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The ligand-receptor-mediated intercellular communication system plays important roles in coordinating developmental and physiological events in multicellular organisms. In plants, CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides and their cognate receptors are thought to be involved in various aspects of the plant life cycle. Although the importance of this communication is broadly recognized, most CLE peptides are yet to be functionally characterized. A major problem in research on small signaling peptide-encoding genes is the limited number of loss-of-function mutants available due to their small gene size. CRISPR/Cas9-mediated gene targeting has the potential to overcome this problem, as it can be used to generate targeted mutations in essentially any gene, regardless of size. Here we generated a series of mutants of CLE-peptide-encoding genes. Newly generated clv3 and cle40 mutants reproduced the expected mutant phenotypes in the shoot apical meristem and root meristem, respectively. Our results show that CRISPR/Cas9-mediated gene targeting is a powerful tool for genetic analyses, even of small genes. We also report a novel mutant for CLE44 [which is thought to encode a tracheary elements differentiation inhibitory factor (TDIF)] and show that CLE44 contributes to vascular development. The bioresources presented here will be a powerful tool for further characterization of CLE peptides.

Importin 13 (Imp13) is a bidirectional nuclear transporter of proteins involved in a range of important cellular processes, with an N-terminally truncated inhibitory isoform (tImp13) specifically expressed in testis. To gain insight into tImp13 function, we performed a yeast-2-hybrid screen from a human testis cDNA library, identifying for the first time a suite of interactors with roles in diverse cellular process. We validated the interaction of tImp13 with Eukaryotic translation initiation factor 4γ2 (EIF4G2) and High mobility group containing protein 20A (HMG20A), benchmarking that with glucocorticoid receptor (GR), a known Imp13 interactor expressed in testis. Coimmunoprecipitation assays indicated association of both tImp13 and Imp13 with EIF4G2, HMG20A and GR. Quantitative confocal microscopic analysis revealed the ability of tImp13 to inhibit the nuclear localisation of EIF4G2, HMG20A and GR, as well as that of Imp13 to act as a nuclear exporter for both EIF4G2 and HMG20A, and as a nuclear importer for GR. The physiological relevance of these results was highlighted by the cytoplasmic localisation of EIF4G2, HMG20A and GR in pachytene spermatocytes/round spermatids in the murine testis where tImp13 is present at high levels, in contrast to the nuclear localisation of HMG20A and GR in spermatogonia, where tImp13 is largely absent. Interestingly, Imp13, EIF4G2, HMG20A and GR were found together in the acrosome vesicle of murine epididymal spermatozoa. Collectively, our findings show, for the first time, that tImp13 may have a functional role in the mature spermatozoa, in addition to that in the meiotic germ cells of the testis.

The homeostasis of meristems in flowering plants is maintained by cell-to-cell communication via CLE (CLAVATA3/EMBRYO SURROUNDING REGION-related) peptide hormones. In contrast, cell signals that regulate meristem activity remains elusive in bryophytes that maintain apical meristems in the gametophyte (haploid) body and undergo a gametophyte-dominant life cycle. We here show that MpCLE1 confines the proliferative activity of gametophytic meristem and affects the overall size of gametangiophores (reproductive organs) in Marchantia polymorpha, which is in sharp contrast with the meristem-promoting function of its ortholog TDIF/CLE41/CLE44 in Arabidopsis vascular meristems. Expression analysis suggests that MpCLE1 and its receptor gene MpTDR are expressed in distinct patterns across the apical meristem. These data suggest that local CLE peptide signaling may have had a role in regulating cell proliferation in the shoot meristem in the ancestral land plant and acts in both sporophytic and gametophytic meristems of extant plants., Author summary Land plants undergo an alternation of generations where both haploid and diploid phases develop multicellular bodies. Their growth relies on the activity of meristems at the growing tips of their bodies. Here we show a CLE peptide hormone acts as an intercellular signal controlling proliferative activity in the apical meristem of Marchantia polymorpha. Our finding reveals a general association of CLE peptide signaling with meristem homeostasis, a feature that evolved in the ancestral land plant, in both haploid and diploid phases.

The Spalt-like 4 (Sall4) zinc finger protein is a critical transcription factor for pluripotency in embryonic stem cells (ESCs). It is also involved in the formation of a variety of organs, in mice, and humans. We report the essential roles of Sall4 in mouse primordial germ cell (PGC) specification. PGC specification is accompanied by the activation of the stem cell program and repression of the somatic cell program in progenitor cells. Conditional inactivation of Sall4 during PGC specification led to a reduction in the number of PGCs in embryonic gonads. Sall4del/del PGCs failed to translocate from the mesoderm to the endoderm and underwent apoptosis. In Sall4del/del PGC progenitors, somatic cell program genes (Hoxa1 and Hoxb1) were derepressed, while activation of the stem cell program was not impaired. We demonstrated that in differentiated ESCs, Sall4 bound to these somatic cell program gene loci, which are reportedly occupied by Prdm1 in embryonic carcinoma cells. Given that Sall4 and Prdm1 are known to associate with the histone deacetylase repressor complex, our findings suggest that Sall4 suppresses the somatic cell program possibly by recruiting the repressor complex in conjunction with Prdm1; therefore, it is essential for PGC specification. Stem Cells 2015;33:289–300

Cell-to-cell communication is crucial for the coherent functioning of multicellular organisms, and they have evolved intricate molecular mechanisms to achieve such communication. Small, secreted peptide hormones participate in cell-to-cell communication to regulate various physiological processes. One such family of plant peptide hormones is the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-related (CLE) family, whose members play crucial roles in the differentiation of shoot and root meristems. Recent biochemical and genetic studies have characterized various CLE signaling modules, which include CLE peptides, transmembrane receptors, and downstream intracellular signaling components. CLE signaling systems are conserved across the plant kingdom but have divergent modes of action in various developmental processes in different species. Moreover, several CLE peptides play roles in symbiosis, parasitism, and responses to abiotic cues. Here we review recent studies that have provided new insights into the mechanisms of CLE signaling.

The family of interferon-induced transmembrane protein (Ifitm/mil/fragilis) genes encodes cell surface proteins that may modulate cell adhesion and influence cell differentiation. Mouse Ifitm1 and -3, which are expressed in primordial germ cells (PGCs), are implicated to have roles in germ cell development, but the specific functions have been unclear. Our results show that Ifitm1 activity is required for PGC transit from the mesoderm into the endoderm, and that it appears to act via a repulsive mechanism, such that PGCs avoid Ifitm1-expressing tissues. In contrast, Ifitm3, which is expressed in migratory PGCs, is sufficient to confer autonomous PGC-like homing properties to somatic cells. These guidance activities are mediated by the N-terminal extracellular domain of the specific IFITM, which cannot be substituted by that of another family member. Complex homo- and/or heterotypic intercellular interactions among various IFITMs in PGCs and neighboring cells may underpin coordinated germ cell guidance in mice.

A well-established experimental paradigm to analyze gene function in development is to elucidate the impact of gain and loss of gene activity on cell differentiation, tissue modelling, organogenesis, and morphogenesis. This chapter describes the experimental protocols to study gene function by means of electroporation and lipofection to manipulate genetic activity in whole embryos and fetal organs in vitro. These techniques allow for more precise control of the timing, with reference to developmental age or stage, and the cell/tissue-specificity of the changes in gene activity. They provide an alternative strategy that can expedite the analysis of gene function before resorting to the conventional means of transgenesis and gene targeting in the whole organism.

A well-established experimental paradigm to analyze gene function in development is to elucidate the impact of gain and loss of gene activity on cell differentiation, tissue modelling, organogenesis, and morphogenesis. This chapter describes the experimental protocols to study gene function by means of electroporation and lipofection to manipulate genetic activity in whole embryos and fetal organs in vitro. These techniques allow for more precise control of the timing, with reference to developmental age or stage, and the cell/tissue-specificity of the changes in gene activity. They provide an alternative strategy that can expedite the analysis of gene function before resorting to the conventional means of transgenesis and gene targeting in the whole organism.

Dullard/Ctdnep1 is a member of the serine/threonine phosphatase family of the C-terminal domain of eukaryotic RNA polymerase II. Embryos lacking Dullard activity fail to form primordial germ cells (PGCs). In the mouse, the formation of PGCs is influenced by BMP4 and WNT3 activity. Although Dullard is reputed to negatively regulate BMP receptor function, in this study we found mutations in Dullard had no detectable effect on BMP4 and p-Smad activity. Furthermore Dullard mutations did not influence the dosage-dependent inductive effect of Bmp4 in PGC formation. However, Dullard may function as a positive regulator of WNT signalling. Combined loss of one copy each of Dullard and Wnt3 had a synergistic effect on the reduction of PGC numbers in the compound heterozygous embryo. In addition, loss of Dullard function was accompanied by down-regulation of WNT/β-catenin signalling activity and a reduction in the level of Dishevelled 2 (Dvl2). Therefore, Dullard may play a role in the fine-tuning of WNT signalling activity by modulating the expression of ligands/antagonists and the availability of Dvl2 protein during specification of the germ cell lineage.

SummaryThe Y-linked gene Sry regulates mammalian sex determination in bipotential embryonic gonads. Here, we report that the transcription factors Six1 and Six4 are required for male gonadal differentiation. Loss of Six1 and Six4 together, but neither alone, resulted in a male-to-female sex-reversal phenotype in XY mutant gonads accompanied by a failure in Sry activation. Decreased gonadal precursor cell formation at the onset of Sry expression and a gonadal size reduction in both sexes were also found in mutant embryos. Forced Sry transgene expression in XY mutant gonads rescued testicular development but not the initial disruption to precursor growth. Furthermore, we identified two downstream targets of Six1/Six4 in gonadal development, Fog2 (Zfpm2) and Nr5a1 (Ad4BP/Sf1). These two distinct Six1/Six4-regulated pathways are considered to be crucial for gonadal development. The regulation of Fog2 induces Sry expression in male sex determination, and the regulation of Nr5a1 in gonadal precursor formation determines gonadal size.

WNT signaling activity is involved in the regulation of many cellular functions, including proliferation, migration, cell fate specification, maintenance of pluripotency and induction of tumorigenicity. Here we summarize recent progress towards understanding the regulation of canonical WNT/β-catenin signaling activity through feedback regulatory loops involving the ligands, agonists and antagonists, the availability of intracellular pools of active β-catenin and the cross-regulation of the WNT activity by β-catenin independent pathway. We also review recent findings on the role of WNT/β-catenin signaling in tissue lineage differentiation during embryogenesis and the maintenance and self renewal of embryo-derived stem cells in vitro.

The translocon-associated protein (TRAP, also termed the signal sequence receptor) complex is required for the efficient translocation of secretory and membrane proteins in the endoplasmic reticulum, and is also involved in the endoplasmic reticulum stress-mediated unfolded protein response pathway. To investigate the roles of Trap-γ, a TRAP complex subunit, we generated Trap-γ knockout mice and found that mutant pups died soon after birth because of retarded embryonic organ growth, especially in the lung. The mutant placentae showed severe vascular network malformation in the labyrinth and significant reductions in blood space areas, which had an adverse effect on intrauterine embryonic growth. Placental malformation was already found by the mid-gestation-stage mutant placenta, with poor vascular endothelial cell proliferation in the chorionic plate region and increased apoptotic cell death in the labyrinth. Thus, Trap-γ appears to be required for vascular network formation in murine placental development. Developmental Dynamics 240:394–403, 2011. © 2011 Wiley-Liss, Inc.

Mouse embryos lacking Lhx1 (Lim1) activity display defective gastrulation and are deficient of primordial germ cells (PGCs) (Tsang et al. [2001] International Journal of Developmental Biology 45:549-555). To dissect the specific role of Lhx1 in germ cell development, we studied embryos with conditional inactivation of Lhx1 activity in epiblast derivatives, which, in contrast to completely null embryos, develop normally through gastrulation before manifesting a head truncation phenotype. Initially, PGCs are localized properly to the definitive endoderm of the posterior gut in the conditional mutant embryos, but they depart from the embryonic gut prematurely. The early exit of PGCs from the gut is accompanied by the failure to maintain a strong expression of Ifitm1 in the mesoderm enveloping the gut, which may mediate the repulsive activity that facilitates the retention of PGCs in the hindgut during early organogenesis. Lhx1 therefore may influence the localization of PGCs by modulating Ifitm1-mediated repulsive activity.

The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells.

Importin-mediated transport of cargoes is known to be a key mechanism for nucleo-cytoplasmic trafficking of molecules. Ipo13, which is a member of Importin-β gene family, encodes two transcripts by utilizing different transcription start sites. In the mouse, the full-length transcript (L-Ipo13) is expressed in the primordial germ cells in the embryo and is later expressed predominantly at the pachytene phase of meiosis in both male and female germ cells. The shorter transcript (TS-Ipo13) is only expressed in the germ cells in the adult testis. Activity of L-Ipo13, but not TS-Ipo13, mediates the nuclear accumulation of ubiquitin-conjugating enzyme 9 (UBC9), a cargo of human IPO13. This finding is consistent with the progressive accumulation of UBC9 in the nucleus of the meiotic germ cells after the onset of L-Ipo13 expression. Following siRNA knockdown of IPO13 activity in the fetal ovary, fewer germ cells are found to progress to the late-pachytene stage of meiosis and nuclear accumulation of UBC9 is reduced. Our findings strongly implicate a stage-specific role of IPO13 in nuclear-cytoplasmic translocation of cargoes that accompanies meiotic differentiation of the mouse germ cells.