SUMMARYClathrin has previously been implicated in Drosophila male fertility and spermatid individualization. To understand further the role of membrane transport in this process, we analyzed the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm. The reproductive defects of aux males were rescued by male germ cell-specific expression of aux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, this rescue depends on both the clathrin-binding and J domains, suggesting that the ability of Aux to bind clathrin and the Hsc70 ATPase is essential for sperm formation. aux mutant spermatids show a deficit in formation of the plasma membrane during elongation, which probably disrupts the subsequent coordinated migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. By contrast, in aux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its colocalization with AP-1 was disrupted. Based on these results, we propose that Golgi-derived clathrin-positive vesicles are normally required for sustaining the plasma membrane increase necessary for spermatid differentiation. Our data suggest that Aux participates in forming these Golgi-derived clathrin-positive vesicles and that Aux, therefore, has a role in the secretory pathway.

Numerous previous studies demonstrated that gene expression was influenced by histone modifications. However, little information is available about the relation of histone methylation with embryonic gene expression. Here, we examine the significance of histone H3 dimethyl-lysine 4 (H3K4me2) during mouse zygotic genome activation (ZGA) by inhibiting demethylation with the specific histone H3 lysine 4 demethylase inhibitor bisguanidine 1c (1c). A 1c treatment of one-cell embryos did not significantly affect the level of eIF-4C transcripts but did affect Oct4 levels by the two-cell stage. Furthermore, 1c treatment significantly inhibited cleavage of the embryos to the four-cell stage (from 82.7% to 18.2%), and the inhibitory effect was identified to be irreversible. These results suggest that histone methylation may be closely correlated with the formation of a transcriptionally repressive state during ZGA and that the repressive state actually dictates the appropriate pattern of gene expression required for further development.

Abstract. Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos. Key words: Gene expression, Histone H3 methylation, Mouse, Somatic cell nuclear transfer, U2afbp-rs, Xist (J. Reprod. Dev. 54: [233][234][235][236][237][238] 2008) eprogramming of differentiated somatic nuclei to a totipotent state by transfer into oocyte cytoplasm is not efficient. Although in some experiments a considerable proportion of reconstructed embryos might reach the blastocyst stage, the potential developmental ability to full-term is very limited. To improve the efficiency of nuclear transfer (NT), it is necessary to understand the mechanisms of the reprogramming process.The reprogramming events following transfer of somatic nuclei into oocyte cytoplasm occur at the epigenetic level. Histone acetylation and methylation are two important epigenetic modalities [1][2][3][4]. It is known that acetylation of histone plays important roles in transcription activity [5] and histone methylation at K9 associates with gene silencing [6,7]. Recently, histone H3K4 di-methylation (H3K4me2) has been found to be a positive chromatin marker associated with promoters of active genes [8,9]. Similar studies have found that H3K4me3 is enriched at fully activated promoter regions, while H3K4me2 is correlated with the basal transcriptionpermissive state [10]. Demethylation of H3K4 catalyzed by lysinespecific demethylase 1 (LSD1) is associated with transcriptional repression [11]. In addition, H3K4 di-and trimethylation of reprogrammed somatic genomes may be independent of gene activity and may represent one of the major events that occur during somatic genome reprogramming towards a transcriptional activation-permissive state [12].In early embryonic development, activation of the embryonic genome is a crucial event for onset of transcription during preimplantation development [1...

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BackgroundThe J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.ResultsLike mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures.ConclusionIn support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.