4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation

• Text
• PDF

Abstract

Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atrium^m58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress–mediated Notch activation localizes to the endocardium. Deleting endocardium via the cloche^sk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress–activated Notch signaling to modulate trabeculation.

Authors

Juhyun Lee, Peng Fei, René R. Sevag Packard, Hanul Kang, Hao Xu, Kyung In Baek, Nelson Jen, Junjie Chen, Hilary Yen, C.-C. Jay Kuo, Neil C. Chi, Chih-Ming Ho, Rongsong Li, Tzung K. Hsiai

A collagen VI–dependent pathogenic mechanism for Hirschsprung’s disease

• Text
• PDF

Abstract

Hirschsprung’s disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR — named Holstein — that contains an untargeted transgenic insertion upstream of the collagen-6α4 (Col6a4) gene. This insertion induces eNCC-specific upregulation of Col6a4 expression that increases total collagen VI protein levels in the extracellular matrix (ECM) surrounding both the developing and the postnatal ENS. Increased collagen VI levels during development mainly result in slower migration of eNCCs. This appears to be due to the fact that collagen VI is a poor substratum for supporting eNCC migration and can even interfere with the migration-promoting effects of fibronectin. Importantly, for a majority of patients in a HSCR cohort, the myenteric ganglia from the ganglionated region are also specifically surrounded by abundant collagen VI microfibrils, an outcome accentuated by Down syndrome. Collectively, our data thus unveil a clinically relevant pathogenic mechanism for HSCR that involves cell-autonomous changes in ECM composition surrounding eNCCs. Moreover, as COL6A1 and COL6A2 are on human Chr.21q, this mechanism is highly relevant to the predisposition of patients with Down syndrome to HSCR.

Authors

Rodolphe Soret, Mathilde Mennetrey, Karl F. Bergeron, Anne Dariel, Michel Neunlist, Franziska Grunder, Christophe Faure, David W. Silversides, Nicolas Pilon, for the Ente-Hirsch study group

Lymph flow regulates collecting lymphatic vessel maturation in vivo

• Text
• PDF

Abstract

Fluid shear forces have established roles in blood vascular development and function, but whether such forces similarly influence the low-flow lymphatic system is unknown. It has been difficult to test the contribution of fluid forces in vivo because mechanical or genetic perturbations that alter flow often have direct effects on vessel growth. Here, we investigated the functional role of flow in lymphatic vessel development using mice deficient for the platelet-specific receptor C-type lectin–like receptor 2 (CLEC2) as blood backfills the lymphatic network and blocks lymph flow in these animals. CLEC2-deficient animals exhibited normal growth of the primary mesenteric lymphatic plexus but failed to form valves in these vessels or remodel them into a structured, hierarchical network. Smooth muscle cell coverage (SMC coverage) of CLEC2-deficient lymphatic vessels was both premature and excessive, a phenotype identical to that observed with loss of the lymphatic endothelial transcription factor FOXC2. In vitro evaluation of lymphatic endothelial cells (LECs) revealed that low, reversing shear stress is sufficient to induce expression of genes required for lymphatic valve development and identified GATA2 as an upstream transcriptional regulator of FOXC2 and the lymphatic valve genetic program. These studies reveal that lymph flow initiates and regulates many of the key steps in collecting lymphatic vessel maturation and development.

Authors

Daniel T. Sweet, Juan M. Jiménez, Jeremy Chang, Paul R. Hess, Patricia Mericko-Ishizuka, Jianxin Fu, Lijun Xia, Peter F. Davies, Mark L. Kahn

GATA2 is required for lymphatic vessel valve development and maintenance

• Text
• PDF

Abstract

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.

Authors

Jan Kazenwadel, Kelly L. Betterman, Chan-Eng Chong, Philippa H. Stokes, Young K. Lee, Genevieve A. Secker, Yan Agalarov, Cansaran Saygili Demir, David M. Lawrence, Drew L. Sutton, Sebastien P. Tabruyn, Naoyuki Miura, Marjo Salminen, Tatiana V. Petrova, Jacqueline M. Matthews, Christopher N. Hahn, Hamish S. Scott, Natasha L. Harvey

Transcription factor ISL1 is essential for pacemaker development and function

• Text
• PDF

Abstract

The sinoatrial node (SAN) maintains a rhythmic heartbeat; therefore, a better understanding of factors that drive SAN development and function is crucial to generation of potential therapies, such as biological pacemakers, for sinus arrhythmias. Here, we determined that the LIM homeodomain transcription factor ISL1 plays a key role in survival, proliferation, and function of pacemaker cells throughout development. Analysis of several Isl1 mutant mouse lines, including animals harboring an SAN-specific Isl1 deletion, revealed that ISL1 within SAN is a requirement for early embryonic viability. RNA-sequencing (RNA-seq) analyses of FACS-purified cells from ISL1-deficient SANs revealed that a number of genes critical for SAN function, including those encoding transcription factors and ion channels, were downstream of ISL1. Chromatin immunoprecipitation assays performed with anti-ISL1 antibodies and chromatin extracts from FACS-purified SAN cells demonstrated that ISL1 directly binds genomic regions within several genes required for normal pacemaker function, including subunits of the L-type calcium channel, Ank2, and Tbx3. Other genes implicated in abnormal heart rhythm in humans were also direct ISL1 targets. Together, our results demonstrate that ISL1 regulates approximately one-third of SAN-specific genes, indicate that a combination of ISL1 and other SAN transcription factors could be utilized to generate pacemaker cells, and suggest ISL1 mutations may underlie sick sinus syndrome.

Authors

Xingqun Liang, Qingquan Zhang, Paola Cattaneo, Shaowei Zhuang, Xiaohui Gong, Nathanael J. Spann, Cizhong Jiang, Xinkai Cao, Xiaodong Zhao, Xiaoli Zhang, Lei Bu, Gang Wang, H.S. Vincent Chen, Tao Zhuang, Jie Yan, Peng Geng, Lina Luo, Indroneal Banerjee, Yihan Chen, Christopher K. Glass, Alexander C. Zambon, Ju Chen, Yunfu Sun, Sylvia M. Evans

DNA methylation directs functional maturation of pancreatic β cells

• Text
• PDF

Abstract

Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to prevent hyperglycemia and inhibit insulin secretion under fasting conditions to protect against hypoglycemia. β cells lack this functional capability at birth and acquire glucose-stimulated insulin secretion (GSIS) during neonatal life. Here, we have shown that during postnatal life, the de novo DNA methyltransferase DNMT3A initiates a metabolic program by repressing key genes, thereby enabling the coupling of insulin secretion to glucose levels. In a murine model, β cell–specific deletion of Dnmt3a prevented the metabolic switch, resulting in loss of GSIS. DNMT3A bound to the promoters of the genes encoding hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) — both of which regulate the metabolic switch — and knockdown of these two key DNMT3A targets restored the GSIS response in islets from animals with β cell–specific Dnmt3a deletion. Furthermore, DNA methylation–mediated repression of glucose-secretion decoupling genes to modulate GSIS was conserved in human β cells. Together, our results reveal a role for DNA methylation to direct the acquisition of pancreatic β cell function.

Authors

Sangeeta Dhawan, Shuen-ing Tschen, Chun Zeng, Tingxia Guo, Matthias Hebrok, Aleksey Matveyenko, Anil Bhushan

Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome

• Text
• PDF

Abstract

Individuals with an inherited deficiency in gonadotropin-releasing hormone (GnRH) have impaired sexual reproduction. Previous genetic linkage studies and sequencing of plausible gene candidates have identified mutations associated with inherited GnRH deficiency, but the small number of affected families and limited success in validating candidates have impeded genetic diagnoses for most patients. Using a combination of exome sequencing and computational modeling, we have identified a shared point mutation in semaphorin 3E (SEMA3E) in 2 brothers with Kallmann syndrome (KS), which causes inherited GnRH deficiency. Recombinant wild-type SEMA3E protected maturing GnRH neurons from cell death by triggering a plexin D1–dependent (PLXND1-dependent) activation of PI3K-mediated survival signaling. In contrast, recombinant SEMA3E carrying the KS-associated mutation did not protect GnRH neurons from death. In murine models, lack of either SEMA3E or PLXND1 increased apoptosis of GnRH neurons in the developing brain, reducing innervation of the adult median eminence by GnRH-positive neurites. GnRH neuron deficiency in male mice was accompanied by impaired testes growth, a characteristic feature of KS. Together, these results identify SEMA3E as an essential gene for GnRH neuron development, uncover a neurotrophic function for SEMA3E in the developing brain, and elucidate SEMA3E/PLXND1/PI3K signaling as a mechanism that prevents GnRH neuron deficiency.

Authors

Anna Cariboni, Valentina André, Sophie Chauvet, Daniele Cassatella, Kathryn Davidson, Alessia Caramello, Alessandro Fantin, Pierre Bouloux, Fanny Mann, Christiana Ruhrberg

Taspase1-dependent TFIIA cleavage coordinates head morphogenesis by limiting Cdkn2a locus transcription

• Text
• PDF

Abstract

Head morphogenesis requires complex signal relays to enable precisely coordinated proliferation, migration, and patterning. Here, we demonstrate that, during mouse head formation, taspase1-mediated (TASP1-mediated) cleavage of the general transcription factor TFIIA ensures proper coordination of rapid cell proliferation and morphogenesis by maintaining limited transcription of the negative cell cycle regulators p16Ink4a and p19Arf from the Cdkn2a locus. In mice, loss of TASP1 function led to catastrophic craniofacial malformations that were associated with inadequate cell proliferation. Compound deficiency of Cdkn2a, especially p16Ink4a deficiency, markedly reduced the craniofacial anomalies of TASP1-deficent mice. Furthermore, evaluation of mice expressing noncleavable TASP1 targets revealed that TFIIA is the principal TASP1 substrate that orchestrates craniofacial morphogenesis. ChIP analyses determined that noncleaved TFIIA accumulates at the p16Ink4a and p19Arf promoters to drive transcription of these negative regulators. In summary, our study elucidates a regulatory circuit comprising proteolysis, transcription, and proliferation that is pivotal for construction of the mammalian head.

Authors

Shugaku Takeda, Satoru Sasagawa, Toshinao Oyama, Adam C. Searleman, Todd D. Westergard, Emily H. Cheng, James J. Hsieh

Periderm prevents pathological epithelial adhesions during embryogenesis

• Text
• PDF

Abstract

Appropriate development of stratified, squamous, keratinizing epithelia, such as the epidermis and oral epithelia, generates an outer protective permeability barrier that prevents water loss, entry of toxins, and microbial invasion. During embryogenesis, the immature ectoderm initially consists of a single layer of undifferentiated, cuboidal epithelial cells that stratifies to produce an outer layer of flattened periderm cells of unknown function. Here, we determined that periderm cells form in a distinct pattern early in embryogenesis, exhibit highly polarized expression of adhesion complexes, and are shed from the outer surface of the embryo late in development. Mice carrying loss-of-function mutations in the genes encoding IFN regulatory factor 6 (IRF6), IκB kinase-α (IKKα), and stratifin (SFN) exhibit abnormal epidermal development, and we determined that mutant animals exhibit dysfunctional periderm formation, resulting in abnormal intracellular adhesions. Furthermore, tissue from a fetus with cocoon syndrome, a lethal disorder that results from a nonsense mutation in IKKA, revealed an absence of periderm. Together, these data indicate that periderm plays a transient but fundamental role during embryogenesis by acting as a protective barrier that prevents pathological adhesion between immature, adhesion-competent epithelia. Furthermore, this study suggests that failure of periderm formation underlies a series of devastating birth defects, including popliteal pterygium syndrome, cocoon syndrome, and Bartsocas-Papas syndrome.

Authors

Rebecca J. Richardson, Nigel L. Hammond, Pierre A. Coulombe, Carola Saloranta, Heidi O. Nousiainen, Riitta Salonen, Andrew Berry, Neil Hanley, Denis Headon, Riitta Karikoski, Michael J. Dixon

TSHZ1-dependent gene regulation is essential for olfactory bulb development and olfaction

• Text
• PDF

Abstract

The olfactory bulb (OB) receives odor information from the olfactory epithelium and relays this to the olfactory cortex. Using a mouse model, we found that development and maturation of OB interneurons depends on the zinc finger homeodomain factor teashirt zinc finger family member 1 (TSHZ1). In mice lacking TSHZ1, neuroblasts exhibited a normal tangential migration to the OB; however, upon arrival to the OB, the neuroblasts were distributed aberrantly within the radial dimension, and many immature neuroblasts failed to exit the rostral migratory stream. Conditional deletion of Tshz1 in mice resulted in OB hypoplasia and severe olfactory deficits. We therefore investigated olfaction in human subjects from families with congenital aural atresia that were heterozygous for TSHZ1 loss-of-function mutations. These individuals displayed hyposmia, which is characterized by impaired odor discrimination and reduced olfactory sensitivity. Microarray analysis, in situ hybridization, and ChIP revealed that TSHZ1 bound to and regulated expression of the gene encoding prokineticin receptor 2 (PROKR2), a G protein–coupled receptor essential for OB development. Mutations in PROKR2 lead to Kallmann syndrome, characterized by anosmia and hypogonadotrophic hypogonadism. Our data indicate that TSHZ1 is a key regulator of mammalian OB development and function and controls the expression of molecules involved in human Kallmann syndrome.

Authors

Daniela Ragancokova, Elena Rocca, Anne M.M. Oonk, Herbert Schulz, Elvira Rohde, Jan Bednarsch, Ilse Feenstra, Ronald J.E. Pennings, Hagen Wende, Alistair N. Garratt