Primordial Germ Cell Cryopreservation and Revival of <em>Drosophila</em> Strains

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and ...

Kyoto Institute of Technology

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.

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

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.

Humanized Flies and Resources for Cross-Species Study.

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.

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

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.

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

There is an urgent need to cryopreserve Drosophila stocks that have been maintained as living cultures for a long time. Long-term culture increases the risk of accidental loss and of unwanted genetic alteration. Here, we report that cryopreserved primordial germ cells (PGCs) can produce F1 progeny when transplanted into hosts. The cryopreserved donor PGCs could form germline stem cells in host gonads and contributed to continuous offspring production. Furthermore, the ability to produce offspring did not appear to vary with either differences between donor strains or cryopreservation duration. Therefore, we propose that our cryopreservation method is feasible for long-term storage of various Drosophila strains. These results underscore the potential usefulness of our cryopreservation method for backing up living stocks to avoid either accidental loss or genetic alteration.

Offspring production from cryopreserved primordial germ cells in Drosophila.

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.

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

Flower breeding, tropical and subtropical  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  are reported in many insect taxa,  infection in  is unclear. There is only a report of no  detected in a laboratory strain of brown morph. This PCR diagnosis study revealed no  infection in  males collected from the wild in the Ryukyu Islands. We concluded that  black morph in the Ryukyu Islands is not infected with  .

Toshiyuki Takano-Shimizu-Kouno

KYOTO Drosophila Stock Center

Kyoto Institute of Technology

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains

Toshiyuki Takano-Shimizu-Kouno

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Kyoto Institute of Technology

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains

KYOTO Drosophila Stock Center