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The mechanisms regulating endothelial cell response to hemodynamic forces required for heart valve development, especially valve remodeling, remain elusive. Tie1, an endothelial specific receptor tyrosine kinase, is up-regulated by oscillating shear stress and is required for lymphatic valve development. In this study, we demonstrate that valvular endothelial Tie1 is differentially expressed in a dynamic pattern predicted by disturbed flow during valve remodeling. Following valvular endocardial specific deletion of Tie1 in mice, we observed enlarged aortic valve leaflets, decreased valve stiffness and valvular insufficiency. Valve abnormalities were only detected in late gestation and early postnatal mutant animals and worsened with age. The mutant mice developed perturbed extracellular matrix (ECM) deposition and remodeling characterized by increased glycosaminoglycan and decreased collagen content, as well as increased valve interstitial cell expression of Sox9, a transcription factor essential for normal ECM maturation during heart valve development. This study provides the first evidence that Tie1 is involved in modulation of late valve remodeling and suggests that an important Tie1-Sox9 signaling axis exists through which disturbed flows are converted by endocardial cells to paracrine Sox9 signals to modulate normal matrix remodeling of the aortic valve.
Copyright © 2019. Published by Elsevier Inc.
The endocardium interacts with the myocardium to promote proliferation and morphogenesis during the later stages of heart development. However, the role of the endocardium in early cardiac ontogeny remains under-explored. Given the shared origin, subsequent juxtaposition, and essential cell-cell interactions of endocardial and myocardial cells throughout heart development, we hypothesized that paracrine signaling from the endocardium to the myocardium is crucial for initiating early differentiation of myocardial cells. To test this, we generated an , endocardial-specific ablation model using the diphtheria toxin receptor under the regulatory elements of the genomic locus (). Early treatment of mouse embryoid bodies with diphtheria toxin efficiently ablated endocardial cells, which significantly attenuated the percentage of beating EBs in culture and expression of early and late myocardial differentiation markers. The addition of Bmp2 during endocardial ablation partially rescued myocyte differentiation, maturation and function. Therefore, we conclude that early stages of myocardial differentiation rely on endocardial paracrine signaling mediated in part by Bmp2. Our findings provide novel insight into early endocardial-myocardial interactions that can be explored to promote early myocardial development and growth.
© 2019. Published by The Company of Biologists Ltd.
In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet cell types: α, β, δ, and γ; when and how the Neurog3 cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3 cells co-expressing Myt1 (i.e., Myt1Neurog3) were biased toward β cell fate, while those not simultaneously expressing Myt1 (Myt1Neurog3) favored α fate. Myt1 manipulation only marginally affected α versus β cell specification, suggesting Myt1 as a marker but not determinant for islet-cell-type specification. The Myt1Neurog3 cells displayed higher Dnmt1 expression and enhancer methylation at Arx, an α-fate-promoting gene. Inhibiting Dnmts in pancreatic progenitors promoted α cell specification, while Dnmt1 overexpression or Arx enhancer hypermethylation favored β cell production. Moreover, the pancreatic progenitors contained distinct Arx enhancer methylation states without transcriptionally definable sub-populations, a phenotype independent of Neurog3 activity. These data suggest that Neurog3-independent methylation on fate-determining gene enhancers specifies distinct endocrine-cell programs.
Published by Elsevier Inc.
During mouse pancreas organogenesis, endocrine cells are born from progenitors residing in an epithelial plexus niche. After a period in a lineage-primed state, progenitors become endocrine committed via upregulation of We find that the to transition is associated with distinct stages of an epithelial egression process: narrowing the apical surface of the cell, basalward cell movement and eventual cell-rear detachment from the apical lumen surface to allow clustering as nascent islets under the basement membrane. Apical narrowing, basalward movement and transcriptional upregulation still occur without Neurog3 protein, suggesting that morphogenetic cues deployed within the plexus initiate endocrine commitment upstream or independently of Neurog3. Neurog3 is required for cell-rear detachment and complete endocrine-cell birth. The ROCK-nmMyoII pathway coordinates epithelial-cell morphogenesis and the progression through -expressing states. NmMyoII is necessary for apical narrowing, basalward cell displacement and upregulation, but all three are limited by ROCK activity. We propose that ROCK-nmMyoII activity, gene-dose and Notch signaling integrate endocrine fate allocation with epithelial plexus growth and morphogenesis, representing a feedback control circuit that coordinates morphogenesis with lineage diversification in the endocrine-birth niche.
© 2018. Published by The Company of Biologists Ltd.
The transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and onecut1 (Oc1) are coexpressed in multipotent pancreatic progenitors (MPCs), but their expression patterns diverge in hormone-expressing cells, with Oc1 expression being extinguished in the endocrine lineage and Pdx1 being maintained at high levels in β-cells. We previously demonstrated that cooperative function of these two factors in MPCs is necessary for proper specification and differentiation of pancreatic endocrine cells. In those studies, we observed a persistent decrease in expression of the β-cell maturity factor MafA. We therefore hypothesized that Pdx1 and Oc1 cooperativity in MPCs impacts postnatal β-cell maturation and function. Here our model of Pdx1-Oc1 double heterozygosity was used to investigate the impact of haploinsufficiency for both of these factors on postnatal β-cell maturation, function, and adaptability. Examining mice at postnatal day (P) 14, we observed alterations in pancreatic insulin content in both Pdx1 heterozygotes and double heterozygotes. Gene expression analysis at this age revealed significantly decreased expression of many genes important for glucose-stimulated insulin secretion (e.g., Glut2, Pcsk1/2, Abcc8) exclusively in double heterozygotes. Analysis of P14 islets revealed an increase in the number of mixed islets in double heterozygotes. We predicted that double-heterozygous β-cells would have an impaired ability to respond to stress. Indeed, we observed that β-cell proliferation fails to increase in double heterozygotes in response to either high-fat diet or placental lactogen. We thus report here the importance of cooperation between regulatory factors early in development for postnatal islet maturation and adaptability.
The optic neuroepithelial continuum of vertebrate eye develops into three differentially growing compartments: the retina, the ciliary margin (CM), and the retinal pigment epithelium (RPE). Neurofibromin 2 (Nf2) is strongly expressed in slowly expanding RPE and CM compartments, and the loss of mouse Nf2 causes hyperplasia in these compartments, replicating the ocular abnormalities seen in human NF2 patients. The hyperplastic ocular phenotypes were largely suppressed by heterozygous deletion of Yap and Taz, key targets of the Nf2-Hippo signaling pathway. We also found that, in addition to feedback transcriptional regulation of Nf2 by Yap/Taz in the CM, activation of Nf2 expression by Mitf in the RPE and suppression by Sox2 in retinal progenitor cells are necessary for the differential growth of the corresponding cell populations. Together, our findings reveal that Nf2 is a key player that orchestrates the differential growth of optic neuroepithelial compartments during vertebrate eye development.
Copyright © 2017 Elsevier Inc. All rights reserved.
Apicobasal polarity is known to affect epithelial morphogenesis and cell differentiation, but it remains unknown how these processes are mechanistically orchestrated. We find that ligand-specific EGFR signalling via PI(3)K and Rac1 autonomously modulates apicobasal polarity to enforce the sequential control of morphogenesis and cell differentiation. Initially, EGF controls pancreatic tubulogenesis by negatively regulating apical polarity induction. Subsequently, betacellulin, working via inhibition of atypical protein kinase C (aPKC), causes apical domain constriction within neurogenin3 endocrine progenitors, which results in reduced Notch signalling, increased neurogenin3 expression, and β-cell differentiation. Notably, the ligand-specific EGFR output is not driven at the ligand level, but seems to have evolved in response to stage-specific epithelial influences. The EGFR-mediated control of β-cell differentiation via apical polarity is also conserved in human neurogenin3 cells. We provide insight into how ligand-specific EGFR signalling coordinates epithelial morphogenesis and cell differentiation via apical polarity dynamics.
Cerebellar growth and foliation require the Hedgehog-driven proliferation of granule cell precursors (GCPs) in the external granule layer (EGL). However, that increased or extended GCP proliferation generally does not elicit ectopic folds suggests that additional determinants control cortical expansion and foliation during cerebellar development. Here, we find that genetic loss of the serine-threonine kinase Liver Kinase B1 (Lkb1) in GCPs increased cerebellar cortical size and foliation independent of changes in proliferation or Hedgehog signaling. This finding is unexpected given that Lkb1 has previously shown to be critical for Hedgehog pathway activation in cultured cells. Consistent with unchanged proliferation rate of GCPs, the cortical expansion of Lkb1 mutants is accompanied by thinning of the EGL. The plane of cell division, which has been implicated in diverse processes from epithelial surface expansions to gyrification of the human cortex, remains unchanged in the mutants when compared to wild-type controls. However, we find that Lkb1 mutants display delayed radial migration of post-mitotic GCPs that coincides with increased cortical size, suggesting that aberrant cell migration may contribute to the cortical expansion and increase foliation. Taken together, our results reveal an important role for Lkb1 in regulating cerebellar cortical size and foliation in a Hedgehog-independent manner.
Copyright © 2017 Elsevier Inc. All rights reserved.
Spatiotemporal balancing of cellular proliferation and differentiation is crucial for postnatal tissue homoeostasis and organogenesis. During embryonic development, pancreatic progenitors simultaneously proliferate and differentiate into the endocrine, ductal and acinar lineages. Using in vivo clonal analysis in the founder population of the pancreas here we reveal highly heterogeneous contribution of single progenitors to organ formation. While some progenitors are bona fide multipotent and contribute progeny to all major pancreatic cell lineages, we also identify numerous unipotent endocrine and ducto-endocrine bipotent clones. Single-cell transcriptional profiling at E9.5 reveals that endocrine-committed cells are molecularly distinct, whereas multipotent and bipotent progenitors do not exhibit different expression profiles. Clone size and composition support a probabilistic model of cell fate allocation and in silico simulations predict a transient wave of acinar differentiation around E11.5, while endocrine differentiation is proportionally decreased. Increased proliferative capacity of outer progenitors is further proposed to impact clonal expansion.
Three-dimensional tissue-structural relationships are not well captured by typical thin-section histology, posing challenges for the study of tissue physiology and pathology. Moreover, while recent progress has been made with intact methods for clearing, labeling, and imaging whole organs such as the mature brain, these approaches are generally unsuitable for soft, irregular, and heterogeneous tissues that account for the vast majority of clinical samples and biopsies. Here we develop a biphasic hydrogel methodology, which along with automated analysis, provides for high-throughput quantitative volumetric interrogation of spatially-irregular and friable tissue structures. We validate and apply this approach in the examination of a variety of developing and diseased tissues, with specific focus on the dynamics of normal and pathological pancreatic innervation and development, including in clinical samples. Quantitative advantages of the intact-tissue approach were demonstrated compared to conventional thin-section histology, pointing to broad applications in both research and clinical settings.