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Salivary gland branching morphogenesis is regulated by the functional integration of neuronal signaling, but the underlying mechanisms are not fully understood in aging accelerated klotho‐deficient (Kl−/−) mice. Here, we investigated whether the neuropeptides substance P (SP) and neuropeptide Y (NPY) affect the branching morphogenesis of embryonic salivary glands in aging Kl−/− mice. In the salivary glands of embryonic Kl−/− mice, morphological analysis and immunostaining revealed that epithelial bud formation, neuronal cell proliferation/differentiation, and the expression of the salivary gland functional marker ZO‐1 were decreased in embryonic ductal cells. Incubation with SP/NPY at E12‐E13d promoted branching morphogenesis, parasympathetic innervation, and epithelial proliferation in salivary glands of embryonic Kl−/− mice. The ERK inhibitor U0126 specifically inhibited neuronal substance‐induced epithelial bud formation in the embryonic salivary gland. RNA‐seq profiling analysis revealed that the expression of fibroblast growth factors/fibroblast growth factors (FGFs/FGFRs) and their receptors was significantly regulated by SP/NPY treatment in the embryonic salivary gland (E15). The FGFR inhibitor BGJ389 inhibited new branching formation induced by SP and NPY treatment and ERK1/2 expression. These results showed that aging may affect virtually the development of salivary gland by neuronal dysfunction. The neuropeptides SP/NPY induced embryonic salivary gland development through FGF/FGFR/ERK1/2‐mediated signaling. [ABSTRACT FROM AUTHOR]

The genetic basis of vertebrate emergence during metazoan evolution has remained largely unknown. Understanding vertebrate‐specific genes, such as the tight junction protein Occludin (Ocln), may help answer this question. Here, it is shown that mammary glands lacking Ocln exhibit retarded epithelial branching, owing to reduced cell proliferation and surface expansion. Interestingly, Ocln regulates mitotic spindle orientation and function, and its loss leads to a range of defects, including prolonged prophase and failed nuclear and/or cytoplasmic division. Mechanistically, Ocln binds to the RabGTPase‐11 adaptor FIP5 and recruits recycling endosomes to the centrosome to participate in spindle assembly and function. FIP5 loss recapitulates Ocln null, leading to prolonged prophase, reduced cell proliferation, and retarded epithelial branching. These results identify a novel role in OCLN‐mediated endosomal trafficking and potentially highlight its involvement in mediating membranous vesicle trafficking and function, which is evolutionarily conserved and essential. [ABSTRACT FROM AUTHOR]

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases. [Display omitted] • 24 zebrafish mutants with intrahepatic biliary network defects were identified. • Unsupervised classification algorithms sorted the mutants into subgroups. • Computational analysis revealed the detailed network structures in the mutants. • This work provides computational and biological toolsets for biliary system biology. [ABSTRACT FROM AUTHOR]

How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from multipotent stem cells (MaSCs), which are progressively replaced by unipotent progenitors by birth. However, the lack of specific markers for early fate specification has prevented the delineation of the features and spatial localization of MaSC-derived lineage-committed progenitors. Here, using single-cell RNA sequencing from E13.5 to birth, we produced an atlas of matched mouse mammary epithelium and mesenchyme and reconstructed the differentiation trajectories of MaSCs toward basal and luminal fate. We show that murine MaSCs exhibit lineage commitment just prior to the first sprouting events of mammary branching morphogenesis at E15.5. We identify early molecular markers for committed and multipotent MaSCs and define their spatial distribution within the developing tissue. Furthermore, we show that the mammary embryonic mesenchyme is composed of two spatially restricted cell populations, and that dermal mesenchyme-produced FGF10 is essential for embryonic mammary branching morphogenesis. Altogether, our data elucidate the spatiotemporal signals underlying lineage specification of multipotent MaSCs, and uncover the signals from mesenchymal cells that guide mammary branching morphogenesis. Synopsis: The timing of lineage specification during mammary gland morphogenesis remains debated. Here, combined single-cell sequencing and ex vivo imaging reveal the earliest cell fate markers and mesenchymal cues instructing mouse embryonic mammary branching. Single-cell RNA-sequencing across embryonic mammary development from E13.5 to birth shows distinct lineage-committed and undifferentiated hybrid embryonic mammary progenitors. Newly identified early fate decision markers indicate that luminal and basal progenitors are spatially segregated at E15.5, before branching morphogenesis starts. The embryonic mammary stroma is composed of two regionalized and molecularly distinct mesenchymal cell populations. FGF10 produced by the dermal mesenchyme is essential for embryonic mammary branching. A longitudinal single-cell transcriptome atlas uncovers earliest cell-fate markers and mesenchymal cues instructing mouse mammary gland development. [ABSTRACT FROM AUTHOR]

The silica-based cell walls of diatoms are prime examples of genetically controlled, species-specific mineral architectures. The physical principles underlying morpho-genesis of their hierarchically structured silica patterns are not understood, yet such insight could indicate novel routes toward synthesizing functional inorganic materials. Recent advances in imaging nascent diatom silica allow rationalizing possible mechanisms of their pattern formation. Here, we combine theory and experiments on the model diatom Thalassiosira pseudonana to put forward a minimal model of branched rib patterns--a fundamental feature of the silica cell wall. We quantitatively recapitulate the time course of rib pattern morphogenesis by accounting for silica biochemistry with autocatalytic formation of diffusible silica precursors followed by conversion into solid silica. We propose that silica deposition releases an inhibitor that slows down up-stream precursor conversion, thereby implementing a self-replicating reaction--diffusion system different from a classical Turing mechanism. The proposed mechanism highlights the role of geometrical cues for guided self-organization, rationalizing the instructive role for the single initial pattern seed known as the primary silicification site. The mechanism of branching morphogenesis that we characterize here is possibly generic and may apply also in other biological systems. [ABSTRACT FROM AUTHOR]

Abstract The genetic basis of vertebrate emergence during metazoan evolution has remained largely unknown. Understanding vertebrate‐specific genes, such as the tight junction protein Occludin (Ocln), may help answer this question. Here, it is shown that mammary glands lacking Ocln exhibit retarded epithelial branching, owing to reduced cell proliferation and surface expansion. Interestingly, Ocln regulates mitotic spindle orientation and function, and its loss leads to a range of defects, including prolonged prophase and failed nuclear and/or cytoplasmic division. Mechanistically, Ocln binds to the RabGTPase‐11 adaptor FIP5 and recruits recycling endosomes to the centrosome to participate in spindle assembly and function. FIP5 loss recapitulates Ocln null, leading to prolonged prophase, reduced cell proliferation, and retarded epithelial branching. These results identify a novel role in OCLN‐mediated endosomal trafficking and potentially highlight its involvement in mediating membranous vesicle trafficking and function, which is evolutionarily conserved and essential.

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.

Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin β1 and inhibits its downstream signaling, and Integrin β1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin β1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.

Background: Kidney development is regulated by cellular interactions between the ureteric epithelium, mesenchyme, and stroma. Previous studies demonstrate essential roles for stromal β‐catenin in kidney development. However, how stromal β‐catenin regulates kidney development is not known. We hypothesize that stromal β‐catenin modulates pathways and genes that facilitate communications with neighboring cell populations to regulate kidney development. Results: We isolated purified stromal cells with wild type, deficient, and overexpressed β‐catenin by fluorescence‐activated cell sorting and conducted RNA Sequencing. A Gene Ontology network analysis demonstrated that stromal β‐catenin modulates key kidney developmental processes, including branching morphogenesis, nephrogenesis and vascular formation. Specific stromal β‐catenin candidate target genes that may mediate these effects included secreted, cell‐surface and transcriptional factors that regulate branching morphogenesis and nephrogenesis (Wnts, Bmp, Fgfr, Tcf/Lef) and secreted vascular guidance cues (Angpt1, VEGF, Sema3a). We validated established β‐catenin targets including Lef1 and novel candidate β‐catenin targets including Sema3e which have unknown roles in kidney development. Conclusions: These studies advance our understanding of gene and biological pathway dysregulation in the context of stromal β‐catenin misexpression during kidney development. Our findings suggest that during normal kidney development, stromal β‐catenin may regulate secreted and cell‐surface proteins to communicate with adjacent cell populations. Key Findings: β‐catenin in the renal stroma plays an essential role during kidney development. However, how stromal β‐catenin regulates kidney development is unknown. We identified differentially expressed genes and biological pathways in the renal stroma in mouse models with stabilized or deficient stromal β‐catenin. Our data suggest that stromal β‐catenin may regulate branching morphogenesis, nephrogenesis and vascular development through the regulation of secreted and cell‐surface proteins to facilitate cell‐to‐cell communication during development. Among the differentially expressed genes, we identified and validated candidate target genes that have known roles in kidney development, and other novel targets with unexplored roles. [ABSTRACT FROM AUTHOR]

Collective migration is essential for development, wound repair, and cancer metastasis. For most collective systems, "leader cells" determine both the direction and the power of the migration. It has remained unclear, however, how the highly polarized vertebrate epithelium migrates directionally during branching morphogenesis. We show here that, unlike in other systems, front-rear polarity of the mammary epithelium is set up by preferential cell proliferation in the front in response to the FGF10 gradient. This leads to frontal stratification, loss of apicobasal polarity, and leader cell formation. Leader cells are a dynamic population and move faster and more directionally toward the FGF10 signal than do follower cells, partly because of their intraepithelial protrusions toward the signal. Together, our data show that directional migration of the mammary epithelium is a unique multistep process and that, despite sharing remarkable cellular and molecular similarities, vertebrate and invertebrate epithelial branching are fundamentally distinct processes.

Airway smooth muscle is best known for its role as an airway constrictor in diseases such as asthma. However, its function in lung development is debated. A prevalent model, supported by in vitro data, posits that airway smooth muscle promotes lung branching through peristalsis and pushing intraluminal fluid to branching tips. Here, we test this model in vivo by inactivating Myocardin, which prevented airway smooth muscle differentiation. We found that Myocardin mutants show normal branching, despite the absence of peristalsis. In contrast, tracheal cartilage, vasculature, and neural innervation patterns were all disrupted. Furthermore, airway diameter is reduced in the mutant, counter to the expectation that the absence of smooth muscle constriction would lead to a more relaxed and thereby wider airway. These findings together demonstrate that during development, while airway smooth muscle is dispensable for epithelial branching, it is integral for building the tracheal architecture and promoting airway growth.

The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum (RT-PCR analysis). The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms. [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]

Loss of FGF signaling leads to defects in salivary gland branching, but the mechanisms underlying this phenotype remain largely unknown. We disrupted expression of Fgfr1 and Fgfr2 in salivary gland epithelial cells and found that both receptors function coordinately in regulating branching. Strikingly, branching morphogenesis in double knockouts is restored by Fgfr1 and Fgfr2 (Fgfr1/2) knock-in alleles incapable of engaging canonical RTK signaling, suggesting that additional FGF-dependent mechanisms play a role in salivary gland branching. Fgfr1/2 conditional null mutants showed defective cell-cell and cell-matrix adhesion, both of which have been shown to play instructive roles in salivary gland branching. Loss of FGF signaling led to disordered cell-basement membrane interactions in vivo as well as in organ culture. This was partially restored upon introducing Fgfr1/2 wild-type or signaling alleles that are incapable of eliciting canonical intracellular signaling. Together, our results identify non-canonical FGF signaling mechanisms that regulate branching morphogenesis through celladhesion processes. [ABSTRACT FROM AUTHOR]

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis. [ABSTRACT FROM AUTHOR]

Electronic cigarette usage has significantly expanded among young people and pregnant women in the last decade. Although there are already some data regarding the short- and long-term consequences of e-cigarettes on human health, their effect on embryo and lung development still needs to be fully disclosed. In this sense, this study describes, for the first time, the impact of electronic cigarette aerosol on early lung development. For this purpose, ex vivo chick (Gallus gallus) embryonic lungs were cultured in vitro for 48 h in e-cigarette aerosol exposed-medium or unexposed medium. Chick lung explants were also cultured in a cigarette smoke-exposed medium for comparison purposes. Lung explants were morphologically analyzed to assess the impact on lung growth. Additionally, TNF-α levels were determined in the supernatant as a marker of pro-inflammatory response. The results suggest that electronic cigarette aerosol impairs lung growth and promotes lung inflammation. However, its impact on early lung growth seems less detrimental than conventional cigarette smoke. This work provides significant data regarding the impact of e-cig aerosol, adding to the efforts to fully understand its effect on embryo development. The validation of these effects may eventually lead to new tobacco control recommendations for pregnant women. • E-cig aerosol impairs lung growth but to a lesser extent than c-cig smoke; • TNF-α induction was similar upon e-cig aerosol or c-cig smoke exposure; • The impact on lung growth was not due to the difference in nicotine content. [ABSTRACT FROM AUTHOR]

Abstract Background The Bcl-3 protein is an atypical member of the inhibitor of -κB family that has dual roles as a transcriptional repressor and a coactivator for dimers of NF-κB p50 and p52. Bcl-3 is expressed in mammary adenocarcinomas and can promote tumorigenesis and survival signaling and has a key role in tumor metastasis. In this study, we have investigated the role of Bcl-3 in the normal mammary gland and impact on tumor pathology. Methods We utilized bcl-3 −/− mice to study mammary gland structure in virgins and during gestation, lactation and early involution. Expression of involution-associated genes and proteins and putative Bcl-3 target genes was examined by qRT-PCR and immunoblot analysis. Cell autonomous branching morphogenesis and collagen I invasion properties of bcl-3 −/− organoids were tested in 3D hydrogel cultures. The role of Bcl-3 in tumorigenesis and tumor pathology was also assessed using a stochastic carcinogen-induced mammary tumor model. Results Bcl-3 −/− mammary glands demonstrated reduced branching complexity in virgin and pregnant mice. This defect was recapitulated in vitro where significant defects in bud formation were observed in bcl-3 −/− mammary organoid cultures. Bcl-3 −/− organoids showed a striking defect in protrusive collective fibrillary collagen I invasion associated with reduced expression of Fzd1 and Twist2. Virgin and pregnant bcl-3 −/− glands showed increased apoptosis and rapid increases in lysosomal cell death and apoptosis after forced weaning compared to WT mice. Bcl-2 and Id3 are strongly induced in WT but not bcl-3 −/− glands in early involution. Tumors in WT mice were predominately adenocarcinomas with NF-κB activation, while bcl-3 −/− lesions were largely squamous lacking NF-κB and with low Bcl-2 expression. Conclusions Collectively, our results demonstrate that Bcl-3 has a key function in mammary gland branching morphogenesis, in part by regulation of genes involved in extracellular matrix invasion. Markedly reduced levels of pro-survival proteins expression in bcl-3 null compared to WT glands 24 h post-weaning indicate that Bcl-3 has a role in moderating the rate of early phase involution. Lastly, a reduced incidence of bcl-3 −/− mammary adenocarcinomas versus squamous lesions indicates that Bcl-3 supports the progression of epithelial but not metaplastic cancers.

Triclosan (TCS) is a commonly used antibacterial agent present in personal care and household products. Recently, there have been increasing concerns about the association between children’s health and TCS exposure during gestation, but the toxicological effects of TCS exposure on embryonic lung development remain undetermined. In this study, through using an ex vivo lung explant culture system, we found that prenatal exposure to TCS resulted in impaired lung branching morphogenesis and altered proximal-distal airway patterning. These TCS-induced dysplasias are accompanied by significantly reduced proliferation and increased apoptosis within the developing lung, as a consequence of activated Bmp4 signaling. Inhibition of Bmp4 signaling by Noggin partially rescues the lung branching morphogenesis and cellular defects in TCS-exposed lung explants. In addition, we provided in vivo evidence that administration of TCS during gestation leads to compromised branching formation and enlarged airspace in the lung of offspring. Thus, this study provides novel toxicological information on TCS and indicated a strong/possible association between TCS exposure during pregnancy and lung dysplasia in offspring.

Localization of multiple hydrogels is expected to develop the structure of 3D tissue models in a location‐specific manner. Here, 3D tissue morphogenesis is spatially guided by localizing different hydrogel conditions at different parts of a tissue. To achieve the localization, a unit‐based scaffold is developed with a unique frame design to trap hydrogel solutions inside their designated units. An optimal range of unit dimensions and surface wettabilities enables a solution trapping up to several cubic millimeters without any need for chemical additives. This capability allows spatial organization of biomolecular compositions and physical conditions of hydrogels, as well as the relative position of biological samples (cells, spheroids, and reconstituted tissues) within the scaffold. Successful localization of branching development on reconstituted human epithelial tissues is achieved by localizing growth factors or cross‐linked matrix proteins within hydrogels, demonstrating a direct dependence on local hydrogel conditions. Unlike 3D‐bioprinting or microfluidic techniques, this scaffold‐based localization of hydrogels requires only a manual pipetting and no specialized tools, making it ready‐to‐use for researchers from any field. This localization technique provides a new promising route to spatially control morphogenesis, differentiation, and other developmental processes within 3D organoids or tissue models for practical biomedical applications in the future. [ABSTRACT FROM AUTHOR]

Branched epithelial networks are generated through an iterative process of elongation and bifurcation. We sought to understand bifurcation of the mammary epithelium. To visualize this process, we utilized three-dimensional (3D) organotypic culture and time-lapse confocal microscopy.We tracked cell migration during bifurcation and observed local reductions in cell speed at the nascent bifurcation cleft. This effect was proximity dependent, as individual cells approaching the cleft reduced speed, whereas cells exiting the cleft increased speed. As the cells slow down, they orient both migration and protrusions towards the nascent cleft, while cells in the adjacent branches orient towards the elongating tips. We next tested the hypothesis that TGF-β signaling controls mammary branching by regulating cell migration. We first validated that addition of TGF-β1 (TGFB1) protein increased cleft number, whereas inhibition of TGF-β signaling reduced cleft number. Then, consistent with our hypothesis, we observed that pharmacological inhibition of TGF-β1 signaling acutely decreased epithelial migration speed. Our data suggest a model for mammary epithelial bifurcation in which TGF-β signaling regulates cell migration to determine the local sites of bifurcation and the global pattern of the tubular network. [ABSTRACT FROM AUTHOR]

The major lactiferous ducts of the human breast branch out and end at terminal ductal lobular units (TDLUs). Despite their functional and clinical importance, the three-dimensional (3D) architecture of TDLUs has remained undetermined. Our quantitative and volumetric imaging of healthy human breast tissue demonstrates that highly branched TDLUs, which exhibit increased proliferation, are uncommon in the resting tissue regardless of donor age, parity, or hormonal contraception. Overall, TDLUs have a consistent shape and branch parameters, and they contain a main subtree that dominates in bifurcation events and exhibits a more duct-like keratin expression pattern. Simulation of TDLU branching morphogenesis in three dimensions suggests that evolutionarily conserved mechanisms regulate mammary gland branching in humans and mice despite their anatomical differences. In all, our data provide structural insight into 3D anatomy and branching of the human breast and exemplify the power of volumetric imaging in gaining a deeper understanding of breast biology. [Display omitted] • Breast terminal ductal lobular unit (TDLU) branching varies regardless of age or parity • TDLUs have a main branch that dominates in bifurcations and has duct-like features • Highly branched TDLUs proliferate more and may contain more S100A4+ stromal cells • Human and mouse mammary glands appear to branch by the same principles Paavolainen, Peurla et al. studied intact terminal ductal lobular unit (TDLU) structures to quantitatively assess the branching morphology of the human breast in three dimensions. The study provides a framework for investigating the role of TDLU architecture and cell-type-specific anatomical niches in breast homeostasis and malignant progression. [ABSTRACT FROM AUTHOR]

Background: Technical advances in whole tissue imaging and clearing have allowed 3D reconstruction of exocrine uterine glands deep-seated in the endometrium. However, there are limited gland structure analysis platforms to analyze these imaging data sets. Here, we present a pipeline for segmenting and analyzing uterine gland shape., Results: Using our segmentation methodology, we derive metrics to describe gland length, shape, and branching patterns. We then quantify gland behavior with respect to organization around the embryo and proximity of each gland to the uterine lumen. We apply this image analysis pipeline to uterine glands at the peri-implantation time points of a mouse pregnancy. Our analysis reveals that at the time of embryo or egg entry into the uterus, glands show changes in length, tortuosity, and proximity to the uterine lumen while gland branch number stays the same. Eventually, these shape changes aid in reorganization of the glands around the embryo implantation site. We further apply our analysis pipeline to human and guinea pig uterine glands, extending feasibility to other mammalian species., Conclusion: This work serves as a resource for researchers to extract quantitative and reproducible morphological features from three-dimensional uterine gland images to reveal insights about functional and structural patterns., (© 2024 The Author(s). Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

During airway branching morphogenesis, focal regions of FGF-10 expression in the pulmonary mesenchyme are thought to provide a local guidance cue, which promotes chemotactically the directional outgrowth of the airway epithelium. Here, however, we show that an ectopic source of FGF-10 induces epithelial buckling morphogenesis and the formation of multiple new supernumerary buds. FGF-10-induced budding can be modulated by altered epithelial tension and luminal fluid pressure. Increased tension suppresses the formation of ectopic branches, while a collapse of the embryonic airway promotes more expansive buckling and additional FGF-10-induced supernumerary buds. Our results indicate that a focal source of FGF-10 can promote epithelial buckling and suggest that the overall branching pattern cannot be explained entirely by the templated expression of FGF-10. Both FGF-10-mediated cell behaviors and exogenous mechanical forces must be integrated to properly shape the bronchial tree.

Background: The Bcl-3 protein is an atypical member of the inhibitor of -κB family that has dual roles as a transcriptional repressor and a coactivator for dimers of NF-κB p50 and p52. Bcl-3 is expressed in mammary adenocarcinomas and can promote tumorigenesis and survival signaling and has a key role in tumor metastasis. In this study, we have investigated the role of Bcl-3 in the normal mammary gland and impact on tumor pathology.Methods: We utilized bcl-3-/- mice to study mammary gland structure in virgins and during gestation, lactation and early involution. Expression of involution-associated genes and proteins and putative Bcl-3 target genes was examined by qRT-PCR and immunoblot analysis. Cell autonomous branching morphogenesis and collagen I invasion properties of bcl-3-/- organoids were tested in 3D hydrogel cultures. The role of Bcl-3 in tumorigenesis and tumor pathology was also assessed using a stochastic carcinogen-induced mammary tumor model.Results: Bcl-3-/- mammary glands demonstrated reduced branching complexity in virgin and pregnant mice. This defect was recapitulated in vitro where significant defects in bud formation were observed in bcl-3-/- mammary organoid cultures. Bcl-3-/- organoids showed a striking defect in protrusive collective fibrillary collagen I invasion associated with reduced expression of Fzd1 and Twist2. Virgin and pregnant bcl-3-/- glands showed increased apoptosis and rapid increases in lysosomal cell death and apoptosis after forced weaning compared to WT mice. Bcl-2 and Id3 are strongly induced in WT but not bcl-3-/- glands in early involution. Tumors in WT mice were predominately adenocarcinomas with NF-κB activation, while bcl-3-/- lesions were largely squamous lacking NF-κB and with low Bcl-2 expression.Conclusions: Collectively, our results demonstrate that Bcl-3 has a key function in mammary gland branching morphogenesis, in part by regulation of genes involved in extracellular matrix invasion. Markedly reduced levels of pro-survival proteins expression in bcl-3 null compared to WT glands 24 h post-weaning indicate that Bcl-3 has a role in moderating the rate of early phase involution. Lastly, a reduced incidence of bcl-3-/- mammary adenocarcinomas versus squamous lesions indicates that Bcl-3 supports the progression of epithelial but not metaplastic cancers. [ABSTRACT FROM AUTHOR]

A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys.

We analyze evolutionary dynamics in a confluent, branching cellular population, such as in a growing duct, vasculature, or in a branching microbial colony. We focus on the coarse-grained features of the evolution and build a statistical model that captures the essential features of the dynamics. Using simulations and analytic approaches, we show that the survival probability of strains within the growing population is sensitive to the branching geometry: Branch bifurcations enhance survival probability due to an overall population growth (i.e., "inflation"), while branch termination and the small effective population size at the growing branch tips increase the probability of strain extinction. We show that the evolutionary dynamics may be captured on a wide range of branch geometries parameterized just by the branch diameter N 0 and branching rate b. We find that the survival probability of neutral cell strains is largest at an "optimal" branching rate, which balances the effects of inflation and branch termination. We find that increasing the selective advantage s of the cell strain mitigates the inflationary effect by decreasing the average time at which the mutant cell fate is determined. For sufficiently large selective advantages, the survival probability of the advantageous mutant decreases monotonically with the branching rate. [ABSTRACT FROM AUTHOR]

Branching morphogenesis is the process that gives rise to branched structures in several organs, such as the lung, the kidney, and the mammary gland. Although morphologically well described, the exact mechanisms driving branch elongation and bifurcation are still poorly understood. Signaling cues from the stroma and extracellular matrix have an important role in driving branching morphogenesis. Organoid models derived from primary mammary epithelial cells have emerged as a powerful tool to gain insight into branching morphogenesis of the mammary gland. However, current available mammary organoid culture protocols result in morphologically simple structures which do not resemble the complex branched structure of the in vivo mammary gland. Supplementation of growth factors to mammary organoids cultured in basement membrane extract or collagen I were shown to induce bud formation and elongation but are not sufficient to drive true branching events. Here, we present an improved culture approach based on 3D primary mammary epithelial cell culture to develop branched organoids with a complex morphology. By alternating the addition of fibroblast growth factor 2 and epidermal growth factor to mammary organoids cultured in a basement membrane extract matrix enriched with collagen type I fibers, we obtain complex mammary organoid structures with primary, secondary, and tertiary branches over a period of 15–20 days. Mammary organoid structures grow >1 mm in size and show an elongated and branched shape which resembles in vivo mammary gland morphology. This novel branched mammary organoid model offers many possibilities to study the mechanisms of branching in the developing mammary gland. [ABSTRACT FROM AUTHOR]

Epithelial–mesenchymal interaction is required for normal growth, morphogenetic patterning, and cellular differentiation in developing lungs. Various signaling pathways have been defined in establishing the patterning of this branched organ. The phosphoinositide-3-kinase (PI3K) signaling plays an important role in disease pathogenesis but remains largely uncharacterized in embryonic development. In this study, we activated a specific catalytic subunit of PI3K catalytic enzymes, Class IA p110α (p110α), in the embryonic lung mesenchyme using the Dermo1-Cre mouse. Activation of p110α promoted branching morphogenesis and blocked club cell differentiation in both proximal and distal airways. Mechanistically, the LIM homeodomain gene Islet-1 (Isl1), fibroblast growth factor 10 (Fgf10), and SRY (sex-determining region Y)-box9 (Sox9) were found to be downstream targets of p110α. The significantly increased expressions of Isl1, Fgf10, and Sox9 resulted in the stimulation of branching in mutant lungs. Activation of p110α-mediated signaling also increased the expression of phosphatase and tensin homolog deleted on chromosome 10 (Pten) and hairy/enhancer of split 1 (Hes1), which in turn blocked club cell differentiation. Thus, the signaling pathway by which PI3K/p110α-regulated epithelial–mesenchymal interactions may entail Isl1–Fgf10–Sox9 and Pten–Hes1 networks, which consequently regulate branching morphogenesis and club cell differentiation, respectively.

Branching morphogenesis is the process that gives rise to branched structures in several organs, such as the lung, the kidney, and the mammary gland. Although morphologically well described, the exact mechanisms driving branch elongation and bifurcation are still poorly understood. Signaling cues from the stroma and extracellular matrix have an important role in driving branching morphogenesis. Organoid models derived from primary mammary epithelial cells have emerged as a powerful tool to gain insight into branching morphogenesis of the mammary gland. However, current available mammary organoid culture protocols result in morphologically simple structures which do not resemble the complex branched structure of the in vivo mammary gland. Supplementation of growth factors to mammary organoids cultured in basement membrane extract or collagen I were shown to induce bud formation and elongation but are not sufficient to drive true branching events. Here, we present an improved culture approach based on 3D primary mammary epithelial cell culture to develop branched organoids with a complex morphology. By alternating the addition of fibroblast growth factor 2 and epidermal growth factor to mammary organoids cultured in a basement membrane extract matrix enriched with collagen type I fibers, we obtain complex mammary organoid structures with primary, secondary, and tertiary branches over a period of 15–20 days. Mammary organoid structures grow >1 mm in size and show an elongated and branched shape which resembles in vivo mammary gland morphology. This novel branched mammary organoid model offers many possibilities to study the mechanisms of branching in the developing mammary gland.

The role of the local microenvironment in influencing cell behavior is central to both normal development and cancer formation. Here, we show that sprouty 1 (SPRY1) modulates the microenvironment to enable proper mammary branching morphogenesis. This process occurs through negative regulation of epidermal growth factor receptor (EGFR) signaling in mammary stroma. Loss of SPRY1 resulted in up-regulation of EGFR-extracellular signal-regulated kinase (ERK) signaling in response to amphiregulin and transforming growth factor alpha stimulation. Consequently, stromal paracrine signaling and ECM remodeling is augmented, leading to increased epithelial branching in the mutant gland. By contrast, down-regulation of EGFR-ERK signaling due to gain of Sprouty function in the stroma led to stunted epithelial branching. Taken together, our results show that modulation of stromal paracrine signaling and ECM remodeling by SPRY1 regulates mammary epithelial morphogenesis during postnatal development.

Branching morphogenesis is an important process in shaping the arborized structures of several organs. However, the driving force that directs this process from progenitor pools remains incompletely understood. In this lineage tracing study, we investigated the role of Id2+ embryonic progenitor cells in branching organs such as the pancreas, kidney, mammary gland, thyroid gland, and salivary gland. We found that a subset of Id2+ distal progenitor cells in the embryonic pancreas and kidney can give rise to multiple lineages of progeny cells during branching morphogenesis. Id2-labeled cells also supported the postnatal development of the mammary glands. However, Id2+ cells did not contribute to the development of the salivary and thyroid glands. We found the Id2+ cells located in the tip progenitor pools of pancreas and kidney have self-renewal potential and contribute descendants to multiple epithelial cell lineages. Our findings enrich the current model of distal progenitor pools driving branching morphogenesis and provide a new marker to investigate the regularity of branching in these organs. [ABSTRACT FROM AUTHOR]

Fluid secretion by exocrine glandular organs is essential to the survival of mammals. Each glandular unit within the body is uniquely organized to carry out its own specific functions, with failure to establish these specialized structures resulting in impaired organ function. Here, we review glandular organs in terms of shared and divergent architecture. We first describe the structural organization of the diverse glandular secretory units (the end-pieces) and their fluid transporting systems (the ducts) within the mammalian system, focusing on how tissue architecture corresponds to functional output. We then highlight how defects in development of end-piece and ductal architecture impacts secretory function. Finally, we discuss how knowledge of exocrine gland structure-function relationships can be applied to the development of new diagnostics, regenerative approaches and tissue regeneration. [ABSTRACT FROM AUTHOR]

The shaping of tissues and organs in many animals relies on interactions between the epithelial cell layer and its underlying mesoderm-derived tissues. Inductive signals, such as receptor tyrosine kinase (RTK) signaling emanating from mesoderm, act on cells of the epithelium to initiate three-dimensional changes. However, how tissues are shaped in a diploblastic animal with no mesoderm remains largely unknown. In this study, the jellyfish Cladonema pacificum was used to investigate branch formation. The tentacles on its medusa stage undergo branching, which increases the epithelial surface area available forcarrying nematocytes, thereby maximizing prey capture. Pharmacological and cellular analyses of the branching process suggest a two-step model for tentacle branch formation, in which mitogen-activated protein kinase kinase signaling accumulates interstitial cells in the future branch-forming region, and fibroblast growth factor signaling regulates branch elongation. This study highlights an essential role for these pluripotent stem cells in the tissue-shaping morphogenesis of a diploblastic animal. In addition, it identifies a mechanism involving RTK signaling and cell proliferative activity at the branch tip for branching morphogenesis that is apparently conserved across the animal kingdom. [ABSTRACT FROM AUTHOR]

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development. [ABSTRACT FROM AUTHOR]

Here we present an experimental model for human luminal progenitor cells that enables single, primary cells isolated from normal tissue to generate complex branched structures resembling the ductal morphology of low‐grade carcinoma of no special type. Thereby, we find that ductal structures are generated through invasive branching morphogenesis via matrix remodeling and identify reduced actomyosin contractility as a prerequisite for invasion. In addition, we show that knockout of E‐cadherin causes a dissolution of duct formation as observed in invasive lobular carcinoma, a subtype of invasive carcinomas where E‐cadherin function is frequently lost. Thus, our model shows that invasive capacity can be elicited from normal luminal cells in specific environments, which results in low‐grade no special type morphology. This assay offers a platform to investigate the dynamics of luminal cell invasion and unravel the impact of genetic and non‐genetic aberrations on invasive morphology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

In investigating fetal organ development, fetus of the rabbit was one of the furthermost frequently handled as a model in animal investigational research. During the second half of gestation in rabbit fetuses thorax volume (TV) was the predictive factor for assessment of normal development. Concerning these fetuses, evaluation of lung development was more applicable in the 20th–29th gestational days for experiments. A large internal surface area in which the inspired air and capillary blood got in close contact to each other was specified by the lung tissue. Consequently, an efficient gas exchange was occurred. A cycle of separate but overlapping developmental processes happened to reach this target. The anlage of the left and right lungs appeared as an outpouching of the foregut endoderm during organogenesis. A repetitive process of outgrowth and branching pattern (branching morphogenesis) was occurred in each lung bud. Consequently, all of the future airways were formed mainly during the pseudoglandular stage. Repetitive airspace septation would lead to tremendous increase of the gas exchange surface area in both late fetal and postnatal lung development leading to the formation of alveoli. The first air-blood barriers were appeared and surfactant production began during the canalicular stage.

Despite years of positive animal data, hepatocyte growth factor (HGF) has never been developed into a useful pharmaceutical, primarily due to its poor pharmacological properties. CM1021 is a fusion protein containing the K1 loop of HGF and the human IgG1 Fc region. The experiments described here demonstrate that CM1021 has the biological properties of HGF and the pharmacological properties of a monoclonal antibody. CM1021 stimulates scattering and branching morphogenesis in MDCK cells and stimulates liver growth in vivo. Unlike HGF, it is available via intraperitoneal injection and has an estimated half-life similar to an antibody.

In this protocol, we focused on analyzing internal branches of Drosophila class IV neurons. These neurons are characterized by their highly branched axons and dendrites and intricately tile the larval body. As Drosophila larvae progress through developmental stages, the dendritic arbors of Class IV neurons undergo notable transformations. As Drosophila larvae develop, their Class IV dendritic arbors grow. In the initial 24 h after egg laying (AEL), the dendrites are smaller than segments. During the subsequent 24 h of the first instar larval stage, dendritic arbors outpace segment growth, achieving tiling. After 48 h, arbors and segments grow concurrently. Epidermal cells near Class IV dendrites expand in proportion to segment growth. This observation suggested that Class IV cells might grow via branch dilation-uniformly elongating branches, akin to Class I cells [1,2]. To understand whether the class IV complex arbor structure is formed by dilation or simply from growing tips, we developed this protocol to introduce a systematic approach for quantitatively assessing the growth dynamics of internal branches. Key features • This protocol employs imaging the same neuron over different development times • Drosophila embryo and larvae genotype is ;;ppkCD4-tdGFP, which explicitly tags class IV neurons • This protocol for the preparation of agar pads to mount and image Drosophila larvae is adapted from Monica Driscoll's method • Neurons are imaged without the use of anesthetics and for a short duration of time • This technique involves the use of a spinning disk confocal microscope., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

In investigating fetal organ development, fetus of the rabbit was one of the furthermost frequently handled as a model in animal investigational research. During the second half of gestation in rabbit fetusesthorax volume (TV) was the predictive factor for assessment of normal development. Concerning these fetuses, evaluation of lung development was more applicable in the 20th–29th gestational days for experiments. A large internal surface area in which the inspired air and capillary blood got in close contact to each other was specified by the lung tissue. Consequently, an efficient gas exchange was occurred. A cycle of separate but overlapping developmental processes happened to reach this target. The anlage of the left and right lungs appeared as an outpouching of the foregut endoderm during organogenesis. A repetitive process of outgrowth and branching pattern (branching morphogenesis) was occurred in each lung bud. Consequently, all of the future airways were formed mainly during the pseudoglandular stage. Repetitive airspace septation would lead to tremendous increase of the gas exchange surface area in both late fetal and postnatal lung development leading to the formation of alveoli. The first air-blood barriers were appeared and surfactant production began during the canalicular stage. [ABSTRACT FROM AUTHOR]

Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha -expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha +basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha +domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent 'tethering' to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis. [Display omitted] • Shh/Smo signaling is restricted to distal shha + basal epidermal cells and adjacent pre-osteoblasts in developing zebrafish fins. • Sustained Shh/Smo signaling promotes ray branching morphogenesis in all unpaired and paired fins. • Shh/Smo largely is dedicated to ray branching throughout caudal fin development. • Collectively distal-moving, shha + basal epidermal cells slow while transiently associated with underlying pre-osteoblasts. • Shh/Smo may tether pre-osteoblasts to laterally-splitting shha-expressing basal epidermis, thereby partitioning ray-forming cell pools. [ABSTRACT FROM AUTHOR]

A common developmental process, called branching morphogenesis, generates the epithelial trees in a variety of organs, including the lungs, kidneys, and glands. How branching morphogenesis can create epithelial architectures of very different shapes and functions remains elusive. In this review, we compare branching morphogenesis and its regulation in lungs and kidneys and discuss the role of signaling pathways, the mesenchyme, the extracellular matrix, and the cytoskeleton as potential organ-specific determinants of branch position, orientation, and shape. Identifying the determinants of branch and organ shape and their adaptation in different organs may reveal how a highly conserved developmental process can be adapted to different structural and functional frameworks and should provide important insights into epithelial morphogenesis and developmental disorders.