Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

University of California San Francisco

Nucleic acid aptamers are finding increasing applications in biology, especially as therapeutic candidates and diagnostic components. An important characteristic in meeting the needs of these applications is improved stability in physiological fluids, which is most often accomplished with chemical modification or unnatural nucleotides. In an alternative approach we have specified the design of a multivalent circular DNA aptamer topology that encompasses a number of properties relevant to nucleic acid therapeutic candidates, especially the ability to multitask by combining different activities together within a modular structure. Improved stability in blood products, greater conformational stability, antidoting by complementary circular antiaptamers, heterovalency, transcription factor decoy activity and minimal unintended effects upon the cellular innate immune response are desirable properties that are described here. Multitasking by circular DNA aptamers could similarly find applications in diagnostics and biomaterials, where the combination of interchangeable modules might generate new functions, such as anticoagulation coupled with reversible cell capture as, described here. These results provide a platform for further exploration of multivalent circular aptamer properties, especially in novel combinations of nucleic acid therapeutic modes.

Multitasking by multivalent circular DNA aptamers.

Division abnormally delayed (Dally) is one of two glycosylphosphatidylinositol (GPI)-linked heparan sulfate proteoglycans in Drosophila. Numerous studies have shown that it influences Decapentaplegic (Dpp) and Wingless signaling. It has been generally assumed that Dally affects signaling by directly interacting with these growth factors, primarily through its heparan sulfate (HS) chains. To understand the functional contributions of HS chains and protein core we have (1) assessed the growth factor binding properties of purified Dally using surface plasmon resonance, (2) generated a form of Dally that is not HS modified and evaluated its signaling capacity in vivo. Purified Dally binds directly to FGF2, FGF10, and the functional Dpp homolog BMP4. FGF binding is abolished by preincubation with HS, but BMP4 association is partially HS-resistant, suggesting the Dally protein core contributes to binding. Cell binding and co-immunoprecipitation studies suggest that non-HS-modified Dally retains some ability to bind Dpp or BMP4. Expression of HS-deficient Dally in vivo showed it does not promote signaling as well as wild-type Dally, yet it can rescue several dally mutant phenotypes. These data reveal that heparan sulfate modification of Dally is not required for all in vivo activities and that significant functional capacity resides in the protein core.

The function of a Drosophila glypican does not depend entirely on heparan sulfate modification.

Heparan sulfate proteoglycans are essential for biological processes regulated by fibroblast growth factors (FGFs). Heparan sulfate (HS) regulates the activity of FGFs by acting as a coreceptor at the cell surface, enhancing FGF-FGFR affinity, and being a storage reservoir for FGFs in the extracellular matrix (ECM). Here we demonstrate a critical role for heparanase during mouse submandibular gland (SMG) branching morphogenesis. Heparanase, an endoglycosidase, colocalized with perlecan in the basement membrane and in epithelial clefts of SMGs. Inhibition of heparanase activity in organ culture decreased branching morphogenesis, and this inhibition was rescued specifically by FGF10 and not by other FGFs. By contrast, exogenous heparanase increased SMG branching and MAPK signaling and, surprisingly, when isolated epithelia were cultured in a three-dimensional ECM with FGF10, it increased the number of lateral branches and end buds. In a solid-phase binding assay, an FGF10-FGFR2b complex was released from the ECM by heparanase. In addition, surface plasmon resonance (SPR) analysis showed that FGF10 and the FGF10-FGFR2b complex bound to purified perlecan HS and could be released by heparanase. We used the FGF10-FGFR2b complex as a probe for HS in SMGs, and it colocalized with perlecan in the basement membrane and partly colocalized with syndecan 1 in the epithelium, and binding was reduced by treatment with heparanase. In summary, our results show heparanase releases FGF10 from perlecan HS in the basement membrane, increasing MAPK signaling, epithelial clefting, and lateral branch formation, which results in increased branching morphogenesis.

Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis.

Perlecan is a large multidomain proteoglycan that is essential for normal cartilage development. In this study, perlecan was localized in the pericellular matrix of hypertrophic chondrocytes in developing human cartilage rudiments. Perlecan immunopurified from medium conditioned by cultured human fetal chondrocytes was found to be substituted with heparan sulfate (HS), chondroitin sulfate (CS), and keratan sulfate (KS). Ligand and carbohydrate engagement (LACE) assays demonstrated that immunopurified chondrocyte-derived perlecan formed HS-dependent ternary complexes with fibroblast growth factor (FGF) 2 and either FGF receptors (FGFRs) 1 or 3; however, these complexes were not biologically active in the BaF32 cell system. Chondrocyte-derived perlecan also formed HS-dependent ternary complexes with FGF18 and FGFR3. The proliferation of BaF32 cells expressing FGFR3 was promoted by chondrocyte-derived perlecan in the presence of FGF18, and this activity was reduced by digestion of the HS with either heparinase III or mammalian heparanase. These data suggest that FGF2 and -18 bind to discrete structures on the HS chains attached to chondrocyte-derived perlecan which modulate the growth factor activities. The presence and activity of mammalian heparanase may be important in the turnover of HS and subsequent signaling required for the establishment and maintenance of functional osteo-chondral junctions in long bone growth.

Heparan sulfate-dependent signaling of fibroblast growth factor 18 by chondrocyte-derived perlecan.

Parasympathetic nerves are a vital component of the progenitor cell niche during development, maintaining a pool of progenitors for organogenesis. Injured adult organs do not regenerate after parasympathectomy, and there are few treatments to improve organ regeneration, particularly after damage by therapeutic irradiation. Here we show that restoring parasympathetic function with the neurotrophic factor neurturin increases epithelial organ regeneration after damage. We use mouse salivary gland explant culture containing fluorescently labelled progenitors, and injure the tissue with irradiation. The progenitors survive, parasympathetic function is diminished and epithelial apoptosis reduces the expression of neurturin, which increases neuronal apoptosis. Treatment with neurturin reduces neuronal apoptosis, restores parasympathetic function and increases epithelial regeneration. Furthermore, adult human salivary glands damaged by irradiation also have reduced parasympathetic innervation. We propose that neurturin will protect the parasympathetic nerves from damage and improve organ regeneration. This concept may be applicable for other organs where parasympathetic innervation influences their function.

Parasympathetic stimulation improves epithelial organ regeneration.

Nerfs parasympathiques sont une composante essentielle de la niche de cellules souches au cours du d&eacute;veloppement, le maintien d&#39;un pool de g&eacute;niteurs de l&#39;organogen&egrave;se. Organes adultes bless&eacute;s ne se r&eacute;g&eacute;n&egrave;rent pas apr&egrave;s parasympathectomy, et il existe peu de traitements pour am&eacute;liorer la r&eacute;g&eacute;n&eacute;ration de l&#39;orgue, particuli&egrave;rement apr&egrave;s les dommages caus&eacute;s par l&#39;irradiation th&eacute;rapeutique. Nous d&eacute;montrons que retour fonction parasympathique avec le neurturine de facteur neurotrophique augmente la r&eacute;g&eacute;n&eacute;ration &eacute;pith&eacute;liale orgue apr&egrave;s avarie. Nous utiliser souris glande salivaire explant culture contenant des prog&eacute;niteurs fluorescent &eacute;tiquet&eacute;s et blesser les tissus avec l&#39;irradiation. Les anc&ecirc;tres de survivre, fonction parasympathique est diminu&eacute;e et l&#39;apoptose &eacute;pith&eacute;liale r&eacute;duit l&#39;expression de neurturine, ce qui augmente l&#39;apoptose neuronale. Traitement avec neurturine r&eacute;duit l&#39;apoptose neuronale, restaure la fonction parasympathique et augmente la r&eacute;g&eacute;n&eacute;ration &eacute;pith&eacute;liale. En outre, adultes humains des glandes salivaires, endommag&eacute;s par une irradiation ont &eacute;galement r&eacute;duit innervation parasympathique. Nous vous proposons cette neurturine va prot&eacute;ger les nerfs parasympathiques de dommages et am&eacute;liorer la r&eacute;g&eacute;n&eacute;ration de l&#39;orgue. Ce concept peut s&#39;appliquer pour les autres organes o&ugrave; l&#39;innervation parasympathique influe sur leur fonction.

La stimulation parasympathique am&eacute;liore la r&eacute;g&eacute;n&eacute;ration &eacute;pith&eacute;liale orgue.

Nervi parasimpatici sono una componente vitale della nicchia delle cellule progenitrici durante lo sviluppo, il mantenimento di un pool di progenitori per organogenesi. Organi adulti feriti non rigenerare dopo parasympathectomy, e ci sono alcuni trattamenti per migliorare la rigenerazione di organi, particolarmente dopo danno da irradiazione terapeutica. Qui vi mostriamo che ripristino funzione parasimpatico con il neurturin di fattore neurotrophic aumenta la rigenerazione epiteliale organo dopo danni. Noi utilizzare mouse delle ghiandole salivari espianto cultura contenente progenitori fluorescente coniugati e ferire il tessuto con irradiazione. I progenitori di sopravvivere, &egrave; diminuita funzione parasimpatico e apoptosi epiteliale riduce l&#39;espressione di neurturin, che aumenta l&#39;apoptosi neuronale. Trattamento con neurturin riduce l&#39;apoptosi neuronale, ripristina la funzione parasimpatico e aumenta la rigenerazione epiteliale. Inoltre, adulti umani ghiandole salivari danneggiati dall&#39;irradiazione hanno anche ridotto innervazione parasimpatico. Proponiamo che neurturin sar&agrave; proteggere i nervi parasimpatici da danni e migliorare la rigenerazione di organi. Questo concetto pu&ograve; essere applicabile per altri organi dove innervazione parasimpatico influenza la loro funzione.

Stimolazione parasimpatico migliora la rigenerazione epiteliale organo.

Our understanding of vertebrate salivary gland organogenesis has been largely informed by the study of the developing mouse submandibular gland (SMG), which will be the major focus of this review. The mouse SMG has been historically used as a model system to study epithelial-mesenchymal interactions, growth factor-extracellular matrix (ECM) interactions, and branching morphogenesis. SMG organogenesis involves interactions between a variety of cell types and their stem/progenitor cells, including the epithelial, neuronal, and mesenchymal cells, and their ECM microenvironment, or niche. Here, we will review recent literature that provides conceptual advances in understanding the molecular mechanisms of salivary gland development. We will describe SMG organogenesis, introduce the model systems used to study development, and outline the key signaling pathways and cellular processes involved. We will also review recent research focusing on the identification of stem/progenitor cells in the SMG and how they are directed along a series of cell fate decisions to form a functional gland. The mechanisms that drive SMG organogenesis provide a template to regenerate functional salivary glands in patients who suffer from salivary hypofunction due to irreversible glandular damage after irradiation or removal of tumors. Additionally, these mechanisms may also control growth and development of other organ systems.

Salivary gland organogenesis.

A fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs, tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to form a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate tubulogenesis in the developing salivary gland. We show that vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(-) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organogenesis.

Article	Year
Multitasking by multivalent circular DNA aptamers. Chembiochem : a European journal of chemical biology\| PubMed ID: 16482500	2006
The function of a Drosophila glypican does not depend entirely on heparan sulfate modification. Developmental biology\| PubMed ID: 17055473	2006
Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis. Development (Cambridge, England)\| PubMed ID: 17959718	2007
Heparan sulfate-dependent signaling of fibroblast growth factor 18 by chondrocyte-derived perlecan. Biochemistry\| PubMed ID: 20507176	2010
Parasympathetic stimulation improves epithelial organ regeneration. Nature communications\| PubMed ID: 23422662	2013
Salivary gland organogenesis. Wiley interdisciplinary reviews. Developmental biology\| PubMed ID: 23801668	2012
Parasympathetic innervation regulates tubulogenesis in the developing salivary gland. Developmental cell\| PubMed ID: 25158854	2014

Sarah M. Knox

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