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encodes a transcription factor that is anchored in the endoplasmic reticulum (ER) and activated during the unfolded protein response (UPR) to protect cells from ER stress. Deletion of the isoform activating transcription factor 6α (ATF6α) and its paralog ATF6β results in embryonic lethality and notochord dysgenesis in nonhuman vertebrates, and loss-of-function mutations in ATF6α are associated with malformed neuroretina and congenital vision loss in humans. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated this hypothesis using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a small-molecule ATF6 agonist and, conversely, inhibited ATF6 using induced pluripotent stem cells from patients with mutations. We found that ATF6 suppressed pluripotency, enhanced differentiation, and unexpectedly directed mesodermal cell fate. Our findings reveal a role for ATF6 during differentiation and identify a new strategy to generate mesodermal tissues through the modulation of the ATF6 arm of the UPR.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
In the immature lung, inflammation and injury disrupt the epithelial-mesenchymal interactions required for normal development. Innate immune signaling and NF-κB activation disrupt the normal expression of multiple mesenchymal genes that play a key role in airway branching and alveolar formation. To test the role of the NF-κB pathway specifically in lung mesenchyme, we utilized the mesenchymal Twist2-Cre to drive expression of a constitutively active inhibitor of NF-κB kinase subunit β (IKKβca) mutant in developing mice. Embryonic Twist2-IKKβca mice were generated in expected numbers and appeared grossly normal. Airway branching also appeared normal in Twist2-IKKβca embryos, with airway morphometry, elastin staining, and saccular branching similar to those in control littermates. While Twist2-IKKβca lungs did not contain increased levels of Il1b, we did measure an increased expression of the chemokine-encoding gene Ccl2. Twist2-IKKβca lungs had increased staining for the vascular marker platelet endothelial cell adhesion molecule 1. In addition, type I alveolar epithelial differentiation appeared to be diminished in Twist2-IKKβca lungs. The normal airway branching and lack of Il1b expression may have been due to the inability of the Twist2-IKKβca transgene to induce inflammasome activity. While Twist2-IKKβca lungs had an increased number of macrophages, inflammasome expression remained restricted to macrophages without evidence of spontaneous inflammasome activity. These results emphasize the importance of cellular niche in considering how inflammatory signaling influences fetal lung development.
Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). With the use of Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. With the use of an experimental coculture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of three-dimensional structures. Both LPS and tyrosine kinase inhibition reduced three-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme.
Copyright © 2017 the American Physiological Society.
BACKGROUND - Cleft palate occurs in up to 1:1,000 live births and is associated with mutations in multiple genes. Palatogenesis involves a complex choreography of palatal shelf elongation, elevation, and fusion. Transforming growth factor β (TGFβ) and bone morphogenetic protein 2 (BMP2) canonical signaling is required during each stage of palate development. The type III TGFβ receptor (TGFβR3) binds all three TGFβ ligands and BMP2, but its contribution to palatogenesis is unknown.
RESULTS - The role of TGFβR3 during palate formation was found to be during palatal shelf elongation and elevation. Tgfbr3(-) (/) (-) embryos displayed reduced palatal shelf width and height, changes in proliferation and apoptosis, and reduced vascular and osteoblast differentiation. Abnormal vascular plexus organization as well as aberrant expression of arterial (Notch1, Alk1), venous (EphB4), and lymphatic (Lyve1) markers was also observed. Decreased osteoblast differentiation factors (Runx2, alk phos, osteocalcin, col1A1, and col1A2) demonstrated poor mesenchymal cell commitment to the osteoblast lineage within the maxilla and palatal shelves in Tgfbr3(-) (/) (-) embryos. Additionally, in vitro bone mineralization induced by osteogenic medium (OM+BMP2) was insufficient in Tgfbr3(-) (/) (-) palatal mesenchyme, but mineralization was rescued by overexpression of TGFβR3.
CONCLUSIONS - These data reveal a critical, previously unrecognized role for TGFβR3 in vascular and osteoblast development during palatogenesis.
© 2014 Wiley Periodicals, Inc.
Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.
Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Maxillary hypoplasia occurs due to insufficient maxillary intramembranous ossification, leading to poor dental occlusion, respiratory obstruction and cosmetic deformities. Conditional deletion of Jagged1 (Jag1) in cranial neural crest (CNC) cells using Wnt1-cre; Jagged1(f/f) (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using μCT evaluation. Jag1CKO maxillas revealed altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification. In vitro bone cultures on Jag1CKO mouse embryonic maxillary mesenchymal (MEMM) cells demonstrated decreased mineralization that was also associated with diminished induction of osteoblast determinants. BMP receptor expression was dysregulated in the Jag1CKO MEMM cells suggesting that these cells were unable to respond to BMP-induced differentiation. JAG1-Fc rescued in vitro mineralization and osteoblast gene expression changes. These data suggest that JAG1 signaling in CNC-derived MEMM cells is required for osteoblast development and differentiation during maxillary ossification.
Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
The neural crest is a multipotent stem cell population that arises from the dorsal aspect of the neural tube and generates both non-ectomesenchymal (melanocytes, peripheral neurons and glia) and ectomesenchymal (skeletogenic, odontogenic, cartilaginous and connective tissue) derivatives. In amniotes, only cranial neural crest generates both classes, with trunk neural crest restricted to non-ectomesenchyme. By contrast, it has been suggested that anamniotes might generate derivatives of both classes at all axial levels, with trunk neural crest generating fin osteoblasts, scale mineral-forming cells and connective tissue cells; however, this has not been fully tested. The cause and evolutionary significance of this cranial/trunk dichotomy, and its absence in anamniotes, are debated. Recent experiments have disputed the contribution of fish trunk neural crest to fin osteoblasts and scale mineral-forming cells. This prompted us to test the contribution of anamniote trunk neural crest to fin connective tissue cells. Using genetics-based lineage tracing in zebrafish, we find that these fin mesenchyme cells derive entirely from the mesoderm and that neural crest makes no contribution. Furthermore, contrary to previous suggestions, larval fin mesenchyme cells do not generate the skeletogenic cells of the adult fin, but persist to form fibroblasts associated with adult fin rays. Our data demonstrate that zebrafish trunk neural crest does not generate ectomesenchymal derivatives and challenge long-held ideas about trunk neural crest fate. These findings have important implications for the ontogeny and evolution of the neural crest.
Sonic hedgehog (Shh) signal, mediated by the Gli family of transcription factors, plays an essential role in the growth and patterning of the limb. Through analysis of the early limb bud transcriptome, we identified a posteriorly-enriched gene, Hyaluronic Acid Synthase 2 (Has2), which encodes a key enzyme for the synthesis of hyaluronan (HA), as a direct target of Gli transcriptional regulation during early mouse limb development. Has2 expression in the limb bud is lost in Shh null and expanded anteriorly in Gli3 mutants. We identified an ∼3kb Has2 promoter fragment that contains two strong Gli-binding consensus sequences, and mutation of either site abrogated the ability of Gli1 to activate Has2 promoter in a cell-based assay. Additionally, this promoter fragment is sufficient to direct expression of a reporter gene in the posterior limb mesenchyme. Chromatin immunoprecipitation of DNA-Gli3 protein complexes from limb buds indicated that Gli3 strongly binds to the Has2 promoter region, suggesting that Has2 is a direct transcriptional target of the Shh signaling pathway. We also showed that Has2 conditional mutant (Has2cko) hindlimbs display digit-specific patterning defects with longitudinally shifted phalangeal joints and impaired chondrogenesis. Has2cko limbs show less capacity for mesenchymal condensation with mislocalized distributions of chondroitin sulfate proteoglycans (CSPGs), aggrecan and link protein. Has2cko limb phenotype displays striking resemblance to mutants with defective chondroitin sulfation suggesting tight developmental control of HA on CSPG function. Together, our study identifies Has2 as a novel downstream target of Shh signaling required for joint patterning and chondrogenesis.
Copyright © 2013 Elsevier Inc. All rights reserved.
Hindgut-derived endoderm can differentiate into rectal, prostatic, and bladder phenotypes. Stromal-epithelial interactions are crucial for this development; however, the precise mechanisms by which epithelium responds to stromal cues remain unknown. We have previously reported ectopic expression of peroxisome proliferator-activated receptor-γ2 (PPARγ2) increased androgen receptor expression and promoted differentiation of mouse prostate epithelium. PPARγ is also implicated in urothelial differentiation. Herein we demonstrate that knockdown of PPARγ2 in benign human prostate epithelial cells (BHPrEs) promotes urothelial transdifferentiation. Furthermore, in vitro and in vivo heterotypic tissue regeneration models with embryonic bladder mesenchyme promoted urothelial differentiation of PPARγ2-deficient BHPrE cells, and deficiency of both PPARγ isoforms 1 and 2 arrested differentiation. Because PTEN deficiency is cooperative in urothelial pathogenesis, we engineered BHPrE cells with combined knockdown of PPARγ and PTEN and performed heterotypic recombination experiments using embryonic bladder mesenchyme. Whereas PTEN deficiency alone induced latent squamous differentiation in BHPrE cells, combined PPARγ and PTEN deficiency accelerated the development of keratinizing squamous metaplasia (KSM). We further confirmed via immunohistochemistry that gene expression changes in metaplastic recombinants reflected human urothelium undergoing KSM. In summary, these data suggest that PPARγ isoform expression provides a molecular basis for observations that adult human epithelium can be transdifferentiated on the basis of heterotypic mesenchymal induction. These data also implicate PPARγ and PTEN inactivation in the development of KSM.
Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
BACKGROUND - To generate the mature intestine, splanchnic mesoderm diversifies into six different tissue layers each with multiple cell types through concurrent and complex morphogenetic events. Hindering the progress of research in the field is the lack of a detailed description of the fundamental morphological changes that constitute development of the intestinal mesoderm.
RESULTS - We used immunofluorescence and morphometric analyses of wild-type and Tg(tie1:H2B-eYFP) quail embryos to establish a comprehensive timeline of mesodermal development in the avian intestine. The following landmark features were analyzed from appearance of the intestinal primordium through generation of the definitive structure: radial compartment formation, basement membrane dynamics, mesothelial differentiation, mesenchymal expansion and growth patterns, smooth muscle differentiation, and maturation of the vasculature. In this way, structural relationships between mesodermal components were identified over time.
CONCLUSIONS - This integrated analysis presents a roadmap for investigators and clinicians to evaluate diverse experimental data obtained at individual stages of intestinal development within the longitudinal context of intestinal morphogenesis.
Copyright © 2012 Wiley Periodicals, Inc.