1. The Acrosomal Protein Dickkopf-Like 1 (DKKL1) Is Not Essential For Fertility OBJECTIVE: To determine the role of Dkkl1 on mouse development, viability, and fertility. DESIGN: Prospective experimental study. SETTING: Government research institution. ANIMAL(S): Mice of C57BL/6 and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies. INTERVENTION(S): Mice were constructed that lacked a functional Dkkl1 gene. MAIN OUTCOME MEASURE(S): Deletion of the gene was confirmed by DNA, RNA, and protein analyses;in vivo fertility was examined by continuous mating scheme. RESULT(S): Previous studies have shown that Dkkl1, a gene unique to mammals, is expressed predominantly, if not exclusively, in developing spermatocytes, and the DKKL1 protein accumulates in the acrosome of mature sperm. Subsequent studies (reported in the accompanying article) demonstrate that Dkkl1 also is expressed in the trophectoderm/placental lineage. Taken together, these results strongly suggested that DKKL1 protein is required for terminal differentiation either of trophoblast giant cells or of sperm, both of which are directly involved in fertility. To challenge this hypothesis, conditional targeted mutagenesis was used to ablate the Dkkl1 gene in mice. Surprisingly, Dkkl1 nullizygous embryos developed into viable, fertile adults, despite the fact that they failed to produce any portion of the DKKL1 protein. CONCLUSION(S): DKKL1 is a mammalian-specific acrosomal protein that is not essential either for development or fertility. 2. The Acrosomal Protein Dickkopf-Like 1 (DKKL1) Facilitates Sperm Penetration Of The Zona Pellucida OBJECTIVE: To determine the role of Dkkl1 in mouse development, viability, and fertility. DESIGN: Prospective experimental study. SETTING: Government research institution. ANIMAL(S): Mice of C57BL/6, B6D2F1/J, and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies. INTERVENTION(S): Expression of the Dkkl1 gene was characterized during early mouse development, and the effects of Dkkl1 ablation on reproduction and fertility were characterized in vitro and in vivo. MAIN OUTCOME MEASURE(S): Dkkl1 RNA expression was determined by Northern blotting hybridization as well as quantitative reverse transcriptase-polymerase chain reaction assays. In vitro fertilization assays were used to assess fertility of sperm from male mice lacking functional Dkkl1. RESULT(S): Dkkl1 is a gene unique to mammals that is expressed primarily in developing spermatocytes and its product localized in the acrosome of mature sperm. Here we show that Dkkl1 also is expressed in the trophectoderm/placental lineage. Surprisingly, embryos lacking DKKL1 protein developed into viable, fertile adults. Nevertheless, the ability of sperm that lacked DKKL1 protein to fertilize wild-type eggs was severely compromised in vitro. Because this defect could be overcome either by removal of the zona pellucida or by the presence of wild-type sperm, Dkkl1, either directly or indirectly, facilitates the ability of sperm to penetrate the zona pellucida. Penetration of the zona pellucida by Dkkl1(-) sperm was delayed in vivo as well as in vitro, but the delay in vivo was compensated by other factors during preimplantation development. Accordingly, Dkkl1-/- males offer an in vitro fertilization model for identifying factors that may contribute to infertility. CONCLUSION(S): DKKL1 is a mammalian-specific, acrosomal protein that strongly affects in vitro fertilization, although the effect is attenuated in vivo. 3. Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. Inthis study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway. 4. Checkpoint Kinase-1 Prevents Cell Cycle Exit Linked To Terminal Cell Differentiation. Trophoblast stem (TS) cells proliferate in the presence of fibroblast growth factor-4, but in its absence, they differentiate into polyploid trophoblast giant (TG) cells that remain viable but nonproliferative. Differentiation is coincident with expression of the CDK-specific inhibitors p21 and p57;of which p57 is essential for switching from mitotic cell cycles to endocycles. Here we show that, in the absence of induced DNA damage, checkpoint kinase-1 (CHK1), an enzyme essential for preventing mitosis in response to DNA damage, functions as a mitogen-dependent protein kinase that prevents premature differentiation of TS cells into TG cells by suppressing expression of p21 and p57, but not p27, the CDK-inhibitor that regulates mitotic cell cycles. CHK1 phosphorylates p21 and p57 proteins at specific sites, thereby targeting them for degradation by the 26S proteasome. TG cells lack CHK1, and restoring CHK1 activity in TG cells suppresses expression of p57 and restores mitosis. Thus, CHK1 is part of a 'G2 restriction point'that prevents premature cell cycle exit in cells programmed for terminal differentiation, a role that CHK2 cannot play.

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DePamphilis, Melvin L (2016) Genome Duplication: The Heartbeat of Developing Organisms. Curr Top Dev Biol 116:201-29
DePamphilis, M L (2016) Genome Duplication at the Beginning of Mammalian Development. Curr Top Dev Biol 120:55-102
Huang, Yi-Yuan; Kaneko, Kotaro J; Pan, Haiyan et al. (2015) Geminin Is Essential to Prevent DNA Re-Replication-Dependent Apoptosis in Pluripotent Cells, but not in Differentiated Cells. Stem Cells :
de Renty, Christelle; DePamphilis, Melvin L; Ullah, Zakir (2014) Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis. PLoS One 9:e97434
Kaneko, Kotaro J; Depamphilis, Melvin L (2013) TEAD4 establishes the energy homeostasis essential for blastocoel formation. Development 140:3680-90
Zielke, Norman; Edgar, Bruce A; DePamphilis, Melvin L (2013) Endoreplication. Cold Spring Harb Perspect Biol 5:a012948
Depamphilis, Melvin L; de Renty, Christelle M; Ullah, Zakir et al. (2012) ""The Octet"": Eight Protein Kinases that Control Mammalian DNA Replication. Front Physiol 3:368
Lee, Chrissie Y; Johnson, Ronald L; Wichterman-Kouznetsova, Jennifer et al. (2012) High-throughput screening for genes that prevent excess DNA replication in human cells and for molecules that inhibit them. Methods 57:234-48
Ullah, Zakir; DePamphilis, Melvin L (2012) Checkpoint kinase-1: one actor playing two roles in ""maintaining genomic integrity"". Cell Cycle 11:1873
Ullah, Zakir; de Renty, Christelle; DePamphilis, Melvin L (2011) Checkpoint kinase 1 prevents cell cycle exit linked to terminal cell differentiation. Mol Cell Biol 31:4129-43

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