Extensive Branching Morphogenesis Research Articles

Morphogenesis of the kidney and lung requires branch-tip directed activity of the Adamts18 metalloprotease

Adamts18 encodes a secreted metalloprotease restricted to branch-tip progenitor pools directing the morphogenesis of multiple mammalian organs. Adamts18 was targeted to explore a potential role in branching morphogenesis. In the kidney, an arborized collecting system develops through extensive branching morphogenesis of an initial epithelial outgrowth of the mesonephric duct, the ureteric bud. Adamts18 mutants displayed a weakly penetrant phenotype: duplicated ureteric outgrowths forming enlarged, bi-lobed kidneys with an increased nephron endowment. In contrast, Adamts18 mutants showed a fully penetrant lung phenotype: epithelial growth was markedly reduced and early secondary branching scaled to the reduced length of the primary airways. Furthermore, there was a pronounced delay in the appearance of differentiated cell types in both proximal and distally positions of the developing airways. Adamts18 is closely related to Adamts16. In the kidney but not the lung, broad epithelial Adamts16 expression overlaps Adamts18 in branch tips. However, compound Adamts16/18 mutants displayed a comparable low penetrance duplicated ureteric phenotype, ruling out a possible role for Adamts16 as a functional modifier of the Adamts18 kidney phenotype. Given the predicted action of secreted Adamts18 metalloprotease, and broad expression of Adamts18 in branching organ systems, these findings suggest distinct requirements for matrix modelling in the morphogenesis of epithelial networks.

Role of the mTOR signalling pathway in salivary gland development.

Development of the salivary gland is characterized by extensive branching morphogenesis. Although various molecules have been implicated in salivary gland development, the role of the mammalian target of rapamycin (mTOR) signalling pathway, including both mTOR complexes 1 and 2 (mTORC1 and 2), in salivary gland development is unknown. Here, we examined protein expression levels related to the mTOR signalling pathway using an exvivo submandibular salivary gland (SMG) organ culture. We showed that branching buds in the salivary glands were substantially decreased and phosphorylation of mTORC1 signalling pathway related proteins (mTOR, p70 ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1) was inhibited by rapamycin (an mTOR inhibitor). In addition, AKT, which is an upstream protein kinase of mTORC1 and is downstream of mTORC2, is inhibited by LY294002 (a phosphatidylinositol 3-kinase inhibitor), but not by rapamycin. Moreover, rapamycin-treated ICR neonatal mice exhibited a reduction in both body weight and salivary glands compared with vehicle-treated neonatal mice. The present data indicate that the mTOR signalling pathway, including both mTORC1 and mTORC2, plays a critical role in salivary gland development both in exvivo SMG organ culture and ICR neonatal mice invivo.

Grhl2 regulation of SPINT1 expression controls salivary gland development

Development of the salivary gland is characterized by extensive branching morphogenesis and lumen formation, the latter of which is closely associated with differentiation into acinar and ductal cells. Although various molecules, including signaling and cell adhesion molecules, have been implicated in salivary gland development, transcription factors (TFs) regulating the expression of those molecules and morphological development of the gland are largely unknown. Here we show that knockdown of the epithelial TF, Grainyhead-like 2 (Grhl2), with siRNA in developing mouse submandibular salivary gland (SMG) cultured ex vivo resulted in retardation of epithelial development. This retardation was concomitant with suppression of gene expression for the cell adhesion molecules, such as E-cadherin and the extracellular protease inhibitor SPINT1, and with the disorganized deposition of the basal lamina protein laminin. ChIP-PCR demonstrated the binding of Grhl2 protein to the Spint1 gene in the SMG. Notably, addition of recombinant SPINT1 protein in cultured SMG overcame the suppressive effects of Grhl2 siRNA on epithelial development and laminin deposition. These findings show that Grhl2 regulation of SPINT1 expression controls salivary gland development.

FGF7 signals are relayed to autocrine EGF family growth factors to induce branching morphogenesis of mouse salivary epithelium

The Matrigel-embedded epithelium of the mouse submandibular gland undergoes branching morphogenesis when cultured in medium supplemented with fibroblast growth factor 7 (FGF7) and lysophosphatidic acid (LPA), whereas it elongates a stalk with limited branching in medium with only FGF7. Because LPA is a well-known activator of epidermal growth factor (EGF) signaling, we hypothesized the involvement of autocrine EGF family growth factors in the branching morphogenesis. Reverse transcriptase polymerase chain reaction studies showed that three members, Tgfa, Hbegf,and Nrg1 of the EGF family were expressed in the epithelium cultured with FGF7 + LPA as well as in the epithelium freshly isolated from the rudiments. All the growth factors induced extensive branching morphogenesis in the Matrigel-embedded epithelium in the presence of LPA. Tyrphostin AG112, an inhibitor of EGF signaling, severely impaired branching morphogenesis induced by FGF7 + LPA without exogenous addition of EGF family growth factors to the culture medium. The shaking cultures, which were expected to decrease the concentration of autocrine growth factors near the epithelium by promoting their diffusion, significantly reduced branching morphogenesis induced by FGF7 + LPA. Autocrine EGF family growth factors are involved in epithelial branching morphogenesis induced by FGF7 + LPA.

Mammary ductal elongation and myoepithelial migration are regulated by the composition of the extracellular matrix.

Mammary branching morphogenesis occurs over a period of weeks deep inside an adipocyte-rich stroma. The adipocytes contain light-scattering lipid droplets that limit the depth of penetration of visible light. Organotypic culture methods were developed to enable high-resolution optical monitoring of branching morphogenesis ex vivo. A challenge has been to identify the best culture conditions to model specific developmental events. We recently demonstrated that collagen I induces protrusive invasion in both normal and neoplastic mammary epithelium. In this study, we observed that the abundance of collagen I fibrils correlated strongly with invasive behaviour, even when the collagen I concentration was identical. We found that the extent of fibril assembly was experimentally manipulable by varying the incubation time at 4°C following pH neutralization. We next tested the capacity of collagen I fibrils to induce invasive behaviour when presented in combination with basement membrane proteins (Matrigel). We found that epithelial organoids in mixed gels of collagen I and basement membrane proteins exhibited more extensive branching morphogenesis but did not initiate protrusions into the matrix. Organoids in pure Matrigel produced many small epithelial buds that were bare of myoepithelial cells. Surprisingly, organoids in mixed gels of collagen I and Matrigel produced fewer epithelial buds, the buds elongated further, and the elongating buds remained covered by myoepithelial cells. Our mixed gels therefore provide a more physiologically accurate model of mammary branching morphogenesis. Our results also suggest that changes in the composition of the extracellular matrix could induce migration of epithelial cells past myoepithelial coverage.

The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling

The maintenance of organs and their regeneration in case of injury are crucial to the survival of all animals. High rates of tissue turnover and nearly unlimited regenerative capabilities make planarian flatworms an ideal system with which to investigate these important processes, yet little is known about the cell biology and anatomy of their organs. Here we focus on the planarian excretory system, which consists of internal protonephridial tubules. We find that these assemble into complex branching patterns with a stereotyped succession of cell types along their length. Organ regeneration is likely to originate from a precursor structure arising in the blastema, which undergoes extensive branching morphogenesis. In an RNAi screen of signaling molecules, we identified an EGF receptor (Smed-EGFR-5) as a crucial regulator of branching morphogenesis and maintenance. Overall, our characterization of the planarian protonephridial system establishes a new paradigm for regenerative organogenesis and provides a platform for exploring its functional and evolutionary homologies with vertebrate excretory systems.

Abstract LB-39: Phenotypic switching and molecular anatomy of malignant progression (E-M-T/M-E-T).

Abstract Proliferation and invasion are two fundamental phenotypes required for malignancy while phenotypic switching is required for malignant progression. Thus, during malignant progression primary tumor cells switch from proliferative to invasive phenotypes and switch again to form proliferating metastases. The acquisition of the invasive phenotype through epithelial-mesenchymal-transition (E-M-T) is a crucial step in tumor progression, as is mesenchymal-epithelial-transition (M-E-T) or switching to become tumor metastases. Using a reductionist approach, we have been able to reproduce these steps through a combination of in vitro and in vivo methods: Beginning with prostate and ovarian carcinoma cells, we show that the majority are epithelial (E) cells, grow as acini in three dimensional (3D) Matrigel and are nonresponsive to hepatocyte growth factor/scatter factor (HGF/SF). These E cells grow in soft agar and are tumorigenic in immunocompromised mice. However, there is a second cell population which also grows as acini in 3D Matrigel, but display extensive branching morphogenesis when HGF/SF is present. These subclones are mesenchymal (M), are highly invasive in response to HGF/SF, but do not grow in soft agar and display low or no tumorigenicity in vivo. We show that M cells can originate in vitro from the non-invasive E subclones. However, M subclones undergo M-E-T only in vivo, at low frequency mimicking tumor progression to proliferating metastases. We show that E-M-T switching events occur with loss of E-Cadherin and the gain of c-MET expression. Expression of β-Catenin does not change with phenotypic switching, but with the loss of E-Cadherin, its localization shifts from the cell membrane to nuclear/cytoplasmic location, activating β-Catenin dependent expression. Moreover, depleting E-Cadherin in E cells triggers nuclear/cytoplasmic shift of β-Catenin and enhanced HGF/SF-c-Met signaling, while ectopic expression of E-Cadherin in M cells restores E phenotype and decreases HGF/SF-Met signaling. Importantly, we show that phenotypic switching occurs with distinct changes in karyotype that dictate changes in the chromosome transcriptome and the expression of genes that account for changes to M or to E phenotypes, thereby implicating chromosome instability as a key mechanism for phenotypic switching. We conclude that chromosome instability generates the transcriptome diversity in vitro or in vivo, which in the appropriate environment, leads to expansion of clonal variants responsible for phenotypic switching. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-39. doi:10.1158/1538-7445.AM2011-LB-39

An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.

Branching morphogenesis during development of placental villi

The placenta forms a complex interface between the mother and fetus during development that is designed for efficient nutrient exchange. A large surface area is created by extensive branching morphogenesis of the trophoblast-derived epithelium to create a villous network, called the labyrinth in rodents. These villi are subsequently vascularized with an elaborate capillary network. Morphogenesis begins with selection of a subset of trophoblast cells in the basal layer of the chorion that express the Gcm1 transcription factor. These cells leave the cell cycle and undergo cell shape changes that initiate a process of involution to create primary villi into which fetal blood vessels grow. Much less is known about the regulation of subsequent events in branching, certainly compared with other organs. However, over 60 different mouse mutants have defects during later labyrinth development. Some of these mutant genes encode components of signaling pathways such as the fibroblast growth factor and Wnt pathways that play evolutionarily conserved roles in other branched organs, These mutants represent a still largely untapped resource as most of them have not been studied in detail in relation to placental morphogenesis.

The von Hippel-Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells.

Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G(0)) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.

The role of growth factors in the development and growth of the prostate and seminal vesicle

Summary Mesenchymal-epithelial interactions play a critical role in the development of the male urogenital tract in that mesenchyme induces and specifies patterns of epithelial morphogenesis, regulates epithelial proliferation, promotes epithelial cytodifferentiation, and induces and specifies epithelial functional or biochemical activity. Prostatic epithelial growth and development is dependent upon androgens. Analysis of tissue recombinants prepared with normal (wild-type) urogenital sinus mesenchyme plus Tfm (testicular feminization) epithelium has demonstrated that androgen-induced prostatic epithelial growth and ductal branching morphogenesis are elicited via paracrine influences from the androgen-receptor-positive mesenchyme. Since KGF has recently emerged as a prototypic paracrine mediator of mesenchymal-epithelial interactions, androgen-dependent prostatic development was investigated to determine the possible role of KGF using a serum-free organ culture method. For this purpose newborn rat ventral prostates (VP) were incubated in vitro in a serum basal-free medium supplemented with testosterone (T: 10 −8 M), human recombinant KGF, or a neutralizing monoclonal antibody to KGF (anti-KGF), alone or in combination. When T (10 −8 M) was added to the basal media, VPs grew and underwent extensive branching morphogenesis similar to that in situ . The anti-KGF (60 μg/ml) inhibited T-induced VP growth (DNA content) by 33% and reduced ductal endbuds by 62% after 6 days of culture. Neonatal rat VPs grown without T exhibited reduced growth and branching morphogenesis in comparison to those grown with T. When KGF (50 ng/ml) was added to the T-deficient media, VP growth was stimulated to a level approaching that elicited by T. Ductal endbuds were elevated from 23 (without T) to 60 (without T + KGF) per expiant which represents 73% of that observed in cultures grown with T. Given the facts: a) that KGF is produced by mesenchymal/fibroblastic cells and acts on epithelia; b) that androgens stimulate development and growth of the prostate via androgen-receptor-mediated processes: and c) that androgen receptors (AR) are only detected in the mesenchyme of the rat VP at birth, our data suggests that androgens regulate epithelial growth and development of the VP by acting on the AR+ mesenchymal cells to elicit the synthesis and secretion of KGF which in turn stimulates epithelial growth and development.

Morphogenetic and Proliferative Effects of Testosterone and Insulin on the Neonatal Mouse Seminal Vesicle in Vitro*

Effects of testosterone (T) and insulin on epithelial branching morphogenesis were investigated in cultured seminal vesicles (SVs) of neonatal mice. SVs from 0-day-old male mice were cultured for 0.5-6 days in serum-free chemically defined medium in the presence of T (10(-7) M), insulin (10 micrograms/ml), or T plus insulin or in medium lacking both hormones. Without the addition of both hormones, SVs failed to grow, based on DNA and protein contents, and did not show any epithelial branching morphogenesis. T induced a 2.5-fold increase in protein content in SVs cultured for 6 days and elicited modest epithelial branching morphogenesis. Insulin increased the protein content of cultured SV rudiments as much as T, but failed to elicit epithelial branching morphogenesis. The combination of both hormones induced a 4.3-fold increase in protein content in cultured SVs and elicited more extensive epithelial branching morphogenesis than T alone. Epithelial and mesenchymal DNA contents in SVs declined slightly during the first 12 h of culture in all treatment groups. The epithelial DNA content in SVs grown with insulin alone remained constant thereafter, while that of SVs grown with T alone or in combination with insulin increased 1.2- and 2.6-fold, respectively. In the absence of both hormones, the mesenchymal DNA content remained constant in SVs grown for 6 days after the initial decline in DNA content. In contrast, mesenchymal DNA content was increased to the same degree (1.4-fold) by either T or insulin alone or in combination. The labeling index with [3H]thymidine of SV epithelium and mesenchyme grown under the hormonal conditions described above corroborated the results of epithelial and mesenchymal DNA contents. These data indicate that insulin by itself has no effect on epithelial proliferation and branching morphogenesis in the neonatal mouse SV, but, instead, amplifies the morphogenetic and proliferative effects of androgen on the developing mouse SV, thus eliciting extensive branching morphogenesis. Both insulin and T have a slight (nonsynergistic) effect on proliferation of SV mesenchyme. Analysis of androgen metabolism in developing mouse SVs indicated that dihydrotestosterone was the major product when T was used as substrate in SVs grown under all hormonal conditions. The rate of DHT production per 100 mg protein was not significantly different among the different treatment groups. Higher levels of 5 alpha-androstane-3 alpha,17 beta-diol were detected in SVs grown in the absence vs. the presence of T regardless of the presence or absence of insulin.(ABSTRACT TRUNCATED AT 400 WORDS)