DLK1 and DLK2 characterization in mouse salivary gland development

Abstract

Organogenesia seinalizazio bide ezberdinez gidatutako prozesu konplexua da. Askotan organu ezberdinen garapena seinalizazio faktore berdinez dago erregulatuta. Gainera, seinalizazio bideen ikerketa garapena ezagutarazteko erabiltzeaz aparte, minbizia eta ehunen birsorkuntza zelan gertatzen diren jakiteko ere balio digu.Lan honetan, listu-guruinaren garapena aztertu dugu. Listu-guruinaren organogenesia adrakatze morfogenesia deituriko prozesuaren bidez ematen da. Garapen prozesu hau beste organo batzuetan ere ematen da, esaterako, birikietan, giltzurrunetan eta ugatz guruinetan.NOTCH seinale bidea, garapenean dauden ehunetan, seinalerik aztertuenetako bat da, non orain dela gutxi deskribatu diren DLK1 eta DLK2 lotugai ez kanonikoak dauden. Gure lanaren helbururik nagusiena, DLK1en eta DLK2ren funtzioa ezagutzea izan zen. Batetik NOTCH seinalizazioan duten efektuak argituz eta bestetik baraila azpiko listu-guruinaren (SMG: submandibular salivary gland) garapenean jokatzen duen rola aztertuz. Hau egiteko, sagu enbrioien SMGen hazkunde organotipikoak egin genituen, sDLK1 edo DAPTrekin ( ¿-sekretasaren inibitzaile erreaktiboa). Bestalde, Dlk1 (-/-) saguarekin ere esperimentuak egin genituen. Gure emaitzek DLK1 eta DLK2 lotugaiak garapeneko listu-guruinaren maila altuetan adierazten direla erakutsi zuten. Aldiz, sagu helduko listu-guruinetan DLK1-2ren adierazpen mailak murriztu egiten dira, emaitza hauek DLK1-2 SMGaren morfogenesian parte hartu dezaketelaren ideia eman zigun. Bestalde, enbrioien SMG hazkuntzetan, sDLK1 edo DAPTrekin inhibituriko NOTCH seinale bideak, listu-guruinaren adarkatze morfogenesia kaltetzeaz aparte, inerbazioa murriztu eta azinoen barneko zelula progenitore epitelialen apoptosia eragiten zuen. NOTCH seinalearen inhibitzaileek SMGaren inerbazioa oztopatzen dutenez, karbakola (CCh) erabili genuen, inerbazio kolinergikoaren ordezko gisa bezala, modu honetan SMGaren garapena berreskuratzeko. CChak SMGaren adarkadura morfogenesia partzialki laguntzen zuela ikusi genuen, baina NOTCH seinale bidearen inhibizioa eteten zenean baino ez. Eraberean, konturatu ginen NOTCH inhibizioak sortutako morfogenesi akasduna ez zela bakarrik inerbazioa kaltetuaren ondorioz, isolatutako epitelioen morfogenesian DLK1ek eta DAPTk eragina zutela ikusi baigenuen.Dlk1 (-/-) saguaren listu-guruinen analisiarekin, DLK1en gabeziak listu-guruinaren tamaina murrizten duela ondorioztatu genuen. Gainera, pilokarpina bidezko estimulazioaren ondoren denboran neurtutako listu jariakina murriztuta dago sagu KOean. Sagu DLK1 KOaren listu-guruinen histologia eta TEMko argazkiek SMG normalak eta osasuntsuak aurkeztu zituzten, baina ama/progenitore zelulen populazioa handituta agertzen zen.Orokorrean, lan honek NOTCH seinalizazioko lotugai ez kanonikoek, DLK1 eta DLK2, saguen listu-guruinen garapenean duten garrantzia deskribatzen du. Ondorio gisa, DLK1en adierazpen mailak garapenean dauden guruinen ama/progenitore zelulen sorkuntzaren sustapen eta eragozpen arteko mekanismoaren oreka finean parte hartzen du. THESIS ABSTRACT:The organogenesis is a complex process orchestrated by different signaling pathways. The development of different organs is often regulated by the same signaling factors. The study of signaling pathways is not only to understand how organs develop, but also as a clue to investigate cancer and tissue regeneration, since these factors ale also implicated in these processes. In this work, we studied the salivary gland development. The salivary gland organogenesis occurs by a process called branching morphogenesis. This process also occurs in other organs such as mammary gland, lung and kidney. One of the most studied signaling pathways in organogenesis of developing tissues is NOTCH, where the non-canonical ligands DLK1 and DLK2 have been recently described. The main goal of our work was to elucidate the function of DLK1 and DLK2 in terms of NOTCH signaling and their role in the submandibular salivary gland (SMG) development. For this purpose, we used an in vitro organotypic culture of SMG rudiments with sDLK1 or DAPT (inhibitor reagent of ¿-secretase). For our study, we also performed experiments with null-Dlk1 mice. Our results demonstrate that these ligands are highly expressed in the development of the mouse SMG and function as NOTCH signaling inhibitors. In adult mice, DLK1-2 expression decrease, what make us think the possible role of DLK1-2 in the morphogenesis of the SMG. In SMG cultures, the inhibition of NOTCH signaling pathway, by either sDLK1 or DAPT, reduced SMG branching morphogenesis, impaired innervation and produced apoptosis in the inner epithelial progenitor cells of the developing end buds. As inhibitors of NOTCH disrupted SMG innervation, we employed a cholinergic activation reagent, carbachol (CCh), to rescue the development of the SMG. We found that CCh managed to partially recover the branching of the SMG, but only when the inhibitory effect ceased. In this context, we realized that the impaired SMG branching morphogenesis by NOTCH signaling inhibition was not only a consequence of the reduced innervation, but a DLK1 or DAPT direct effect on the SMG epithelia morphogenesis.From the analysis of the Dlk1 (-/-) mice, we concluded that DLK1 absence results in a reduced salivary gland size. As a consequence these mice produced less saliva in time after pilocarpine stimulation. DLK1-KO mice SMG histology and transmission electron microscopy showed a healthy and normal SMG, although the number of epithelial stem/progenitor cells amount increased.In overall, this work describes the importance and mechanisms of function of NOTCH non-canonical ligands DLK1 and DLK2 in the development of the salivary gland. In conclusion, we propose that the levels of DLK1 in the normal development of the salivary gland need a fine balance between the control mechanisms of inhibiting and promoting stem cells.