Apoptosis during embryonic tissue remodeling is accompanied by cell senescence

Results

Interdigital tissue regression and cell senescence

Several programmed cell death processes in the embryo are accompanied by cell senescence [19–21], which is characterized by the irreversible loss of replicating ability. The arrest of proliferation in senescent cells is often associated with the overactivation of β-galacto-sidase (“senescence-associated β-gal”), up-regulation of the tumor suppressor gene p21 (inhibitor of cyclin-dependent kinases), and the production of secreted factors, which are collectively termed the senescence-associated secretory phenotype (SASP) that reinforce and propagate senescence in autocrine and paracrine manners [42].

The interdigital tissue expresses p21 [43] and in the course of regression undergoes growth arrest [5, 44]. Therefore, we examined markers of cell senescence. We observed that most recognized markers of senescence were activated in regressing interdigits. Figure 1 A-C shows that β-gal activity at pH6 was very high, and it constituted a precise marker of the regressing process. p21 was expressed at moderate levels in the interdigital mesoderm but it was up-regulated during regression (Table 1). At difference of p21, its usual regulator in other senescence processes, p53 was down-regulated in the course of regression. However, p63 and p73, which are the other two members of the p53 family of tumor suppressor genes implicated in the control of mesodermal limb apoptosis [15], are also up-regulated in the course of interdigit remodeling. In addition, many characteristic members of SASP appeared intensely up-regulated. These factors included the following: cytokines, such as Interleukin 8 L1 (K60) and Interleukin 8 L2 (EMF-1); growth factors, such as Igf1, IgfBP5, HGF, Tgfβ2 and AREGB (Amphiregulin B); metalloproteinases (MMP) such as, MMP2 (gelatinase A), MMP9, and ADAMTS9; and members of the TNF signaling pathway, including Fas (CD95), Tnfrsf21 (DR6), and Tnfrsf 23 (Table 1). Notably, some of these factors, such as ADAMTS9 or Tgfβs, play a role in promoting the regression of the interdigit webs [45, 46].

Figure 1. 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).

Table 1. Transcriptional modifications of cell cycle regulators and SASP components during the regression of the third interdigit

		Expression Fold Changes
Gene	GenBank	6 id.	7.5 id.	8 id.
Cell Cycle Regulation
p21	AF513031	1.00 ± 0.05	1.30 ± 0.30*	3.01 ± 0.10***
p53	NM_205264	1.00 ± 0.06	0.57 ± 0.03**	0.40 ± 0.17**
p63	NM_204351	1.00 ± 0.03	2.42 ± 0.37**	4.73 ± 0.88***
p73	XM_417545	1.00 ± 0.04	1.67 ± 0.27*	2.37 ± 0.61*
Senescence-Associated Secretory Phenotype (SASP)
IL1b	NM_204524	1.01 ± 0.06	1.70 ± 0.73	1.26 ± 0.43
IL6	NM_204628	1.00 ± 0.02	1.72 ± 0.70	1.39 ± 0.30
IL8L1	NM_205018	1.00 ± 0.04	11.10 ± 3.51*	20.59 ± 5.18*
IL8L2	NM_205498	1.02 ± 0.08	1.93 ± 0.22**	3.12 ± 0.46*
CXCL12	NM_204510	1.00 ± 0.03	0.43 ± 0.04***	0.53 ± 0.10**
AREGB	NM_001031537	1.05 ± 0.19	2.23 ± 0.43*	2.46 ± 0.71*
HGF	NM_001030370	1.01 ± 0.08	1.98 ± 0.20**	1.85 ± 0.37*
TGFβ2	XM_003640970	1.03 ± 0.07	1.85 ± 0.25*	2.53 ± 0.53*
IGF1	NM_001004384	1.00 ± 0.01	5.72 ± 1.62*	7.86 ± 2.20*
IGFBP5	XM_004942886	1.00 ± 0.02	7.70 ± 1.86**	19.36 ± 4.44**
IGFBP7	XM_420577	1.00 ± 0.04	0.86 ± 0.11	0.66 ± 0.07
MMP2	NM_204420	1.01 ± 0.06	2.79 ± 0.45**	4.47 ± 0.78*
MMP9	NM_204667	1.00 ± 0.02	2.49 ± 0.44**	4.33 ± 0.91*
Adamts9	XM_414417	1.00 ± 0.04	6.70 ± 1.11**	22.66 ± 5.92*
Adam17	NM_001008682	1.00 ± 0.04	1.14 ± 0.13	1.26 ± 0.07
FAS	XM_421659	1.02 ± 0.04	1.88 ± 0.18***	1.81 ± 0.24*
FASLG	NM_001031559	1.01 ± 0.06	0.78 ± 0.16	1.59 ± 0.22
TNFRSF21	NM_001031103	1.00 ± 0.02	0.83 ± 0.12	1.62 ± 0.25*
TNFRSF23	NM_204386	1.03 ± 0.14	1.92 ± 0.31*	3.28 ± 0.88*
The values are compared with the basal interdigital expression prior to tissue regression (6 id).
*** p < 0,001;
** p < 0.01;
* p< 0.05.

Expression of Btg/Tob genes in interdigital tissue

Members of the Btg/Tob gene family of tumor suppressor genes are expressed in early embryo, including in the limb bud [24, 25]. We investigate the potential implication of these genes in interdigit remodeling because of the role of this gene family in the regulation of proliferation of cancer cells and neural stem cells. Four members of the family, Btg1, Btg2, Tob1 and Tob2, exhibited interdigital expression domains during the course of tissue remodeling in chick and mouse embryos (Fig. 2). Btg3, which is a member of the family that lacks known representative in avian species, is also expressed in the mouse interdigits. Figure 2 shows that not all genes exhibited identical patterns of expression in the autopodial tissues, but all were included in the zones of interdigital cell death.

Figure 2. (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.

Quantitative expression analysis using QPCR in interdigital tissue samples revealed that, the expression level of Btg/Tob genes, except for Tob2, were up-regulated as regression progressed in chick and mouse embryos (Fig. 2E and K). We compared gene expression levels in the chick and mouse legs with the embryonic duck leg, which is a model of physiological syndactyly, to select the most suitable member of the family for subsequent functional analysis in interdigit regression. Btg1, Btg2 and Tob1 were expressed in the duck leg interdigits, but Btg2 was downregulated in the developing duck interdigits in contrast to being upregulated in chick and mouse embryos (Fig. 2N). The presence of BTG2 protein in the interdigit was also confirmed using immunolabeling in tissue sections and isolated cells (Fig. 2L and M).

We analyzed the regulation of Btg2 in interdigit explants and mesodermal cell cultures after treatments with growth factors and signals present in autopodial tissues during interdigital tissue regression to examine the association between Btg2 and the signaling pathways that are proposed to control interdigital cell death. Expression was down-regulated by treatments with FGF2 (0.32± 0.05 x; means ± S.D.; p<0.05) and IGF1 (0.48± 0.12 x; means ± S.D.; p<0.05) which are both survival signals for the interdigital mesoderm. Conversely, expression was up-regulated after treatments with IGFBP5 which binds and neutralizes IGF1 (2.13± 0.20 x; means ± S.D.; p<0.05) and with the IGF1 antagonist AG1024 (2.0 ± 0.09x ; means ± S.D.; p<0.05). Btg2 expression was also up-regulated by addition of H₂O₂ in the culture medium to increase oxidative stress (1.49± 0.04x; means ± S.D.; p<0.01).

Btg2 overexpression in limb mesodermal progenitors increases oxidative stress and induces cell death and cell senescence

The extent of cell death and cell cycle profiles were analyzed using flow cytometry after propidium iodide staining in mesodermal progenitors overexpressing Btg2 (Fig. 3). Senescence was also evaluated using flow cytometry measurement of CFSE dye dilution [40] (Fig. 3 B and B′).

Figure 3. (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.

Cells overexpressing Btg2 exhibited a twofold increase in cell death compared to control cells (212.70 ± 36.70 x; means ± S.D. considering the number of dead cells in the control cultures as 100%; p<0.05) and S-phase arrest of cell cycle progression (Fig. 3A). CSFE dye dilution experiments revealed that 15% of mesodermal progenitors that overexpressed Btg2 did not divide during our 48-hr time period in this study (15,20 ± 0.05, vs 0.20 ± 0.01 in control cultures; means ± S.D.; p<0.01; Fig. 3 B-B′).

QPCR analysis confirmed the changes induced by Btg2 at transcriptional level in limb mesodermal progenitors (Table 2). Bcl2, which protects from mitochondrial permeabilization (antiapoptotic), was down-regulated, and Bak, which induces mitochondrial permeability (proapoptotic), was up-regulated. Members of the MAF gene family of transcription factors were recently implicated in interdigital cell death [15] and these genes were also up-regulated by Btg2. The expression of the cell cycle regulator p21 was up-regulated more than threefold and most components of the interdigit secretome, including: Interleukin 8 L1, Interleukin 8 L2, HGF, Igf1, Tgfβ2, AREGB, MMP2, MMP9, Tnfrsf21, and Fas, were also up-regulated (Table 2). Considering the central function of p21 in cell senescence we analyzed whether silencing of this factor modulates the pro-senescence influence of Btg2 overexpression. As shown in Table 2 up-regulation of most SASP components induced by Btg2 were attenuated or abrogated when the cells were transfected with hBtg2 in combination with sh-p21.

Table 2. Transcriptional gene regulation in skeletal progenitors overexpressing Btg2 gene (hBtg2) and Btg2 in combination with sh-p21 (hBtg2 + sh-p21) compared with control cells transfected with empty vectors

Gene	GenBank	hBtg2(Fold changes vs Control)	hBtg2 + sh-p21(Fold changes vs Control)
Cell Cycle Regulation
p21	AF513031	3.24 ± 0.63**	0.54 ± 0.03***##
p53	NM_205264	1.06 ± 0.11	0.68 ± 0.04
p63	NM_204351	0.83 ± 0.05	0.74 ± 0.07
p73	XM_417545	0.94 ± 0.10	1.19 ± 0.14
Bcl2	NM_205339	0.58 ± 0.03***	0.75 ± 0.04 ##
Bak	NM_001030920	3.20 ± 0.97*	3.81 ± 0.16*
MAFA	NM_205025	2.57 ± 0.17***	1.48 ± 0.14*###
MAFB	NM_001030852	1.64 ± 0.16**	1.18 ± 0.10 #
IL8L1	NM_205018	6.36 ± 1.00***	3.24 ± 0.35**#
IL8L2	NM_205498	6.07 ± 1.58**	6.42 ± 1.03**
AREGB	NM_001031537	3.01 ± 0.44***	1.76 ± 0.29*#
HGF	NM_001030370	5.46 ± 0.56***	3.00 ± 0.16**##
TGFβ2	XM_003640970	2.00 ± 0.35*	0.91 ± 0.05 #
IGF1	NM_001004384	4.16 ± 0.58***	2.16 ± 0.14**#
IGFBP5	XM_004942886	0.75 ± 0.07	0.62 ± 0.03
MMP2	NM_204420	1.52 ± 0.15*	1.56 ± 0.04**
MMP9	NM_204667	1.46 ± 0.13*	1.29 ± 0.13
Adamts9	XM_414417	1.05 ± 0.08	1.46 ± 0.11*##
FAS	XM_421659	2.39 ± 0.42**	1.53 ± 0.12**#
FASLG	NM_001031559	1.11 ± 0.14	0.98 ± 0.13
TNFRSF21	NM_001031103	1.95 ± 0.29**	0.94 ± 0.10 ##
TNFRSF23	NM_204386	1.02 ± 0.11	0.77 ± 0.11
*** p < 0,001;
** p < 0.01;
* p< 0.05 statistical significance vs control cells;
### p< 0,001;
## p < 0.01;
# p< 0.05 statistical significance vs hBtg2.

Btg2 increases oxidative stress in HeLa cells [47]. Therefore, we explored whether overexpression of Btg2 in limb mesodermal progenitors was accompanied by changes in oxidative stress. We detected a threefold increase in lipid oxidation and a twofold increase in carbonylated proteins in 2-day cultures of limb mesodermal progenitors over-expressing Btg2 (Fig. 3C).

Overexpression of Btg2 inhibits limb outgowth in vivo

We overexpressed Btg2 in the mesenchymal limb progenitors of day 2 embryos to investigate whether the antiproliferative and proapoptotic effects of Btg2 were also induced in vivo. Electroporation at this stage is very efficient and devoid of undesired side effects [37]. This stage precedes the initiation of interdigital cell death by 4 days, but it is well known that the response of the limb mesoderm to death-inducing and survival signals is maintained during the entire period of morphogenesis [48]. Control of transfection using a GFP construct confirmed the mesodermal distribution of the electroporated genes (Fig 4B, D). Figures 4A and C show that the size of the transfected limbs was reduced compared to the contralateral control limbs. Histological analysis demonstrated the presence of a moderate number of TUNEL positive cells in the experimental limb that were not observed in the control contralateral limb (Fig. 4 D′). The number of anti-p-H3 positive mesodermal dividing cells in experimental limbs decreased 33% compared to control limbs (67,4 ± 3,6; mean values ± S.D. per field considering the number of p-H3 positive cells in the control limb as 100%; Fig. 4 E-F). Cells positive for β-gal labeling also appeared increased in experimental limbs in relation with their contralateral control (Fig. 5). As observed in the in vitro experiments these changes were accompanied by up-regulation of p21 (1,47 ± 0.19 x; mean± S.D.; p<0.05)

Figure 4. Pictures illustrate chick embryos 48 hr after electroporation of GFP /Btg2 constructs in the right wing bud at 2 id. (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).

Figure 5. (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).

Discussion

Previous studies support the coexistence of different cell death effectors that are able to replace one another during the elimination of the interdigital mesoderm [13, 49, 50]. These death effectors include caspase-driven apoptosis [12], lysosomal-driven cell death [13], and oxidative stress-driven cell death [8, 16, 51]. Our findings add changes in the characteristics of senescence in the regressing interdigits to these factors. We detected an intense up-regulation of p21, which is a characteristic feature of developmental senescence [21], that was accompanied by an up-regulation of characteristic components of the senescence-associated secretory phenotype (SASP) including Interleukin 8 L1, Interleukin 8 L2, Igf1, IgfBP5, HGF, Tgfβ2, AREGB (Amphiregulin B), the metalloproteinases MMP2, MMP9 and ADAMTS9; and members of the TNF signaling superfamily (Fas, Tnfrsf21, and Tnfrsf 23). SASP components in adult and tumoral tissues function as a hierarchical network that establishes functional links between regressive changes. SASP functions include the induction of low-level inflammation accompanied by the activation of matrix proteases, the disruption of stem cell function, and the liberation of oxidizing molecules [52–54]. These processes, together with programmed apoptosis, are central events during physiological interdigit tissue regression.

We also detected an intense activation of β-gal in the regressing interdigits. This feature is a hallmark of cell senescence [21, 34]. Notably, the labeling pattern of β-gal activity in the interdigits closely correlated with progression of cell death during interdigit remodeling.

This correlation was also observed in other areas of embryonic cell death including the closure of the lens, limb AER, and the developing heart (Figures 5 and 6).

Figure 6. 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.

Our finding also revealed that members of the Btg/Tob gene family of antiproliferative, tumor suppressor factors [55] were expressed in the interdigital tissue, which suggests a redundant participation in the control of tissue remodeling. The Tob and Btg genes have evolutionarily conserved domains in their amino terminal regions that contribute to their antiproliferative activities [23, 56, 57]. Furthermore, each family member participates in distinct physiological processes via a variety of molecular mechanisms, including regulation of DNA-binding of sequence-specific transcription factors, interaction with intracellular regulatory factors, and modulation of mRNA turnover [31]. Btg2, which is the founding member of the Btg/Tob gene family, replicated the molecular and cellular changes in limb mesoderm during interdigital tissue remodeling, as observed in tumoral tissues [26, 28]. These changes were induced by gain-of-function experiments in vivo and in vitro and included apoptosis, transcriptional modification of cell senescence characteristics, arrest of cell cycle progression, and oxidative stress. The proapoptotic influence was associated at the transcriptional level with the down-regulation of Bcl-2 and the up-regulation of Bak. Therefore, the expression ratio between Bcl2 and Bak, which determines cell fate (apoptosis vs survival, [58]), was shifted toward apoptosis. Cell cycle arrest is accompanied by intense up-regulation of p21, as observed in other senescence processes [19, 21]. Notably, 15% of cells over-expressing Btg2 did not replicate during the 48 hr period that was analyzed in this study, which supports the induction of cell senescence. Btg2 overexpression also induced the expression of the SASP components in the interdigit, including Interleukin 8L1, Interleukin 8L2, Igf1, HGF, Tgfβ2, AREGB, MMP2, MMP9, Tnfrsf21, and FAS. Our findings show that p21 exerts a central role in these effects. Hence, up-regulation of many components of the SASP transcriptome by overexpression of Btg2 is abrogated or reduced when p21 is silenced by co-transfection with a sh-RNAi against p21.

Oxidative stress plays a central role in interdigit regression [8, 16, 51]. Our study indicate that overexpression of Btg2 doubled the level of protein carbonylation and tripled the level of lipid peroxidation. We also demonstrated that Btg2 was up-regulated when oxidative stress increased in limb mesoderm in cultures containing H₂O₂. This finding suggests that the local increase in ROS observed in the regressing interdigit [51] establishes a positive feedback loop that promotes and/or maintains the expression of this antiproliferative factor.

One remarkable feature of the regulation of interdigital cell death is the combined participation of various death mechanisms. The coordination of these dying routes is not known. This study provides a new mechanism that promotes cell senescence and apoptosis. We demonstrate that Btg2 was down-regulated in cultures treated with FGF2 or IGF1, which are major antiapoptotic factors implicated in interdigital cell death [59–63] and up-regulated after chemically blockade of IGF signaling and when mesodermal progenitors were cultured in the presence of IGF binding protein-5. The interdigital expression of various IGF binding proteins that antagonize IGF1 [59] and the decay of FGF production by the AER at the end of limb morphogenesis together with the local increase in ROS may all contribute to the physiological up-regulation of Btg2 in the regressing interdigit.

In summary, this study highlights the contribution of a senescence-like process during the regression of interdigital tissue and delineates the involvement of Btg2 in cooperation with p21, in the promotion of cell cycle arrest, caspase-dependent cell death, and transcriptional up-regulation of components of the senescence-associated secretory phenotype. Our unpublished observations also indicate that other members of this family, such as Btg1 and Tob1 also promote cell death in the limb mesoderm, which explains the absence of an overt digit phenotype in mice that are deficient for members of this gene family.

Acknowledgments

Thanks are due to Montse Fernandez-Calderon, Sonia Perez-Mantecón and Susana Dawalibi for technical assistance.

Funding

This work was supported by a Grant (BFU2014-54026P) from the Spanish Science and Innovation Ministry to J.M.H.

Conflicts of Interest

The authors declare that they have no conflicts of interest with the contents of this article.

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