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NATIONAL INSTITUTE OF GENETICS
Mammalian Development Laboratory

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RESEARCH

Reserch

  1. Genetic technologies in the mouse
  2. Molecular mechanisms of somite segmentation
  3. Germ cell development
  4. The roles of Notch signaling in cardiovascular development

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3.Germ cell development

Objectives and significance

Germ cells are the only cells that can transmit genetic information to the next generation, and it is becoming clear that they have specific strategies for development, differentiation, and survival, which are totally different from those of somatic cells. However, many questions still remain unanswered for the understanding of their identity at the molecular level. The sexual differentiation of germ cells in mammals starts after they are taken into the gonads in the embryonic stage. An RNA-binding protein, Nanos2 is involved in this process. Without Nanos2, male germ cells may enter female differentiation pathway. In addition, Nanos2 is expressed in spermatogenic stem cells during spermatogenesis, and plays an important role in the maintenance of stem cells; however, the accompanying molecular mechanisms are unknown.

Background : Expression and function of Nanos genes in mouse

The mouse genome has three Nanos genes, Nanos1-3. Nanos1 is expressed in both embryonic and adult brains and also in adult testes. However, Nanos1 knockout mice show no abnormalities and are fertile. In contrast, Nanos2 and Nanos3 are expressed in the embryonic germ cells and a deficiency in these genes results in the loss of germ cells. Nanos3 is activated in the early PGCs. The number of PGCs is greatly reduced in the knockout animals, which results in a complete loss of germ cells in both male and female (Fig. 1).
In contrast, the expression of Nanos2 is restricted to the male germ cells after their colonization of the gonads and the lack of Nanos2 leads to male specific sterility (Fig. 1).

Fig.1.Phenotypes of Nanos2-KO and Nanos3-KO mice

Project1:Expression and function of Nanos3

The migrating PGCs undergo apoptosis in Nanos3-null embryos. We assessed whether the Bax-dependent apoptotic pathway is responsible for this cell death by knocking out the Bax gene together with the Nanos3 gene. However, the Bax elimination did not completely rescue PGC apoptosis in Nanos3-null embryos, and only a portion of the PGCs survived in the double knockout embryo. We further established a mouse line, Nanos3-Cre-pA, to undertake lineage analysis and our results indicate that most of the Nanos3-null PGCs die rather than differentiate into somatic cells, irrespective of the presence or absence of Bax. In addition, a small number of surviving PGCs in Nanos3/Bax-null mice are maintained and differentiate as male and female germ cells in the adult gonads. Our findings thus suggest that heterogeneity exists in the PGC populations and that Nanos3 maintains the germ cell lineage by suppressing both Bax-dependent and Bax-independent apoptotic pathways. However, the detailed molecular mechanism of Nanos3 action remains to be uncovered.

Project 2: Function of Nanos2 on sexual differentiation of germ cells

Germ cells are sexually bi-potential at the migrating stage and their sex-specific differentiation begins after their colonization into the gonads at around E10.5 (Fig. 2). Once committed to the normal developmental process, female germ cells enter meiosis at E13.5 whereas male germ cells undergo cell-cycle arrest at G1/G0 and never enter meiosis during the embryonic stages of development. Retinoic acid (RA) is identified as the meiosis-inducing factor that induces Stra8 expression, required for the premeiotic replication in female germ cells. In male gonads, Cyp26b1, an enzyme to destroy RA works as a meiosis-inhibiting factor up to E13.5. Cys26b1 expression becomes gradually reduced after E13.5. In turn, Nanos2 begins to be expressed in male germ cells from E13.5 and no Stra8 expression is observed. In the absence of Nanos2, however, Stra8 expression is observed and germ cells enter meiotic process. These observations strongly indicate that Nanos2 is required to suppress Stra8 expression for preventing male germ cells from entering meiosis. As the molecular mechanism, we have recently shown that Nanos2 interacts with the CCR4-NOT deadenylation complex and the Nanos2/CCR4-NOT complex has a deadenylase activity in vitro. We propose that NANOS2-interacting RNAs may be degraded through NANOS2-mediated deadenylation.
However direct target RNAs of Nanos2 are not yet identified.

Fig. 2.Sexual development of germ cells

研究課題3:平滑筋細胞の発生のメカニズム

The continuation of the spermatogenic process throughout life relies on the proper regulation of the stem cell maintenance and differentiation. A stem cell functions during steady state of mouse spermatogenesis might reside in the undifferentiated spermatogonia, especially in a recently proposed specific compartment called “actual stem cells”. Nanos2 plays an essential role in the maintenance of these cells. Conditional disruption of postnatal Nanos2 results in the depletion of spermatogonial cell reserves (Fig. 3), whereas mouse testes in which Nanos2 has been overexpressed show an accumulation of spermatogonia with undifferentiated properties. Moreover, lineage tracing experiments revealed that endogenous Nanos2-expressing cells give rise to all spermatogenic cell types over long periods, indicating that a Nanos2-expressing subpopulation of the undifferentiated spermatogonia functions as stem cells in vivo. Nanos2 is expressed in these actual stem cells and functions to maintain this undifferentiated stem cell population in part by suppressing their differentiation. We need to know the mechanism of regulation of Nanos2 expression (i.e. its induction & maintenance) in the spermatogonial stem cells. In addition, the target RNAs critical for Nanos2’s function have to be identified.

Fig. 3.Germ cell-loss phenotype of Nanos2 –KO after birth


Project 4:Cyclic gene expression changes in Sertoli cells

In mammalian testes, Sertoli cells create microenvironments essential for spermatogenesis and support the differentiation of male germ cells. It has been known that Sertoli cells cyclically change their gene expression coincident with the alteration of an interacting germ cell group. In seminiferous tubules, twelve stages (stage I-XII) of differentiating germ cell groups can be distinguished, and each group of cells synchronously progresses to the next stage. The association between gene expression in Sertoli cells and germ cell groups suggests the presence of stage-specific microenvironments. Although some stage-regulated genes in Sertoli cells were identified, so far it remains unclear, how many and what kinds of genes show cyclic expression in Sertoli cells, how their periodicity is regulated and whether the periodic expression of these genes have any biological functions. We are trying to examine the importance of stage-regulated gene expression in Sertoli cells in spermatogenesis by focusing on transcription factors and signaling pathways.





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