Conserved roles of fibroblast growth factor receptor 2 signaling in the regulation of inner cell mass development in bovine blastocysts

Akizawa, Hiroki; Nagatomo, Hiroaki; Odagiri, Haruka; Kohri, Nanami; Yamauchi, Nobuhiko; Yanagawa, Yuchio; Nagano, Masashi; Takahashi, Masashi; Kawahara, Manabu

doi:10.1002/mrd.22646

Cited by 14 publications

(9 citation statements)

References 48 publications

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“…Total RNA extraction, cDNA synthesis, and subsequent PCR analysis were performed according to a previous study 18 . Briefly, TE portions of embryos were mechanically dissected under the stereomicroscope.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA extraction, cDNA synthesis, and subsequent PCR analysis were performed according to a previous study. 18 Briefly, TE portions of embryos were mechanically dissected under the stereomicroscope. TE portions from 20 blastocysts, five D10 on-gel-cultured embryos, and two D14 on-gel-cultured embryos per biological replicate were pooled and served for RNA was extracted using a ReliaPrep RNA cell extraction kit (Promega) according to the manufacturer's instructions.…”

Section: Quantitative Pcr (Qpcr)mentioning

confidence: 99%

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Deciphering two rounds of cell lineage segregations during bovine preimplantation development

et al. 2021

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Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this "ongel" culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic

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Section: Methodsmentioning

confidence: 99%

Section: Quantitative Pcr (Qpcr)mentioning

confidence: 99%

Deciphering two rounds of cell lineage segregations during bovine preimplantation development

et al. 2021

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“…Proportion of putative zygotes that reached the blastocyst stage, day 7.5 (N = 8) a and subsequent downregulation of Nanog transcription. In cattle, knockdown of FGFR2 does not affect expression of epiblast and hypoblast markers NANOG and GATA6 but decreases the hypoblast marker HNF4A [45]. Also, addition of FGF4 [20] and FGF2 [26] can promote hypoblast formation.…”

Section: Vehiclementioning

confidence: 99%

Role of yes-associated protein 1, angiomotin, and mitogen-activated kinase kinase 1/2 in development of the bovine blastocyst†

Negrón‐Pérez

Hansen

2017

Biology of Reproduction

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The morula-stage embryo is transformed into a blastocyst composed of epiblast, hypoblast, and trophectoderm (TE) through mechanisms that, in the mouse, involve the Hippo signaling and mitogen-activated kinase (MAPK) pathways. Using the cow as an additional model, we tested the hypotheses that TE and hypoblast differentiation were regulated by the Hippo pathway regulators, yes-associated protein 1 (YAP1) and angiomotin (AMOT), and MAPK kinase 1/2 (MAPK1/2). The presence of YAP1 and CDX2 in the nucleus and cytoplasm of MII oocytes and embryos was evaluated by immunofluorescence labeling. For both molecules, localization changed from cytoplasmic to nuclear as development advanced. Inhibition of YAP1 activity, either by verteporfin or a YAP1 targeting GapmeR, reduced the percent of zygotes that became blastocysts, the proportion of blastocysts that hatched and numbers of CDX2+ cells in blastocysts. Moreover, the YAP1-targeting GapmeR altered expression of 15 of 91 genes examined in the day 7.5 blastocyst. Treatment of embryos with an AMOT targeting GapmeR did not affect blastocyst development or hatching but altered expression of 16 of 91 genes examined at day 7.5 and reduced the number of CDX2+ nuclei and YAP1+ nuclei in blastocysts at day 8.5 of development. Inhibition of MAPK1/2 with PD0325901 did not affect blastocyst development but increased the number of epiblast cells. Results indicate a role for YAP1 and AMOT in function of TE in the bovine blastocyst. YAP1 can also affect function of the epiblast and hypoblast, and MAPK signaling is important for inner cell mass differentiation by reducing epiblast numbers.

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“…Briefly, an shRNA containing antisense/sense regions, a 19‐bp loop (5′‐CTGTGAAGCCACAGATGGG‐3’), and a 6‐bp terminator element (5′‐TTTTTT‐3’), was designed to target nucleotides 665–683 of the CCN2 mRNA sequence (NCBI Reference Sequence NM_174030), and ligated downstream of the U6 promoter in the pBAsi/mU6 Neo vector (Stratagene, CA, USA). Embryos injected with pBAsi/mU6 Neo plasmids lacking the shRNA insert (empty vector) were used as controls, as previously described(Akizawa et al., ; Nagatomo et al., ). Twelve hours after insemination, the synthesized shRNA‐expression constructs (diluted to a final concentration of 10 ng/ml with mSOFai medium) were injected into denuded presumptive zygotes using a FemtoJet injection device (Eppendorf, Hamburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Significance of CCN2 expression in bovine preimplantation development

Akizawa

Yanagawa

Nagano

et al. 2018

Animal Science Journal

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In mammalian preimplantation development, the first cell lineage segregation occurs during the blastocyst stage, when the inner cell mass and trophectoderm (TE) differentiate. Species-specific analyses are essential to elucidate the molecular mechanisms that underlie this process, since they differ between various species. We previously showed that the reciprocal regulation of CCN2 and TEAD4 is required for proper TE differentiation in bovine blastocysts; however, the function of CCN2 during early embryogenesis has remained otherwise elusive. The present study assessed the spatiotemporal expression dynamics of CCN2 in bovine embryos, and evaluated how changes to CCN2 expression (using a CCN2 knockdown (KD) blastocyst model) regulate the expression of pluripotency-related genes such as OCT4 and NANOG. The conducted quantitative PCR analysis revealed that CCN2 mRNA was expressed in bovine oocytes (at the metaphase stage of their second meiosis) and embryos.Similarly, immunostaining detected both cytoplasmic and nuclear CCN2 at all analyzed oocyte and embryonic stages. Finally, both OCT4 and NANOG expression levels were shown to be significantly reduced in CCN2 KD blastocysts. Together, these results demonstrate that bovine CCN2 exhibits unique expression patterns during preimplantation development, and is required for the proper expression of key regulatory genes in bovine blastocysts. K E Y W O R D Sblastocyst, bovine, CCN2, cell-lineage specification, pluripotency-related genes

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Conserved roles of fibroblast growth factor receptor 2 signaling in the regulation of inner cell mass development in bovine blastocysts

Cited by 14 publications

References 48 publications

Deciphering two rounds of cell lineage segregations during bovine preimplantation development

Deciphering two rounds of cell lineage segregations during bovine preimplantation development

Role of yes-associated protein 1, angiomotin, and mitogen-activated kinase kinase 1/2 in development of the bovine blastocyst†

Significance of CCN2 expression in bovine preimplantation development

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