such gene, Rack 1, and found that it plays an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.

The Drosophila Neprilysin 4 gene is essential for sperm function following sperm transfer to females.

Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. Here, we focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. We examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, we induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, we conclude that the Nep4 gene is essential for sperm function following sperm transfer to females.

Identification of CR43467 encoding a long non-coding RNA as a novel genetic interactant with dFIG4, a CMT-causing gene.

The eye imaginal disc-specific knockdown of dFIG4, a Drosophila homolog of FIG4 that is one of the Charcot-Marie-Tooth disease (CMT)-causing genes, induces an aberrant adult compound eye morphology, the so-called rough eye phenotype. We previously performed modifier screening on the dFIG4 knockdown-induced rough eye phenotype and identified several genes, including CR18854, encoding a long non-coding RNA (lncRNA) as genetic interactants with dFIG4. In the present study, in more extensive genetic screening, we found that the deletion of a gene locus encoding both Odorant rector 46a (Or46a) and lncRNA CR43467 effectively suppressed the rough eye phenotype induced by the knockdown of dFIG4. Both genes were located on the same locus, but oriented in opposite directions. In order to identify which of these genes is responsible for the suppression of the rough eye phenotype, we established a CR43467-specific knockdown line using the CRISPR-dCas9 system. By using this system, we demonstrated that the CR43467 gene, but not the Or46a gene, genetically interacted with the dFIG4 gene. The knockdown of CR43467 rescued the reductions in the length of synaptic branches and number of boutons at neuromuscular junctions induced by the knockdown of dFIG4. The vacuole enlargement phenotype induced by the fat body-specific dFIG4 knockdown was also effectively suppressed by the knockdown of CR43467. The knockdown of CR43467 also suppressed the rough eye phenotype induced by other peripheral neuropathy-related genes, such as dCOA7, dHADHB, and dPDHB. We herein identified another gene encoding lncRNA, CR43467 as a genetic interactant with the CMT-causing gene.

Humanized Flies and Resources for Cross-Species Study.

The completion of whole-genome sequences has greatly broadened our understanding of genes and genomes. The availability of model organism databases facilitates the sharing of information. However, it is still challenging to predict the pathogenicity of missense mutations, and it is more difficult to evaluate the functional impact of noncoding variants. What is more, it is a primary question to understand what variants interact to express phenotypes. Powerful genetic tools and resources available in Drosophila now make it much easier to replace endogenous genes with exogenous DNA. This allows us to directly investigate and compare the functions of orthologs, variants, and fragments in a single genetic background, the value of which should be widely appreciated. To take one example, we are currently studying so-called ultra-conserved elements, which have been conserved over hundreds of millions of years of vertebrate evolution. Many highly conserved elements are in noncoding regions and are thought to play a pivotal role in gene regulation. We generated transgenic fly lines carrying human ultra-conserved elements for enhancer reporter assay and indeed observed the reporter expression in one or more tissues of embryos and larvae in all elements tested. Currently, transgenic human-ORF lines expressing human genes under the control of GAL4/UAS system are also been developed, which will greatly facilitate the cross-species in Drosophila. In this chapter, I introduce useful tools and resources available in Drosophila to nonspecialists, encouraging their further use in many applications.

A novel cell death gene acts to repair patterning defects in Drosophila melanogaster.

Cell death is a mechanism utilized by organisms to eliminate excess cells during development. Here, we describe a novel regulator of caspase-independent cell death, Mabiki (Mabi), that is involved in the repair of the head patterning defects caused by extra copies of bicoid in Drosophila melanogaster. Mabiki functions together with caspase-dependent cell death mechanisms to provide robustness during development.

Two types of cis-trans compensation in the evolution of transcriptional regulation.

Because distant species often share similar macromolecules, regulatory mutations are often considered responsible for much of their biological differences. Recently, a large portion of regulatory changes has been attributed to cis-regulatory mutations. Here, we examined an alternative possibility that the putative contribution of cis-regulatory changes was, in fact, caused by compensatory action of cis- and trans-regulatory elements. First, we show by stochastic simulations that compensatory cis-trans evolution maintains the binding affinity of a transcription factor at a constant level, thereby spuriously exaggerating the contribution of cis-regulatory mutations to gene expression divergence. This exaggeration was not observed when changes in the binding affinity were compensated by variable transcription factor concentration. Second, using reciprocal introgressions of Drosophila, we show that relative expression of heterozygous alleles from two distinct species often varied significantly between different species backgrounds, indicating the possible action of cis-trans compensation. Taken together, we propose that cis-trans hybrid incompatibilities are accumulating much faster than generally considered.