Epithelial-mesenchymal Interactions Research Articles

Notch Signaling: Linking Embryonic Lung Development and Asthmatic Airway Remodeling.

Lung development is mediated by assorted signaling proteins and orchestrated by complex mesenchymal-epithelial interactions. Notch signaling is an evolutionarily conserved cell-cell communication mechanism that exhibits a pivotal role in lung development. Notably, both aberrant expression and loss of regulation of Notch signaling are critically linked to the pathogenesis of various lung diseases, in particular, pulmonary fibrosis, lung cancer, pulmonary arterial hypertension, and asthmatic airway remodeling; implying that precise regulation of intensity and duration of Notch signaling is imperative for appropriate lung development. Moreover, evidence suggests that Notch signaling links embryonic lung development and asthmatic airway remodeling. Herein, we summarized all-recent advances associated with the mechanistic role of Notch signaling in lung development, consequences of aberrant expression or deletion of Notch signaling in linking early-impaired lung development and asthmatic airway remodeling, and all recently investigated potential therapeutic strategies to treat asthmatic airway remodeling.

Immunoexpression of transforming growth factor-β3 (TGF-β3) and receptor III of β transforming growth factor (TGF β-RIII) in pediatric patients with complete or incomplete non-syndromic cleft palate

ABSTRACT Introduction Transforming growth factor β3 (TGF-β3), and receptor III of transforming growth factor β (TGFβ-RIII), regulate epithelial mesenchymal interactions. The lack of TGF-β3 and TGFβ-RIII expression causes defects in palatal fusion. Objective To observe the immunoexpression of TGF-β3 and TGFβ-RIII in pediatric patients with non-syndromic complete or incomplete cleft palate (CP). Material and methods The study design was observational, descriptive, prospective and transversal. Samples were taken from 20 complete and incomplete CP patients from five to 28 months of age. Samples of mucoperiostium were removed from the cleft in the palate during palatoplasty. In order to perform immunohistochemistry, samples were processed with VENTANA BenchMark Ultra equipment, using TGF-β3 and TGFβ-RIII antibody. Immunoexpression was evaluated by means of global cell impression. Results TGF-β3 immunoexpression was greater in epithelial cells than in fibroblasts of patients with complete and incomplete CP; however, TGFβ-RIII immunoexpression was greater within the fibroblasts than in epithelial cells of patients with complete cleft palate. Conclusions It was concluded that the lack of TGFβ-RIII expression in the epithelium may be related to the lack of fusion of the palatal shelves. It would be of great interest to perform a more in-depth analysis of the expressions of TGF-β3 and TGFβ-RIII within different cell populations.

Human lung branching morphogenesis is orchestrated by the spatiotemporal distribution of ACTA2, SOX2, and SOX9.

Lung morphogenesis relies on a number of important processes, including proximal-distal patterning, cell proliferation, migration and differentiation, as well as epithelial-mesenchymal interactions. In mouse lung development, SOX2+ cells are localized in the proximal epithelium, whereas SOX9+ cells are present in the distal epithelium. We show that, in human lung, expression of these transcription factors differs, in that during the pseudoglandular stage distal epithelial progenitors at the tips coexpress SOX2 and SOX9. This double-positive population was no longer present by the canalicular stages of development. As in mouse, the human proximal epithelial progenitors express solely SOX2 and are surrounded by smooth muscle cells (SMCs) both in the proximal airways and at the epithelial clefts. Upon Ras-related C3 botulinum toxin substrate 1 inhibition, we noted decreased branching, as well as increased SMC differentiation, attenuated peristalsis, and a reduction in the distal double-positive SOX2/SOX9 progenitor cell population. Thus, the presence of SOX2/SOX9 double-positive progenitor cells in the distal epithelium during the pseudoglandular stage of human lung development appears to be critical to proximal-distal patterning and lung branching. Moreover, SMCs promote a SOX2 proximal phenotype and seem to suppress the SOX9+ population.

Activity of msx-1 type genes during craniofacial development

Summary: MSX-1 gene is a member of the MSX homeoboxgene family and it has different functions during the organogenesis process. This homeobox gene isexpressed in sites where epithelium-mesenchyme interactions are required. It also seems to have animportant activity controlling dental and craniofacial development, as investigated in human andmurine models. The following article reviews severasl studies in order to identify MSX.1relationships with dental and craniofacial development. Key words: Odontogenesis. MSX-1.Craniofacial development. Cleft palate. Tooth agenesis.

IKKβ Activation in the Fetal Lung Mesenchyme Alters Lung Vascular Development but Not Airway Morphogenesis

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 inconsidering how inflammatory signaling influences fetal lung development.

Engineering human cell spheroids to model embryonic tissue fusion in vitro.

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.

255 Bone morphogenetic proteins are critical factors for hair follicle epithelial–mesenchymal interactions in androgenetic alopecia

255 Bone morphogenetic proteins are critical factors for hair follicle epithelial–mesenchymal interactions in androgenetic alopecia

Hairless controls hair fate decision via Wnt/β–catenin signaling

The hairless (Hr) gene plays a central role in the hair cycle, considering that mutations in the gene result in hair loss with the exception of a few vibrissae after the first hair growth cycle in both mice and humans. This study examinedthe uncommon phenotype and using microarray analyses and functional studies, we found that β-catenin was mediated by Hr. Progenitor keratinocytes from the bulge region differentiate into both epidermis and sebaceous glands, and fail to adopt the hair keratinocytes fate in the mutant scalp, due to the decreased Wnt/β-catenin signaling in the absence of the hairless protein. This may be attributed to the dysfunction of normal epithelial-mesenchymal interactions in the hair follicle (HF).

Exosomes Mediate Epithelium-Mesenchyme Crosstalk in Organ Development.

Organ development requires complex signaling by cells in different tissues. Epithelium and mesenchyme interactions are crucial for the development of skin, hair follicles, kidney, lungs, prostate, major glands, and teeth. Despite myriad literature on cell-cell interactions and ligand-receptor binding, the roles of extracellular vesicles in epithelium-mesenchyme interactions during organogenesis are poorly understood. Here, we discovered that ∼100 nm exosomes were secreted by the epithelium and mesenchyme of a developing tooth organ and diffused through the basement membrane. Exosomes were entocytosed by epithelium or mesenchyme cells with preference by reciprocal cells rather than self-uptake. Exosomes reciprocally evoked cell differentiation and matrix synthesis: epithelium exosomes induce mesenchyme cells to produce dentin sialoprotein and undergo mineralization, whereas mesenchyme exosomes induce epithelium cells to produce basement membrane components, ameloblastin and amelogenenin. Attenuated exosomal secretion by Rab27a/b knockdown or GW4869 disrupted the basement membrane and reduced enamel and dentin production in organ culture and reduced matrix synthesis and the size of the cervical loop, which harbors epithelium stem cells, in Rab27aash/ash mutant mice. We then profiled exosomal constituents including miRNAs and peptides and further crossed all epithelium exosomal miRNAs with literature-known miRNA Wnt regulators. Epithelium exosome-derived miR135a activated Wnt/β-catenin signaling and escalated mesenchymal production of dentin matrix proteins, partially reversible by Antago-miR135a attenuation. Our results suggest that exosomes may mediate epithelium-mesenchyme crosstalk in organ development, suggesting that these vesicles and/or the molecular contents they are transporting may be interventional targets for treatment of diseases or regeneration of tissues.

Wound-Healing Studies in Cornea and Skin: Parallels, Differences and Opportunities.

The cornea and the skin are both organs that provide the outer barrier of the body. Both tissues have developed intrinsic mechanisms that protect the organism from a wide range of external threats, but at the same time also enable rapid restoration of tissue integrity and organ-specific function. The easy accessibility makes the skin an attractive model system to study tissue damage and repair. Findings from skin research have contributed to unravelling novel fundamental principles in regenerative biology and the repair of other epithelial-mesenchymal tissues, such as the cornea. Following barrier disruption, the influx of inflammatory cells, myofibroblast differentiation, extracellular matrix synthesis and scar formation present parallel repair mechanisms in cornea and skin wound healing. Yet, capillary sprouting, while pivotal in proper skin wound healing, is a process that is rather associated with pathological repair of the cornea. Understanding the parallels and differences of the cellular and molecular networks that coordinate the wound healing response in skin and cornea are likely of mutual importance for both organs with regard to the development of regenerative therapies and understanding of the disease pathologies that affect epithelial-mesenchymal interactions. Here, we review the principal events in corneal wound healing and the mechanisms to restore corneal transparency and barrier function. We also refer to skin repair mechanisms and their potential implications for regenerative processes in the cornea.

Tongue morphogenesis through epithelial-mesenchymal interaction in mouse embryos

Tongue morphogenesis through epithelial-mesenchymal interaction in mouse embryos

Quiescent adult stem cells in murine teeth are regulated by Shh signaling.

The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed inthe enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.

Dissection and Culture of Mouse Embryonic Kidney.

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant. One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant. One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

Tooth development is initiated by epithelial-mesenchymal interactions via basement membrane (BM) and growth factors. In the present study, we found that nephronectin (Npnt), a component of the BM, is highly expressed in the developing tooth. Npnt localizes in the BM on the buccal side of the tooth germ and shows an expression pattern opposite that of the dental epithelial stem cell marker Sox2. To identify the roles of Npnt during tooth development, we performed knockdown and overexpression experiments using ex vivo organ and dental epithelial cell cultures. Our findings showed that loss of Npnt induced ectopic Sox2-positive cells and reduced tooth germ size. Over expression of Npnt showed increased proliferation, whereas the number of Sox2-positive cells was decreased in dental epithelial cells. Npnt contains 5 EGF-like repeat domains, as well as an RGD sequence and MAM domain. We found that the EGF-like repeats are critical for Sox2 expression and cell proliferation. Furthermore, Npnt activated the EGF receptor (EGFR) via the EGF-like repeat domains and induced the PI3K-Akt signaling pathway. Our results indicate that Npnt plays a critical scaffold role in dental epithelial stem cell differentiation and proliferation, and regulates Sox2 expression during tooth development.

Shh… Sweat gland in progress!

Shh… Sweat gland in progress!

The innate immune response in fetal lung mesenchymal cells targets VEGFR2 expression and activity.

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 Vegfr2 Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express Vegfr2 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.

Foxc1 and Foxc2 in the Neural Crest Are Required for Ocular Anterior Segment Development.

PurposeThe large Forkhead (Fox) transcription factor family has essential roles in development, and mutations cause a wide range of ocular and nonocular disease. One member, Foxc2 is expressed in neural crest (NC)-derived periocular mesenchymal cells of the developing murine eye; however, its precise role in the development, establishment, and maintenance of the ocular surface has yet to be investigated.MethodsTo specifically delete Foxc2 from NC-derived cells, conditional knockout mice for Foxc2 (NC-Foxc2−/−) were generated by crossing Foxc2F mice with Wnt1-Cre mice. Similarly, we also generated compound NC-specific mutations of Foxc2 and a closely related gene, Foxc1 (NC-Foxc1−/−;NC-Foxc2−/−) in mice.ResultsNeural crest-Foxc2−/− mice show abnormal thickness in the peripheral-to-central corneal stroma and limbus and displaced pupils with irregular iris. The neural crest-specific mutation in Foxc2 also leads to ectopic neovascularization in the cornea, as well as impaired ocular epithelial cell identity and corneal conjunctivalization. Compound, NC-specific Foxc1; Foxc2 homozygous mutant mice have more severe defects in structures of the ocular surface, such as the cornea and eyelids, accompanied by significant declines in the expression of another key developmental factor, Pitx2, and its downstream effector Dkk2, which antagonizes canonical Wnt signaling.ConclusionsThe neural crest-Foxc2 mutation is associated with corneal conjunctivalization, ectopic corneal neovascularization, and disrupted ocular epithelial cell identity. Furthermore, Foxc2 and Foxc1 cooperatively function in NC-derived mesenchymal cells to ensure proper morphogenesis of the ocular surface via the regulation of Wnt signaling. Together, Foxc2 is required in the NC lineage for mesenchymal-epithelial interactions in corneal and ocular surface development.

Abstract P4-04-01: A paradigm shift for insulin-like growth factor-1 receptor function in triple negative breast cancers

Abstract Loss of insulin growth factor (IGF) signaling in the mammary gland at puberty leads to delayed elongation and defects in terminal end bud (TEB) proliferation. Moreover, IGF signaling is essential for epithelial mesenchymal interactions in mammary bud morphogenesis. We have generated and characterized a mouse line containing both MMTV-Wnt1 and MMTV-dnIGF-1R (kinase dead-IGF-1R) transgenes (referred to subsequently as bigenic mice) that leads to increased mammary tumor initiation and metastasis1. Recent analyses of TCGA human datasets support the relevance of our model in human triple negative breast cancers (TNBCs) that have reduced IGF-1R expression and increased expression of Wnt1 and the Frizzled co-receptor Lrp6. We have identified alterations in a number of target genes that potentially regulate metastasis in the bigenic primary tumors including increases in interleukin-6 (IL-6), Nanog, Slug, and decreases in E-cadherin expression as assessed by Qiagen specific qPCR and single gene qPCR. Furthermore, a subset of anti-inflammatory chemokines and cytokines were assessed by a targeted qPCR array revealing increases in interleukin-4, interleukin-10, and C-C motif chemokine ligand-2 and decreases in tumor necrosis factor-alpha and interleukin-1 in the bigenic tumors suggesting a tumor associated macrophage phenotype. By flow cytometry analysis, the ratio of CD4+ T cells to CD8+ T cells is increased in the bigenic tumors suggesting a tumor promoting immune microenvironment. However, increased expression of IL-6 in the bigenic tumors is specific to the luminal tumor cells, and inhibition of IGF-1R in the MCF7 human breast cancer cell line results in increases in both IL-6 and Slug expression suggesting the tumor cells activate EMT and a pro-tumorigenic microenvironment in response to decreased IGF-1R. Taken together, our data suggest IGF-1R acts as a negative regulator of mediators that promote invasion and metastasis in TNBCs.

Functional Salivary Gland Regenerative Therapy for Oral Health

Salivary glands maintain oral health and homeostasis via functional interactions with many organs, including the teeth and the tongue. Dysfunction of salivary glands causes many problems, such as dental caries, bacterial infection, and swallowing dysfunction. Current regenerative therapy for salivary gland tissue repair and whole salivary gland replacement is currently a novel therapeutic concept that may result in full recovery of salivary gland function. The salivary glands arise from reciprocal epithelial and mesenchymal interactions. We developed a novel three-dimensional cell manipulation method that can reproduce organogenesis via the epithelial-mesenchymal interaction. The bioengineered salivary glands develop correct structure and successfully secrete saliva into the oral cavity via reestablishment of the afferent-efferent neural network. The bioengineered salivary glands also improve dry mouth symptoms, such as bacterial infections and swallowing dysfunction. This review summarizes recent findings and technological advances in salivary gland regenerative therapy.