MECHANISMS OF TROPHOBLAST LINEAGE FORMATION IN MAMMALIAN EMBRYOS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1019019
• Project Start Date: Jun 1, 2019
• Project End Date: May 31, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
ANIMAL SCIENCE

Non Technical Summary
Completion of proposed studies will provide novelinsights into the molecular mechanisms that govern trophoblast lineage development in the pre- and postimplantation embryo. Knowledge gained from studying TFAP2C function in mouse embryos and stemcells will be directly relevant tounderstanding dairy cattle infertility and human pregnancy loss.Such work will allow us to gain a greater comprehension of theetiology of early pregnancy loss and specific placental disorders that may arise inpreimplantation embryosbecause of a defective trophoblast cell lineage.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3840	1050	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1050 - Developmental biology;

Keywords

preimplantation embryo

trophoblast lineage formation and placentation

early embryonic loss and miscarriage

transcription factor

assisted reproductive technologies

Goals / Objectives
Specific Aim 1: To determine the role of TFAP2C in mouse trophoblast lineage formation. Hypothesis: In preimplantation embryos TFAP2C induces a trophoblast cell-fate by positively regulating the formation of polar-outside cells and negatively regulating the establishment of apolar-inside cells. Using TFAP2C loss of and gain of function, and time-lapse microscopy combined with lineage tracing approaches we will assay for effects on: (a) cell polarity and HIPPO signaling, (b) the frequency of symmetric vs. asymmetric cell divisions, and (c) allocation to the trophectoderm and inner cell mass (ICM).Specific Aim 2: To define the molecular mechanism by which TFAP2C induces a trophoblast gene expression program in mice. Hypothesis: TFAP2C establishes a trophoblast gene expression program by forming a regulatory complex with transcription factor TEAD4 and the HIPPO signaling protein YAP1. Using loss of function and gain of function approaches, co-immunoprecipitation, ChIP, and gene expression analysis in preimplantation embryos and Tfap2c-inducible ES cells we will assay for: (a) physical and functional interactions between TFAP2C, TEAD4, YAP1, and other co-regulators, (b) effects on transcriptional regulation of Cdx2 and Elf5, and (c) effects on ES cell to trophoblast lineage conversion.Specific Aim 3: Determine the importance of the TFAP2C-mediated mechanisms in mouse preimplantation embryos on subsequent postimplantation trophoblast development. Hypothesis: TFAP2C-mediated establishment of the transcriptional program in early embryos is essential for proper development of trophoblast progenitor cells in postimplantation embryos. We will test this by transiently perturbing Tfap2c expression during preimplantation and determining the effects of transient loss of TFAP2C on postimplantation placental development. We will assay for effects on: (a) formation of the extraembryonic ectoderm and ectoplacental cone, (b) gene expression of trophoblast progenitors within the ectoplacental cone and chorion, and (c) differentiation of trophoblast progenitors towards specialized cell-types.Specific Aim 4: Elucidate the role of TFAP2C in human trophoblast lineage development. Hypothesis: TFAP2C is required for trophoblast lineage formation and differentiation into functional syncytiotrophoblasts. We will test this by manipulating TFAP2C expression in human ES cells and inducing them to undergo trophoblast lineage differentiation. We will assay for effects on: (a) conversion of ES cells into trophoblast-like cells, (b) activation of CDX2 expression and other markers of human early trophoblast lineage cells, and (c) pregnancy hormone production and cellular invasion.

Project Methods
Mouse studies A combination of cellular, molecular, and biochemical approaches will be utilized in mouse preimplantation embryos and ES cells. Traditional knock in/out approaches are NOT suitable for the proposed studies in preimplantation embryos because of the inability to precisely increase and decrease the levels of TFAP2C during the window of preimplantation development. Moreover, the application of CRIPSR-dCas9 transcriptional interference is relatively new and has not been well tested in the preimplantation embryo. A tremendously powerful and well-established approach for manipulating gene function during preimplantation is through microinjection of mRNA and siRNA. Using this strategy, we can precisely manipulate the levels of TFAP2C in zygotes and cleavage stage embryos. Consideration of sex as a biological variable: All of our proposed studies involve using preimplantation embryos derived from mated mice. The ratio of male to female embryos is approximately 50:50. Published work in our laboratory has already shown that the TFAP2C loss-of-function phenotype induced by RNAi is uniform in greater than 85% of the embryos, suggesting that male and female embryos exhibit a similar phenotype. However, in the postimplantation embryo experiments we will genotype embryos and placentae to determine if there is a phenotypic difference between male and females. Molecular studies in embryos will be complemented with functional and biochemical experiments in TFAP2C-inducible ES cells. These ES cells were obtained from Coriell Cell repositories (Camden, NJ). They contain a tetracycline controllable Tfap2c transgene that was inserted into the ROSA26 locus. Removal of tetracycline from the culture media induces expression of TFAP2C and causes transdifferentiation into trophoblast stem (TS) -like cells. The TFAP2C expressed also contains a FLAG-tag that can be used for protein immunoprecipitation (IP) studies. These cells are an ideal system for examining physical and function interactions between TFAP2C and other transcriptional regulators such as TEAD4 and YAP1.Human ES cell studies To complement the mechanistic studies performed in mouse embryos and ES cells, TFAP2C loss-of-function and gain-of-function studies will be performed in human ES cells undergoing differentiation into trophoblast cells. Consideration of sex as a biological variable: A male and female human ES cell will be used in these studies to discern whether there is an effect of sex on TFAP2C-dependent trophoblast lineage formation. The ES cells were acquired from WiCell (Madison, WI). When these cells are treated with BMP4 and inhibitors of activin A and FGF2 signaling (A83-01/PD173074) they transdifferentiate into trophoblast cells that resemble syncytiotrophoblast cells. Functional studies will be performed using lentiviral transduction of TFAP2C shRNAs (loss-of-function) and TFAP2C cDNA (gain-of-function). The Knott laboratory is very well accomplished with using this approach to manipulate gene expression in stem cells.Overall Impact: We will provide novel insights into the regulatory role of TFAP2C in trophoblast lineage formation, and how TFAP2C-dependent mechanisms affect subsequent placental development. Importantly, proposed research will provide a wealth of new information on the role of TFAP2C at the cellular, molecular, and conceptus level. This will be highly relevant to understanding the molecular basis of preimplantation embryo arrest, early miscarriage, and clinical reproductive disorders in cattle and humans that may arise because of a defective trophoblast lineage.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Scientific community; Society for the Study of Reproduction (SSR), graduate students and post-docs, international scientists in China and Argentina. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and post-docs were provided training through the Reproductive and Developmental Sciences (RDSP) program at MSU. Trainees particiated in seminars, journal clubs, and research in progress (RIP) talks. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Four papers were published related to Aims 1 and 2 (Karesek et al., 2020, Ashry et al., 2020, Savy et al., 2020, and Cao et al., 2019).

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Cao Z, Zhang L, Hong R, Li Y, Wang Y, Qi X, Ning W, Gao D, Xu T, Ma Y, Yu T, Knott JG, Sathanawongs, Zhang Y. METTL3-mediated m6A methylation negatively modulates autophagy to support porcine blastocyst development. Biol Reprod 2020 (Accepted)
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Cao Z, Zhang D, Wang Y, Tong X, Avalos L, Khan I, Gao D, Xu T, Zhang L, Knott JG, Zhang Y. Identification and functional annotation of m6A methylation modification in granulosa cells during antral follicle development in pigs. Animal Reproduction Science. 2020 August, Volume 219, 106510.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ashry M, Rajput SK, Folger JK, Yang C, Knott JG, Smith GW. (Co-corresponding Author). Follistatin treatment modifies DNA methylation of the CDX2 gene in bovine preimplantation embryos. Mol Reprod and Dev. 2020 Aug 10. Doi: 10.1002/mrd.23409.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Karasek C, Ashry M, Driscoll CS, Knott JG. A tale of two cell-fates: Role of the Hippo signaling pathway and transcription factors in early lineage formation in mouse preimplantation embryos. Mol Human Reprod. 2020 July 9; gaaa052,�https://doi.org/10.1093/molehr/gaaa052.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Zhang L, Qi X, Ning W, Shentu L, Guo T, Zhang X, Li Y, Ma Y, Yu T, Knott JG,�Cao Z, Zhang Y. Single-cell transcriptome profiling revealed that vitrification of somatic cloned porcine blastocysts causes substantial perturbations in gene expression. Front. Genet. 2020 July 24; 11:640. doi: 10.3389/fgene.2020.00640. eCollection 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ock SA,�Knott JG, Choi I. (Co-corresponding Author) Involvement of CDKN1A (p21) in Cellular Senescence in Response to Heat and Irradiation Stress During Preimplantation Development. Cell Stress Chaperones. 2020 May;25(3):503-508. doi: 10.1007/s12192-020-01090-4. Epub 2020 Apr 6.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Savy V, Alberio V, Canel NG, Ratner LD, Gismondi MI, Ferraris SF, Fernandez-Mart�n R,�Knott JG, Bevacqua RJ, Salamone DF. CRISPR-on for Activation of Endogenous SMARCA4 and TFAP2C Expression in Bovine Embryos. Reproduction. 2020 Jun;159(6):767-778. doi: 10.1530/REP-19-0517.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Rajput SK, Yang C, Ashry M, Folger JK,�Knott JG, Smith GW. Role of Bone Morphogenetic Protein Signaling in Bovine Early Embryonic Development and Stage Specific Embryotropic Actions of follistatin. Biol Reprod. 2020 Apr 15;102(4):795-805. doi: 10.1093/biolre/ioz235.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Cao Z, Xu T, Tong X, Wang Y, Zhang D, Gao D, Zhang L, Ning W1, Qi X, Ma Y, Yu T, Knott JG, Zhang Y. Maternal Yes-Associated Protein Participates in Porcine Blastocyst Development via Modulation of Trophectoderm Epithelium Barrier Function. Cells. 2019 Dec 11;8(12). pii: E1606.

Progress 06/01/19 to 09/30/19

Outputs
Target Audience:The target audience includes the scientific community, the medical community and inferility specialists. Students, post-docs, and faculty are key members of the target audience. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The projecthas provided multiple learning opportunities fortrainees. These include instruction on properexperimental design, giving oral presentations. My grad students and post-docsparticipate inweekly and annual activities as part of thereproductive and developmental sciences program (RDSP). These include weekly researchmeetings, monthlyjournal clubs, and yearly researchmeetings. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period the students and post-doc will work to complete experiments outlined in Aims 1 and 2.

Impacts
What was accomplished under these goals? During the reporting periodgraduate students and post-doc worked on preliminary experiments for work outlined inaims 1 and 2. They learnedbasic laboratory techniques requiredfor each aim. These includemolecular cloning, superovulation of mice, embryo isolation, embryo culture, micromanipulation of embryos, andimmunofluorescence. In addition, mypost-doc optimized a protein co-immunoprecipiatation protocol for masspect experiments proposed in Aim 2. He also learned how to grow embryonic stem cells.

Publications