In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.A llocation of blastomeres to outside and inside positions during preimplantation mammalian development initiates specification of the first two cell lineages, the trophectoderm (TE) and the inner cell mass (ICM) (1, 2). Successful progression of TE and ICM fate specification and proper development of the preimplantation embryo depends on differential transcriptional programs that are instigated and maintained within the outer and inner cells. Gene-KO studies in mice showed TEAD4 as the master orchestrator of the TE-specific transcriptional program (3-5). TEAD4-null embryos do not mature to the blastocyst stage and TEAD4-null blastomeres lack expression of TE-specific master regulators like CDX2, GATA3, and EOMES (3, 4). However, they maintain expression of ICM-specific factors like OCT4 and NANOG.Interestingly, TEAD4 expression is maintained both in cells of TE and ICM lineages, as well as in the TE-derived trophoblast stem cells (TSCs) and ICM-derived ES cells (ESCs) (5, 6). Thus, questions are raised as to how TEAD4 selectively orchestrates the TE/TSC-specific transcriptional program but not the ICM/ ESC-specific transcriptional program. The current model predicts that the presence vs. the absence of a TEAD4 cofactor, yesassociated protein (YAP), modulates TEAD4 function at its target genes in outer vs. inner blastomeres (6), leading to the segregation of the TE and ICM lineages. However, YAP-null mouse embryos do not show preimplantation developmental defects (7), indicating that, unlike TEAD4, YAP function is dispensable during TE and ICM fate determination. It is proposed that another YAP-related cofactor, WWTR1 (i.e., TAZ), could compensate for the absence of YAP during early development (6). However, the mode of TAZ function during TE and ICM specification is unknown. Furthermore, direct targets of TEAD4 have not been identified in the TE or in trophoblast cells. Thus, definitive experiments have not been performed to conclude that loss of cofactor function/recruitmen...
Sexual stimulation induces rapid secretion of cortisol and prolactin (PRL) in stallions. Experiment 1 was designated to determine whether stallions associated location and(or) procedure with previous sexual stimulation in that location. After a control period on d 1, four stallions were exposed to an estrous mare for 5 min on d 2. On d 3, 4, 5, and 6, the same procedure was followed with no mare present. Concentrations of PRL and cortisol increased (P less than .05) after mare exposure on d 2 but did not vary (P greater than .05) on d 1, 3, 4, 5, or 6. In Exp. 2, six stallions were used to determine the short-term effects of 1) sexual stimulation, 2) acute physical exercise, 3) restraint via a twitch (twitching), 4) epinephrine administration, and 5) no stimulation on plasma concentrations of PRL and cortisol. Stallions received one treatment per day separated by 2 d of no treatment. Concentrations of cortisol increased (P less than .05) within 10 min after sexual stimulation, exercise, twitching, and epinephrine administration but not during control bleedings. Concentrations of PRL increased (P less than .05) immediately after sexual stimulation, exercise, and twitching but not after epinephrine administration or during control bleeding. In Exp. 3, the same five treatments were administered to six geldings. Concentrations of cortisol increased (P less than .05) after epinephrine administration, exercise, and twitching but not after sexual stimulation or during control bleedings. Concentrations of PRL increased (P less than .05) after exercise and sexual stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)—three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C—and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.
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