Epithelial Organogenesis Research Articles

Linking chemical signaling to mechanical components to determine tissue shape in the Drosophila wing imaginal disc

During organogenesis in the Drosophila wing imaginal disc, the regulation of epithelial cell height and curvature is crucial in developing correct tissue shapes. This requires the interplay between mechanical forces and morphogen-mechanogen pathways, at both the cell and tissue levels. Morphogens, such as Decapentaplegic (Dpp), regulate cell growth and division, as well as mechanogen activity. Mechanogens, such as RhoGTPases, are small diffusible molecules that regulate mechanical components, such as actin and myosin, to coordinate cell shape and tissue geometry. Even though the effect of morphogens in regulating mechanogens is critical for proper tissue formation, insufficient work has been done to understand this in the context of epithelial organogenesis. In this study, a combination of experimental and mathematical modeling approaches are used to study the linkage between Dpp and Rho1, Cdc42 in the wing imaginal disc. By using experiments, a new regulation between Dpp/pMad and Cdc42 has been identified, as well as the interaction between Cdc42 and Rho1. A mathematical model has been developed by using a system of reaction-diffusion equations to model Dpp, Rho1, and Cdc42 dynamics, as well as the newly identified regulations. In particular, the signaling model uses a discrete node structure from a multi-scale subcellular element model. We apply this model to investigate the robustness of the proposed networks, in addition to the effect of tissue geometry on the concentration profiles of both the morphogens and mechanogens.

Endothelial Wnts control mammary epithelial patterning via fibroblast signaling

Endothelial and fibroblast niches are crucial for epithelial organs. How these heterotypic cells interact is of great interest. In this study, we reveal an axis of signaling in which fibroblasts relay Wnt signals from the endothelial niche to organize epithelial patterning. We generate an Axin2-membrane GFP (mGFP) reporter mouse and observe robust Wnt/β-catenin signaling activities in fibroblasts surrounding the mammary epithelium. To enable cell-type-specific gene manipulation invitro, we establish an organoid system via coculture of endothelial cells (ECs), fibroblasts, and mammary epithelial cells. Deletion of β-catenin in fibroblasts impedes epithelium branching, and ECs are responsible for the activation of Wnt/β-catenin signaling in fibroblasts. Invivo, EC deletion of Wntless inhibits Wnt/β-catenin signaling activity in fibroblasts, rendering a reduction in epithelial branches. These findings highlight the significance of the endothelial niche in tissue patterning, shedding light on the interactive mechanisms in which distinct niche components orchestrate epithelial organogenesis and tissue homeostasis.

Epithelial organ shape is generated by patterned actomyosin contractility and maintained by the extracellular matrix.

Epithelial sheets define organ architecture during development. Here, we employed an iterative multiscale computational modeling and quantitative experimental approach to decouple direct and indirect effects of actomyosin-generated forces, nuclear positioning, extracellular matrix, and cell-cell adhesion in shaping Drosophila wing imaginal discs. Basally generated actomyosin forces generate epithelial bending of the wing disc pouch. Surprisingly, acute pharmacological inhibition of ROCK-driven actomyosin contractility does not impact the maintenance of tissue height or curved shape. Computational simulations show that ECM tautness provides only a minor contribution to modulating tissue shape. Instead, passive ECM pre-strain serves to maintain the shape independent from actomyosin contractility. These results provide general insight into how the subcellular forces are generated and maintained within individual cells to induce tissue curvature. Thus, the results suggest an important design principle of separable contributions from ECM prestrain and actomyosin tension during epithelial organogenesis and homeostasis.

2155-P: Peripheral Nervous System Innervation of the Developing Pancreas

Pancreatic organogenesis is a complex, multistep process whereby multiple cell types must migrate, proliferate, and differentiate in a strict spatiotemporal manner to ultimately form the complete organ. Although pancreatic epithelial organogenesis is well characterized, the peripheral nervous system (PNS) innervating the organ is poorly understood. Although the PNS has been shown to be critical in the formation of other budding exocrine organs, and therefore likely plays a role in the formation of the pancreas, there is a dearth of information on innervation of this developing organ. To this end, our lab has taken advantage of an explant culture system and single-cell RNA-sequencing (scRNAseq) to characterize the nerves supplying the developing pancreas. Imaging of whole pancreata reveals that peripheral nerves develop in parallel with the pancreas, with intrapancreatic ganglia being present from early stages. Using scRNAseq analysis we have begun to decipher the timing of pancreatic PNS progenitor cell differentiation, and the potential signaling pathways guiding axonal growth and morphogenesis. Continued investigation of the mechanisms guiding innervation of the pancreas, deciphering how nerve-epithelial interactions are established and defining signaling pathways activated by the nerves will allow us to discover the role of the PNS in pancreatic morphogenesis, as well as mis-regulation during disease. Disclosure S. de la O: None. L. Byrnes: None. D.M. Wong: None. S. Knox: None. J.B. Sneddon: Advisory Panel; Spouse/Partner; Akouos. Advisory Panel; Self; Encellin. Advisory Panel; Spouse/Partner; Encellin. Consultant; Self; Semma Therapeutics, Inc.

Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland.

The tear-producing lacrimal gland is a tubular organ that protects and lubricates the ocular surface. The lacrimal gland possesses many features that make it an excellent model in which to investigate tubulogenesis, but the cell types and lineage relationships that drive lacrimal gland formation are unclear. Using single-cell sequencing and other molecular tools, we reveal novel cell identities and epithelial lineage dynamics that underlie lacrimal gland development. We show that the lacrimal gland from its earliest developmental stages is composed of multiple subpopulations of immune, epithelial and mesenchymal cell lineages. The epithelial lineage exhibits the most substantial cellular changes, transitioning through a series of unique transcriptional states to become terminally differentiated acinar, ductal and myoepithelial cells. Furthermore, lineage tracing in postnatal and adult glands provides the first direct evidence of unipotent KRT5+ epithelial cells in the lacrimal gland. Finally, we show conservation of developmental markers between the developing mouse and human lacrimal gland, supporting the use of mice to understand human development. Together, our data reveal crucial features of lacrimal gland development that have broad implications for understanding epithelial organogenesis.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Taming the young and restless—epigenetic gene regulation in pancreas and beta‐cell precursors

The in vivo assessment of epigenetic changes during mouse pancreatic beta‐cell differentiation reveals surprising differences to directed, in vitro differentiation of human embryonic stem cells. New findings reported in this issue of The EMBO Journal further identify Ezh2 as a critical determinant of endocrine progenitor number and could instruct improved protocols for stem cell-based therapies.

Growth factor–heparan sulfate “switches” regulating stages of branching morphogenesis

The development of branched epithelial organs, such as the kidney, mammary gland, lung, pancreas, and salivary gland, is dependent upon the involvement and interaction of multiple regulatory/modulatory molecules, including soluble growth factors, extracellular matrix components, and their receptors. How the function of these molecules is coordinated to bring about the morphogenetic events that regulate iterative tip-stalk generation (ITSG) during organ development remains to be fully elucidated. A common link to many growth factor-dependent morphogenetic pathways is the involvement of variably sulfated heparan sulfates (HS), the glycosaminoglycan backbone of heparan sulfate proteoglycans (HSPG) on extracellular surfaces. Genetic deletions of HS biosynthetic enzymes (e.g., C5-epimerase, Hs2st), as well as considerable in vitro data, indicate that variably sulfated HS are essential for kidney development, particularly in Wolffian duct budding and early ureteric bud (UB) branching. A role for selective HS modifications by enzymes (e.g., Ext, Ndst, Hs2st) in stages of branching morphogenesis is also strongly supported for mammary gland ductal branching, which is dependent upon a set of growth factors similar to those involved in UB branching. Taken together, these studies provide support for the notion that the specific spatio-temporal HS binding of growth factors during the development of branched epithelial organs (such as the kidney, mammary gland, lung and salivary gland) regulates these complex processes by potentially acting as "morphogenetic switches" during the various stages of budding, branching, and other developmental events central to epithelial organogenesis. It may be that two or more growth factor-selective HS interactions constitute a functionally equivalent morphogenetic switch; this may help to explain the paucity of severe branching phenotypes with individual growth factor knockouts.

Control of Cell Identity in Pancreas Development and Regeneration

The endocrine and exocrine cells in the adult pancreas are not static, but can change their differentiation state in response to injury or stress. This concept of cells in flux means that there may be ways to generate certain types of cells (such as insulin-producing β-cells) and prevent formation of others (such as transformed neoplastic cells). We review different aspects of cell identity in the pancreas, discussing how cells achieve their identity during embryonic development and maturation, and how this identity remains plastic, even in the adult pancreas.

Pancreatic Mesenchyme Regulates Epithelial Organogenesis throughout Development

Author SummaryEmbryonic development is a highly complex process that requires tight orchestration of cellular proliferation, differentiation, and migration as cells grow within loosely aggregated mesenchyme and more organized epithelial sheets to form organs and tissues. In addition to intrinsic cell-autonomous signals, these events are further regulated by environmental cues provided by neighboring cells. Prior work demonstrated a critical role for the surrounding mesenchyme in guiding epithelial growth during the early stages of pancreas development. However, it remained unclear whether the mesenchyme also guided the later stages of pancreas organogenesis when the functional exocrine and endocrine cells are formed. Here, we show that specific genetic ablation of the mesenchyme at distinct developmental stages in vivo results in the formation of a smaller, misshapen pancreas. Loss of the mesenchyme profoundly impairs the expansion of both endocrine and exocrine pancreatic progenitors, as well as the proliferative capacity of maturing cells, including insulin-producing beta-cells. Thus, our studies reveal unappreciated roles for the mesenchyme in guiding the formation of the epithelial pancreas throughout development. The results suggest that identifying the specific mesenchymal signals might help to optimize cell culture protocols that aim to achieve the differentiation of stem cells into insulin-producing beta cells.

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).

Regulation of Growth by Ploidy in Caenorhabditis elegans

Some animals, such as the larvae of Drosophila melanogaster, the larvae of the Appendicularian chordate Oikopleura, and the adults of the nematode Caenorhabditis elegans, are unusual in that they grow largely by increases in cell size. The giant cells of such species are highly polyploid, having undergone repeated rounds of endoreduplication. Since germline polyploid strains tend to have large cells, it is often assumed that endoreduplication drives cell growth, but this remains controversial. We have previously shown that adult growth in C. elegans is associated with the endoreduplication of nuclei in the epidermal syncitium, hyp 7. We show here that this relationship is causal. Manipulation of somatic ploidy both upwards and downwards increases and decreases, respectively, adult body size. We also establish a quantitative relationship between ploidy and body size. Finally, we find that TGF-beta (DBL-1) and cyclin E (CYE-1) regulate body size via endoreduplication. To our knowledge, this is the first experimental evidence establishing a cause-and-effect relationship between somatic polyploidization and body size in a metazoan.

A Structural Model for the Membrane-bound Form of the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family involved in the regulation of cellular proliferation and differentiation. Its juxtamembrane domain (JX), the region located between the transmembrane and kinase domains, plays important roles in receptor trafficking. Two sorting signals, a PXXP motif and a 658LL659 motif, are responsible for basolateral sorting in polarized epithelial cells, and a 679LL680 motif targets the ligand-activated receptor for lysosomal degradation. To understand the regulation of these signals, we characterized the structural properties of recombinant JX domain in aqueous solution and in dodecylphosphocholine (DPC) detergent. JX is inherently unstructured in aqueous solution, albeit a nascent helix encompasses the lysosomal sorting signal. In DPC micelles, structures derived from NMR data showed three amphipathic, helical segments. A large, internally inconsistent group of long range nuclear Overhauser effects suggest a close proximity of the helices, and the presence of significant conformational averaging. Models were determined for the average JX conformation using restraints representing the translational restriction due to micelle-surface adsorption, and the helix orientations were determined from residual dipolar couplings. Two equivalent average structural models were obtained that differ only in the relative orientation between first and second helices. In these models, the 658LL659 and 679LL680 motifs are located in the first and second helices and face the micelle surface, whereas the PXXP motif is located in a flexible helix-connecting region. The data suggest that the activity of these signals may be regulated by their membrane association and restricted accessibility in the intact receptor.

PEA3 transcription factors are expressed in tissues undergoing branching morphogenesis and promote formation of duct-like structures by mammary epithelial cells in vitro

The genetic program that controls reciprocal tissue interactions during epithelial organogenesis is still poorly understood. Erm, Er81 and Pea3 are three highly related transcription factors belonging to the Ets family, within which they form the PEA3 group. Little information is yet available regarding the function of these transcription factors. We have previously used in situ hybridization to compare their expression pattern during critical stages of murine embryogenesis [Oncogene 15 (1997), 937; Mech. Dev. 108 (2001), 191]. In this study, we have examined the expression of PEA3 group members during organogenesis of the lung, salivary gland, kidney, and mammary gland. In all of these developmental settings, we observed a tight correlation between branching morphogenesis and the expression of specific members of the PEA3 group. To assess the functional relevance of these findings, Erm and Pea3 were overexpressed in the TAC-2.1 mammary epithelial cell line, which has the ability to form branching duct-like structures when grown in collagen gels. We found that overexpression of Erm and Pea3 markedly enhances branching tubulogenesis of TAC-2.1 cells and also promotes their invasion into a collagen matrix. Collectively, these findings suggest that the differential expression of PEA3 group transcription factors has an important role in the regulation of branching morphogenesis and raise the question of their implication in branching signaling.

Grainy head controls apical membrane growth and tube elongation in response to Branchless/FGF signalling.

Epithelial organogenesis involves concerted movements and growth of distinct subcellular compartments. We show that apical membrane enlargement is critical for lumenal elongation of the Drosophila airways, and is independently controlled by the transcription factor Grainy head. Apical membrane overgrowth in grainy head mutants generates branches that are too long and tortuous without affecting epithelial integrity, whereas Grainy head overexpression limits lumenal growth. The chemoattractant Branchless/FGF induces tube outgrowth, and we find that it upregulates Grainy head activity post-translationally, thereby controlling apical membrane expansion to attain its key role in branching. We favour a two-step model for FGF in branching: first, induction of cell movement and apical membrane growth, and second, activation of Grainy head to limit lumen elongation, ensuring that branches reach and attain their characteristic lengths.

Patterning through Differential Endoreduplication in Epithelial Organogenesis of the Chordate, Oikopleura dioica

The contributions that control of cell proliferation and cell growth make to developmental regulation of organ and body size remain poorly explored, particularly with respect to endocycles in polyploid tissues. The epithelium of the marine chordate Oikopleura dioica is composed of a fixed number of cells grouped in territories according to gene-specific expression and nuclear sizes and shapes. As the animal grows 10-fold during the life cycle, epithelial cells increase in size differentially as a function of their spatial position. We show that this cellular pattern reflected differences in ploidy levels ranging from 34 to 1300 C. The diverse ploidy levels in defined cellular fields resulted both from different timing of entry into endocycles and from cell-specific regulation of endocycle lengths. Throughout the life cycle, differential cell size and ploidy increases were accompanied by field-specific profiles of progressive reductions in G-phase duration. Endocycles were asynchronous among cells of a given epithelial territory, but at the resolution of individual cells, both DNA replication timing and ploidy levels were bilaterally symmetric. The transparent, accessible, oikoplastic epithelium is a model of choice for the study of endoreduplication in the context of pattern formation and growth.

Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis.

Branching morphogenesis is central to epithelial organogenesis. In the developing kidney, the epithelial ureteric bud invades the metanephric mesenchyme, which directs the ureteric bud to undergo repeated branching. A soluble factor(s) in the conditioned medium of a metanephric mesenchyme cell line is essential for multiple branching morphogenesis of the isolated ureteric bud. The identity of this factor had proved elusive, but it appeared distinct from factors such as HGF and EGF receptor ligands that have been previously implicated in branching morphogenesis of mature epithelial cell lines. Using sequential column chromatography, we have now purified to apparent homogeneity an 18 kDa protein, pleiotrophin, from the conditioned medium of a metanephric mesenchyme cell line that induces isolated ureteric bud branching morphogenesis in the presence of glial cell-derived neurotrophic factor. Pleiotrophin alone was also found to induce the formation of branching tubules in an immortalized ureteric bud cell line cultured three-dimensionally in an extracellular matrix gel. Consistent with an important role in ureteric bud morphogenesis during kidney development, pleiotrophin was found to localize to the basement membrane of the developing ureteric bud in the embryonic kidney. We suggest that pleiotrophin could act as a key mesenchymally derived factor regulating branching morphogenesis of the ureteric bud and perhaps other embryonic epithelial structures.

A role for Timeless in epithelial morphogenesis during kidney development.

Central to the process of epithelial organogenesis is branching morphogenesis into tubules and ducts. In the kidney, this can be modeled by a very simple system consisting of isolated ureteric bud (UB) cells, which undergo branching morphogenesis in response to soluble factors present in the conditioned medium of a metanephric mesenchyme cell line. By employing a targeted screen to identify transcription factors involved early in the morphogenetic program leading to UB branching, we identified the mammalian ortholog of Timeless (mTim) as a potential immediate early gene (IEG) important in this process. In the embryo, mTim was found to be expressed in patterns very suggestive of a role in epithelial organogenesis with high levels of expression in the developing lung, liver, and kidney, as well as neuroepithelium. In the embryonic kidney, the expression of mTim was maximal in regions of active UB branching, and a shift from the large isoform of mTim to a smaller isoform occurred as the kidney developed. Selective down-regulation of mTim resulted in profound inhibition of embryonic kidney growth and UB morphogenesis in organ culture. A direct effect on the branching UB was supported by the observation that down-regulation of mTim in the isolated UB (cultured in the absence of mesenchyme) resulted in marked inhibition of morphogenesis, suggesting a key role for Tim in the epithelial cell morphogenetic pathway leading to the formation of branching tubules.

Expression of the MUC 6 mucin gene in development of the human kidney and male genital ducts.

The MUC 6 mucin cDNA was isolated from a human stomach cDNA library and has been shown to be expressed in a number of other tissues in the gastrointestinal tract, including the gallbladder, pancreas, and parts of the ileum and colon. Here we establish that MUC 6 is expressed transiently in the nephrogenic zone of the kidney in the early mid-trimester of development. MUC 6 transcripts were detected in the epithelium of ureteric buds at 13 weeks and at lower levels from 17 to 23 weeks of gestation. Traces of MUC 6 mRNA were seen in the collecting ducts but not elsewhere in the developing kidney, and MUC 6 glycoprotein was detected in the epithelium of ureteric buds and collecting ducts. MUC 6 transcripts were absent from adult kidney. This pattern of expression of MUC 6 in the developing kidney suggests a role in epithelial organogenesis. MUC 6 transcripts were also present at low levels in mid-trimester epididymal epithelium.

Delayed Mammary Tumor Progression in Muc-1 Null Mice

The mucin gene, Muc-1, encodes a high molecular weight integral membrane glycoprotein that is present on the apical surface of most simple secretory epithelial cells. Muc-1 is highly expressed and aberrantly glycosylated by most carcinomas and metastatic lesions. Numerous functions have been proposed for this molecule, including protection of the epithelial cell surface, an involvement in epithelial organogenesis, and a role in tumor progression. Mice deficient in Muc-1 were generated using homologous recombination in embryonic stem cells. These mice appeared to develop normally and were healthy and fertile. However, the growth rate of primary breast tumors induced by polyoma middle T antigen was found to be significantly slower in Muc-1 deficient mice. This suggests that Muc-1 plays an important role in the progression of mammary carcinoma.