Bhavwanti Sheth

Adaptive Responses by Mouse Early Embryos to Maternal Diet Protect Fetal Growth but Predispose to Adult Onset Disease

Abstract Poor maternal nutrition during pregnancy can alter postnatal phenotype and increase susceptibility to adult cardiovascular and metabolic diseases. However, underlying mechanisms are largely unknown. Here, we show that maternal low protein diet (LPD), fed exclusively during mouse preimplantation development, leads to offspring with increased weight from birth, sustained hypertension, and abnormal anxiety-related behavior, especially in females. These adverse outcomes were interrelated with increased perinatal weight being predictive of later adult overweight and hypertension. Embryo transfer experiments revealed that the increase in perinatal weight was induced within blastocysts responding to preimplantation LPD, independent of subsequent maternal environment during later pregnancy. We further identified the embryo-derived visceral yolk sac endoderm (VYSE) as one mediator of this response. VYSE contributes to fetal growth through endocytosis of maternal proteins, mainly via the multiligand megalin (LRP2) receptor and supply of liberated amino acids. Thus, LPD maintained throughout gestation stimulated VYSE nutrient transport capacity and megalin expression in late pregnancy, with enhanced megalin expression evident even when LPD was limited to the preimplantation period. Our results demonstrate that in a nutrient-restricted environment, the preimplantation embryo activates physiological mechanisms of developmental plasticity to stablize conceptus growth and enhance postnatal fitness. However, activation of such responses may also lead to adult excess growth and cardiovascular and behavioral diseases.

Sustained TL1A expression modulates effector and regulatory T-cell responses and drives intestinal goblet cell hyperplasia

The tumor necrosis factor (TNF) superfamily protein TNF-like 1A (TL1A) is the ligand for death receptor 3 (DR3). TL1A is induced on activated dendritic cells (DCs) and its expression has been linked to human inflammatory bowel disease. To address how TL1A might influence intestinal inflammation, we generated transgenic mice that constitutively express TL1A on DCs. TL1A transgenic mice developed striking goblet cell hyperplasia in the ileum that was associated with elevated interleukin (IL)-13 levels in the small intestine. IL-13- and IL-17-producing small intestinal lamina propria T cells were increased in TL1A transgenic mice. TL1A also enhanced regulatory T (Treg) cell turnover in vivo and directly stimulated Treg cell proliferation in vitro. The presence of TL1A attenuated the ability of Treg cells to suppress conventional T cells, an effect that required DR3 signaling in either conventional T cells or Treg cells. Our findings identify mechanisms by which chronic DR3 signaling could promote pathogenesis in inflammatory bowel disease.

Mouse early extra-embryonic lineages activate compensatory endocytosis in response to poor maternal nutrition

Mammalian extra-embryonic lineages perform the crucial role of nutrient provision during gestation to support embryonic and fetal growth. These lineages derive from outer trophectoderm (TE) and internal primitive endoderm (PE) in the blastocyst and subsequently give rise to chorio-allantoic and visceral yolk sac placentae, respectively. We have shown maternal low protein diet exclusively during mouse preimplantation development (Emb-LPD) is sufficient to cause a compensatory increase in fetal and perinatal growth that correlates positively with increased adult-onset cardiovascular, metabolic and behavioural disease. Here, to investigate early mechanisms of compensatory nutrient provision, we assessed the influence of maternal Emb-LPD on endocytosis within extra-embryonic lineages using quantitative imaging and expression of markers and proteins involved. Blastocysts collected from Emb-LPD mothers within standard culture medium displayed enhanced TE endocytosis compared with embryos from control mothers with respect to the number and collective volume per cell of vesicles with endocytosed ligand and fluid and lysosomes, plus protein expression of megalin (Lrp2) LDL-family receptor. Endocytosis was also stimulated using similar criteria in the outer PE-like lineage of embryoid bodies formed from embryonic stem cell lines generated from Emb-LPD blastocysts. Using an in vitro model replicating the depleted amino acid (AA) composition found within the Emb-LPD uterine luminal fluid, we show TE endocytosis response is activated through reduced branched-chain AAs (leucine, isoleucine, valine). Moreover, activation appears mediated through RhoA GTPase signalling. Our data indicate early embryos regulate and stabilise endocytosis as a mechanism to compensate for poor maternal nutrient provision.

Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.

Epigenetic regulation of histone modifications and Gata6 gene expression induced by maternal diet in mouse embryoid bodies in a model of developmental programming

BackgroundDietary interventions during pregnancy alter offspring fitness. We have shown mouse maternal low protein diet fed exclusively for the preimplantation period (Emb-LPD) before return to normal protein diet (NPD) for the rest of gestation, is sufficient to cause adult offspring cardiovascular and metabolic disease. Moreover, Emb-LPD blastocysts sense altered nutrition within the uterus and activate compensatory cellular responses including stimulated endocytosis within extra-embryonic trophectoderm and primitive endoderm (PE) lineages to protect fetal growth rate. However, these responses associate with later disease. Here, we investigate epigenetic mechanisms underlying nutritional programming of PE that may contribute to its altered phenotype, stabilised during subsequent development. We use embryonic stem (ES) cell lines established previously from Emb-LPD and NPD blastocysts that were differentiated into embryoid bodies (EBs) with outer PE-like layer.ResultsEmb-LPD EBs grow to a larger size than NPD EBs and express reduced Gata6 transcription factor (regulator of PE differentiation) at mRNA and protein levels, similar to Emb-LPD PE derivative visceral yolk sac tissue in vivo in later gestation. We analysed histone modifications at the Gata6 promoter in Emb-LPD EBs using chromatin immunoprecipitation assay. We found significant reduction in histone H3 and H4 acetylation and RNA polymerase II binding compared with NPD EBs, all markers of reduced transcription. Other histone modifications, H3K4Me2, H3K9Me3 and H3K27Me3, were unaltered. A similar but generally non-significant histone modification pattern was found on the Gata4 promoter. Consistent with these changes, histone deacetylase Hdac-1, but not Hdac-3, gene expression was upregulated in Emb-LPD EBs.ConclusionsFirst, these data demonstrate ES cells and EBs retain and propagate nutritional programming adaptations in vitro, suitable for molecular analysis of mechanisms, reducing animal use. Second, they reveal maternal diet induces persistent changes in histone modifications to regulate Gata6 expression and PE growth and differentiation that may affect lifetime health.

Society for Reproductive Biology Founders’ Lecture 2003. The making of an embryo: short-term goals and long-term implications

During early development, the eutherian mammalian embryo forms a blastocyst comprising an outer trophectoderm epithelium and enclosed inner cell mass (ICM). The short-term goal of blastocyst morphogenesis, including epithelial differentiation and segregation of the ICM, is mainly regulated autonomously and comprises a combination of temporally controlled gene expression, cell polarisation, differentiative cell divisions and cell-cell interactions. This aspect of blastocyst biogenesis is reviewed, focusing, in particular, on the maturation and role of cell adhesion systems. Early embryos are also sensitive to their environment, which can affect their developmental potential in diverse ways and may lead to long-term consequences relating to fetal or postnatal growth and physiology. Some current concepts of embryo-environment interactions, which may impact on future health, are also reviewed.

Insulin and branched-chain amino acid depletion during mouse preimplantation embryo culture programmes body weight gain and raised blood pressure during early postnatal life

Mouse maternal low protein diet exclusively during preimplantation development (Emb-LPD) is sufficient to programme altered growth and cardiovascular dysfunction in offspring. Here, we use an in vitro model comprising preimplantation culture in medium depleted in insulin and branched-chain amino acids (BCAA), two proposed embryo programming inductive factors from Emb-LPD studies, to examine the consequences for blastocyst organisation and, after embryo transfer (ET), postnatal disease origin. Two-cell embryos were cultured to blastocyst stage in defined KSOM medium supplemented with four combinations of insulin and BCAA concentrations. Control medium contained serum insulin and uterine luminal fluid amino acid concentrations (including BCAA) found in control mothers from the maternal diet model (N-insulin + N-bcaa). Experimental medium (three groups) contained 50% reduction in insulin and/or BCAA (L-insulin + N-bcaa, N-insulin + L-bcaa, and L-insulin + N-bcaa). Lineage-specific cell numbers of resultant blastocysts were not affected by treatment. Following ET, a combined depletion of insulin and BCAA during embryo culture induced a non sex-specific increase in birth weight and weight gain during early postnatal life. Furthermore, male offspring displayed relative hypertension and female offspring reduced heart/body weight, both characteristics of Emb-LPD offspring. Combined depletion of metabolites also resulted in a strong positive correlation between body weight and glucose metabolism that was absent in the control group. Our results support the notion that composition of preimplantation culture medium can programme development and associate with disease origin affecting postnatal growth and cardiovascular phenotypes and implicate two important nutritional mediators in the inductive mechanism. Our data also have implications for human assisted reproductive treatment (ART) practice.

Tight junctions during development

During early development, tight junction biogenesis and the differentiation of the first epithelium in the blastocyst is critical for embryonic patterning and organization. Here, we discuss the programme of exactly timed transcription, translation, and post-translational modification of specific junctional proteins that regulates the stepwise membrane assembly of tight junctions during cleavage in the mouse model. Underlying mechanisms that coordinate these processes are discussed along with newly emerging data from other mammalian species. In the mouse embryo, junction assembly follows the establishment of cell polarity at the 8-cell stage and is characterized by sequential membrane delivery of JAM-1, ZO-1α− and Rab13, cingulin and ZO-2 followed by ZO-1α+ and occludin. Tight junction assembly occurs over three developmental stages; compaction, first differentiative division and cavitation. Post-translational modification of occludin, the late expression of ZO-1α+ isoform and their intracellular colocalisation may all contribute to the rapid coordinated delivery of these two proteins to the membrane, resulting in the final sealing of the tight junction followed by blastocoel cavitation. This coordinated delivery of these two tight junction-associated proteins may therefore provide a rate limiting step for the sealing of tight junctions and regulated timing of blastocoel cavitation. Taken together, our studies in mouse, human and bovine embryos suggest that defects in the tightly controlled programming of early development may contribute to reduced embryo viability.

Cell Junctions and Cell Interactions in Animal and Human Blastocyst Development

The first differentiation event in mammalian development is the generation of the outer trophectoderm epithelium of the blastocyst, which is responsible for blastocoel cavity formation and for vectorial transport between the maternal environment and the embryo interior where the inner cell mass (ICM) resides. Blastomeres within the trophectoderm lineage interact via four types of epithelial intercellular junctions: the E-cadherin/catenin (or adherens junction) system, gap junctions, tight junctions (or zonula occludens), and desmosomes. These membrane complexes collectively contribute to the integrity, signaling activity, polarized functioning, and stability of the developing epithelium, therefore, they are crucial elements in blastocyst morphogenesis and viability. Moreover, particularly in the mouse model, analysis of the mechanisms of junction formation and activation, commonly regulated by the pattern of cell interactions provides insight into the timing and coordination of epithelial differentiation.