Roles of the Rlim-Rex1 axis during X chromosome inactivation in mice.

In female mice, the gene dosage from X chromosomes is adjusted by a process called X chromosome inactivation (XCI) that occurs in two steps. An imprinted form of XCI (iXCI) that silences the paternally inherited X chromosome (Xp) is initiated at the 2- to 4-cell stages. As extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI require the lncRNA Xist, which is expressed from the X to be inactivated. The X-linked E3 ubiquitin ligase Rlim (Rnf12) in conjunction with its target protein Rex1 (Zfp42), a critical repressor of Xist, have emerged as major regulators of iXCI. However, their roles in rXCI remain controversial. Investigating early mouse development, we show that the Rlim-Rex1 axis is active in pre-implantation embryos. Upon implantation Rex1 levels are downregulated independently of Rlim specifically in epiblast cells. These results provide a conceptual framework of how the functional dynamics between Rlim and Rex1 ensures regulation of iXCI but not rXCI in female mice.

hnRNPL expression dynamics in the embryo and placenta.

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a conserved RNA binding protein (RBP) that plays an important role in the alternative splicing of gene transcripts, and thus in the generation of specific protein isoforms. Global deficiency in hnRNPL in mice results in preimplantation embryonic lethality at embryonic day (E) 3.5. To begin to understand the contribution of hnRNPL-regulated pathways in the normal development of the embryo and placenta, we determined hnRNPL expression profile and subcellular localization throughout development. Proteome and Western blot analyses were employed to determine hnRNPL abundance between E3.5 and E17.5. Histological analyses supported that the embryo and implantation site display distinct hnRNPL localization patterns. In the fully developed mouse placenta, nuclear hnRNPL was observed broadly in trophoblasts, whereas within the implantation site a discrete subset of cells showed hnRNPL outside the nucleus. In the first-trimester human placenta, hnRNPL was detected in the undifferentiated cytotrophoblasts, suggesting a role for this factor in trophoblast progenitors. Parallel in vitro studies utilizing Htr8 and Jeg3 cell lines confirmed expression of hnRNPL in cellular models of human trophoblasts. These studies [support] coordinated regulation of hnRNPL during the normal developmental program in the mammalian embryo and placenta.

SLC20a1/PiT-1 is required for chorioallantoic placental morphogenesis.

The placenta mediates the transport of nutrients, such as inorganic phosphate (Pi), between the maternal and fetal circulatory systems. The placenta itself also requires high levels of nutrient uptake as it develops to provide critical support for fetal development. This study aimed to determine placental Pi transport mechanisms using in vitro and in vivo models. We observed that Pi (P33) uptake in BeWo cells is sodium dependent and that SLC20A1/Slc20a1 is the most highly expressed placental sodium-dependent transporter in mouse (microarray), human cell line (RT-PCR) and term placenta (RNA-seq), supporting that normal growth and maintenance of the mouse and human placenta requires SLC20A1/Slc20a1. Slc20a1 wild-type (Slc20a1+/+) and knockout (Slc20a1-/-) mice were produced through timed intercrosses and displayed yolk sac angiogenesis failure as expected at E10.5. E9.5 tissues were analyzed to test whether placental morphogenesis requires Slc20a1. At E9.5, the developing placenta was reduced in size in Slc20a1-/-. Multiple structural abnormalities were also observed in the Slc20a1-/-chorioallantois. We determined that monocarboxylate transporter 1 protein (MCT1+) cells were reduced in developing Slc20a1-/-placenta, confirming that Slc20a1 loss reduced trophoblast syncytiotrophoblast 1 (SynT-I) coverage. Next, we examined the cell type-specific Slc20a1 expression and SynT molecular pathways in silico and identified Notch/Wnt as a pathway of interest that regulates trophoblast differentiation. We further observed that specific trophoblast lineages express Notch/Wnt genes that associate with endothelial cell tip-and-stalk cell markers. In conclusion, our findings support that Slc20a1 mediates the symport of Pi into SynT cells, providing critical support for their differentiation and angiogenic mimicry function at the developing maternal-fetal interface.

Interaction of extraembryonic microglia and neonatal brain development.

Microglia are resident immune cells in the central nervous system (CNS), which, in a healthy state, promote CNS homeostasis and respond to CNS injury. In contrast, microglia are also implicated in pathological conditions where they may contribute to neural damage. Primitive microglia arise from extraembryonic progenitors in the yolk sac (YS). The extraembryonic origins of primitive microglia are distinct from other tissue macrophages. The YS is the first site of hematopoiesis in development. Uniquely, microglial pregenital cells in the mouse derive from an early myeloid branch of the hematopoietic lineage in the YS. Microglia are critical in several key stages of physiological brain development, including embryonic vasculogenesis, immunosurveillance, and neurogenesis. Abnormal microglial function has been linked to neurodevelopmental and neurodegenerative diseases, although mechanistic roles in disease etiology remain incompletely understood. Knowledge of species-specific differences between human, murine and other animal models is also critical to understanding translational relevance to human health and disease as biomedical understanding of the importance of primitive microglia advances. This significance drives the importance of understanding, comparatively, the extraembryonic origins and developmental mechanisms whereby human primitive microglia differentiate and migrate to inform translational research. A better understanding of the molecular drivers may lead to biomarkers and/or preventative or therapeutic measures for neonatal brain development and neurodegenerative diseases. Herein, the role of microglia in neonatal brain development is discussed, current understandings of the developmental origins of microglia are described, the ontogeny and phylogeny of microglia, and implications of in vitro microglia-like cell differentiation, with a specific interest on neurodegenerative diseases, are reviewed. Together, these emphasize the importance of leveraging the extraembryonic origins of microglia to not only better understand neurodevelopment and neurodegenerative diseases, but also to develop protective measures that are specific to human microglia.

Cellular Complexity of Hemochorial Placenta: Stem Cell Populations, Insights from scRNA-seq, and SARS-CoV-2 Susceptibility.

Purpose of Review: The placenta is a transient organ that forms de novo and serves a critical role in supporting fetal growth and development. Placental oxygen, nutrients, and waste are transported through processes that depend on vascular structure and cell type-specific expression and localization of membrane transporters. Understanding how the placenta develops holds great significance for maternal-fetal medicine. The purpose of this review is to examine current information regarding placental progenitor populations. Recent Findings: Recent advancements in single-cell RNA sequencing (scRNA-seq) provide unprecedented depth for the investigation of cell type-specific gene expression patterns in the placenta. Thus far, several mouse placenta scRNA-seq studies have been conducted which produced and analyzed transcriptomes of placental progenitors and cells of the fully developed placenta between embryonic day (E) 7.0 and E12.5. Together with human placenta scRNA-seq data which, in part, has been produced through coordinated research campaigns in the scientific community to understand the potential for SARS-CoV-2 infection, these mammalian studies lend fundamental insight into the cellular and molecular composition of hemochorial placentae found in both mouse and human. Summary: Single-cell placenta research has advanced understanding of tissue-resident stem cells and molecules that are poised to support maternal-fetal communication and nutrient transport. Herein, we provide context for these recent findings by reviewing placental anatomy and cell populations, and discuss recent scRNA-seq mouse placenta findings. Further research is needed to evaluate the utility of placental stem cells in the development of new therapeutic approaches for the treatment of wound healing and disease.

Adapter Protein RapGEF1 Is Required for ERK1/2 Signaling in Response to Elevated Phosphate in Vascular Smooth Muscle Cells.

The sodium-dependent phosphate transporter, SLC20A1, is required for elevated inorganic phosphate (Pi) induced vascular smooth muscle cell (VSMC) matrix mineralization and phenotype transdifferentiation. Recently, elevated Pi was shown to induce ERK1/2 phosphorylation through SLC20A1 by Pi uptake-independent functions in VSMCs, suggesting a cell signaling response to elevated Pi. Previous studies identified Rap1 guanine nucleotide exchange factor (RapGEF1) as an SLC20A1-interacting protein and RapGEF1 promotes ERK1/2 phosphorylation through Rap1 activation. In this study, we tested the hypothesis that RapGEF1 is a critical component of the SLC20A1-mediated Pi-induced ERK1/2 phosphorylation pathway. Co-localization of SLC20A1 and RapGEF1, knockdown of RapGEF1 with siRNA, and small molecule inhibitors of Rap1, B-Raf, and Mek1/2 were investigated. SLC20A1 and RapGEF1 were co-localized in peri-membranous structures in VSMCs. Knockdown of RapGEF1 and small molecule inhibitors against Rap1, B-Raf, and Mek1/2 eliminated elevated Pi-induced ERK1/2 phosphorylation. Knockdown of RapGEF1 inhibited SM22α mRNA expression and blocked elevated Pi-induced downregulation of SM22α mRNA. Together, these data suggest that RapGEF1 is required for SLC20A1-mediated elevated Pi signaling through a Rap1/B-Raf/Mek1/2 cell signaling pathway, thereby promoting ERK1/2 phosphorylation and inhibiting SM22α gene expression in VSMCs.

Deficient spermiogenesis in mice lacking Rlim.

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile. Here, we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis.

Human SMAD4 Genomic Variants Identified in Individuals with Heritable and Early-Onset Thoracic Aortic Disease.

Thoracic aortic aneurysms (TAAs) that progress to acute thoracic aortic dissections (TADs) are life threatening vascular events that have been associated with altered transforming growth factor (TGF) ß signaling. In addition to TAA, multiple genetic vascular disorders, including hereditary hemorrhagic telangiectasia (HHT), involve altered TGFß signaling and vascular malformations. Due to the importance of TGFß, genomic variant databases have been curated for activin receptor-like kinase 1 (ALK1) and endoglin (ENG). This case report details seven variants in SMAD4 that are associated with either heritable or early onset aortic dissections and compares them to pathogenic exon variants in gnomAD v2.1.1. The TAA and TAD variants were identified through whole exome sequencing of 346 unrelated heritable thoracic aortic disease (HTAD) and 355 individuals of early onset (age ≤ 56 years old) of thoracic aortic dissection (ESTAD). An allele frequency filter of less than 0.05% was applied in the Genome Aggregation Database (gnomAD exome v2.1.1) with a combined annotation dependent depletion score (CADD) greater than 20. These seven variants also have a higher REVEL score (>0.2), indicating pathogenic potential. Further in vivo and in vitro analysis is needed to evaluate how these variants affect mRNA stability and SMAD4 protein activity in association with thoracic aortic disease.

Inorganic Phosphate in the Pathogenesis of Pregnancy-Related Complications.

Inorganic phosphate (Pi) is an essential nutrient that fulfills critical roles in human health. It enables skeletal ossification, supports cellular structure and organelle function, and serves key biochemical roles in energetics and molecular signaling. Pi homeostasis is modulated through diet, intestinal uptake, renal reabsorption, and mobilization of stores in bone and extracellular compartments. Disrupted Pi homeostasis is associated with phosphate wasting, mineral and bone disorders, and vascular calcification. Mechanisms of Pi homeostasis in pregnancy remain incompletely understood. The study presented herein examined biological fluid Pi characteristics over the course of gestation. Correlations with gestation age, pregnancy number, preterm birth, preeclampsia, diabetes mellitus, and placental calcification were evaluated during the last trimester. The results support that maternal urinary Pi levels increased during the third trimester of pregnancy. Reduced levels were observed with previous pregnancy. Amniotic fluid Pi levels decreased with gestation while low second trimester levels associated with preterm birth. No significant difference in urinary Pi levels was observed between preeclampsia and controls (8.50 ± 2.74 vs. 11.52 ± 2.90 mmol/L). Moreover, increased maternal urinary Pi was associated with preexisting diabetes mellitus in preeclampsia. Potential confounding factors in this study are maternal age at delivery and body mass index (BMI)-information which we do not have access to for this cohort. In conclusion, Pi levels provide clinical information regarding the pathogenesis of pregnancy-related complications, supporting that phosphate should be examined more closely and in larger populations.

New Frontiers in Placenta Tissue Imaging.

The placenta is a highly vascularized organ with unique structural and metabolic complexities. As the primary conduit of fetal support, the placenta mediates transport of oxygen, nutrients, and waste between maternal and fetal blood. Thus, normal placenta anatomy and physiology is absolutely required for maintenance of maternal and fetal health during pregnancy. Moreover, impaired placental health can negatively impact offspring growth trajectories as well as increase the risk of maternal cardiovascular disease later in life. Despite these crucial roles for the placenta, placental disorders, such as preeclampsia, intrauterine growth restriction (IUGR), and preterm birth, remain incompletely understood. Effective noninvasive imaging and image analysis are needed to advance the obstetrician's clinical reasoning toolkit and improve the utility of the placenta in interpreting maternal and fetal health trajectories. Current paradigms in placental imaging and image analysis aim to improve the traditional imaging techniques that may be time-consuming, costly, or invasive. In concert with conventional clinical approaches such as ultrasound (US), advanced imaging modalities can provide insightful information on the structure of placental tissues. Herein we discuss such imaging modalities, their specific applications in structural, vascular, and metabolic analysis of placental health, and emerging frontiers in image analysis research in both preclinical and clinical contexts.

Vascular Mineralocorticoid Receptor: Evolutionary Mediator of Wound Healing Turned Harmful by Our Modern Lifestyle.

The mineralocorticoid receptor (MR) is indispensable for survival through its critical role in maintaining blood pressure in response to sodium scarcity or bleeding. Activation of MR by aldosterone in the kidney controls water and electrolyte homeostasis. This review summarizes recent advances in our understanding of MR function, specifically in vascular endothelial and smooth muscle cells. The evolving roles for vascular MR are summarized in the areas of (i) vascular tone regulation, (ii) thrombosis, (iii) inflammation, and (iv) vascular remodeling/fibrosis. Synthesis of the data supports the concept that vascular MR does not contribute substantially to basal homeostasis but rather, MR is poised to be activated when the vasculature is damaged to coordinate blood pressure maintenance and wound healing. Specifically, MR activation in the vascular wall promotes vasoconstriction, inflammation, and exuberant vascular remodeling with fibrosis. A teleological model is proposed in which these functions of vascular MR may have provided a critical evolutionary survival advantage in the face of mechanical vascular injury with bleeding. However, modern lifestyle is characterized by physical inactivity and high fat/high sodium diet resulting in diffuse vascular damage. Under these modern conditions, diffuse, persistent and unregulated activation of vascular MR contributes to post-reproductive cardiovascular disease in growing populations with hypertension, obesity, and advanced age.

Placental Vascular Calcification and Cardiovascular Health: It Is Time to Determine How Much of Maternal and Offspring Health Is Written in Stone.

Vascular calcification is the deposition of calcium phosphate minerals in vascular tissue. Vascular calcification occurs by both active and passive processes. Extent and tissue-specific patterns of vascular calcification are predictors of cardiovascular morbidity and mortality. The placenta is a highly vascularized organ with specialized vasculature that mediates communication between two circulatory systems. At delivery the placenta often contains calcified tissue and calcification can be considered a marker of viral infection, but the mechanisms, histoanatomical specificity, and pathophysiological significance of placental calcification are poorly understood. In this review, we outline the current understanding of vascular calcification mechanisms, biomedical consequences, and therapeutic interventions in the context of histoanatomical types. We summarize available placental calcification data and clinical grading systems for placental calcification. We report on studies that have examined the association between placental calcification and acute adverse maternal and fetal outcomes. We then review the intersection between placental dysfunction and long-term cardiovascular health, including subsequent occurrence of maternal vascular calcification. Possible maternal phenotypes and trigger mechanisms that may predispose for calcification and cardiovascular disease are discussed. We go on to highlight the potential diagnostic value of placental calcification. Finally, we suggest avenues of research to evaluate placental calcification as a research model for investigating the relationship between placental dysfunction and cardiovascular health, as well as a biomarker for placental dysfunction, adverse clinical outcomes, and increased risk of subsequent maternal and offspring cardiovascular events.

Increased Calcific Aortic Valve Disease in response to a diabetogenic, procalcific diet in the LDLr-/-ApoB100/100 mouse model.

OBJECTIVE: Calcific aortic valve disease (CAVD) is a major cause of aortic stenosis (AS) and cardiac insufficiency. Patients with type II diabetes mellitus (T2DM) are at heightened risk for CAVD, and their valves have greater calcification than nondiabetic valves. No drugs to prevent or treat CAVD exist, and animal models that might help identify therapeutic targets are sorely lacking. To develop an animal model mimicking the structural and functional features of CAVD in people with T2DM, we tested a diabetogenic, procalcific diet and its effect on the incidence and severity of CAVD and AS in the, LDLr-/-ApoB100/100 mouse model. RESULTS: LDLr-/-ApoB100/100 mice fed a customized diabetogenic, procalcific diet (DB diet) developed hyperglycemia, hyperlipidemia, increased atherosclerosis, and obesity when compared with normal chow fed LDLr-/-ApoB100/100 mice, indicating the development of T2DM and metabolic syndrome. Transthoracic echocardiography revealed that LDLr-/-ApoB100/100 mice fed the DB diet had 77% incidence of hemodynamically significant AS, and developed thickened aortic valve leaflets and calcification in both valve leaflets and hinge regions. In comparison, normal chow (NC) fed LDLr-/-ApoB100/100 mice had 38% incidence of AS, thinner valve leaflets and very little valve and hinge calcification. Further, the DB diet fed mice with AS showed significantly impaired cardiac function as determined by reduced ejection fraction and fractional shortening. In vitro mineralization experiments demonstrated that elevated glucose in culture medium enhanced valve interstitial cell (VIC) matrix calcium deposition. CONCLUSIONS: By manipulating the diet we developed a new model of CAVD in T2DM, hyperlipidemic LDLr-/-ApoB100/100 that shows several important functional, and structural features similar to CAVD found in people with T2DM and atherosclerosis including AS, cardiac dysfunction, and inflamed and calcified thickened valve cusps. Importantly, the high AS incidence of this diabetic model may be useful for mechanistic and translational studies aimed at development of novel treatments for CAVD.

Loss of PiT-2 results in abnormal bone development and decreased bone mineral density and length in mice.

Normal bone mineralization requires phosphate oversaturation in bone matrix vesicles, as well as normal regulation of phosphate metabolism via the interplay among bone, intestine, and kidney. In turn, derangement of phosphate metabolism greatly affects bone function and structure. The type III sodium-dependent phosphate transporters, PiT-1 and PiT-2, are believed to be important in tissue phosphate metabolism and physiological bone formation, but their requirement and molecular roles in bone remain poorly investigated. In order to decipher the role of PiT-2 in bone, we examined normal bone development, growth, and mineralization in global PiT-2 homozygous knockout mice. PiT-2 deficiency resulted in reduced vertebral column, femur, and tibia length as well as mandibular dimensions. Micro-computed tomography analysis revealed that bone mineral density in the mandible, femur, and tibia were decreased, indicating that maintenance of bone function and structure is impaired in both craniofacial and long bones of PiT-2 deficient mice. Both cortical and trabecular thickness and mineral density were reduced in PiT-2 homozygous knockout mice compared with wild-type mice. These results suggest that PiT-2 is involved in normal bone development and growth and plays roles in cortical and trabecular bone metabolism feasibly by regulating local phosphate transport and mineralization processes in the bone. Further studies that evaluate bone cell-specific loss of PiT-2 are now warranted and may yield insight into complex mechanisms of bone development and growth, leading to identification of new therapeutic options for patients with bone diseases.

YY1 Is Required for Posttranscriptional Stability of SOX2 and OCT4 Proteins.

Yinyang1 (YY1) participates in protein-DNA, protein-RNA, and protein-protein interactions and regulates developmental processes and disease mechanisms. YY1 interactions regulate a range of important biological functions, including oocyte maturation, epithelial to mesenchymal transition, and vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that YY1 is required for inner cell mass (ICM) lineage commitment during preimplantation development. In this study, we document gene expression patterns and protein localization of key transcription factors in Yy1 global, tissue-specific, and dsRNA-mediated knockout/down embryos. YY1 protein was found in cells of preimplantation and peri-implantation embryos, and adult tissues where two isoforms are observed. In the absence of YY1, OCT4 and SOX2 protein were lost in the ICM during preimplantation and naive neuroectoderm during gastrulation stages, yet no difference in Oct4 or Sox2 mRNA levels was observed. The loss of OCT4 and SOX2 protein occurred specifically in cells that normally express both OCT4 and SOX2 protein. These observations support a role for YY1 meditating and/or regulating the interaction of OCT4 and SOX2 at a posttranscriptional level. Our results suggest that distinct mechanisms of YY1-mediated molecular regulation are present in the early embryo, and may offer insight to promote lineage commitment in in vitro cell lines.

Runx2 deletion in smooth muscle cells inhibits vascular osteochondrogenesis and calcification but not atherosclerotic lesion formation.

AIMS: Vascular smooth muscle cells (SMCs) are major precursors contributing to osteochondrogenesis and calcification in atherosclerosis. Runt-related transcription factor-2 (Runx2) has been found essential for SMC differentiation to an osteochondrogenic phenotype and subsequent calcification in vitro. A recent study using a conditional targeting allele that produced a truncated Runx2 protein in SMCs of ApoE-/- mice showed reduced vascular calcification, likely occurring via reduction of receptor activator of nuclear factor-κB ligand (RANKL), macrophage infiltration, and atherosclerotic lesion formation. Using an improved conditional Runx2 knockout mouse model, we have elucidated new roles for SMC-specific Runx2 in arterial intimal calcification (AIC) without effects on atherosclerotic lesion size. METHODS AND RESULTS: We used an improved targeting construct to generate LDLr-/- mice with floxed-Runx2 alleles ( LDLr-/- :Runx2 f/f ) such that Cre-mediated recombination ( LDLr-/- :Runx2 ΔSM ) does not produce functional truncated Runx2 protein, thereby avoiding off-target effects. We found that both LDLr-/- :Runx2 f/f and LDLr-/- :Runx2 ΔSM mice fed with a high fat diet developed atherosclerosis. SMC-specific Runx2 deletion did not significantly reduce atherosclerotic lesion size, macrophage number, or expression of RANKL, MCP-1, and CCR2. However, it significantly reduced AIC by 50%. Mechanistically, Sox9 and type II collagen were unaltered in vessels of LDLr-/- :Runx2 ΔSM mice compared to LDLr-/- :Runx2 f/f counterparts, while type X collagen, MMP13 and the osteoblastic marker osteocalcin were significantly reduced. CONCLUSIONS: SMC autonomous Runx2 is required for SMC differentiation towards osteoblast-like cells, SMC-derived chondrocyte maturation and AIC in atherosclerotic mice. These effects were independent of systemic lipid metabolism, RANKL expression, macrophage infiltration, and atheromatous lesion progression.

Slc20a2 deficiency results in fetal growth restriction and placental calcification associated with thickened basement membranes and novel CD13 and lamininα1 expressing cells.

The essential nutrient phosphorus must be taken up by the mammalian embryo during gestation. The mechanism(s) and key proteins responsible for maternal to fetal phosphate transport have not been identified. Established parameters for placental phosphate transport match those of the type III phosphate transporters, Slc20a1 and Slc20a2. Both members are expressed in human placenta, and their altered expression is linked to preeclampsia. In this study, we tested the hypothesis that Slc20a2 is required for placental function. Indeed, complete deficiency of Slc20a2 in either the maternal or embryonic placental compartment results in fetal growth restriction. We found that Slc20a2 null mice can reproduce, but are subviable; ∼50% are lost prior to weaning age. We also observed that 23% of Slc20a2 deficient females develop pregnancy complications at full term, with tremors and placental abnormalities including abnormal vascular structure, increased basement membrane deposition, abundant calcification, and accumulation of novel CD13 and lamininα1 positive cells. Together these data support that Slc20a2 deficiency impacts both maternal and neonatal health, and Slc20a2 is required for normal placental function. In humans, decreased levels of placental Slc20a1 and Slc20a2 have been correlated with early onset preeclampsia, a disorder that can manifest from placental dysfunction. In addition, preterm placental calcification has been associated with poor pregnancy outcomes. We surveyed placental calcification in human preeclamptic placenta samples, and detected basement membrane-associated placental calcification as well as a comparable lamininα1 positive cell type, indicating that similar mechanisms may underlie both human and mouse placental calcification.

Identification of a novel isoform of the leukemia-associated MLLT1 (ENL/LTG19) protein.

Genome wide transcriptional profiles offer abundant information regarding mRNA levels in specific tissues, organs or developmental stages. Although these data sets do not offer spatial or cell type-specific information, they can be extremely useful for gene discovery when analyzed by the appropriate techniques. Previously, we proposed and validated the use of combinatorial dataset analysis techniques to identify novel genes required during pre-implantation development. Now we build upon this work to identify genes that have dynamic expression during gametogenesis. Here we present detailed analysis of the expression pattern of leukemia-associated, myeloid/lymphoid or mixed-lineage leukemia; translocated to 1 (Mllt1) gene. We document a novel splice isoform of Mllt1 and confirm that both Mllt1 mRNA isoforms are translated. We provide data supporting that MLLT1 protein isoforms display distinct stage-specific expression during spermiogenesis and adult tissues. Finally, we evaluated genes neighboring the Mllt1 locus, and show dynamic stage specific expression patterns of other genes Catsperd, Prr22, Rfx2 and Slc25a41. We document testes expressed alternative isoforms of Prr22 and Rfx2. These results indicate that transcriptome data mining, combined with specific expression analysis provides a wealth of novel gene expression information.

Loss of PiT-1 results in abnormal endocytosis in the yolk sac visceral endoderm.

PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE.