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• Research Paper Volume 7, Issue 11 pp 974-985

  Apoptosis during embryonic tissue remodeling is accompanied by cell senescence

  Relevance score: 6.3702703

  Carlos I. Lorda-Diez, Beatriz Garcia-Riart, Juan A. Montero, Joaquín Rodriguez-León, Juan A Garcia-Porrero, Juan M. Hurle

  Keywords: programmed cell death, senescence, limb development, β-galactosidase, syndactyly, SASP, INZ

  Published in Aging on November 14, 2015

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  This study re-examined the dying process in the interdigital tissue during the formation of free digits in the developing limbs. We demonstrated that the interdigital dying process was associated with cell senescence, as deduced by induction of β-gal activity, mitotic arrest, and transcriptional up-regulation of p21 together with many components of the senescence-associated secretory phenotype. We also found overlapping domains of expression of members of the Btg/Tob gene family of antiproliferative factors in the regressing interdigits. Notably, Btg2 was up-regulated during interdigit remodeling in species with free digits but not in the webbed foot of the duck. We also demonstrate that oxidative stress promoted the expression of Btg2, and that FGF2 and IGF1 which are survival signals for embryonic limb mesenchyme inhibited Btg2 expression. Btg2 overexpression in vivo and in vitro induced all the observed changes during interdigit regression, including oxidative stress, arrest of cell cycle progression, transcriptional regulation of senescence markers, and caspase-mediated apoptosis. Consistent with the central role of p21 on cell senescence, the transcriptional effects induced by overexpression of Btg2 are attenuated by silencing p21. Our findings indicate that cell senescence and apoptosis are complementary processes in the regression of embryonic tissues and share common regulatory signals.

  

  β-gal activity in the course of interdigit tissue regression in the embryonic chick. Longitudinal vibratome sections of chick limb autopods at days 7 (A), 7.5 (B), and 8 id (C).

  
  
  

  

  (A-D) in situ hybridizations showing the expression of Btg1 (A), Btg2 (B), Tob1(C) and Tob2 (D) in the chick autopod during interdigit regression. Note that, in addition to the interdigital domains, Tob2, Btg2 and Tob1 are also expressed in the developing interphalangeal joints (arrows). (E) Expression level of Btg and Tob genes in interdigital tissue of chick leg during the course of remodeling. The chart shows QPCR-evaluated fold changes in the expression of Btg1, Btg2, Tob1 and Tob2 in the third interdigit of the chick leg bud at 7.5 and 8 id compared with their expression levels prior to the onset of tissue regression (6 id). (F-J) in situ hybridizations showing the expression of the Btg/Tob genes in the developing mouse autopod. (K) chart is a comparative QPCR analysis of the interdigital expression of Btg/Tob genes at day 13 p.c. (light columns) versus day 13,5 pc (dark columns). (L-M) immunostaining for BTG2 (green) combined with phalloidin (red) in vibratome sections of the third interdigit (M), and in cultured mesodermal progenitors (M). Note that the protein is expressed in the cytoplasm and nuclei. Scale bar in L = 100μm; Bar in M = 20μm. (N) shows a QPCR analysis of the expression of Btg1, Btg2 and Tob1 in the third interdigit of embryonic duck leg at equivalent stages of that of the chick in E. Unlike in the chick (compare with E), Btg2 becomes down-regulated and Tob1 is not up-regulated over the course of tissue remodeling. ***p < 0,001; ** p < 0.01; * p< 0.05.

  
  
  

  

  (A) Graphic illustrations comparing the proportion of cells at different cell cycle stages in control (white columns) versus Btg2-overexpressing mesodermal progenitors (grey columns) after 48 hr of culture as evaluated using flow cytometry after PI staining. (B-B′) Illustrates the dilution of CFSE labeling after 48 hr of culture in a representative sample of three distinct experiments. Blue: control cells; Green: Btg2 overexpressing; Yellow: control cells maintained at 4°C. NP (non-proliferating) marks the area of the plot of no proliferation, deduced from the absence of CFSE dilution in control cells maintained at 4°C. P (proliferating) marks the area of the cytometry plot of cells in proliferation. The lower dilution of CFSE in the proliferating cell population (P) of Btg2-overexpressing cells (green) in comparison with control cells (blue) indicates a reduced proliferation rate. B′: detailed view of the Btg2-overexpressing cells in B, isolated from the other values to appreciate that a significant portion (15%) of experimental cells are non-proliferating. (C) Graphic representation of the levels of lipid oxidation and carbonylated proteins in control and Btg2-overexpressing limb mesodermal progenitors. * p< 0.05.

  
  
  

  

  (A) Dorsal view of a chick embryo showing the reduced size of the experimental limb (Exp) compared with the contralateral control (Cont). (B) Mesodermal expression of GFP in the limb 48 hr after electroporation. (C) HE stained transverse section of an experimental embryo to show the different size of the experimental limb (exp) compared with the contralateral limb (cont). (D) and (D′), show the expression of GFP (D) and the presence of TUNEL-positive apoptotic cells (arrows in (D′) in a correlative section of the experimental limb in (C). E-F, are correlative sections of the embryo in (C) after immunolabeling with anti-p-H3. Note the reduced number of positive cells in the experimental limb (F) compared with the control limb (E).

  
  
  

  

  (A) Transverse vibratome section of an experimental embryo 48 hr after electroporation of Btg2. Note the abundance of cells positive for β-gal activity in the electroporated limb (*). (B) is a detailed view of the limb electroporated with Btg2. As previously reported, β-gal activity is intense in the AER of both control and experimental limbs, in the mesonephros (M) and in the notochord (N).

  
  
  

  

  Selection of areas of embryonic programmed cell death showing the parallelism between the distribution of neutral red vital staining (A′, B′, C′) and β-gal activity (A, B C). A-A′: cell death (arrows) during the closure of the lens in chick embryos at 2.5 id. B-B′ cell death in the AER in the embryonic limb at id 3.5. C-C′, cell death in the root of the main arteries of the embryonic heart at id 7.5.