Salivary Gland Development in Culture.

Salivary glands are branching organs which develop by bud and cleft formation to create an organ with a large surface area. The epithelium and mesenchyme signal back and forth to control this branching process, with additional cues provided by the parasympathetic nerves and blood vessels that surround the developing branches. This branching morphogenesis can be recapitulated successfully in organ culture , allowing access to the tissue to follow development and manipulate the tissue interactions, and signals. To culture glands, the filter-grid method has been widely used, allowing the development of salivary glands cultured as a whole organ, or the gland epithelium in isolation, or with the surrounding craniofacial tissue in a cranial slice. Here, we describe the methods for each approach and show the applicability of culturing glands from a wide variety of species: mouse , snake, and human. The resulting samples and data from these cultures can be employed for morphological and molecular analysis, with some examples described in this chapter, bringing valuable knowledge to our understanding of branching morphogenesis.

Comparing development and regeneration in the submandibular gland highlights distinct mechanisms.

A common question in organ regeneration is the extent to which regeneration recapitulates embryonic development. To investigate this concept, we compared the expression of two highly interlinked and essential genes for salivary gland development, Sox9 and Fgf10, during submandibular gland development, homeostasis and regeneration. Salivary gland duct ligation/deligation model was used as a regenerative model. Fgf10 and Sox9 expression changed during regeneration compared to homeostasis, suggesting that these key developmental genes play important roles during regeneration, however, significantly both displayed different patterns of expression in the regenerating gland compared to the developing gland. Regenerating glands, which during homeostasis had very few weakly expressing Sox9-positive cells in the striated/granular ducts, displayed elevated expression of Sox9 within these ducts. This pattern is in contrast to embryonic development, where Sox9 expression was absent in the proximally developing ducts. However, similar to the elevated expression at the distal tip of the epithelium in developing salivary glands, regenerating glands displayed elevated expression in a subpopulation of acinar cells, which during homeostasis expressed Sox9 at lower levels. A shift in expression of Fgf10 was observed from a widespread mesenchymal pattern during organogenesis to a more limited and predominantly epithelial pattern during homeostasis in the adult. This restricted expression in epithelial cells was maintained during regeneration, with no clear upregulation in the surrounding mesenchyme, as might be expected if regeneration recapitulated development. As both Fgf10 and Sox9 were upregulated in proximal ducts during regeneration, this suggests that the positive regulation of Sox9 by Fgf10, essential during development, is partially reawakened during regeneration using this model. Together these data suggest that developmentally important genes play a key role in salivary gland regeneration but do not precisely mimic the roles observed during development.

Inhibition of Aurora Kinase B activity disrupts development and differentiation of salivary glands.

Little is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB) during development, using embryonic salivary glands (E-SGs) as a model. AURKB is a serine/threonine kinase that regulates key events in mitosis, which makes it an attractive target for tailored anticancer therapy. Many reports have elaborated on the role of AURKB in neoplasia and cancer; however, no previous study has shown its role during organ development. Our previous experiments have highlighted the essential requirement for AURKB during adult exocrine regeneration. To investigate if AURKB is similarly required for progression during embryonic development, we pharmacologically inhibited AURKB in developing submandibular glands (SMGs) at embryonic day (E)13.5 and E16.5, using the highly potent and selective drug Barasertib. Inhibition of AURKB interfered with the expansion of the embryonic buds. Interestingly, this effect on SMG development was also seen when the mature explants (E16.5) were incubated for 24 h with another cell cycle inhibitor Aphidicolin. Barasertib prompted apoptosis, DNA damage and senescence, the markers of which (cleaved caspase 3, γH2AX, SA-ßgal and p21, respectively), were predominantly seen in the developing buds. In addition to a reduction in cell cycling and proliferation of the epithelial cells in response to AURKB inhibition, Barasertib treatment led to an excessive generation of reactive oxygen species (ROS) that resulted in downregulation of the acinar differentiation marker Mist1. Importantly, inhibition of ROS was able to rescue this loss of identity, with Mist1 expression maintained despite loss of AURKB. Together, these data identify AURKB as a key molecule in supporting embryonic development and differentiation, while inhibiting senescence-inducing signals during organogenesis.

Development of the Vestibular Lamina in Human Embryos: Morphogenesis and Vestibule Formation.

The vestibular lamina (VL) is a transient developmental structure that forms the lip furrow, creating a gap between the lips/cheeks and teeth (oral vestibule). Surprisingly, little is known about the development of the VL and its relationship to the adjacent dental lamina (DL), which forms the teeth. In some congenital disorders, such as Ellis-van Creveld (EVC) syndrome, development of the VL is disrupted and multiple supernumerary frenula form, physically linking the lips and teeth. Here, we assess the normal development of the VL in human embryos from 6.5 (CS19) to 13 weeks of development, showing the close relationship between the VL and DL, from initiation to differentiation. In the anterior lower region, the two structures arise from the same epithelial thickening. The VL then undergoes complex morphogenetic changes during development, forming a branched structure that separates to create the vestibule. Changing expression of keratins highlight the differentiation patterns in the VL, with fissure formation linked to the onset of filaggrin. Apoptosis is involved in removal of the central portion of the VL to create a broad furrow between the future cheek and gum. This research forms an essential base to further explore developmental defects in this part of the oral cavity.

FGF10 is an essential regulator of tracheal submucosal gland morphogenesis.

Mucus secretion and mucociliary clearance are crucial processes required to maintain pulmonary homeostasis. In the trachea and nasal passages, mucus is secreted by submucosal glands (SMGs) that line the airway, with an additional contribution from goblet cells of the surface airway epithelium. The SMG mucus is rich in mucins and antimicrobial enzymes. Defective tracheal SMGs contribute to hyper-secretory respiratory diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease, however little is known about the signals that regulate their morphogenesis and patterning. Here, we show that Fgf10 is essential for the normal development of murine tracheal SMGs, with gland development arresting at the early bud stage in the absence of FGF10 signalling. As Fgf10 knockout mice are lethal at birth, inducible knockdown of Fgf10 at late embryonic stages was used to follow postnatal gland formation, confirming the essential role of FGF10 in SMG development. In heterozygous Fgf10 mice the tracheal glands formed but with altered morphology and restricted distribution. The reduction in SMG branching in Fgf10 heterozygous mice was not rescued with time and resulted in a reduction in overall tracheal mucus secretion. Fgf10 is therefore a key signal in SMG development, influencing both the number of glands and extent of branching morphogenesis, and is likely, therefore, to play a role in aspects of SMG-dependent respiratory health.

Mapping the distribution of stem/progenitor cells across the mouse middle ear during homeostasis and inflammation.

The middle ear epithelium is derived from neural crest and endoderm, which line distinct regions of the middle ear cavity. Here, we investigate the distribution of putative stem cell markers in the middle ear, combined with an analysis of the location of label-retaining cells (LRCs) to create a map of the middle ear mucosa. We show that proliferating cells and LRCs were associated with specific regions of the ear epithelium, concentrated in the hypotympanum at the base of the auditory bulla and around the ear drum. Sox2 was widely expressed in the endodermally derived ciliated pseudostratified epithelium of the hypotympanum. This part of the middle ear showed high levels of Wnt activity, as indicated by the expression of Axin2, a readout of Wnt signalling. Keratin 5 showed a more restricted expression within the basal cells of this region, with very little overlap between the Sox2- and keratin 5-positive epithelium, indicating that these genes mark distinct populations. Little expression of Sox2 or keratin 5 was observed in the neural crest-derived middle ear epithelium that lined the promontory, except in cases of otitis media when this epithelium underwent hyperplasia. This study lays the foundation for furthering our understanding of homeostasis and repair in the middle ear.

Multiple Cranial Organ Defects after Conditionally Knocking Out Fgf10 in the Neural Crest.

Fgf10 is necessary for the development of a number of organs that fail to develop or are reduced in size in the null mutant. Here we have knocked out Fgf10 specifically in the neural crest driven by Wnt1cre. The Wnt1creFgf10fl/fl mouse phenocopies many of the null mutant defects, including cleft palate, loss of salivary glands, and ocular glands, highlighting the neural crest origin of the Fgf10 expressing mesenchyme surrounding these organs. In contrast tissues such as the limbs and lungs, where Fgf10 is expressed by the surrounding mesoderm, were unaffected, as was the pituitary gland where Fgf10 is expressed by the neuroepithelium. The circumvallate papilla of the tongue formed but was hypoplastic in the conditional and Fgf10 null embryos, suggesting that other sources of FGF can compensate in development of this structure. The tracheal cartilage rings showed normal patterning in the conditional knockout, indicating that the source of Fgf10 for this tissue is mesodermal, which was confirmed using Wnt1cre-dtTom to lineage trace the boundary of the neural crest in this region. The thyroid, thymus, and parathyroid glands surrounding the trachea were present but hypoplastic in the conditional mutant, indicating that a neighboring source of mesodermal Fgf10 might be able to partially compensate for loss of neural crest derived Fgf10.

Apoptosis-associated protein expression in human salivary gland morphogenesis.

OBJECTIVE: Salivary gland (SG) development is based on branching morphogenesis, in which programmed cell death has been proposed to play a role in cell signalling and organ shaping. In the mouse salivary gland apoptosis has been suggested to play a key role in lumen formation, removing the central cells of the epithelial stalks. Here we analyse the expression of several anti- and pro-regulators of apoptosis during human SG development in a range of developmental stages. DESIGN: Foetal SGs obtained from the University of São Paulo were analysed by immunohistochemistry to assess the expression of apoptosis-associated proteins: caspases (caspase-6, -7, -9 and cleaved caspase-3), Bcl-2 family members (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x and Bcl-xL), Survivin (BIRC5), Cytochrome C and Apaf-1. RESULTS: Nuclear expression of Bax and Bak was identified in presumptive luminal areas at initial stages, while Bcl-xL showed the most relevant anti-apoptotic activity. Caspase-6, -7 and -9 were expressed during all stages, while interestingly cleaved caspase-3 showed no prominent expression, indicating that caspase-7 is the main effector. Apoptosome complex components Apaf-1 and Cytochrome C, as well as survivin were all positive in developing glands. CONCLUSIONS: The particular expression pattern of several apoptotic regulators in human SG development suggests the existence of a fundamental role for apoptosis during duct formation. The absence of Bad and Bid expressions indicates that the instrinsic pathway is more active then the extrinsic during human gland formation. The subcellular localisation of intrinsic-apoptosis proteins correlated with apoptotic activity, but also suggested additional non-apoptotic functions.

Development of human minor salivary glands: expression of mucins according to stage of morphogenesis.

The formation of salivary glands entails the proliferation of epithelial cells from the stomatodeum into the underlying ectomesenchyme, culminating in a complex network of ducts and acinar bulbs. The extent to which mucins regulate this process is unknown, but they appear to mediate luminal space formation and maturation. Our aim was to examine mucin expression patterns during the morphogenesis of human salivary glands. Mucin expression - MUC1, 2, 3, 4, 5AC, 5B, 6, and 16 - was analyzed in specimens of developing human salivary glands, obtained from fetuses at 4-24 weeks' gestation, and fully developed salivary glands by immunohistochemistry. Expression patterns were analyzed qualitatively according to the development stage of the salivary glands. Mucins 1, 3, 4, 5B, and 16 were expressed during salivary gland development - being stronger in all ductal segments by the final phases of branching morphogenesis and in mature glands. Acinar cells were negative for most mucins, including MUC1 in mature salivary glands. Mucins 2, 5AC, and 6 were not expressed. Mucins MUC1, 3, 4, 5B, and 16 are expressed in developing human salivary glands and in mature glands, suggesting important roles in the maturation and maintenance of the ductal network.