Nuclear factor I A (NFIA) is a transcription factor that belongs to the NFI family. Truncating variants or intragenic deletion of the NFIA gene are known to cause the human neurodevelopmental disorder known as NFIA‐related disorder, but no patient heterozygous for a missense mutation has been reported. Here, we document two unrelated patients with typical phenotypic features of the NFIA‐related disorder who shared a missense variant p.Lys125Glu (K125E) in the NFIA gene. Patient 1 was a 6‐year‐old female with global developmental delay, corpus callosum anomaly, macrocephaly, and dysmorphic facial features. Patient 2 was a 14‐month‐old male with corpus callosum anomaly and macrocephaly. By using Drosophila and zebrafish models, we functionally evaluated the effect of the K125E substitution. Ectopic expression of wild‐type human NFIA in Drosophila caused developmental defects such as eye malformation and premature death, while that of human NFIA K125E variant allele did not. nfia‐deficient zebrafish embryos showed defects of midline‐crossing axons in the midbrain/hindbrain boundary. This impairment of commissural neurons was rescued by expression of wild‐type human NFIA, but not by that of mutant variant harboring K125E substitution. In accordance with these in vivo functional analyses, we showed that the K125E mutation impaired the transcriptional regulation of HES1 promoter in cultured cells. Taken together, we concluded that the K125E variant in the NFIA gene is a loss‐of‐function mutation.

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.

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.

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.