Development
Impaired plasma membrane localization of ubiquitin ligase complex underlies 3-M syndrome development
Abstract
3-M primordial dwarfism is an inherited disease characterized by severe pre- and postnatal growth retardation and by mutually exclusive mutations in three genes, CUL7, OBSL1, and CCDC8. The mechanism underlying 3-M dwarfism is not clear. We showed here that CCDC8, derived from a retrotransposon Gag protein in placental mammals, exclusively localized on the plasma membrane and was phosphorylated by CK2 and GSK3. Phosphorylation of CCDC8 resulted in its binding first with OBSL1, and then CUL7, leading to the membrane assembly of the 3-M E3 ubiquitin ligase complex. We identified LL5β, a plasma membrane protein that regulates cell migration, as a substrate of 3-M ligase. Wnt inhibition of CCDC8 phosphorylation or patient-derived mutations in 3-M genes disrupted membrane localization of the 3-M complex and accumulated LL5β. Deletion of Ccdc8 in mice impaired trophoblast migration and placental development, resulting in intrauterine growth restriction and perinatal lethality. These results identified a mechanism regulating cell migration and placental development that underlies the development of 3-M dwarfism.
Authors
Pu Wang, Feng Yan, Zhijun Li, Yanbao Yu, Scott E. Parnell, Yue Xiong
Abstract
Bronchopulmonary dysplasia (BPD) remains a major respiratory illness in extremely premature infants. The biological mechanisms leading to BPD are not fully understood, although an arrest in lung development has been implicated. The current study aimed to investigate the occurrence of autophagy in the developing mouse lung and its regulatory role in airway branching and terminal sacculi formation. We found 2 windows of epithelial autophagy activation in the developing mouse lung, both resulting from AMPK activation. Inhibition of AMPK-mediated autophagy led to reduced lung branching in vitro. Conditional deletion of beclin 1 (Becn1) in mouse lung epithelial cells (Becn1Epi-KO), either at early (E10.5) or late (E16.5) gestation, resulted in lethal respiratory distress at birth or shortly after. E10.5 Becn1Epi-KO lungs displayed reduced airway branching and sacculi formation accompanied by impaired vascularization, excessive epithelial cell death, reduced mesenchymal thinning of the interstitial walls, and delayed epithelial maturation. E16.5 Becn1Epi-KO lungs had reduced terminal air sac formation and vascularization and delayed distal epithelial differentiation, a pathology similar to that seen in infants with BPD. Taken together, our findings demonstrate that intrinsic autophagy is an important regulator of lung development and morphogenesis and may contribute to the BPD phenotype when impaired.
Authors
Behzad Yeganeh, Joyce Lee, Leonardo Ermini, Irene Lok, Cameron Ackerley, Martin Post
Abstract
Background: While the human fetal immune system defaults to a program of tolerance, there is concurrent need for protective immunity to meet the antigenic challenges encountered after birth. Activation of T cells in utero is associated with the fetal inflammatory response with broad implications for the health of the fetus and of the pregnancy. However, the characteristics of the fetal effector T cells that contribute to this process are largely unknown. Methods: We analyzed primary human fetal lymphoid and mucosal tissues and performed phenotypic, functional, and transcriptional analysis to identify T cells with pro-inflammatory potential. The frequency and function of fetal-specific effector T cells was assessed in the cord blood of infants with localized and systemic inflammatory pathologies and compared to healthy term controls. Results: We identified a transcriptionally distinct population of CD4+ T cells characterized by expression of the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF). PLZF+ CD4+ T cells were specifically enriched in the fetal intestine, possessed an effector memory phenotype, and rapidly produced pro-inflammatory cytokines. Engagement of the C-type lectin CD161 on these cells inhibited TCR-dependent production of IFNγ in a fetal-specific manner. IFNγ-producing PLZF+ CD4+ T cells were enriched in the cord blood of infants with gastroschisis, a natural model of chronic inflammation originating from the intestine, as well as in preterm birth, suggesting these cells contribute to fetal systemic immune activation. Conclusion: Our work reveals a fetal-specific program of protective immunity whose dysregulation is associated with fetal and neonatal inflammatory pathologies.
Authors
Joanna Halkias, Elze Rackaityte, Sara L. Hillman, Dvir Aran, Ventura F. Mendoza, Lucy R. Marshall, Tippi C. MacKenzie, Trevor D. Burt
Abstract
About 1% of all newborns are affected by congenital heart disease (CHD). Recent findings identify aberrantly functioning cilia as a possible source for CHD. Faulty cilia also prevent the development of proper left-right asymmetry and cause heterotaxy, the incorrect placement of visceral organs. Intriguingly, signaling cascades such as mTor that influence mitochondrial biogenesis also affect ciliogenesis, and can cause heterotaxy-like phenotypes in zebrafish. Here, we identify levels of mitochondrial function as a determinant for ciliogenesis and a cause for heterotaxy. We detected reduced mitochondrial DNA content in biopsies of heterotaxy patients. Manipulation of mitochondrial function revealed a reciprocal influence on ciliogenesis and affected cilia-dependent processes in zebrafish, human fibroblasts and Tetrahymena thermophila. Exome analysis of heterotaxy patients revealed an increased burden of rare damaging variants in mitochondria-associated genes as compared to 1000 Genome controls. Knockdown of such candidate genes caused cilia elongation and ciliopathy-like phenotypes in zebrafish, which could not be rescued by RNA encoding damaging rare variants identified in heterotaxy patients. Our findings suggest that ciliogenesis is coupled to the abundance and function of mitochondria. Our data further reveal disturbed mitochondrial function as an underlying cause for heterotaxy-linked CHD and provide a mechanism for unexplained phenotypes of mitochondrial disease.
Authors
Martin D. Burkhalter, Arthi Sridhar, Pedro Sampaio, Raquel Jacinto, Martina S. Burczyk, Cornelia Donow, Max Angenendt, Competence Network for Congenital Heart Defects Investigators, Maja Hempel, Paul Walther, Petra Pennekamp, Heymut Omran, Susana S. Lopes, Stephanie M. Ware, Melanie Philipp
Abstract
Ciliopathies are clinically overlapping genetic disorders involving structural and functional abnormalities of cilia. Currently, there are no small-molecule drugs available to treat ciliary defects in ciliopathies. Our phenotype-based screen identified the flavonoid eupatilin and its analogs as lead compounds for developing ciliopathy medication. CEP290, a gene mutated in several ciliopathies, encodes a protein that forms a complex with NPHP5 to support the function of the ciliary transition zone. Eupatilin relieved ciliogenesis and ciliary receptor delivery defects resulting from deletion of CEP290. In rd16 mice harboring a blinding Cep290 in-frame deletion, eupatilin treatment improved both opsin transport to the photoreceptor outer segment and electrophysiological responses of the retina to light stimulation. The rescue effect was due to eupatilin-mediated inhibition of calmodulin binding to NPHP5, which promoted NPHP5 recruitment to the ciliary base. Our results suggest that deficiency of a ciliopathy protein could be mitigated by small-molecule compounds that target other ciliary components that interact with the ciliopathy protein.
Authors
Yong Joon Kim, Sungsoo Kim, Yooju Jung, Eunji Jung, Ho Jeong Kwon, Joon Kim
Abstract
Genome-wide association studies have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers, and promoted loss of distal alveoli and airspace enlargement of over twenty percent compared to controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts is conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets, and disruption of fibroblast identity can alter the alveolar stem cell niche leading to emphysematous changes in the murine lung.
Authors
Chaoqun Wang, Nabora S. Reyes de Mochel, Stephanie A. Christenson, Monica Cassandras, Rebecca Moon, Alexis N. Brumwell, Lauren E. Byrnes, Alfred Li, Yasuyuki Yokosaki, Peiying Shan, Julie B. Sneddon, David Jablons, Patty J. Lee, Michael A. Matthay, Harold A. Chapman, Tien Peng
Abstract
SEC24 family members are components of the coat protein complex II (COPII) machinery that interact directly with cargo or with other adapters to ensure proper sorting of secretory cargo into COPII vesicles. SEC24C is 1 of 4 mammalian SEC24 paralogs (SEC24A–D), which segregate into 2 subfamilies on the basis of sequence homology (SEC24A/SEC24B and SEC24C/SEC24D). Here, we demonstrate that postmitotic neurons, unlike professional secretory cells in other tissues, are exquisitely sensitive to loss of SEC24C. Conditional KO of Sec24c in neural progenitors during embryogenesis caused perinatal mortality and microcephaly, with activation of the unfolded protein response and apoptotic cell death of postmitotic neurons in the murine cerebral cortex. The cell-autonomous function of SEC24C in postmitotic neurons was further highlighted by the loss of cell viability caused by disrupting Sec24c expression in forebrain neurons of mice postnatally and in differentiated neurons derived from human induced pluripotent stem cells. The neuronal cell death associated with Sec24c deficiency was rescued in knockin mice expressing Sec24d in place of Sec24c. These data suggest that SEC24C is a major cargo adapter for COPII-dependent transport in postmitotic neurons in developing and adult brains and that its functions overlap at least partially with those of SEC24D in mammals.
Authors
Bo Wang, Joung Hyuck Joo, Rebecca Mount, Brett J. W. Teubner, Alison Krenzer, Amber L. Ward, Viraj P. Ichhaporia, Elizabeth J. Adams, Rami Khoriaty, Samuel T. Peters, Shondra M. Pruett-Miller, Stanislav S. Zakharenko, David Ginsburg, Mondira Kundu
Abstract
Although it has been reported that hypoxia inducible factor 2 α (Hif2a), a major transcriptional factor inducible by low oxygen tension, is expressed in the mouse uterus during embryo implantation, its role in pregnancy outcomes remains unclear. This study aimed to clarify functions of uterine HIF using transgenic mouse models. Mice with deletion of Hif2a in the whole uterus (Hif2a-uKO mice) showed infertility due to implantation failure. Supplementation with progesterone (P4) and leukemia inhibitory factor (LIF) restored decidual growth arrest and aberrant position of implantation sites in Hif2a-uKO mice, respectively, but did not rescue pregnancy failure. Histological analyses in Hif2a-uKO mice revealed persistence of the intact luminal epithelium, which blocked direct contact between stroma and embryo, inactivation of PI3K-AKT pathway (embryonic survival signal), and failed embryo invasion. Mice with stromal deletion of Hif2a (Hif2a-sKO mice) showed infertility with impaired embryo invasion and those with epithelial deletion of Hif2a (Hif2a-eKO mice) showed normal fertility, suggesting the importance of stromal HIF2α in embryo invasion. This was reflected in reduced expression of membrane type 2 metalloproteinase (MT2-MMP), lysyl oxidase (LOX), VEGF, and adrenomedullin (ADM) in Hif2a-uKO stroma at the attachment site, suggesting that stromal HIF2α regulates these mediators to support blastocyst invasion. These findings provide new insight that stromal HIF2α allows trophoblast invasion through detachment of the luminal epithelium and activation of an embryonic survival signal.
Authors
Leona Matsumoto, Yasushi Hirota, Tomoko Saito-Fujita, Norihiko Takeda, Tomoki Tanaka, Takehiro Hiraoka, Shun Akaeda, Hidetoshi Fujita, Ryoko Shimizu-Hirota, Shota Igaue, Mitsunori Matsuo, Hirofumi Haraguchi, Mayuko Saito-Kanatani, Tomoyuki Fujii, Yutaka Osuga
Abstract
The transcription factor GATA6 has been shown to be important for lung development and branching morphogenesis in mouse models, but its role in human lung development is largely unknown. Here, we studied the role of GATA6 during lung differentiation using human pluripotent stem cells. We found that the human stem cell lines most efficient at generating NKX2.1+ lung progenitors express lower endogenous levels of GATA6 during endoderm patterning and that knockdown of GATA6 during endoderm patterning increased the generation of these cells. Complete ablation of GATA6 resulted in the generation of lung progenitors displaying increased cell proliferation with up to a 15-fold expansion compared with control cells, whereas the null cell line displayed a defect in further development into mature lung cell types. Furthermore, transgenic expression of GATA6 at the endoderm anteriorization stage skewed development toward a liver fate at the expense of lung progenitors. Our results suggest a critical dosage effect of GATA6 during human endoderm patterning and a later requirement during terminal lung differentiation. These studies offer an approach of modulating GATA6 expression to enhance the production of lung progenitors from human stem cell sources.
Authors
Chia-Min Liao, Somdutta Mukherjee, Amita Tiyaboonchai, Jean Ann Maguire, Fabian L. Cardenas-Diaz, Deborah L. French, Paul Gadue
Abstract
Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond–Blackfan anaemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anaemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor (RNH1) is an ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 to E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knock down in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.
Authors
Vijaykumar Chennupati, Diogo F.T. Veiga, Kendle M. Maslowski, Nicola Andina, Aubry Tardivel, Eric Chi-Wang Yu, Martina Stilinovic, Cedric Simillion, Michel A. Duchosal, Manfredo Quadroni, Irene Roberts, Vijay G. Sankaran, H. Robson MacDonald, Nicolas Fasel, Anne Angelillo-Scherrer, Pascal Schneider, Trang Hoang, Ramanjaneyulu Allam
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Copyright © 2020 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)