Roles Of The Retinoblastoma Protein Rb And P53 In The Control Of Renal Stem And Progenitor Cells’ Properties In Vitro Direct Implications On Kidney Development

الموضوعات

علوم بحتة وطبيعية

الملخص

Nephron formation necessitates the presence of a tight balance between self-renewaland differentiation of NPCs (Nephron Progenitor cells). Proper nephrogenesis duringembryonic development is, therefore, critical for proper renal function in the adult kidney.Thus, a better understanding of the molecular framework (genes and pathways) regulatingrenal cell development including nephron stem and progenitor cells (NSPCs) is of majorsignificance and will provide a better insight about disease pathogenesis.The Retinoblastoma protein, pRb, is known for its central role in controlling cellcycle progression at the G1-S phase checkpoint. Various studies have also highlightedother functions carried by the Rb pathway such as the regulation of progenitors’differentiation and migration as well as survival of their mature progeny in differenttissues including the brain and the retina. Recent work from our laboratory revealed, forthe first time, that Rb is also required for proper kidney development and control ofnephrogenesis. Hence, we found that Rb negatively regulate nephron progenitors’proliferation, and is indispensable for the survival of immature nephrons in vivo. On theother hand, the role of the tumor suppressor gene, p53, is well investigated in kidneydevelopment. Several studies have shown that p53 is needed, in a dose-dependent manner,to regulate ureteric bud branching, self-renewal and maintenance of nephronstem/progenitor cells inside the cap mesenchyme as well as terminal maturation ofnephron structures.To gain a better understanding of the roles of Rb and p53 in the control ofNSPCs’ development, we have investigated here how each tumor suppressor generegulates the properties of these cells in vitro. We used a well-established protocol,adapted from Brown et. 2011, to isolate and culture renal cells derived from the capmesenchyme (by mild enzymatic digestion and purification) after inducing a deletion ofRb and p53 during mid-development (Aaron C Brown et al., 2011). Then, we assessedrenal cell growth, morphology as well as their rate of proliferation and their expression ofkey developmental markers such as Cited1 and Six2 by immunocytochemistry after 2-3days in culture. To delete Rb and p53, we generated pregnant females carrying Nestin-CreERT2-YFP; Rbfl/fl and Nestin-CreERT2-YFP; p53fl/fl embryos along with heterozygous or wild type control littermates, separately. Then, we induced Rb-p53 gene deletion bysingle tamoxifen treatments administered to these females by oral gavage at E10.5followed by sacrifice at E17.5.Our results showed successful Cre recombination and gene deletion in culturedrenal cells, the majority of which belonged to the cap mesenchyme lineage and expressedthe NSPCs’ markers Cited1 and Six2. Accordingly, more than half of the isolated renalcells co-expressed Nestin and GFP and showed similar morphology between differentgenotypes after 2-3 days in culture. However, we detected, in Rb-/- cultures, a significantand proportional increase in cell proliferation as assessed by cell density, Ki67 labeling, and BrdU incorporation compared with controls. This indicated that Rb negativelycontrols the proliferation of renal cells including NSPCs in primary culture and is consistentwith our recent in vivo findings showing enhanced proliferation inside the nephrogeniczone following the loss of Rb. Moreover, we found that loss of p53 significantly compromisesrenal cell growth in culture as manifested by the sharp reduction in cell density comparedwith p53+/+ cultures. This finding is unlikely associated with a proliferation defect giventhat the rate of proliferation was not affected by the loss of p53. Hence, the mechanismsmediating this effect require further investigation and may be linked to a survival defect among other defects as seen in our in vivo study.This is the first study to assess the roles of Rb and p53 in the control of renal stem andprogenitor cells’ proliferation in vitro. Complementary work in the future may shed morelight on how these genes regulate NSPCs’ differentiation and survival in culture.Altogether, these findings will help provide a better understanding of the cellular andmolecular pathways implicating the Rb and p53 pathways in kidney development.

روابط وملفات

التعريف والنوع

رقم الوثيقة

fd60d520-54cd-4a55-ab48-f0c07e236e6e

رقم العقد

0

نوع الوسائط

Crawler

نوع المحتوى

الرسائل العلمية

صيغة المصدر

رسائل ماجيستير

نوع الملف

pdf text

أسماء الملفات

2026108_1.pdf

بيانات النشر

ألقاب المؤلفين

[{"name_ar":"Ihsan Shadi Hammoura","title_ar":"اعداد","title_en":"Preparation"},{"name_ar":"Noel Ghanem ","title_ar":"اشراف","title_en":"Supervision"}]

اللغة

English

المصدر والدورية

اسم المصدر

Roles Of The Retinoblastoma Protein Rb And P53 In The Control Of Renal Stem And Progenitor Cells’ Properties In Vitro Direct Implications On Kidney Development

المحتوى والصفحات

عدد الصفحات

0

كلمات الباحثين

Cell Culture

إشراف وإعداد

الإشراف

Noel Ghanem

الإعداد

Ihsan Shadi Hammoura

الاقتباسات الببليوغرافية