Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast

Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen‐starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy‐defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down‐regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis.     

MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice

Msh4 (MutS homolog 4) is a member of the mammalian mismatch repair gene family whose members are involved in postreplicative DNA mismatch repair as well as in the control of meiotic recombination. In this report we show that MSH4 has an essential role in the control of male and female meiosis. We demonstrate that MSH4 is present in the nuclei of spermatocytes early in prophase I and that it forms discrete foci along meiotic chromosomes during the zygotene and pachytene stages of meiosis. Disruption of the Msh4 gene in mice results in male and female sterility due to meiotic failure. Although meiosis is initiated in Msh4 mutant male and female mice, as indicated by the chromosomal localization of RAD51 and COR1 during leptonema/zygonema, the chromosomes fail to undergo normal pairing. Our results show that MSH4 localization on chromosomes during the early stages of meiosis is essential for normal chromosome synapsis in prophase I and that it acts in the same pathway as MSH5.     

The Arabidopsis synaptonemal complex protein ZYP1 is required for chromosome synapsis and normal fidelity of crossing over

The duplicated Arabidopsis genes ZYP1a/ZYP1b encode closely related proteins with structural similarity to the synaptonemal complex (SC) transverse filament proteins from other species. Immunolocalization detects ZYP1 foci at late leptotene, which lengthen until at pachytene fluorescent signals extending the entire length of the fully synapsed homologs are observed. Analysis of zyp1a and zyp1b T-DNA insertion mutants indicates that the proteins are functionally redundant. The SC is not formed in the absence of ZYP1 and prophase I progression is significantly delayed suggesting the existence of an intraprophase I surveillance mechanism. Recombination is only slightly reduced in the absence of ZYP1 such that the chiasma frequency at metaphase I is approximately 80% of wild type. Moreover cytological analysis indicates that chiasma distribution within zyp1 bivalents is indistinguishable from wild type, providing evidence that the SC is not required for the imposition of interference. Importantly in the absence of ZYP1, recombination occurs between both homologous and nonhomologous chromosomes suggesting the protein is required to ensure the fidelity of meiotic chromosome associations.     

Mouse Fem1b interacts with the Nkx3.1 homeoprotein and is required for proper male secondary sexual development

Previous studies of epithelial cell growth and differentiation in the prostate gland have identified the homeodomain protein Nkx3.1 as a central regulator of prostate development and carcinogenesis. To understand the molecular mechanisms of Nkx3.1 function, we have used yeast two-hybrid analysis to identify Nkx3.1 interacting proteins, and have isolated Fem1b, a mammalian homolog of the C. elegans sex-determining gene Fem-1. In mice, the Fem1b and Nkx3.1 genes encode proteins that interact in glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays, and are co-expressed in the prostate and testis of neonatal mice. Null mutants for Fem1b generated by gene targeting display defects in prostate ductal morphogenesis and secretory protein expression, similar to phenotypes found in Nkx3.1 mutants. We propose that Fem1b may have a conserved role in the generation of sexual dimorphism through its interaction with Nkx3.1 in the developing prostate gland.     

The yeast kinetochore protein Slk19 is required to prevent aberrant chromosome segregation in meiosis and mitosis

BACKGROUND: Slk19 is a coiled-coil protein, which locates to the kinetochores of S. cerevisiae. Most cells lacking Slk19 undergo incomplete meiosis and form dyads during sporulation. Endogenous chromosomes appeared to be predominantly divided in an equational manner during single-division meiosis of slk19 null mutants. RESULTS: We have monitored the segregation of artificial chromosomes (YACs) in slk19 null mutants during both single-division meiosis and complete meiosis. In contrast to the results obtained with endogenous chromosomes, YACs only rarely undergo equational segregation during single division meiosis, although high rates of aberrant segregation were detected. This accounts for the high frequency of lethal spores among dyads of slk19 delta null mutants. The fraction of slk19 delta cells that were able to form tetrads solely exhibited YAC segregation defects in meiosis II, whereas the segregation of YACs in meiosis I was normal in these cells. This result might indicate that correct chromosome division in meiosis I is a prerequisite for tetrad formation. slk19 null mutants also showed YAC instability in mitosis and reduced survival after the induction of mitotic spindle damage. CONCLUSION: Slk19 is required to avoid aberrant segregation of chromosomes in meiosis I and II and in mitosis. We suggest that the absence of Slk19 leads to uncoupling of chromosome movement from completion of microtubule attachment and resolution of chromosome cohesion.     

Dicer is required for proper liver zonation

A number of genes and their protein products are expressed within the liver lobules in a region‐specific manner and confer heterogeneous metabolic properties to hepatocytes; this phenomenon is known as ‘metabolic zonation’. To elucidate the roles of Dicer, an endoribonuclease III type enzyme required for microRNA biogenesis, in the establishment of liver zonation, we examined the distribution of proteins exhibiting pericentral or periportal localization in hepatocyte‐specific Dicer1 knockout mouse livers. Immunohistochemistry showed that the localization of pericentral proteins was mostly preserved in Dicer1‐deficient livers. However, glutamine synthetase, whose expression is normally confined to a few layers of hepatocytes surrounding the central veins, was expressed in broader pericentral areas. Even more striking was the observation that all the periportal proteins that were examined, including phosphoenolpyruvate carboxykinase, E‐cadherin, arginase 1, and carbamoyl phosphate synthetase‐I, lost their localized expression patterns and were diffusely expressed throughout the entire lobule. Thus, with regard to periportal protein expression, the consequences of Dicer loss were similar to those caused by the disruption of β‐catenin. An analysis of livers deficient in β‐catenin did not identify the down‐regulation of Dicer1 or any microRNAs, indicating that they are not directly activated by β‐catenin. Thus, the present study illustrates that Dicer plays a pivotal role in the establishment of liver zonation. Dicer is essential for the suppression of periportal proteins by Wnt/β‐catenin/TCF signalling, albeit it likely acts in an indirect manner. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii

Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis. The primary phenotype is the mis-segregation of duplicated chromosomes with chromosome loss during nuclear division. This defect is conditional-lethal with respect to temperature, although relatively mild in regard to the preservation of the major microtubule organizing centers. Despite severe DNA loss many of the physical structures associated with daughter budding and the assembly of invasion structures formed and operated normally at the non-permissive temperature before completely arresting. These results suggest there are coordinating mechanisms that govern the timing of these events in the parasite cell cycle. The defect in mutant 13-20C2 was mapped by genetic complementation to Toxoplasma chromosome III and to a specific mutation in the gene encoding an ortholog of nuclear actin-related protein 4. A change in a conserved isoleucine to threonine in the helical structure of this nuclear actin related protein leads to protein instability and cellular mis-localization at the higher temperature. Given the age of this protist family, the results indicate a key role for nuclear actin-related proteins in chromosome segregation was established very early in the evolution of eukaryotes.     

Xeroderma pigmentosum group F protein binds to Eg5 and is required for proper mitosis: implications for XP-F and XFE

The xeroderma pigmentosum group F-cross-complementing rodent repair deficiency group 1 (XPF-ERCC1) complex is a structure-specific endonuclease involved in nucleotide excision repair (NER) and interstrand cross-link (ICL) repair. Patients with XPF mutations may suffer from two forms of xeroderma pigmentosum (XP): XP-F patients show mild photosensitivity and proneness to skin cancer but rarely show any neurological abnormalities, whereas XFE patients display symptoms of severe XP symptoms, growth retardation and accelerated aging. Xpf knockout mice display accelerated aging and die before weaning. These results suggest that the XPF-ERCC1 complex has additional functions besides NER and ICL repair and is essential for development and growth. In this study, we show a partial colocalization of XPF with mitotic spindles and Eg5. XPF knockdown in cells led to an increase in the frequency of abnormal nuclear morphology and mitosis. Similarly, the frequency of abnormal nuclei and mitosis was increased in XP-F and XFE cells. In addition, we showed that Eg5 enhances the action of XPF-ERCC1 nuclease activity. Taken together, these results suggest that the interaction between XPF and Eg5 plays a role in mitosis and DNA repair and offer new insights into the pathogenesis of XP-F and XFE.     

A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes

To promote faithful propagation of the genetic material during sexual reproduction, meiotic chromosomes undergo specialized morphological changes that ensure accurate segregation of homologous chromosomes. The molecular mechanisms that establish the meiotic chromosomal structures are largely unknown. We describe a mutation in a recently identified Histone H2A kinase, nhk-1, in Drosophila that leads to female sterility due to defects in the formation of the meiotic chromosomal structures. The metaphase I arrest and the karyosome, a critical prophase I chromosomal structure, require nucleosomal histone kinase-1 (NHK-1) function. The defects are a result of failure to disassemble the synaptonemal complex and to load condensin onto the mutant chromosomes. Embryos laid by nhk-1-/- mutant females arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis is affected as well. We analyzed the role of Histone H2A phosphorylation with respect to the histone code hypothesis and found that it is required for acetylation of Histone H3 and Histone H4 in meiosis. These studies reveal a critical role for histone modifications in chromosome dynamics in meiosis and mitosis.     

The Iml3 protein of the budding yeast is required for the prevention of precocious sister chromatid separation in meiosis I and for sister chromatid disjunction in meiosis II

The mitotic kinetochore of the budding yeast contains a number of proteins which are required for chromosome transmission but are non-essential for vegetative growth. We show that one such protein, Iml3, is essential for meiosis, in that the absence of this protein results in reduced spore viability, precocious sister chromatid segregation of artificial and natural chromosomes in meiosis I and chromosome non-disjunction in meiosis II.     

Nestin is required for the proper self-renewal of neural stem cells

The intermediate filament protein, nestin, is a widely employed marker of multipotent neural stem cells (NSCs). Recent in vitro studies have implicated nestin in a number of cellular processes, but there is no data yet on its in vivo function. Here, we report the construction and functional characterization of Nestin knockout mice. We found that these mice show embryonic lethality, with neuroepithelium of the developing neural tube exhibiting significantly fewer NSCs and much higher levels of apoptosis. Consistent with this in vivo observation, NSC cultures derived from knockout embryos show dramatically reduced self-renewal ability that is associated with elevated apoptosis but no overt defects in cell proliferation or differentiation. Unexpectedly, nestin deficiency has no detectable effect on the integrity of the cytoskeleton. Furthermore, the knockout of Vimentin, which abolishes nestin's ability to polymerize into intermediate filaments in NSCs, does not lead to any apoptotic phenotype. These data demonstrate that nestin is important for the proper survival and self-renewal of NSCs, and that this function is surprisingly uncoupled from nestin's structural involvement in the cytoskeleton.     

Genome-wide variation in recombination in female meiosis: a risk factor for non-disjunction of chromosome 21

Altered recombination patterns along non-disjoined chromosomes is the first molecular correlate identified for non-disjunction in humans. To understand better the factors related to this correlate, we have asked to what extent is recombination altered in an egg with a disomic chromosome: are patterns limited to the non-disjoined chromosome or do they extend to the entire cell? More specifically, we asked whether there is reduced recombination in the total genome of an egg with a non-disjoined chromosome 21 and no detectable recombination. We chose this subclass of non-disjoined chromosomes to enrich potentially for extremes in recombination. We found a statistically significant cell-wide reduction in the mean recombination rate in these eggs with non-disjoined chromosomes 21; no specific chromosomes were driving this effect. Most importantly, we found that this reduction was consistent with normal variation in recombination observed among eggs. Thus, given that recombination is a multifactorial trait, these data suggest that when the number of genome-wide recombination events is less than some threshold, specific chromosomes may be at an increased risk for non-disjunction. Further studies are required to confirm these results, to determine the importance of genetic and environmental factors that regulate recombination and to determine their impact on non-disjunction.     

Condensin is required for chromosome arm cohesion during mitosis

We describe a novel requirement for the condensin complex in sister chromatid cohesion in Saccharomyces cerevisiae. Strikingly, condensin-dependent cohesion can be distinguished from cohesin-based pairing by a number of criteria. First, condensin is required to maintain cohesion at several chromosomal arm sites but, in contrast to cohesin, is not required at either centromere or telomere-proximal loci. Second, condensin-dependent interlinks are established during mitosis independently of DNA replication and are reversible within a single cell cycle. Third, the loss of condensin-dependent linkages occurs without affecting cohesin levels at the separated URA3 locus. We propose that, during mitosis, robust sister chromatid cohesion along chromosome arms requires both condensinand cohesin-dependent mechanisms, which function independently of each other. We discuss the implications of our results for current models of sister chromatid cohesion.     

Asynchronous chromosome pairing in male meiosis of the rat (Rattus norvegicus)

Premeiotic and meiotic chromosome distribution was studied in rat testes suspensions by a triple-color fluorescent staining protocol which allows simultaneous visual inspection of two chromosomal targets highlighted by FISH together with immunostained SCP3 synaptonemal complex (SC) proteins which are marked by a third, composite color. Triple labeling with rat chromosome (RNO) 4q and 19p specific probes and SCP3 staining disclosed that homologs are separated in premeiotic and leptotene nuclei. Pairing of homologous chromosome regions commenced during early zygotene, with pairing of the small metacentric chromosomes 19 preceding that of the distal region of the long arm of RNO4. Our results show that homolog association occurs during zygotene of rat spermatogenesis, with small and large chromosomes showing a considerable asynchrony. Comparison with pairing progression in meiosis of other mammals suggests that asynchronous chromosome pairing reflects size differences within a complement. This revised version was published online in July 2006 with corrections to the Cover Date.     

Retinoic acid signaling is required for proper morphogenesis of mammary gland.

Retinoic acid (RA), a bioactive chemical compound synthesized from dietary derived vitamin A, has been successfully used as a chemopreventive and chemotherapeutic agent through the regulation of cell proliferation, differentiation, and apoptosis acting via the retinoic acid receptors. Despite two decades of research on the function of retinoic acid, the physiological role of RA in mammary gland development is still not well characterized. In this report, we demonstrate that RA is required for proper morphogenesis of mouse mammary gland in a novel transgenic mouse model system. It was found that inhibition of RA signaling in vivo leads to excessive mammary ductal morphogenesis through upregulation of cyclin D1 and MMP-3 expression. Furthermore, we show that the transgene-induced excessive branching morphogenesis could be reversed by treatment with RA, demonstrating the direct physiological effect of RA signaling in vivo. In addition, we demonstrate that excessive branching morphogenesis in the transgenic mammary gland are cell-autonomous and do not require stromal signals within the transgenic mammary gland. Finally, we provide evidence suggesting that retinoic acid signaling is required for appropriate mammary gland differentiation. Collectively, our data indicate for the first time that retinoic acid signaling is required to maintain the homeostasis of mammary gland morphogenesis.