TFAP2C is a key regulator of intrauterine trophoblast cell invasion and deep hemochorial placentation

The placenta is a transient organ that ensures viviparity and optimal fetal development (1–3). The human and rat possess a hemochorial placenta (4, 5), which is composed of specialized cellular constituents termed trophoblast cells (6). Among the trophoblast cell lineages contributing to the placentation site are invasive trophoblast cells, which in the human are referred to as extravillous trophoblast cells (7). Human and rat placentation sites are characterized by deep intrauterine trophoblast cell invasion (8–10). Invasive trophoblast cells facilitate the erosion of maternal uterine spiral arteries permitting a direct flow of maternal nutrients to trophoblast cells (8, 11–15). Uterine vasculature remodeling is a critical process ensuring a successful pregnancy outcome (13, 16, 17). There is a paucity of knowledge regarding regulatory mechanisms controlling development of the invasive trophoblast cell lineage and trophoblast cell–guided uterine spiral remodeling.

Members of the transcription factor AP-2 (TFAP2) family are key regulators of embryonic and extraembryonic development (18, 19). TFAP2C distinguishes itself as a regulator of the trophoblast cell lineage and hemochorial placentation (20–22). In the mouse, global disruption of the Tfap2c locus results in abnormalities within the trophoblast cell lineage and prenatal lethality before placentation (23, 24). Although TFAP2C is an established regulator of the earliest stages of trophoblast cell lineage development (25–28), placental expression profiles and target gene occupancy implicate TFAP2C in the regulation of multiple processes affecting mammalian placentation and placental function (29–40). Included in this broad spectrum of TFAP2C actions on placentation is its potential involvement in invasive trophoblast cell biology (38–41).

In this report, we present the results of in vivo experimentation directed toward elucidating the physiological role of TFAP2C in the developing hemochorial placenta with a focus on trophoblast cell–guided events within the uterine-placental interface. We show that TFAP2C possesses essential roles during distinct stages of placental development. TFAP2C is an essential regulator of early placental morphogenetic events and reappears in a critical capacity for invasive trophoblast cell lineage development. Finally, elements of TFAP2C actions on placentation are dose dependent.

TFAP2C expression within the placentation site. The rat hemochorial placenta is composed of multiple lineages of trophoblast cells arranged into distinct compartments, including the labyrinth zone, junctional zone, and uterine-placental interface (42, 43) (Figure 1). The labyrinth zone is the site of maternal-fetal nutrient/waste transfer (44), whereas the junctional zone is composed of endocrine cells targeting the maternal environment and the site where invasive trophoblast cells arise (42, 43, 45). Cell composition of the uterine-placental interface is dynamic, including an increase in the infiltration of invasive trophoblast cells as gestation proceeds (9, 38). Intrauterine invasive trophoblast cells invade into the stroma located between uterine blood vessels (interstitial) and within uterine blood vessels (endovascular). Tfap2c transcripts were detected within the uterine-placental interface and increased during pregnancy (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.186471DS1). In situ hybridization was used to determine the distribution of Tfap2c transcripts within the hemochorial placenta. At gestation day (gd) 9.5, Tfap2c was prominently expressed in the ectoplacental cone (Figure 1A). Tfap2c transcripts were also localized to the antimesometrial uterine decidua (Figure 1A). As gestation advanced (gd 11.5), Tfap2c expression was observed within trophoblast cells of the developing placenta (Figure 1B). On gd 18.5, Tfap2c was abundantly expressed within the junctional zone and in invasive trophoblast cells of the uterine-placental interface (Figure 1, C–E). The presence of Tfap2c transcripts in the trophoblast cell lineage was supported by its colocalization with Krt8, a pan-trophoblast cell marker (Figure 1, A–C). Expression of Tfap2c transcripts in invasive trophoblast cells was further demonstrated by colocalization with Prl7b1 transcripts (Figure 1, D and E). The Prl7b1 transcript is an invasive trophoblast cell–specific transcript. The results were consistent with single-cell RNA sequencing of the uterine-placental interface (Supplemental Figure 1B) (38). TFAP2C protein was distributed in invasive trophoblast cells of the uterine-placental interface and throughout the junctional zone, replicating the distribution of Tfap2c transcripts at gd 18.5 (Supplemental Figure 2). Within the invasive trophoblast cell lineage, TFAP2C transcript and protein were localized to both endovascular and interstitial trophoblast cells (Figure 1E and Supplemental Figure 3). Tfap2c transcripts were localized to most trophoblast cell populations within the junctional zone, including a smaller subset of invasive trophoblast progenitor cells (Prl7b1 positive; Figure 1F).

Figure 1

Tfap2c transcript distribution during placenta development. (A–C) Tfap2c transcripts (shown in magenta) were localized to the ectoplacental cone at gestation day (gd) 9.5 (A) and placentation sites at 11.5 (B) and 18.5 (C). Tfap2c transcripts were colocalized with keratin 8 (Krt8) transcripts (cyan). Schematic diagrams representing gd 9.5, 11.5, and 18.5 placentation sites are initially presented for each panel. (D) Colocalization of Tfap2c (magenta) and prolactin family 7, subfamily b, member 1 (Prl7b1) (yellow), transcripts within the gd 18.5 placentation site. Prl7b1 is specifically expressed in the invasive trophoblast cell lineage. Scale bar: 500 μm. (E) High-magnification images showing Tfap2c and Prl7b1 transcript localization in endovascular (arrowhead) and interstitial (arrow) invasive trophoblast cells within the uterine-placental interface. Scale bar: 100 μm. (F) Colocalization of Tfap2c and Prl7b1 transcripts (arrows) within the junctional zone of the gd 15.5 placenta. Scale bar: 50 μm. Colocalization is shown as white. EPC, ectoplacental cone; AMD, antimesometrial decidua; SpA, spiral artery; UPI, uterine-placental interface; P, placenta; JZ, junctional zone; LZ, labyrinth zone.

Global deletion of Tfap2c leads to prenatal lethality. Next, we investigated the in vivo role of Tfap2c in rat placenta development using CRISPR/Cas9 genome editing. DNA-binding and dimerization domains encoded by exon 4 of the Tfap2c gene were targeted (Supplemental Figure 4A). A germline mutant rat was generated possessing a 308 bp deletion within exon 4 (Supplemental Figure 4B). The deletion resulted in a frameshift and a premature stop codon (Supplemental Figure 4C). PCR products corresponding to wild-type and mutant alleles were identified by genotyping (828 bp versus 520 bp, respectively, Supplemental Figure 4D). Null Tfap2c conceptuses (–/–) were obtained by mating heterozygous (+/–) Tfap2c females and males. Breeding results collected throughout pregnancy and postnatally, including Mendelian ratios, are presented in Supplemental Table 1. Null Tfap2c conceptuses were viable until gd 8.5, which temporally coincides with the onset of placental morphogenesis. This observation agrees with the timing of embryonic demise observed following global Tfap2c mouse gene disruption (23, 24). Interestingly, we observed an unexpected reduction in the number of surviving heterozygous pups generated from Tfap2c^+/– × Tfap2c^+/–, which prompted an investigation of placental development in conceptuses possessing a single Tfap2c-mutant allele.

Decreased Tfap2c gene dosage leads to a failure in intrauterine trophoblast cell invasion. To explore possible Tfap2c gene dosage effects, Tfap2c^+/+ females and Tfap2c^+/– males were mated, and pregnancies were assessed at gd 18.5. Tfap2c^+/+ and Tfap2c^+/– placentation sites exhibited a similar organizational structure, including a labyrinth zone, a junctional zone, and a uterine-placental interface (Figure 2A). We next assessed infiltration of invasive trophoblast cells into the uterine-placental interfaces of Tfap2c^+/+ versus Tfap2c^+/– placentation sites by monitoring the distribution of cytokeratin protein expression (9, 38) (Figure 2B) and Prl7b1 mRNA (46) (Figure 2C), both representing indices of invasive trophoblast cells. The uterine-placental interface of Tfap2c^+/– placentation sites were depleted of invasive trophoblast cells (Figure 2, B and C). Both interstitial and endovascular invasive trophoblast cells were diminished in the uterine-placental interface of Tfap2c^+/– placentation sites. This result was supported by quantification of the depth of invasion of cytokeratin-positive cells into the uterine parenchyma (Figure 2D). The depletion of invasive trophoblast cells in the uterine-placental interface of Tfap2c^+/– placentation sites was further supported by diminished expression of several invasive trophoblast cell–associated transcripts (Krt7, Krt8, Krt18, Prl7b1, Prl5a1, Cdkn1c, Cited2, Plac1, Peg3, Tfpi) (38) (Figure 2E). Similar findings were recorded when placentation sites were interrogated from pregnancies generated from Tfap2c^+/– females mated to Tfap2c^+/+ males (Supplemental Figure 5, A–D).

Figure 2

Tfap2c gene dosage effects on placental development. Placentation sites were generated from Tfap2c^+/– male and Tfap2c^+/+ female breeding. (A) Identification of placentation site compartments Tfap2c^+/+ and Tfap2c^+/– using vimentin immunostaining (magenta). (B) Immunohistochemical localization of cytokeratin protein in gestation day (gd) 18.5 Tfap2c^+/+ and Tfap2c^+/– placentation sites (cyan). (C) Distribution of Prl7b1 transcripts in gd 18.5 Tfap2c^+/+ and Tfap2c^+/– placentation sites (yellow). (D) Quantification of trophoblast cell invasion area (magenta dotted line in B) determined by cytokeratin immunostaining within the uterine-placental interface. Data are expressed as mean ± standard error of the mean (SEM). Each data point represents different uterine-placental interface tissues (n = 4) obtained from 4 pregnancies. (E) Reverse transcription quantitative PCR (RT-qPCR) of invasive trophoblast cell–specific transcripts in gd 18.5 Tfap2c^+/+ and Tfap2c^+/– uterine-placental interface tissues. Data are expressed as mean ± SEM. Each data point represents a biological replicate obtained from 6 pregnancies (n = 6). (F) Fetal, placenta, junctional zone, and labyrinth zone weights for gd 18.5 Tfap2c^+/+ and Tfap2c^+/– conceptuses. Data are expressed as mean ± SEM. Each data point represents a biological replicate obtained from 6 pregnancies (Tfap2c^+/+, n = 38; Tfap2c^+/–, n = 32). Scale bars: 500 μm. Unpaired t test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. UPI, uterine-placental interface; JZ, junctional zone; LZ, labyrinth zone; SpA, spiral artery.

However, breeding combinations did differentially affect postnatal heterozygote offspring viability and heterozygote fetal and placental weights. Postnatal heterozygote viability was prominently decreased in female Tfap2c^+/– × male Tfap2c^+/– but not in female Tfap2c^+/+ × male Tfap2c^+/– or female Tfap2c^+/– × male Tfap2c^+/+ breeding combinations (Supplemental Tables 1–3). Genotypic differences in fetal, placenta, junctional zone, and labyrinth zone weights at gd 18.5 were observed among breeding combinations. Mating Tfap2c^+/+ females with Tfap2c^+/– males was associated with significant decreases in heterozygote placenta, junctional zone, and labyrinth zone weights (Figure 2F), whereas only heterozygote fetal weights were significantly decreased by mating Tfap2c^+/– females with Tfap2c^+/+ males (Supplemental Figure 5E). Some of these differences might be associated with Tfap2c gene dosage effects on the maternal environment.

Invasive trophoblast cell–conditional Tfap2c mutagenesis. The impact of TFAP2C dosage on invasive trophoblast cell development prompted a direct analysis of the importance of TFAP2C on the invasive trophoblast cell lineage. We generated an invasive trophoblast cell–specific Tfap2c gene disruption using the Cre/loxP system. CRISPR/Cas9 genome editing was used to insert loxP sites flanking exon 4 of the Tfap2c gene (Figure 3A). Tfap2c-floxed (Tfap2c^fl/fl) rats were mated with Prl7b1-Cre rats (47). Cre recombinase is specifically targeted to invasive trophoblast cells in Prl7b1-Cre rats (47). TFAP2C protein was present in lysates of the Tfap2c^fl/fl uterine-placental interface but not in lysates of the Tfap2c^d/d uterine-placental interface (Figure 3B). Immunostaining supported depletion of TFAP2C from the gd 18.5 uterine-placental interface but not from the JZ of Tfap2c^d/d placentation sites (Figure 3C). Our ability to modulate TFAP2C in the invasive trophoblast cell lineage led us to evaluate a potential role for TFAP2C in regulating the invasive trophoblast cell lineage.

Figure 3

Generation of an invasive trophoblast cell–specific Tfap2c deletion. (A) Schematic of loxP sites inserted into introns flanking exon 4 of the Tfap2c gene. (B) TFAP2C Western blot of the uterine-placental interface of Tfap2c^fl/fl and Tfap2c^d/d gd 18.5 placentation sites. d/d, deleted/deleted (C) TFAP2C immunostaining of Tfap2c^fl/fl and Tfap2c^d/d gd 18.5 placentation sites (left panels: TFAP2C immunostaining, white; right panels: TFAP2C immunostaining, magenta, DAPI, white). Scale bar: 500 μm. Inserts to the right show higher magnification images of uterine-placental interface tissues (top) and junctional zone tissue (bottom) for each placentation site. Scale bar: 100 μm. UPI, uterine-placental interface; JZ, junctional zone; LZ, labyrinth zone; SpA, spiral artery.

TFAP2C-dependent intrauterine trophoblast cell invasion. Intrauterine trophoblast cell invasion was assessed in Tfap2c^fl/fl and Tfap2c^d/d placentation sites. At gd 18.5 invasive trophoblast cells were present throughout the Tfap2c^fl/fl uterine-placental interface but were not detected in the Tfap2c^d/d uterine-placental interface (Figure 4, A–C). Interstitial and endovascular invasive trophoblast cells were absent from the uterine-placental interface of Tfap2c^d/d placentation sites. The depletion of invasive trophoblast cells in the uterine-placental interface of Tfap2c^d/d placentation sites was further supported by diminished expression of several invasive trophoblast cell–associated transcripts (Krt7, Krt8, Krt18, Prl7b1, Prl5a1, Cdkn1c, Cited2, Plac1, Peg3, Tfpi; Figure 4D). In contrast with the global disruption of Tfap2c, migration of invasive trophoblast cells into the uterus was not impaired following conditional monoallelic disruption of Tfap2c (Supplemental Figure 6). Single-cell RNA sequencing and single-nucleus assay for transposase-accessible chromatin-sequencing datasets (38, 41) reveal a network of genes potentially regulated by TFAP2C in rat invasive trophoblast cells that include candidate contributors to remodeling extracellular matrix, cytoskeleton restructuring, regulation of cell movement, and transcriptional regulators that target genes encoding other proteins affecting the invasive trophoblast cell phenotype (Table 1 and Supplemental Data 1). Thus, TFAP2C has a fundamental role in regulating the invasive trophoblast cell lineage, especially movement of invasive trophoblast cells into the uterus, and a list of downstream effectors are available to investigate mechanisms underlying TFAP2C actions.

Figure 4

Characterization of invasive trophoblast cell–specific Tfap2c deletion. (A) Immunohistochemical localization of cytokeratin (cyan) in gd 18.5 Tfap2c^fl/fl and Tfap2c^d/d placentation sites. (B) Distribution of Prl7b1 transcripts (yellow) in gd 18.5 Tfap2c^fl/fl and Tfap2c^d/d placentation sites. Scale bar: 500 μm. Inserts to the right show higher magnification images of the boxed areas within the uterine-placental interface tissues for each placentation site. Scale bar: 100 μm. (C) Quantification of trophoblast cell invasion area (magenta dotted line) determined by cytokeratin immunostaining within the uterine-placental interface. Data are expressed as mean ± standard error of the mean (SEM). Each data point represents different uterine-placental interface tissues obtained from 4 pregnancies (n = 4). (D) RT-qPCR of invasive trophoblast cell–specific transcripts in gd 18.5 Tfap2c^fl/fl and Tfap2c^d/d uterine-placental interface tissues. The left panel corresponds to a schematic of the regions of the placentation site used for analysis. Data are expressed as mean ± SEM. Each data point represents a biological replicate from 6 pregnancies (n = 6). Unpaired t test: *P < 0.05, ***P < 0.001, ****P < 0.0001. UPI, uterine-placental interface; JZ, junctional zone; LZ, labyrinth zone; SpA, spiral artery.

Table 1

Subset of genes expressed in invasive trophoblast cells possessing TFAP2C DNA binding motifs

Invasive trophoblast cells and natural killer cells exhibit a reciprocal distribution within the uterine-placental interface. At midgestation, natural killer (NK) cells are an abundant constituent of the uterine-placental interface (48). As gestation progresses, NK cells become depleted as trophoblast cells invade the uterine-placental interface (48). Interestingly, we observed a retention of NK cells in Tfap2c^d/d uterine-placental interfaces depleted of invasive trophoblast cells (Figure 5A). This observation was further supported by increased expression of NK cell–associated transcripts (Prf1, Klrb1c, Klrb1a, Klrb1, Ncr1; Figure 5B) within Tfap2c^d/d uterine-placental interfaces. These findings reinforce the observation that fundamental changes occur in the Tfap2c^d/d uterine-placental interface and imply that invasive trophoblast cells affect cellular dynamics within the uterine-placental interface.

Figure 5

Tfap2c conditional deletion in trophoblast invasive cells results in the retention of uterine NK cells within the uterine-placental interface. (A) Immunohistochemical localization of cytokeratin (cyan) and perforin (magenta) in gd 18.5 Tfap2c^fl/fl and Tfap2c^d/d placentation sites. (B) RT-qPCR of NK cell–specific transcripts in gd 18.5 Tfap2c^fl/fl and Tfap2c^d/d uterine-placental interface tissues. The left panel corresponds to a schematic of the regions of the placentation site used for analysis. Data are expressed as mean ± standard error of the mean. Each data point represents a biological replicate from 6 pregnancies (n = 6). Unpaired t test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 500 μm. Specimens used in this figure are the same used in Figure 4A. UPI, uterine-placental interface; JZ, junctional zone; LZ, labyrinth zone; SpA, spiral artery.

Invasive trophoblast cells impact prenatal and postnatal growth. We next investigated whether the presence of invasive trophoblast cells within the uterine-placental interface affected prenatal or postnatal growth. Depletion of invasive trophoblast cells as observed in Tfap2c^d/d placentation sites was associated with intrauterine placental growth restriction, including both junctional zone and labyrinth zone compartments, and fetal growth restriction at gd 18.5 (Figure 6A). The adverse effects of invasive trophoblast cell depletion on fetal weight extended to postnatal offspring growth. Depletion of invasive trophoblast cells was associated with decreased postnatal body weight (Figure 6B). The results are consistent with invasive trophoblast cells supporting the nutrient demands required for optimal intrauterine growth. Deficits acquired prenatally were sustained during the first 3 weeks of postnatal life.

Figure 6

Impact of an invasive trophoblast cell–specific Tfap2c deletion on prenatal and postnatal development. (A) Fetal, placenta, junctional zone, and labyrinth zone weights from gd 18.5 Tfap2c^fl/fl and Tfap2c^d/d gd 18.5 conceptuses. Data are expressed as mean ± standard error of the mean (SEM). Each data point represents a biological replicate from 6 pregnancies (Tfap2c^fl/fl, n = 20; Tfap2c^d/d, n = 16). Unpaired t test: *P < 0.05, **P < 0.01, ****P < 0.0001. (B) Postnatal body weight for Tfap2c^fl/fl and Tfap2c^d/d pups. Data are expressed as mean ± SEM from 6 pregnancies (Tfap2c^fl/fl, n = 24; Tfap2c^d/d, n = 29). One-way ANOVA and Tukey’s test: **P < 0.01, ****P < 0.0001. JZ, junctional zone; LZ, labyrinth zone.

Placentation is a critical adaptation ensuring the success of viviparity (5, 49). Gene-manipulated animal models and trophoblast stem cells have provided insights into gene regulatory networks controlling trophoblast cell lineage development and placentation (21). TFAP2C figures prominently in these networks and represents a conserved regulator of placentation (20–22). In the research reported here, we utilized genome-edited rat models to elucidate roles for TFAP2C in placentation. The rat, like the human, is a species exhibiting deep intrauterine trophoblast cell invasion (8–10). TFAP2C is expressed in multiple trophoblast cell lineages of the developing rat placenta, including invasive trophoblast cells residing in the uterine-placental interface. Consistent with earlier reports in the mouse (23, 24), global disruption of Tfap2c in the rat led to prenatal lethality (gd 8.5 to 9.5), prior to the initiation of placenta morphogenesis. Interestingly, decreased gene dosage of Tfap2c (heterozygous) was compatible with pregnancy but affected placentation, including diminished infiltration of invasive trophoblast cells into the uterine-placental interface. A role for TFAP2C in the regulation of intrauterine trophoblast cell invasion was further supported using cell lineage–specific mutagenesis. Tfap2c was conditionally disrupted in the invasive trophoblast cell lineage, which led to a uterine-placental interface devoid of invasive trophoblast cells. Impairment of intrauterine trophoblast cell invasion had consequences, including intrauterine and postnatal growth restriction.

Involvement of TFAP2C in early events dictating trophoblast cell lineage development was reinforced from phenotyping conceptuses possessing a global disruption of the Tfap2c locus. TFAP2C is critical to activation of the trophectoderm transcriptome (36, 50) and development of trophectoderm (25, 26, 51, 52). Trophectoderm represents the earliest stage of trophoblast cell lineage development (53, 54). The importance of TFAP2C in early trophoblast cell lineage development is further exemplified by its utilization in the direct reprogramming of fibroblasts to the trophoblast cell lineage (27, 28).

TFAP2C actions in the rat placentation site were affected by gene dosage. Monoallelic disruption of Tfap2c resulted in the development of a rat uterine-placental interface possessing a diminished number of invasive trophoblast cells. Observing a phenotype in Tfap2c global heterozygotes was intriguing but not entirely new for Tfap2c. Haploinsufficiency of Tfap2c is linked to an increased incidence of peripartum lethality (55). Surviving Tfap2c heterozygotes exhibited postnatal growth restriction (24), development of germ cell tumors (56), and hippocampal dysfunction (57). Placentation is also affected by Tfap2c haploinsufficiency (55). The labyrinth zone compartment of the mouse placenta is disorganized in Tfap2c heterozygotes (55). Interestingly, the invasive trophoblast cell phenotype observed in global heterozygotes reported here was not a feature of conceptuses with a monoallelic disruption of Tfap2c restricted to the invasive trophoblast cell lineage. An explanation for this difference is likely connected to the widespread involvement of TFAP2C in multiple stages of trophoblast cell differentiation (58). This is supported by TFAP2C expression extending to many more trophoblast cells in the junctional zone than express Prl7b1, the gene driving Cre recombinase expression in our conditional mutagenesis rat model. Global Tfap2c haploinsufficiency at a key stage of trophoblast cell lineage development distinct from Prl7b1-expressing trophoblast cells must be relevant and responsive to Tfap2c gene dosage. Thus, Tfap2c gene dosage is utilized in a differentiation stage–specific manner to guide trophoblast cell development.

TFAP2C is utilized at different stages of trophoblast cell development. TFAP2C is critical to the derivation of the trophoblast cell lineage and then repurposed for later stages of placentation (58), including the regulation of invasive trophoblast cell development. TFAP2C does not act alone. Context is critical for the actions of TFAP2C. The presence of other transcription factor partners and stoichiometry influence TFAP2C function during trophoblast cell development (21, 59). A short list of potential TFAP2C partners in invasive trophoblast cell development is available (38, 41).

Finally, we have taken transcript and open chromatin profiles (38, 41) and created a path for elucidating gene involvement in the regulation of invasive trophoblast cell lineage development and its impact on the uterine-placental interface. We provide compelling evidence that TFAP2C is critical in establishing the invasive trophoblast cell lineage. Furthermore, we effectively demonstrate that the presence of invasive trophoblast cells in the uterus has consequences. Deficits in invasive trophoblast cell–guided uterine transformation resulted in intrauterine and postnatal growth restriction. Most importantly, tools are in place to establish a gene regulatory network controlling development and function of the invasive trophoblast cell lineage.

In summary, TFAP2C contributes to the progression of distinct stages of rat placental development. TFAP2C is a driver of early events in trophoblast cell development and reappears later in gestation as an essential regulator of the invasive trophoblast cell lineage. A subset of TFAP2C actions on trophoblast cells are dependent on gene dosage.

Sex as a biological variable. Our study examined male and female animals. Similar findings are reported for both sexes.

Animals and tissue collection. Holtzman Sprague-Dawley rats were acquired from Envigo. Animals were maintained in an environmentally controlled facility (14-hour light/10-hour dark cycle) with food and water available ad libitum. Timed pregnancies were established using virgin female rats (8 to 10 weeks of age) mated with adult male rats (>3 months of age). Mating was confirmed by the presence of sperm in the vaginal lavage and was considered gd 0.5. Pseudopregnant female rats were generated by mating with vasectomized male rats. Detection of a seminal plug was considered day 0.5 of pseudopregnancy. Pregnant rats were euthanized on specific days of gestation. Some placentation sites were frozen intact in dry ice–cooled heptane and stored at –80°C for subsequent histological analyses, whereas others were dissected into uterine-placental interface, junctional zone, and labyrinth zone compartments as previously described (38, 60). Dissected placentation site compartments and fetuses were weighed, frozen in liquid nitrogen, and stored at –80°C for subsequent biochemical analyses.

Generation of global and conditional Tfap2c-mutant rat models. A global mutation at the rat Tfap2c locus was generated using CRISPR/Cas9 genome-editing technology (61). RNA guides targeting exon 4 of the Tfap2c gene (Supplemental Figure 4 and Supplemental Table 4) were assembled with CRISPR RNA, transactivating CRISPR RNA, and Cas9 nuclease (Integrated DNA Technologies). Conditional Tfap2c disruption occurred by the insertion of loxP sites targeted to 5′ and 3′ introns flanking exon 4 (Figure 3, Supplemental Table 4, and Supplemental Table 5). Electroporation of constructs was performed into 1-cell rat embryos using a NEPA21 electroporator (Nepa Gene Co Ltd). Electroporated embryos were transferred to pseudopregnant rats. Offspring were screened for mutations by PCR, and the deletion was confirmed by DNA sequencing (GENEWIZ). A founder mutant animal was backcrossed with wild-type rats to evaluate germline transmission. Genotyping and fetal sex chromosome determination were performed as previously described (47, 61, 62). DNA was extracted from tail-tip biopsies with Red Extract-N-Amp tissue PCR kit (XNAT-1000RXN, MilliporeSigma) and used for genotyping. Primers used for genotyping of global and conditional mutations and sex chromosome determinations are provided (Supplemental Table 6). Body weight measurements were performed on offspring generated from specific intercrosses.

RT-qPCR. Total RNA was isolated from tissues with TRIzol (15596018, Thermo Fisher Scientific). Extracted RNA (1 μg) was used to synthesize complementary DNA using a High-Capacity Reverse Transcription kit (4368814, Applied Biosystems, Thermo Fisher Scientific) and then diluted 1:10 in water. qPCR was performed using PowerSYBR Green PCR Master Mix (4367659, Thermo Fisher Scientific) and transcript-specific primer sets (250 nM). Primer sequences are provided (Supplemental Table 7). QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific) cycling conditions were as follows: an initial step (95°C for 10 minutes), preceded by 40 cycles of 2-step PCR (95°C for 15 seconds, 60°C for 1 minute), and then a dissociation step (95°C for 15 seconds, 60°C for 1 minute) and a sequential increase to (95°C for 15 seconds). Relative mRNA expression was calculated using the ΔΔCt method, and glyceraldehyde-3-phosphate dehydrogenase was used as a reference RNA.

In situ hybridization. In situ hybridization was performed using the RNAscope Multiplex Fluorescent reagent kit version 2 (Advanced Cell Diagnostics, Bio-Techne) according to the manufacturer’s instructions. Images were captured on Nikon 90i upright microscopes with Photometrics CoolSNAP-ES monochrome cameras (Roper). Probes were designed to detect Tfap2c (860171, NM_201420.2, target region: 696–1733), Krt8 (87304-C2, NM_199370.1, target region: 134–1472), and Prl7b1 (860181-C2, NM_153738.1, target region: 28–900).

Immunohistochemistry. Frozen rat placentation sites were sectioned at 10 μm, mounted on slides, and fixed in 4% paraformaldehyde. Sections were blocked with 10% goat serum (50062Z, Thermo Fisher Scientific) and incubated overnight at 4°C with a primary antibody for vimentin (1:300, sc-6260, Santa Cruz Biotechnology), pan-cytokeratin antibody (1:100, F3418, MilliporeSigma), perforin (1:300, TP251, Amsbio), or TFAP2C (1:150, 2320, Cell Signaling Technology). Fluorescence-tagged secondary antibodies, Alexa Fluor 488–conjugated goat anti-mouse IgG (A11001, Thermo Fisher Scientific), and Alexa Fluor 568–conjugated goat anti-rabbit IgG (A11011, Thermo Fisher Scientific) were used to visualize immunostaining. Sections were counterstained with DAPI (1:50,000, D1306, Invitrogen, Thermo Fisher Scientific). Slides were mounted in Fluoromount-G (0100-01, SouthernBiotech) and imaged on Nikon 90i upright microscopes with Photometrics CoolSNAP-ES monochrome cameras. Regions of the uterine-placental interface possessing cytokeratin-positive cells were quantified using ImageJ software (NIH), as previously described by our laboratory (63, 64).

Western blotting. Tissue lysates were processed using the radioimmunoprecipitation assay lysis buffer (sc-24948A, Santa Cruz Biotechnology), and protein concentrations were determined using the DC Protein Assay Kit (5000112, Bio-Rad). Proteins (80 μg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (10600023, GE Healthcare, now Cytiva) for 1 hour at 25 V on a semidry transfer apparatus (Bio-Rad). Membranes were subsequently blocked with 5% milk for 1 hour at room temperature, followed by incubation with antibodies against TFAP2C (1:100, sc-12762, Santa Cruz Biotechnology) and GAPDH (1:5,000, AM4300, Invitrogen, Thermo Fisher Scientific) in 5% milk overnight at 4°C. After primary antibody incubation, the membranes were washed in Tris-buffered saline with Tween 20 (TBS-T) 3 times (10 min/wash) at room temperature. The membranes were then incubated with anti-mouse IgG conjugated to horseradish peroxidase (HRP; 1:5,000, Cell Signaling Technology) in 5% milk for 1 hour at room temperature, washed in TBS-T 3 times (10 min/wash) at room temperature, immersed in Immobilon Crescendo Western HRP Substrate (WBLUR0500, MilliporeSigma), and luminescence-detected using ChemiDoc MP Imaging System (Bio-Rad).

Identification of TFAP2C and potential TFAP2C gene targets in rat invasive trophoblast cells. Uniform manifold approximation and projection plots for Tfap2c and Prl7b1 were generated from rat gd 19.5 uterine-placental interface single-cell RNA sequencing (National Center for Biotechnology [NCBI] Gene Expression Omnibus accession no. GSE206086, ref. 38). Gene expression profiles and open chromatin containing TFAP2C DNA binding motifs identified in rat invasive trophoblast cells (38, 41) were integrated and ascribed function through manual annotation using the UniProt (https://www.uniprot.org) and NCBI (https://www.ncbi.nlm.nih.gov/) databases.

Statistics. Statistical analyses were performed with GraphPad Prism 10.2.3 software. Statistical comparisons were evaluated using Student’s 2-tailed t test or 1-way ANOVA with Tukey’s or Dunnett’s post hoc tests as appropriate. Statistical significance was determined as P < 0.05.

Study approval. All protocols using rats were approved by the University of Kansas Medical Center Animal Care and Use Committee, Kansas City, Kansas, USA (approved protocol 22-01-220).

Data availability. All data and materials for this manuscript are included in the Methods and Supporting Data Values.