Integrin α8 Is Abundant in Human, Rat, and Mouse Trophoblasts

Objective: Integrins exert regulatory functions in placentogenesis. Null mutation of certain integrin α subunits leads to placental defects with subsequent fetal growth restriction or embryonic lethality in mice. So far, the placental role of α8 integrin remains to be determined. Methods: Localization of α8 integrin and its ligands, fibronectin (FN) and osteopontin (OPN), was studied by immunohistochemistry in human, rat, and mouse placenta. The vascularization of the placental labyrinth layer of α8 integrin-deficient mice was determined by CD31 staining. In humans, α8 integrin expression was assessed via real-time polymerase chain reaction in healthy placentas, in the placental pathologies such as intrauterine growth restriction (IUGR), preeclampsia, and HELLP-syndrome (hemolysis, elevated liver enzymes, low platelet count), as well as in primary extravillous trophoblasts (EVT) and villous trophoblasts. Results: In humans, α8 integrin was detected in first and third trimester syncytiotrophoblast and EVT. Although OPN showed the same localization, FN was observed in EVT only. No expressional changes in α8 integrin were detected in the placental pathologies studied. Rodent placenta showed α8 integrin expression in giant cells and in the labyrinth layer. The localization of OPN and FN, however, showed species-specific differences. Knockout of α8 integrin in mice did not cause IUGR, despite some reduction in labyrinth layer vascularization. Conclusion: α8 Integrin is expressed in functional placental compartments among its ligands, OPN and/or FN, across species. Although this may point to a regulatory role in trophoblast function, our data from α8 integrin-deficient mice indicated only mild placental pathology. Thus, the lack of placental α8 integrin seems to be largely compensated for

Biallelic variants in PCDHGC4 cause a novel neurodevelopmental syndrome with progressive microcephaly, seizures, and joint anomalies.

PURPOSE: We aimed to define a novel autosomal recessive neurodevelopmental disorder, characterize its clinical features, and identify the underlying genetic cause for this condition. METHODS: We performed a detailed clinical characterization of 19 individuals from nine unrelated, consanguineous families with a neurodevelopmental disorder. We used genome/exome sequencing approaches, linkage and cosegregation analyses to identify disease-causing variants, and we performed three-dimensional molecular in silico analysis to predict causality of variants where applicable. RESULTS: In all affected individuals who presented with a neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability we identified biallelic disease-causing variants in Protocadherin-gamma-C4 (PCDHGC4). Five variants were predicted to induce premature protein truncation leading to a loss of PCDHGC4 function. The three detected missense variants were located in extracellular cadherin (EC) domains EC5 and EC6 of PCDHGC4, and in silico analysis of the affected residues showed that two of these substitutions were predicted to influence the Ca2+-binding affinity, which is essential for multimerization of the protein, whereas the third missense variant directly influenced the cis-dimerization interface of PCDHGC4. CONCLUSION: We show that biallelic variants in PCDHGC4 are causing a novel autosomal recessive neurodevelopmental disorder and link PCDHGC4 as a member of the clustered PCDH family to a Mendelian disorder in humans

Integrin α8 Is Abundant in Human, Rat, and Mouse Trophoblasts

Objective: Integrins exert regulatory functions in placentogenesis. Null mutation of certain integrin α subunits leads to placental defects with subsequent fetal growth restriction or embryonic lethality in mice. So far, the placental role of α8 integrin remains to be determined. Methods: Localization of α8 integrin and its ligands, fibronectin (FN) and osteopontin (OPN), was studied by immunohistochemistry in human, rat, and mouse placenta. The vascularization of the placental labyrinth layer of α8 integrin-deficient mice was determined by CD31 staining. In humans, α8 integrin expression was assessed via real-time polymerase chain reaction in healthy placentas, in the placental pathologies such as intrauterine growth restriction (IUGR), preeclampsia, and HELLP-syndrome (hemolysis, elevated liver enzymes, low platelet count), as well as in primary extravillous trophoblasts (EVT) and villous trophoblasts. Results: In humans, α8 integrin was detected in first and third trimester syncytiotrophoblast and EVT. Although OPN showed the same localization, FN was observed in EVT only. No expressional changes in α8 integrin were detected in the placental pathologies studied. Rodent placenta showed α8 integrin expression in giant cells and in the labyrinth layer. The localization of OPN and FN, however, showed species-specific differences. Knockout of α8 integrin in mice did not cause IUGR, despite some reduction in labyrinth layer vascularization. Conclusion: α8 Integrin is expressed in functional placental compartments among its ligands, OPN and/or FN, across species. Although this may point to a regulatory role in trophoblast function, our data from α8 integrin-deficient mice indicated only mild placental pathology. Thus, the lack of placental α8 integrin seems to be largely compensated for

Cullin 7 and Fbxw 8 expression in trophoblastic cells is regulated via oxygen tension: implications for intrauterine growth restriction?

Objective:The F-box protein Fbxw8 is a cofactor of Cullin 7 (Cul7), which regulates protein transfer to the proteasome and cell growth. Cul7 or Fbxw8 deficiency is associated with intrauterine growth restriction (IUGR) due to abnormal placental development leading to poor oxygen supply to the fetus. We studied the role of hypoxia for Fbxw8 and Cul7 expression in trophoblastic cells. Methods: Immunomagnetic bead-separated extravillous trophoblast (EVT) and villous trophoblast (VT) and trophoblast cell lines were incubated with 1 or 8% O-2. Fbxw8 and Cul7 expression was determined in IUGR versus matched control placentas. Results: Fbxw8 was expressed uniformly in trophoblasts, whereas Cul7 expression was most prominent in trophoblast cell lines. Hypoxia reduced expression of Cul7 and Fbxw8 in all trophoblastic cells, except for villous trophoblasts. In vivo, Cul7 and Fbxw8 were detected in syncytiotrophoblast cells, VT, and EVT cells. Although no significant changes in expression levels of Fbxw8 or Cul7 were noted in IUGR compared with control placentas, Fbxw8 expression correlated negatively with gestational age in the control, but not in the IUGR group. Conclusion: Fbxw8 and Cul7 expression reveals a complex regulation in trophoblastic cells. Our findings suggest that dysregulation of Cul7 and Fbxw8 expression might affect trophoblast turnover in IUGR

Detection of Expressional Changes Induced by Intrauterine Growth Restriction in the Developing Rat Mammary Gland via Exploratory Pathways Analysis

<div><p>Background</p><p>Intrauterine growth restriction (IUGR) is thought to lead to fetal programming that in turn contributes to developmental changes of many organs postnatally. There is evidence that IUGR is a risk factor for the development of metabolic and cardiovascular disease later in life. A higher incidence of breast cancer was also observed after IUGR. This could be due to changes in mammary gland developmental pathways. We sought to characterise IUGR-induced alterations of the complex pathways of mammary development at the level of the transcriptome in a rat model of IUGR, using pathways analysis bioinformatics.</p><p>Methodology/Principal Findings</p><p>We analysed the mammary glands of Wistar rats with IUGR induced by maternal low protein (LP) diet at the beginning (d21) and the end (d28) of pubertal ductal morphogenesis. Mammary glands of the LP group were smaller in size at d28, however did not show morphologic changes. We identified multiple differentially expressed genes in the mammary gland using Agilent SurePrint arrays at d21 and d28. In silico analysis was carried out using Ingenuity Pathways Analysis. In mammary gland tissue of LP rats at d21 of life a prominent upregulation of WT1 and CDKN1A (p21) expression was observed. Differentially regulated genes were associated with the extracellular regulated kinase (ERK)-1/-2 pathway. Western Blot analysis showed reduced levels of phosphorylated ERK-1/-2 in the mammary glands of the LP group at d21. To identify possible changes in circulating steroid levels, serum LC-Tandem mass-spectrometry was performed. LP rats showed higher serum progesterone levels and an increased corticosterone/dehydrocorticosterone-ratio at d28.</p><p>Conclusions/Significance</p><p>Our data obtained from gene array analysis support the hypothesis that IUGR influences pubertal development of the rat mammary gland. We identified prominent differential regulation of genes and pathways for factors regulating cell cycle and growth. Moreover, we detected new pathways which appear to be programmed by IUGR.</p></div

IPA gene network analysis: Top differentially regulated genes at day 21 and day 28 associated with mammary gland (patho-)physiology.

<p>IPA gene network analysis: Top differentially regulated genes at day 21 and day 28 associated with mammary gland (patho-)physiology.</p

Analysis of extracellular signal-regulated kinase (ERK) -1 and -2 activities via Western blot.

<p>(A) Tissue lysates from lumbar mammary glands at day 21 and day 28 were probed with an antibody recognizing phosphorylated (activated) and total (phosphorylated and non-phosphorylated) ERK1 (also known as p44, upper band 44kDa) and ERK2 (also known as p42, lower band 42kDa). Amidoblack (Ambl) served as control. (B+C) Densitometric analysis presented as the ratio of activated ERK-1/-2 to total ERK at day 21 (B) and day 28 (C). LP =  low protein (white bars); NP =  normal protein (black bars); d =  day; * =  p<0.05, ** =  p<0.01.</p

Analysis of ductal morphogenesis.

<p>A+B) Exemplary transmitted light microscope images of lumbar mammary gland whole mount preparations at day 28 of LP (A) and NP (B). Magnification is indicated by the black bar (10 mm). The percentage of lumbar mammary fat pad occupied by ducto-alveolar structures (“area proportion” in %, C), as well as the area (mm²) occupied by ducto-alveolar structures itself (D) were examined at day 21 (LP n = 13, NP n = 18) and day 28 (LP n = 12, NP n = 10). Furthermore the rate of proliferation was determined immunohistochemically in terminal end buds (TEB) via PCNA-stain (E, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100504#pone.0100504.s001" target="_blank">Figure S1</a>), with LP d21 (n = 6), LP d28 (n = 10), and NP d21 (n = 7), NP d28 (n = 9). LP =  low protein (white bars); NP =  normal protein (black bars); d =  day; * =  p<0.05, ** =  p<0.01; *** =  p<0.001; ns =  not significant.</p

BioVenn diagram analysis of functional gene clusters: Displayed are the results of the comparative analysis of differentially regulated genes using IPA Ingenuity software and consecutive Venn diagram transformation.

<p>The upper section lists differentially regulated genes of the generic networks mammary gland development, reactive oxygen species (ROS), insulin resistance and early mammary adenocarcinoma at day 21 and day 28 as heatmaps. The lower section displays overlapping genes of these generic networks as Venn Diagrams at day 21 (on the left) and day 28 (on the right). The quantity of regulated genes of each network cluster is indicated in the respective small Venn diagrams by number. In the upper section, common genes that are differentially regulated at both day 21 and day 28 are represented by the amount of overlap of the circles and listed in the small table next to the diagram. In contrast, the tables in the lower section of this figure display overlapping genes of three selected pathways as indicated. Red =  up-regulated gene; green =  down-regulated gene; white =  fold-change value of 0.</p