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Intestinal smooth muscle differentiation is a complex physico-biological process involving several different pathways. Here, we investigate the properties of Ca2+ waves in the developing intestinal mesenchyme using GCamp6f expressing mouse embryos and investigate their relationship with smooth muscle differentiation. We find that Ca2+ waves are absent in the pre-differentiation mesenchyme and start propagating immediately following α-SMA expression. Ca2+ waves are abrogated by CaV1.2 and gap-junction blockers, but are independent of the Rho pathway. The myosine light-chain kinase inhibitor ML-7 strongly disorganized or abolished Ca2+ waves, showing that perturbation of the contractile machinery at the myosine level also affected the upstream Ca2+ handling chain. Inhibiting Ca2+ waves and contractility with CaV1.2 blockers did not perturb circular smooth muscle differentiation at early stages. At later stages, CaV1.2 blockers abolished intestinal elongation and differentiation of the longitudinal smooth muscle, leading instead to the emergence of KIT-expressing interstitial cells of Cajal at the gut periphery. CaV1.2 blockers also drove apoptosis of already differentiated, CaV1.2-expressing smooth muscle and enteric neural cells. We provide fundamental new data on Ca2+ waves in the developing murine gut and their relation to myogenesis in this organ. Calcium waves underpin smooth muscle contractions in the gut. The authors examine their developmental dynamics, molecular properties and their impact on differentiation of smooth muscle and interstitial cells of Cajal in the mouse embryo. [ABSTRACT FROM AUTHOR]

Background: Disruption of ALX4 causes autosomal dominant parietal foramina and autosomal recessive frontonasal dysplasia with alopecia, but the mechanisms involving ALX4 in craniofacial and other developmental processes are not well understood. Although mice carrying distinct mutations in Alx4 have been previously reported, the perinatal lethality of homozygous mutants together with dynamic patterns of Alx4 expression in multiple tissues have hindered systematic elucidation of the cellular and molecular mechanisms involving Alx4 in organogenesis and disease pathogenesis. Results: We report generation of Alx4f/f conditional mice and show that tissue‐specific Cre‐mediated inactivation of Alx4 in cranial neural crest and limb bud mesenchyme, respectively, recapitulated craniofacial and limb developmental defects as found in Alx4‐null mice but without affecting postnatal survival. While Alx4‐null mice that survive postnatally exhibited dorsal alopecia, mice lacking Alx4 function in the neural crest lineage exhibited a highly restricted region of hair loss over the anterior skull whereas mice lacking Alx4 in the cranial mesoderm lineage exhibited normal hair development, suggesting that Alx4 plays partly redundant roles in multiple cell lineages during hair follicle development. Conclusion: The Alx4f/f mice provide a valuable resource for systematic investigation of cell type‐ and stage‐specific function of ALX family transcription factors in development and disease. Key Findings: We report successful generation and analysis of Alx4 conditional mice.We demonstrate lineage‐specific requirements of Alx4 in craniofacial, limb, and hair development.Our results demonstrate that Alx4 functions in multiple lineages to control hair follicle development.The Alx4 conditional mice provide a valuable and unique resource for systematic characterization of the cellular and molecular mechanisms involving the ALX family transcription factors in development and disease. [ABSTRACT FROM AUTHOR]

Significant technological progress in the field of wireless devices that were primarily intended for military purposes, has resulted in their common manipulation by the general population. Wi-Fi, mobile phones, and other modern devices offer many advantages to their users. On the other hand, their excessive usage creates an environmental burden, also known as electrosmog. The objective of our current study was the observation of the Wi-Fi radiation effect on the histo-logical structure of the organs in the 9-day-old chicken embryo. On day 9 of incubation, the embryological material was routinely processed for preparation of hematoxylin-eosin, picrosirius red and periodic acid Schiff stained histological sections. Radiation with a frequency of 2.4 GHz and average power density of 300 µW.m−2 applied during the entire development up to the 9th embryonic day did not fundamentally affect general organogenesis. However, in the parenchyma of organs such as the liver, spleen, lungs, kidneys, and gonads, as well as in the developing mesenchyme, obvious vascular congestion of the blood vessels of different caliber was observed. Also, an increase in collagen and glycosaminoglycans production in the cartilaginous matrix and perichondrium of the future bone skeleton as well as developing connective tissue was noted. Although these morphological changes were just subtle, they point to the Wi-Fi radiation's ability to influence the histogenesis of the individual. [ABSTRACT FROM AUTHOR]

Abstract Background During pseudoglandular stage of the human lung development the primitive bronchial buds are initially conformed by simple tubules lined by endoderm-derived epithelium surrounded by mesenchyme, which will progressively branch into airways and start to form distal epithelial saculles. For first time alveolar type II (AT2) pneumocytes appears. This study aims to characterize the genes and microRNAs involved in this differentiation process and decipher its role in the starting alveolar differentiation. Methods Gene and microRNA profiling was performed in human embryonic lungs from 7 to 12 post conception weeks (pcw). Protein expression location of candidate genes were analyzed by immunofluorescense in embryonic lung tissue sections. mRNA/miRNA target pairs were identified using computational approaches and their expression was studied in purified epithelial/mesenchymal cell populations and in isolated tips and stalks from the bronchial tree. Additionally, silencing experiments in human embryonic lung mesenchymal cells and in human embryonic tip-derived lung organoids were performed, as well as organoid differentiation studies. AT2 cell markers were studied by qRT-PCR and by immunofluorescence. The TGFB-β phosphorylated pathways was analyzed with membrane protein arrays. Lung explants were cultured in air/liquid interface with/without peptides. Results We identified 88 differentially expressed genes, including IGFBP3. Although IGFBP3 mRNA was detected in both epithelial and mesenchymal populations, the protein was restricted to the epithelium, indicating post-transcriptional regulation preventing IGFBP3 protein expression in the mesenchyme. MicroRNA profiling identified miR-34a as an IGFBP3 regulator. miR-34a was up-regulated in mesenchymal cells, and its silencing in human embryonic lung mesenchymal cells increased IGFBP3 levels. Additionally, IGFBP3 expression showed a marked downregulation from 7 to 12 pcw, suggesting its involvement in the differentiation process. The differentiation of human tip-derived lung embryonic organoids showed a drastic reduction in IGFBP3, supported by the scRNAseq data. IGFBP3 silencing in organoids activated an alveolar-like differentiation process characterized by stem cell markers downregulation and upregulation of AT2 markers. This process was mediated by TGFβ signalling inhibition and BMP pathway activation. Conclusions The IGFBP3/miR-34a axis restricts IGFBP3 expression in the embryonic undifferentiated lung epithelium, and the progressive downregulation of IGFBP3 during the pseudoglandular stage is required for alveolar differentiation. Graphical abstract

Background: During pseudoglandular stage of the human lung development the primitive bronchial buds are initially conformed by simple tubules lined by endoderm-derived epithelium surrounded by mesenchyme, which will progressively branch into airways and start to form distal epithelial saculles. For first time alveolar type II (AT2) pneumocytes appears. This study aims to characterize the genes and microRNAs involved in this differentiation process and decipher its role in the starting alveolar differentiation. Methods: Gene and microRNA profiling was performed in human embryonic lungs from 7 to 12 post conception weeks (pcw). Protein expression location of candidate genes were analyzed by immunofluorescense in embryonic lung tissue sections. mRNA/miRNA target pairs were identified using computational approaches and their expression was studied in purified epithelial/mesenchymal cell populations and in isolated tips and stalks from the bronchial tree. Additionally, silencing experiments in human embryonic lung mesenchymal cells and in human embryonic tip-derived lung organoids were performed, as well as organoid differentiation studies. AT2 cell markers were studied by qRT-PCR and by immunofluorescence. The TGFB-β phosphorylated pathways was analyzed with membrane protein arrays. Lung explants were cultured in air/liquid interface with/without peptides. Results: We identified 88 differentially expressed genes, including IGFBP3. Although IGFBP3 mRNA was detected in both epithelial and mesenchymal populations, the protein was restricted to the epithelium, indicating post-transcriptional regulation preventing IGFBP3 protein expression in the mesenchyme. MicroRNA profiling identified miR-34a as an IGFBP3 regulator. miR-34a was up-regulated in mesenchymal cells, and its silencing in human embryonic lung mesenchymal cells increased IGFBP3 levels. Additionally, IGFBP3 expression showed a marked downregulation from 7 to 12 pcw, suggesting its involvement in the differentiation process. The differentiation of human tip-derived lung embryonic organoids showed a drastic reduction in IGFBP3, supported by the scRNAseq data. IGFBP3 silencing in organoids activated an alveolar-like differentiation process characterized by stem cell markers downregulation and upregulation of AT2 markers. This process was mediated by TGFβ signalling inhibition and BMP pathway activation. Conclusions: The IGFBP3/miR-34a axis restricts IGFBP3 expression in the embryonic undifferentiated lung epithelium, and the progressive downregulation of IGFBP3 during the pseudoglandular stage is required for alveolar differentiation. [ABSTRACT FROM AUTHOR]

Epithelial–mesenchymal transition (EMT) is defined as a cellular process during which epithelial cells acquire mesenchymal phenotypes and behavior following the downregulation of epithelial features. EMT and its reversed process, the mesenchymal-epithelial transition (MET), and the special form of EMT, the endothelial-mesenchymal transition (EndMT), have been considered as mainstream concepts and general rules driving developmental and pathological processes, particularly cancer. However, discrepancies and disputes over EMT and EMT research have also grown over time. EMT is defined as transition between two cellular states, but it is unanimously agreed by EMT researchers that (1) neither the epithelial and mesenchymal states nor their regulatory networks have been clearly defined, (2) no EMT markers or factors can represent universally epithelial and mesenchymal states, and thus (3) EMT cannot be assessed on the basis of one or a few EMT markers. In contrast to definition and proposed roles of EMT, loss of epithelial feature does not cause mesenchymal phenotype, and EMT does not contribute to embryonic mesenchyme and neural crest formation, the key developmental events from which the EMT concept was derived. EMT and MET, represented by change in cell shapes or adhesiveness, or symbolized by EMT factors, are biased interpretation of the overall change in cellular property and regulatory networks during development and cancer progression. Moreover, EMT and MET are consequences rather than driving factors of developmental and pathological processes. The true meaning of EMT in some developmental and pathological processes, such as fibrosis, needs re-evaluation. EMT is believed to endow malignant features, such as migration, stemness, etc., to cancer cells. However, the core property of cancer (tumorigenic) cells is neural stemness, and the core EMT factors are components of the regulatory networks of neural stemness. Thus, EMT in cancer progression is misattribution of the roles of neural stemness to the unknown mesenchymal state. Similarly, neural crest EMT is misattribution of intrinsic property of neural crest cells to the unknown mesenchymal state. Lack of basic rationale in EMT and related concepts urges re-evaluation of their significance as general rules for understanding developmental and pathological processes, and re-evaluation of their significance in scientific research. [ABSTRACT FROM AUTHOR]

Cranial sutures separate neighboring skull bones and are sites of bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Using single-cell transcriptomics, lineage tracing, and mutant analysis in zebrafish, we uncover key developmental transitions regulating bone formation at sutures during skull expansion. In particular, we identify a subpopulation of mesenchyme cells in the mid-suture region that upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. Moreover, combinatorial mutation of BMP antagonists enriched in this mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data reveal establishment of a non-osteogenic mesenchyme population in the mid-suture region that restricts bone formation through local BMP antagonism, thus ensuring proper suture morphology. Cranial sutures separate neighboring skull bones but how osteogenic activity is controlled at cranial sutures remains unclear. Here, authors employ zebrafish to uncover the cellular and transcriptional basis of growth control during suture formation. [ABSTRACT FROM AUTHOR]

The present study reports the formation of claws in lizards and their cornification. From a flat epidermis with lose mesenchyme, the skin forms undulations that become symmetric domes and later asymmetric for the elongation of their proximal part that gives rise to the outer scale surface. At the tip of digits claws develop in association with a mesenchyme that surrounds the cartilage of the last phalange. Beta‐cells of claws appear 2–3 embryonic stages before those of scales formed in other body regions, including digital scales. The epidermis of the developing claw initially forms 3–4 embryonic layers under which elongated beta‐cells accumulate long bundles of corneous material, immunopositive for CBPs. Claw beta‐keratinocytes incorporate tritiated proline and accumulate numerous, oriented corneous bundles that rapidly occupy the entire cell. The bundles are electron‐dense under transmission electron microscopy, suggesting that, in association to CBPs, other proteins are synthesized and increase the mechanical resistance of claws. Immunolabelling for cysteine‐rich IFKs, HA and HB, shows that these keratins co‐localize with CBPs only in claws but are low to absent in digital scales and adhesive setae. This indicates that the hardness of claws mainly depends from the association between these different proteins rich in glycine and cysteine. [ABSTRACT FROM AUTHOR]

Background Objectives Materials and methods Results and discussion Conclusion The epididymis is important for sperm maturation and without its proper development, male infertility will result. Biomechanical properties of tissues/organs play key roles during their morphogenesis, including the Wolffian duct. It is hypothesized that structural/bulk stiffness of the capsule and mesenchyme/extracellular matrix that surround the duct is a major biomechanical property that regulates Wolffian duct morphogenesis. These data will provide key information as to the mechanisms that regulate the development of this important organ.To measure the structural/bulk stiffness in Pascals (force/area) of the capsule and the capsule and mesenchyme together that surrounds the Wolffian duct during the development. To examine the relative membrane tension of mesenchymal cells during the Wolffian duct development. Since Ptk7 was previously shown to regulate ECM integrity and Wolffian duct elongation and coiling, the hypothesis that Ptk7 regulates structural/bulk stiffness and mesenchymal cell membrane tension was tested.Atomic force microscopy and a microsquisher compression apparatus were used to measure the structural stiffness. Biomechanical properties within the membranes of cells within the capsule and mesenchyme were examined using a membrane‐tension fluorescent probe.The structural stiffness (Pascals) of the capsule and underlying mesenchyme was relatively constant during development, with a significant increase in the capsule at the later stages. However, this increase may reflect the ECM and associated mesenchyme being close to the capsule because the coiling of the duct pushed or compressed them into that space. Keeping the capsule and mesenchyme/ECM at constant stiffness would ensure that the duct will continue to coil under similar biomechanical forces throughout the development. Cells within the capsule and mesenchyme at different Wolffian duct regions during the development had varying degrees of membrane lipid tension. It is hypothesized that the dynamic changes ensure the duct is kept at a constant stiffness regardless of any external forces. Loss of Ptk7 resulted in an increase in stiffness at E18.5, which was presumable due to the loss of integrity of the ECM within the mesenchyme.Biomechanical properties of the capsule and the mesenchyme/extracellular matrix that surround the Wolffian duct play an important role toward Wolffian duct morphogenesis, thereby allowing for the proper development of the epididymis and subsequent male fertility. [ABSTRACT FROM AUTHOR]

Our study examines the immunoexpression patterns of Megalin, Cubilin, Caveolin-1, Gipc1 and Dab2IP in the embryonic development (E) and postnatal (P) mouse kidney, with a focus on differentiating patterns between wild-type (wt) and yotari, Dab1−/− (yot) mice. Immunofluorescence revealed raised immunoexpression of receptors Megalin and Cubilin at the ampulla/collecting ducts and convoluted tubules across all developmental stages, with the most prominent immunoexpression observed in the convoluted tubules and the parietal epithelium of the Bowman's capsule. Quantitative analysis showed a higher percentage of Megalin and Cubilin in wt compared to yot mice at E13.5. Co-expression of Megalin and Cubilin was observed at the apical membrane of convoluted tubules and the parietal layer of the Bowman's capsule. The staining intensity of Megalin varied across developmental stages, with the strongest reactivity observed at the ampulla and collecting ducts at embryonic day (E) 13.5 in wt mice. In contrast, Caveolin-1 exhibited high immunoexpression in the metanephric mesenchyme, blood vessels, and the border area between the metanephric mesenchyme and renal vesicle, with a decrease in immunoexpression as development progressed. Gipc1 showed diffuse cytoplasmic staining in metanephric mesenchyme, convoluted tubules and collecting ducts, with significant differences in immunoexpression between wild-type and yot mice at both investigated embryonic time points. Dab2IP immunofluorescent staining was most prominent in renal vesicle/glomeruli and ampulla/collecting ducts at E13.5, with mild staining intensity observed in the distal convoluted tubules postnatally. Our findings elucidate distinct immunoexpression of patterns and potential parts of these proteins in the development and function of the kidney, highlighting the importance of further investigation into their regulatory mechanisms. [ABSTRACT FROM AUTHOR]

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds. [ABSTRACT FROM AUTHOR]

ABSTRACT Shiga toxin (Stx) is produced by some pathogenic strains of Escherichia coli. To study the impact of Stx on the human intestine, we utilized human intestinal organoids and human intestinal enteroids grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and the underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. The medium used for epithelial cells contains added growth factors, while the mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in the medium lacking growth factors, suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to build up in the medium over time, since daily medium changes were not as effective as medium changes performed every 3 days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using in vitro and in vitro models.IMPORTANCEThese studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing E. coli-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.

Alveologenesis, the final stage in lung development, substantially remodels the distal lung, expanding the alveolar surface area for efficient gas exchange. Secondary crest myofibroblasts (SCMF) exist transiently in the neonatal distal lung and are crucial for alveologenesis. However, the pathways that regulate SCMF function, proliferation and temporal identity remain poorly understood. To address this, we purified SCMFs from reporter mice, performed bulk RNA-seq and found dynamic changes in Hippo-signaling components during alveologenesis. We deleted the Hippo effectors Yap/Taz from Acta2-expressing cells at the onset of alveologenesis, causing a significant arrest in alveolar development. Using single cell RNA-seq, we identified a distinct cluster of cells in mutant lungs with altered expression of marker genes associated with proximal mesenchymal cell types, airway smooth muscle and alveolar duct myofibroblasts. In vitro studies confirmed that Yap/Taz regulates myofibroblastassociated gene signature and contractility. Together, our findings show that Yap/Taz is essential for maintaining functional myofibroblast identity during postnatal alveologenesis. [ABSTRACT FROM AUTHOR]

The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions.We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme. [ABSTRACT FROM AUTHOR]

The mammary gland is a unique organ that undergoes dynamic alterations throughout a female's reproductive life, making it an ideal model for developmental, stem cell and cancer biology research. Mammary gland development begins in utero and proceeds via a quiescent bud stage before the initial outgrowth and subsequent branching morphogenesis. How mammary epithelial cells transit from quiescence to an actively proliferating and branching tissue during embryogenesis and, importantly, how the branch pattern is determined remain largely unknown. Here, we provide evidence indicating that epithelial cell proliferation and onset of branching are independent processes, yet partially coordinated by the Eda signaling pathway. Through heterotypic and heterochronic epithelial-mesenchymal recombination experiments between mouse mammary and salivary gland tissues and ex vivo live imaging, we demonstrate that unlike previously concluded, the mode of branching is an intrinsic property of the mammary epithelium whereas the pace of growth and the density of ductal tree are determined by the mesenchyme. Transcriptomic profiling and ex vivo and in vivo functional studies in mice disclose that mesenchymal Wnt/ß-catenin signaling, and in particular IGF-1 downstream of it critically regulate mammary gland growth. These results underscore the general need to carefully deconstruct the different developmental processes producing branched organs. [ABSTRACT FROM AUTHOR]

Isthmin‐1 (Ism1) was first described to be syn‐expressed with Fgf8 in Xenopus. However, its biological role has not been elucidated until recent years. Despite of accumulated evidence that Ism1 participates in angiogenesis, tumor invasion, macrophage apoptosis, and glucose metabolism, the cognate receptors for Ism1 remain largely unknown. Ism1 deficiency in mice results in renal agenesis (RA) with a transient loss of Gdnf transcription and impaired mesenchyme condensation at E11.5. Ism1 binds to and activates Integrin α8β1 to positively regulate Gdnf/Ret signaling, thus promoting mesenchyme condensation and ureteric epithelium branching morphogenesis. Here, we propose the hypothesis underlying the mechanism by which Ism1 regulates branching morphogenesis during early kidney development. [ABSTRACT FROM AUTHOR]

Abstract Background Intestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture. Methods We established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies. Results The proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture. Conclusions The high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

Epithelial folding is a fundamental biological process that requires epithelial interactions with the underlying mesenchyme. In this issue of Cell , Huycke et al. investigate intestinal villus formation. They discover that water-droplet-like behavior of mesenchymal cells drives their coalescence into uniformly patterned aggregates, which generate forces on the epithelium to initiate folding. [ABSTRACT FROM AUTHOR]

Soft tissue sarcomas (STS) are a diverse group of rare solid tumors of mesenchymal cell origin with distinct clinical and pathological features. They account for less than 1% of all adult malignancies and 15% of pediatric neoplasms. They include over hundreds of different histological subtypes. Many of these subtypes can occur at any age and are not confined to a specific site. Each subtype displays variable clinical behavior. Low incidence, variable presentation, behavior, and long-term outcomes further make it challenging to treat. There are multiple ongoing trials that focus on the anatomic site and histologic subtype to tailor the treatment. Further rarity of each histotype is a major barrier to recruit patients to randomized controlled trials. A multidisciplinary approach is mandatory in all cases of soft tissue sarcomas. The purpose of this review is to thoroughly understand the existing literature on history, incidence, epidemiology, etiology, histology, pathogenesis, diagnostic modalities, prognosis, management, and post treatment surveillance of STS. Uterine sarcomas, gastrointestinal stromal tumors (GIST), and pediatric sarcomas are not included here. It briefly highlights various molecular aberrations, changes in staging as per the American Joint Committee on Cancer (AJCC) 8, drugs that are used off-label in specific subtypes of sarcoma along with the recent advances. The classification of STS is undergoing continuous evolution. A wide variety of subtypes can only be diagnosed accurately with sophisticated molecular diagnostic tests and with the involvement of expert geneticists and pathologists to interpret it. There is no clarity on tailoring the treatment of STS to date. There is always a question on how best we can incorporate chemotherapy and radiotherapy along with surgery as a part of multimodality treatment. The heterogeneity of STS has hindered the development of robust, evidence-based treatment strategies, and our therapeutic approach is neither histology-specific nor widely standardized. Increased knowledge about sarcoma biology could help to discover new and more effective treatment strategies and help overcome the therapeutic challenge imposed by this deadly disease. Continued collaboration among various sarcoma centers globally will be of prime importance to optimize STS management. This will allow studies to be both sufficiently large and reasonably focused to generate evidence that is clinically meaningful in specific STS patient populations. [ABSTRACT FROM AUTHOR]

Conditions for self-organisation via Turing's mechanism in biological systems represented by reaction-diffusion or reaction-cross-diffusion models have been extensively studied. Nonetheless, the impact of tissue stratification in such systems is under-explored, despite its ubiquity in the context of a thin epithelium overlying connective tissue, for instance the epidermis and underlying dermal mesenchyme of embryonic skin. In particular, each layer can be subject to extensively different biochemical reactions and transport processes, with chemotaxis - a special case of cross-diffusion - often present in the mesenchyme, contrasting the solely molecular transport typically found in the epidermal layer. We study Turing patterning conditions for a class of reaction-cross-diffusion systems in bilayered regions, with a thin upper layer and coupled by a linear transport law. In particular, the role of differential transport through the interface is explored together with the presence of asymmetry between the homogeneous equilibria of the two layers. A linear stability analysis is carried out around a spatially homogeneous equilibrium state in the asymptotic limit of weak and strong coupling strengths, where quantitative approximations of the bifurcation curve can be computed. Our theoretical findings, for an arbitrary number of reacting species, reveal quantitative Turing conditions, highlighting when the coupling mechanism between the layered regions can either trigger patterning or stabilize a spatially homogeneous equilibrium regardless of the independent patterning state of each layer. We support our theoretical results through direct numerical simulations, and provide an open source code to explore such systems further. [ABSTRACT FROM AUTHOR]

Wolffian duct (WD) maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in WD development is still undefined. By designing a mesenchyme-specific Ar knockout (ARcKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal WDs and cystic formation at the cranial WDs. Ex vivo culture of ARcKO WDs invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote WD survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA sequencing of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a , Wnt9b , Lama2 , and Lamc2) were associated with morphological and cellular changes in ARcKO male embryos (ie, reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in ARcKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in WD maintenance and morphogenesis in mice. [ABSTRACT FROM AUTHOR]

Background: Intestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture. Methods: We established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies. Results: The proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture. Conclusions: The high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine. [ABSTRACT FROM AUTHOR]

Aim: The study was conducted to systematically analyze the recent peer-reviewed literature related to tumoral pseudoangiomatous stromal hyperplasia (PASH) in males. Materials and methods: Articles published in peer-reviewed journals of repute from 2012 to 2023 were evaluated for eight variables: 1) the age of the patient, 2) clinical presentation, 3) laterality (unilateral or bilateral), 4) associated breast disorder, 5) the presence of systemic co-morbidity, 6) imaging modalities aiding diagnosis, 7) treatment, and 8) recurrence. Results: There were 10 patients ranging in age from 1 year to 67 years (mean: 34.5 years). In 8 cases (80%), there were breast lumps; in 2 cases (20%), there were axillary lumps at presentation. Pain was not a prominent symptom. Six patients (60%) had another breast disorder at presentation and 3 (30%) had significant systemic co-morbidities, including diabetes and renal insufficiency. The greatest dimension of the lumps ranged from 2 cm to 12 cm, with 80% of the lumps being below 5 cm in size. Imaging modalities utilized in evaluation included breast ultrasound (USG), mammograms, computerized tomography (CT), and magnetic resonance imaging (MRI). All the cases were treated surgically, and the final diagnosis was made only after the analysis of the surgical specimen. Recurrence was recorded in 2 (20%) cases. Conclusions: Tumoral PASH is a benign myofibroblastic proliferation disorder rarely reported in males. This condition should be considered a differential diagnosis of breast or axillary lumps, and an evaluation should be initiated accordingly. The recommended course of treatment is adequate surgical excision with regular clinical and radiological follow-up. [ABSTRACT FROM AUTHOR]

Heart valve disease is an important cause of morbidity and mortality among cardiac patients worldwide. However, the pathogenesis of heart valve disease is not clear, and a growing body of evidence hints at the importance of the genetic basis and developmental origins of heart valve disease. Therefore, understanding the developmental mechanisms that underlie the formation of heart valves has important implications for the diagnosis, prevention, and treatment of congenital heart disease. Endothelial to mesenchymal transition is a key step in initiating cardiac valve development. The dynamic changes in the relative localization and proportion of different cell sources in the heart valve mesenchymal population are still not fully understood. Here, we used the Cdh5-CreER;R26R-tdTomato mouse line to trace endocardial cushion-derived endothelial cells to explore the dynamic contribution of these cells to each layer of the valve during valve development. This is beneficial for elaborating on the role of endocardial cells in the process of valve remodeling from a precise angle. [ABSTRACT FROM AUTHOR]

ObjectivesThe mammalian tongue develops from the branchial arches (1-4) and comprises highly organized tissues compartmentalized by mesenchyme/connective tissue that is largely derived from neural crest (NC). This study aimed to understand the roles of tumour suppressor Neurofibromin 2 (Nf2) in NC-derived tongue mesenchyme in regulating Hippo signalling and cell proliferation for the proper development of tongue shape and size.Materials and methodsConditional knockout (cKO) of Nf2 in NC cell lineage was generated using Wnt1-Cre (Wnt1-Cre/Nf2cKO ). Nf2 expression, Hippo signalling activities, cell proliferation and tongue shape and size were thoroughly analysed in different tongue regions and tissue types of Wnt1-Cre/Nf2cKO and Cre- /Nf2fx/fx littermates at various stages (E10.5-E18.5).ResultsIn contrast to many other organs in which the Nf2/Hippo pathway activity restrains growth and cell proliferation and as a result, loss of Nf2 decreases Hippo pathway activity and promotes an enlarged organ development, here we report our observations of distinct, tongue region- and stage-specific alterations of Hippo signalling activity and cell proliferation in Nf2cKO in NC-derived tongue mesenchyme. Compared to Cre- /Nf2fx / fx littermates, Wnt1-Cre/Nf2cKO depicted a non-proportionally enlarged tongue (macroglossia) at E12.5-E13.5 and microglossia at later stages (E15.5-E18.5). Specifically, at E12.5 Nf2cKO mutants had a decreased level of Hippo signalling transcription factor Yes-associated protein (Yap), Yap target genes and cell proliferation anteriorly, while having an increased Yap, Yap target genes and cell proliferation posteriorly, which lead to a tip-pointed and posteriorly widened tongue. At E15.5, loss of Nf2 in the NC lineage resulted in distinct changes in cell proliferation in different regions, that is, high in epithelium and mesenchyme subjacent to the epithelium, and lower in deeper layers of the mesenchyme. At E18.5, cell proliferation was reduced throughout the Nf2cKO tongue.

Introduction: Lipoma is the most common benign soft tissue tumor of mesenchymal origin that may occur anywhere in the human body. This tumor is composed of mature adipose tissue without cellular atypia and is often surrounded by a fibrotic connective tissue capsule. The common locations of this tumor are the buccal mucosa, tongue, lips, gums, and floor of the mouth, in order of abundance. This study deals with a case report of angiolipoma in the anterior labial vestibule of the mandible in a 34-year-old female patient.Case Report: A 34-year-old female patient referred with a complaint of presence of a protrusion in the vestibule in front of the mandible. It was found that there is an exophytic mass, non-sessile, with healthy mucosa on the lesion, with a relatively firm consistency, and a size of 4.5×3.5×1.5 cm in the chin area and labial vestibule of the mandible in the anterior area. After cutting the lesion and exposing it, a red color mixed with yellow color, which was fat tissue, was observed. In the microscopic description, it was stated that a mature fat mass mixed with thick vessels was observed. In some areas of these vessels, there was a thrombus and mixed muscle tissue.Conclusion: Lipoma treatment is only sufficient through a surgical method, and cutting the lesion and its histopathological characteristics have no effect on the prognosis of the lesion due to the absence of pain; therefore, its existence is usually remains hidden from the patient.

Tissue interactions are essential for guiding organ development and regeneration. Hair follicle formation relies on inductive signalling between two tissues, the embryonic surface epithelium and the adjacent mesenchyme. Although previous research has highlighted the hair-inducing potential of the mesenchymal component of the hair follicle - the dermal papilla and its precursor, the dermal condensate - the source and nature of the primary inductive signal before dermal condensate formation have remained elusive. Here, we performed epithelial-mesenchymal tissue recombination experiments using hair-forming back skin and glabrous plantar skin from mouse embryos to unveil that the back skin mesenchyme is inductive even before dermal condensate formation. Moreover, the naïve, unpatterned mesenchyme was sufficient to trigger hair follicle formation even in the oral epithelium. Building on previous knowledge, we explored the hair-inductive ability of the Wnt agonist R-spondin 1 and a Bmp receptor inhibitor in embryonic skin explants. Although R-spondin 1 instigated precocious placode-specific transcriptional responses, it was insufficient for hair follicle induction, either alone or in combination with Bmp receptor inhibition. Our findings pave the way for identifying the hair follicle-inducing cue. [ABSTRACT FROM AUTHOR]

Multinucleated cells, or syncytia, are found in diverse taxa. Their biological function is often associated with the compartmentalization of biochemical or cellular activities within the syncytium. How such compartments are generated and maintained is poorly understood. The sea urchin embryonic skeleton is secreted by a syncytium, and local patterns of skeletal growth are associated with distinct subdomains of gene expression within the syncytium. For such molecular compartments to be maintained and to control local patterns of skeletal growth: (1) the mobility of TFs must be restricted to produce stable differences in the transcriptional states of nuclei within the syncytium; and (2) the mobility of biomineralization proteins must also be restricted to produce regional differences in skeletal growth. To test these predictions, we expressed fluorescently tagged forms of transcription factors and biomineralization proteins in sub-domains of the skeletogenic syncytium. We found that both classes of proteins have restricted mobility within the syncytium and identified motifs that limit their mobility. Our findings have general implications for understanding the functional and molecular compartmentalization of syncytia. [ABSTRACT FROM AUTHOR]

The transcription factor Six2 plays a crucial role in maintaining self-renewing nephron progenitor cap mesenchyme (CM) during metanephric kidney development. In mouse and human, expression at single-cell resolution has detected Six2 in cells as they leave the CM pool and differentiate. The role Six2 may play in these cells as they differentiate remains unknown. Here, we took advantage of the zebrafish pronephric kidney which forms directly from intermediate mesoderm to test six2b function during pronephric tubule development and differentiation. Expression of six2b during early zebrafish development was consistent with a role in pronephros formation. Using morpholino knock-down and CRISPR/Cas9 mutagenesis, we show a functional role for six2b in the development of proximal elements of the pronephros. By 48 h post-fertilization, six2b morphants and mutants showed disrupted pronephric tubule morphogenesis. We observed a lower-than-expected frequency of phenotypes in six2b stable genetic mutants suggesting compensation. Supporting this, we detected increased expression of six2a in six2b stable mutant embryos. To further confirm six2b function, F0 crispant embryos were analyzed and displayed similar phenotypes as morphants and stable mutants. Together our data suggests a conserved role for Six2 during nephrogenesis and a role in the morphogenesis of the proximal tubule. [ABSTRACT FROM AUTHOR]

The evolution of jaws has played a major role in the success of vertebrate expansion into a wide variety of ecological niches. A fundamental, yet unresolved, question in craniofacial biology is about the origin of the premaxilla, the most distal bone present in the upper jaw of all amniotes. Recent reports have suggested that the mammalian premaxilla is derived from embryonic maxillary prominences rather than the frontonasal ectomesenchyme as previously shown in studies of chicken embryos. However, whether mammalian embryonic frontonasal ectomesenchyme contributes to the premaxillary bone has not been investigated and a tool to trace the contributions of the frontonasal ectomesenchyme to facial structures in mammals is lacking. The expression of the Alx3 gene is activated highly specifically in the frontonasal ectomesenchyme, but not in the maxillary mesenchyme, from the beginning of facial morphogenesis in mice. Here, we report the generation and characterization of a novel Alx3 CreERT2 knock-in mouse line that express tamoxifen-inducible Cre DNA recombinase from the Alx3 locus. Tamoxifen treatment of Alx3 CreERT2/+; Rosa26 mTmG/+ embryos at E7.5, E8.5, E9.5, and E10.5, each induced specific labeling of the embryonic medial nasal and lateral nasal mesenchyme but not the maxillary mesenchyme. Lineage tracing of Alx3 CreERT2 -labeled frontonasal mesenchyme from E9.5 to E16.5 clearly showed that the frontonasal mesenchyme cells give rise to the osteoblasts generating the premaxillary bone. Furthermore, we characterize a Dlx1-Cre BAC transgenic mouse line that expresses Cre activity in the embryonic maxillary but not the frontonasal mesenchyme and show that the Dlx1-Cre labeled embryonic maxillary mesenchyme cells contribute to the maxillary bone as well as the soft tissues lateral to both the premaxillary and maxillary bones but not to the premaxillary bone. These results clearly demonstrate the developmental origin of the premaxillary bone from embryonic frontonasal ectomesenchyme cells in mice and confirm the evolutionary homology of the premaxilla across amniotes. [Display omitted] • Embryonic origin of mammalian premaxilla is controversial and not well studied. • Alx3 CreERT2 mice enable specific genetic analysis of frontonasal development. • Lineage tracing shows frontonasal mesenchyme gives rise to mouse premaxillary bone. • Results reject recently proposed unique embryonic origin of mammalian premaxilla. • This paper clarifies embryonic origins and homology of mammalian facial bones. [ABSTRACT FROM AUTHOR]

In this study, we present the first spatial transcriptomic atlas of Atlantic salmon skin using the Visium Spatial Gene Expression protocol. We utilized frozen skin tissue from 4 distinct sites, namely the operculum, pectoral and caudal fins, and scaly skin at the flank of the fish close to the lateral line, obtained from 2 Atlantic salmon (150 g). High-quality frozen tissue sections were obtained by embedding tissue in optimal cutting temperature media prior to freezing and sectioning. Further, we generated libraries and spatial transcriptomic maps, achieving a minimum of 80 million reads per sample with mapping efficiencies ranging from 79.3 to 89.4%. Our analysis revealed the detection of over 80,000 transcripts and nearly 30,000 genes in each sample. Among the tissue types observed in the skin, the epithelial tissues exhibited the highest number of transcripts (unique molecular identifier counts), followed by muscle tissue, loose and fibrous connective tissue, and bone. Notably, the widest nodes in the transcriptome network were shared among the epithelial clusters, while dermal tissues showed less consistency, which is likely attributable to the presence of multiple cell types at different body locations. Additionally, we identified collagen type 1 as the most prominent gene family in the skin, while keratins were found to be abundant in the epithelial tissue. Furthermore, we successfully identified gene markers specific to epithelial tissue, bone, and mesenchyme. To validate their expression patterns, we conducted a meta-analysis of the microarray database, which confirmed high expression levels of these markers in mucosal organs, skin, gills, and the olfactory rosette. [ABSTRACT FROM AUTHOR]

Taste papillae are specialized organs, each of which comprises an epithelial wall hosting taste buds and a core of mesenchymal tissue. In the present study, we report that during early taste papilla development in mouse embryos, bone morphogenetic protein (BMP) signaling mediated by type 1 receptor ALK3 in the tongue mesenchyme is required for epithelial Wnt/ß-catenin activity and taste papilla differentiation. Mesenchyme-specific knockout (cKO) of Alk3 using Wnt1-Cre and Sox10-Cre resulted in an absence of taste papillae at E12.0. Biochemical and cell differentiation analyses demonstrated that mesenchymal ALK3-BMP signaling governed the production of previously unappreciated secretory proteins, i.e. it suppressed those that inhibit and facilitated those that promote taste papilla differentiation. Bulk RNA-sequencing analysis revealed many more differentially expressed genes (DEGs) in the tongue epithelium than in the mesenchyme in Alk3 cKO versus control. Moreover, we detected downregulated epithelial Wnt/ß-catenin signaling and found that taste papilla development in the Alk3 cKO was rescued by the GSK3ß inhibitor LiCl, but not by Wnt3a. Our findings demonstrate for the first time the requirement of tongue mesenchyme in taste papilla cell differentiation. [ABSTRACT FROM AUTHOR]

Morphological development of the lung requires complex signal crosstalk between the mesenchymal and epithelial progenitors. Elucidating the genetic cascades underlying signal crosstalk is essential to understanding lung morphogenesis. Here, we identified Nolz1 as a mesenchymal lineage-specific transcriptional regulator that plays a key role in lung morphogenesis. Nolz1 null mutation resulted in a severe hypoplasia phenotype, including a decreased proliferation of mesenchymal cells, aberrant differentiation of epithelial cells and defective growth of epithelial branches. Nolz1 deletion also downregulated Wnt2, Lef1, Fgf10, Gli3 and Bmp4 mRNAs. Mechanistically, Nolz1 regulates lung morphogenesis primarily through Wnt2 signaling. Loss-of-function and overexpression studies demonstrated that Nolz1 transcriptionally activated Wnt2 and downstream β-catenin signaling to control mesenchymal cell proliferation and epithelial branching. Exogenous Wnt2 could rescue defective proliferation and epithelial branching in Nolz1 knockout lungs. Finally, we identified Fgf9 as an upstream regulator of Nolz1. Collectively, Fgf9-Nolz1-Wnt2 signaling represents a novel axis in the control of lung morphogenesis. These findings are relevant to lung tumorigenesis, in which a pathological function of Nolz1 is implicated. [ABSTRACT FROM AUTHOR]

The formation of Wolffian ducts (WDs) includes three highly coordinated developmental stages, specification, elongation, and maintenance, prior to sexual differentiation. After their initial formation in both sexes, the WD epithelium and mesenchyme have sexually dimorphic fates depending on the presence of androgen action and local genetic programs. Postnatal differentiation of the epithelial lineage into principal, basal, and clear cells is controlled by androgens, lumicrine, and local factors. A lineage progression for epithelial cells is established. Aberrant cell differentiation is one underlying cause of WD abnormalities and associated pathologies in both men and women. Wolffian ducts (WDs) are the paired embryonic structures that give rise to internal male reproductive tract organs. WDs are initially formed in both sexes but have sex-specific fates during sexual differentiation. Understanding WD differentiation requires insights into the process of fate decisions of epithelial and mesenchymal cells, which are tightly coordinated by endocrine, paracrine, and autocrine signals. In this review, we discuss current advances in understanding the fate-decision process of WD epithelial and mesenchymal lineages from their initial formation at the embryonic stage to postnatal differentiation. Finally, we discuss aberrant cell differentiation in WD abnormalities and pathologies and identify opportunities for future investigations. [ABSTRACT FROM AUTHOR]

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Novel genetic and epigenetic factors involved in the development and prognosis of idiopathic pulmonary fibrosis (IPF) have been identified. We previously observed that erythrocyte membrane protein band 4.1-like 3 (EPB41L3) increased in the lung fibroblasts of IPF patients. Thus, we investigated the role of EPB41L3 in IPF by comparing the EPB41L3 mRNA and protein expression of lung fibroblast between patients with IPF and controls. We also investigated the regulation of epithelial–mesenchymal transition (EMT) in an epithelial cell line (A549) and fibroblast-to-myofibroblast transition (FMT) in a fibroblast cell line (MRC5) by overexpressing and silencing EPB41L3. EPB41L3 mRNA and protein levels, as measured using RT-PCR, real-time PCR, and Western blot, were significantly higher in fibroblasts derived from 14 IPF patients than in those from 10 controls. The mRNA and protein expression of EPB41L3 was upregulated during transforming growth factor-β-induced EMT and FMT. Overexpression of EPB41L3 in A549 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of N-cadherin and COL1A1. Treatment with EPB41L3 siRNA upregulated the mRNA and protein expression of N-cadherin. Overexpression of EPB41L3 in MRC5 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of fibronectin and α-SMA. Finally, treatment with EPB41L3 siRNA upregulated the mRNA and protein expression of FN1, COL1A1, and VIM. In conclusion, these data strongly support an inhibitory effect of EPB41L3 on the process of fibrosis and suggest the therapeutic potential of EPB41L3 as an anti-fibrotic mediator. [ABSTRACT FROM AUTHOR]

While the generation of many lineages from pluripotent stem cells has resulted in basic discoveries and clinical trials, the derivation of tissue-specific mesenchyme via directed differentiation has markedly lagged. The derivation of lung-specific mesenchyme is particularly important since this tissue plays crucial roles in lung development and disease. Here we generate a mouse induced pluripotent stem cell (iPSC) line carrying a lung-specific mesenchymal reporter/lineage tracer. We identify the pathways (RA and Shh) necessary to specify lung mesenchyme and find that mouse iPSC-derived lung mesenchyme (iLM) expresses key molecular and functional features of primary developing lung mesenchyme. iLM recombined with engineered lung epithelial progenitors self-organizes into 3D organoids with juxtaposed layers of epithelium and mesenchyme. Co-culture increases yield of lung epithelial progenitors and impacts epithelial and mesenchymal differentiation programs, suggesting functional crosstalk. Our iPSC-derived population thus provides an inexhaustible source of cells for studying lung development, modeling diseases, and developing therapeutics. Mesenchymal lineages play critical roles during development, though these cells are frequently overlooked in directed differentiation models. Here they derive lung-specific mesenchyme from mouse iPSCs and generate organoids consisting of both mesenchymal and epithelial progenitors, observing functional epithelial-mesenchymal crosstalk. [ABSTRACT FROM AUTHOR]

Background: The present study attempted to find out the histological changes of parotid gland in the developing human fetuses. Materials and Methods: Parotid glands from 60 fresh fetuses of gestational weeks ranging from 12weeks to term were studied after staining with Hematoxylene & Eosin, Masson's Trichome stain, Van Gieson's stain and Verhoeff's stains. Results: The gland of the fetus at 12 weeks composed of solid epithelial cords with occasional canalization, surrounded by loose mesenchyme. Canalization of cords was completed at 25 weeks and adult picture attained at 36 weeks. Division of the glandular parenchyma into lobes and lobules by connective tissue septa started at 17 weeks. A well formed capsule was seen around the gland at 20 weeks. A gradual decrease in intra-glandular connective tissue occurred in late gestational age. Variety of cells such as fibroblasts, mesenchymal cells, fibrocytes and lymphocytes were seen. Numerous adipocytes were found within the glandular parenchyma and around the gland. Ductal tree showed gradual differentiation, presence of cilia-like surface projections from epithelia of larger ducts were seen in 14-32 week fetuses. Conclusion: When compared with various authors, some findings of the present study followed the foot-steps of previous workers whereas some were in contrary, the most important difference being the age of full maturation of the glandular architecture. One of the unique finding being the presence of cilia-like surface projections seen in the larger ducts in 14-32 week aged fetuses.

Serious intestinal side-effects that target the NOTCH-HES1 pathway in human cancer differentiation therapy make it necessary to understand the pathway at the human organ level. Herein, we endogenously introduced HES1−/− mutations into human embryonic stem cells (hESCs) and differentiated them into human intestinal organoids (HIO). The HES1−/− hESCs retained ES cell properties and showed gene expression patterns similar to those of wild-type hESCs when they differentiated into definitive endoderm and hindgut. During the formation of the HES1−/− lumen we noted an impaired development of mesenchymal cells in addition to the increased differentiation of secretory epithelium. RNA-Seq revealed that inhibited development of the mesenchymal cells may have been due to a downregulation of WNT5A signaling. Overexpression of HES1 and silencing of WNT5A in the intestinal fibroblast cell line CCD-18Co indicated that HES1 was involved in the activation of WNT5A-induced fibroblast growth and migration, suggesting the likelihood of the Notch pathway in epithelial-mesenchymal crosstalk. Our results facilitated the identification of more precise underlying molecular mechanisms displaying distinct roles in HES1 signaling in stromal and epithelial development in human intestinal mucosa. • HES1 is critical to lumen differentiation and maturation of human intestinal organoid development. • Solely disrupting HES1 was sufficient to promote the fate of secretory cells in human intestinal epithelium. • HES1 involved in subepithelial mesenchymal development by affecting WNT5A expression. [ABSTRACT FROM AUTHOR]

In tetrapods, BMP-signaling coordinates limb outgrowth, skeleton patterning, and apoptosis during the formation of their typical autopod structures, the digits. In addition, inhibition of BMP signaling during mouse limb development leads to the persistence and enlargement of an important signaling center, the apical ectodermal ridge (AER), and consequent digit defects. Interestingly, during fish fin development there is a natural elongation of the AER, rapidly converted into an apical finfold (FF), in which osteoblasts differentiate into dermal fin-rays used in aquatic locomotion. Previous reports have led us to suggest that upregulation of Hox13 genes in the distal fin mesenchyme, caused by the origin of novel enhancer modules, may have caused an increment of the BMP signaling potentially leading to the apoptosis of these osteoblasts precursors of the fin-rays. To explore this hypothesis, we characterized the expression of several components of the BMP signaling in zebrafish lines with distinct FF sizes (bmp2b , smad1 , smoc1 , smoc2, grem1a, msx1b, msx2b, Psamd1/5/9). Our data suggest that the BMP signaling is enhanced in shorter FFs and inhibited in longer FFs, as implied by the differential expression of several components of this network. In addition, we detected an earlier expression of several of these BMP-signaling components associated with the development of short FFs and the opposite tendency during the development of longer FFs. Thus, our results suggest that a heterochronic shift, involving enhanced Hox13 expression and BMP signaling may have caused the reduction of the FF size during the evolutionary transition from fish fins to tetrapod limbs. [ABSTRACT FROM AUTHOR]

The outgrowth of epithelial bud followed by reiterated bifurcations during renal development is driven by the ligand-receptor interactions between the epithelium and the surrounding mesenchyme. Here, by exploring ligand-receptor interactions in E10.5 and E11.5 kidneys by single cell RNA-seq, we find that Isthmin1 (Ism1), a secreted protein, resembles Gdnf expression and modulates kidney branching morphogenesis. Mice deficient for Ism1 exhibit defective ureteric bud bifurcation and impaired metanephric mesenchyme condensation in E11.5 embryos, attributable to the compromised Gdnf/Ret signaling, ultimately leading to renal agenesis and hypoplasia/dysplasia. By HRP-induced proximity labelling, we further identify integrin α8β1 as a receptor of Ism1 in E11.5 kidney and demonstrate that Ism1 promoted cell-cell adhesion through interacting with Integrin α8β1, the receptor whose activation is responsible for Gdnf expression and mesenchyme condensation. Taken together, our work reveals Ism1 as a critical regulator of cell-cell interaction that modulates Gdnf/Ret signaling during early kidney development. Loss of Ism1 in mice results in kidney agenesis and dysplasia that are common human diseases. Here they show that Ism1 is expressed in metanephric mesenchyme and acts as a ligand of Integrin α8β1 to regulate mesenchyme condensation during early renal branching morphogenesis. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor (FGF) signaling is known to play an important role in lung organogenesis. However, we recently demonstrated that FGF10 fails to induce branching in human fetal lungs as is observed in mouse. Our previous human fetal lung RNA sequencing data exhibited increased FGF18 during the pseudoglandular stage of development, suggestive of its importance in human lung branching morphogenesis. Whereas it has been previously reported that FGF18 is critical during alveologenesis, few studies have described its implication in lung branching, specifically in human. Therefore, we aimed to determine the role of FGF18 in human lung branching morphogenesis. Human fetal lung explants within the pseudoglandular stage of development were treated with recombinant human FGF18 in air-liquid interface culture. Explants were analyzed grossly to assess differences in branching pattern, as well as at the cellular and molecular levels. FGF18 treatment promoted branching in explant cultures and demonstrated increased epithelial proliferation as well as maintenance of the double positive SOX2/SOX9 distal bud progenitor cells, confirming its role in human lung branching morphogenesis. In addition, FGF18 treated explants displayed increased expression of SOX9, FN1, and COL2A1 within the mesenchyme, all factors that are important to chondrocyte differentiation. In humans, cartilaginous airways extend deep into the lung up to the 12th generation of branching whereas in mouse these are restricted to the trachea and main bronchi. Therefore, our data suggest that FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells. [ABSTRACT FROM AUTHOR]

Background: Frem1 has been linked to human face shape variation, dysmorphology, and malformation, but little is known about its regulation and biological role in facial development. Results: During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Finally, we found that FREM1 is sufficient to induce cNCC proliferation in a concentration‐dependent manner and that Shh pathway antagonism reduces Frem1 expression during pathogenesis of midfacial hypoplasia. Conclusions: By demonstrating that the Shh signaling pathway regulates Frem1 expression in cNCCs, these findings provide novel insight into the mechanisms underlying variation in midfacial morphogenesis. Key Findings: The Sonic Hedgehog signaling pathway regulates Frem1 expression in cranial neural crest cells.Frem1 is expressed in expansive gradients in the cranial neural crest mesenchyme of the embryonic growth centers that form the midface.FREM1 promotes cranial neural crest cell proliferation in a concentration‐dependent manner.Sonic Hedgehog pathway antagonism reduces Frem1 expression during the pathogenesis of midfacial hypoplasia [ABSTRACT FROM AUTHOR]

Of all the cell types arising from the neural crest, ectomesenchyme is likely the most unusual. In contrast to the neuroglial cells generated by neural crest throughout the embryo, consistent with its ectodermal origin, cranial neural crest-derived cells (CNCCs) generate many connective tissue and skeletal cell types in common with mesoderm. Whether this ectoderm-derived mesenchyme (ectomesenchyme) potential reflects a distinct developmental origin from other CNCC lineages, and/or epigenetic reprogramming of the ectoderm, remains debated. Whereas decades of lineage tracing studies have defined the potential of CNCC ectomesenchyme, these are being revisited by modern genetic techniques. Recent work is also shedding light on the extent to which intrinsic and extrinsic cues determine ectomesenchyme potential, and whether maintenance or reacquisition of CNCC multipotency influences craniofacial repair. • Cranial neural crest provides a vertebrate-specific source of head mesenchyme. • Epigenetic reprogramming facilitates mesenchyme potential of neural crest. • Region- and species-specific contributions of ectomesenchyme to diverse cell types. • Plasticity of neural crest and mesoderm to contribute to craniofacial mesenchyme. • Unclear potential of cranial neural crest cells for enhanced skeletal repair. [ABSTRACT FROM AUTHOR]