During embryonic development, many organs form by extensive branching of epithelia through the formation of clefts and buds. In cleft formation, buds are delineated by the conversion of epithelial cell-cell adhesions to cell-matrix adhesions, but the mechanisms of cleft formation are not clear. Here we identify Btbd7 as a dynamic regulator of branching morphogenesis. Btbd7 provides a mechanistic link between the extracellular matrix and cleft propagation through its highly focal expression leading to local regulation of Snail2 (Slug), E-cadherin, and epithelial cell motility. Inhibition experiments show that Btbd7 is required for branching of embryonic mammalian salivary glands and lungs. Hence Btbd7 is a regulatory gene that promotes epithelial tissue remodelling and formation of branched organs.

A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. AbstrAct Salivary glands form during embryonic development by a complex process that creates compact, highly organized secretory organs with functions essential for oral health. The architecture of these glands is generated by branching morphogenesis, revealed by recent research to involve unexpectedly dynamic cell motility and novel regulatory pathways. Numerous growth factors, extracellular matrix molecules, gene regulatory pathways, and mechanical forces contribute to salivary gland morphogenesis, but local gene regulation and morphological changes appear to play particularly notable roles. Here we review these recent advances and their potential application to salivary gland tissue engineering.

Salivary glands provide saliva to maintain oral health, and a loss of salivary gland function substantially decreases quality-of-life. Understanding the biological mechanisms that generate salivary glands during embryonic development may identify novel ways to regenerate function or design artificial salivary glands. This review article summarizes current research on the process of branching morphogenesis of salivary glands, which creates gland structure during development. We highlight exciting new advances and opportunities in studies of cell-cell interactions, mechanical forces, growth factors, and gene expression patterns to improve our understanding of this important process.

Background Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized. Results We generated transgenic mice expressing photo-convertible KikGR and tracked the movements of individual cells highlighted by red fluorescence in different regions of developing salivary glands. Motility was highest for outer bud epithelial cells adjacent to the basement membrane, lower in inner bud cells, and lowest in duct cells. The highly motile outer cells contacting the basement membrane were pleomorphic, whereas inner cells were rounded. Peripheral cell motility was disrupted by antibodies inhibiting α6+β1 integrins and the non-muscle myosin II inhibitor blebbistatin. Inner bud cell migration was unaffected by these inhibitors, but their rate of migration was stimulated by inhibiting E-cadherin. Conclusions Cell motility in developing salivary glands was highest in cells in contact with the basement membrane. The basement membrane-associated motility of these outer bud cells depended on integrins and myosin II, but not E-cadherin. In contrast, motility of inner bud cells was restrained by E-cadherin. These findings identify the importance of integrin-dependent basement membrane association for the morphology, tissue organization, and lateral motility of morphogenetic epithelial cells.

During organ development, local changes in gene expression govern morphogenesis and cell fate. We have generated a microanatomical atlas of epithelial gene expression of embryonic salivary glands. The mouse submandibular salivary gland first appears as a single mass of epithelial cells surrounded by mesenchyme, and it undergoes rapid branching morphogenesis to form a complex secretory organ with acini connected to an extensive ductal system. Using laser capture microdissection, we collected samples from 14 distinct epithelial locations at embryonic days 12.5, 13.5, 14, and 15, and characterized their gene expression by microarray analysis. These microarray results were evaluated by qPCR of biological replicates and by comparisons of the gene expression dataset with published expression data. Using this gene expression atlas to search for novel regulators of branching morphogenesis, we found a substantial reduction in mRNA levels of GSK3β at the base of forming clefts. This unexpected finding was confirmed by immunostaining, and inhibition of GSK3β activity enhanced salivary gland branching. This first microanatomical expression atlas of a developing gland characterizes changes in local gene expression during salivary gland development and differentiation, which should facilitate the identification of key genes involved in tissue morphogenesis.

Background-Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized.

Human high molecular weight-melanoma associated Ag (HMW-MAA) mimics have been shown to elicit HMW-MAA-specific humoral immune responses that appear to be clinically beneficial. This finding has stimulated interest in characterizing the mechanism(s) underlying the ability of the elicited Abs to exert an anti-tumor effect. To address this question, in the present study, we have generated HMW-MAA-specific Abs by sequentially immunizing rabbits with the peptide P763.74, which mimics the HMW-MAA determinant recognized by mAb 763.74, and with HMW-MAA+ melanoma cells. HMW-MAA-specific Abs isolated from immunized rabbits mediated cell-dependent cytotoxicity but did not mediate complement-dependent cytotoxicity of HMW-MAA+ melanoma cells. These Abs also effectively inhibited spreading, migration and Matrigel invasion of HMW-MAA+ melanoma cells. Besides contributing to our understanding of the role of HMW-MAA in the biology of melanoma cells, these results suggest that both immunological and nonimmunological mechanisms underlie the beneficial clinical effects associated with the induction of HMW-MAA-specific Abs in melanoma patients immunized with a HMW-MAA mimic.

Peptide mimics isolated from phage display peptide libraries by panning with self-tumor-associated Ag (TAA)-specific mAbs are being evaluated as immunogens to implement active specific immunotherapy. Although TAA-specific mAb are commonly used to isolate peptide mimics, no information is available regarding the Ab characteristics required to isolate immunogenic TAA peptide mimics. To address this question, we have used mAb 763.74 and mAb GH786, which recognize the same or spatially close antigenic determinant(s) of the human high m.w.-melanoma-associated Ag (HMW-MAA), although with different affinity. mAb 763.74 affinity is higher than that of mAb GH786. Panning of phage display peptide libraries with mAb 763.74 and mAb GH786 resulted in the isolation of peptides P763.74 and PGH786, respectively. When compared for their ability to induce HMW-MAA-specific immune responses in BALB/c mice, HMW-MAA-specific Ab titers were significantly higher in mice immunized with P763.74 than in those immunized with PGH786. The HMW-MAA-specific Ab titers were markedly increased by a booster with HMW-MAA-bearing melanoma cells, an effect that was significantly higher in mice primed with P763.74 than in those primed with PGH786. Lastly, P763.74, but not PGH786, induced a delayed-type hypersensitivity response to HMW-MAA-bearing melanoma cells. These findings suggest that affinity for TAA is a variable to take into account when selecting mAb to isolate peptide mimics from a phage display peptide library.