DNA damage precedes apoptosis during the regression of the interdigital tissue in vertebrate embryos

DNA damage independent of caspase activation accompanies programmed cell death in different vertebrate embryonic organs. We analyzed the significance of DNA damage during the regression of the interdigital tissue, which sculpts the digits in the embryonic limb. Interdigit remodeling involves oxidative stress, massive apoptosis and cell senescence. Phosphorylation of H2AX mediated by ATM precedes caspase dependent apoptosis and cell senescence during interdigit regression. The association of ?H2AX with other downstream DNA repair factors, including MDC1, Rad50 and 53BP1 suggests a defensive response of cells against DNA damage. The relative distribution of cells ?H2AX-only positive, TUNEL-only positive, and cells double positive for both markers is consistent with a sequence of degenerative events starting by damage of the DNA. In support of this interpretation, the relative number of ?H2AX-only cells increases after caspase inhibition while the relative number of TUNELonly cells increases after inhibition of ATM. Furthermore, cultured interdigits survived and maintained intense chondrogenic potential, even at advanced stages of degeneration, discarding a previous commitment to die. Our findings support a new biological paradigm considering embryonic cell death secondary to genotoxic stimuli, challenging the idea that considers physiological cell death a cell suicide regulated by an internal death clock that pre-programmes degeneration

Interdigital cell death in the embryonic limb is associated with depletion of Reelin in the extracellular matrix

Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which has important functions in the developing nervous system, including neuronal survival; however, the significance of Reelin in other systems has received very little attention. We show that reelin expression becomes intensely downregulated in both the chick and mouse interdigits preceding the establishment of the areas of interdigital cell death. Furthermore, fibroblast growth factors, which are cell survival signals for the interdigital mesoderm, intensely upregulated reelin expression, while BMPs, which are proapototic signals, downregulate its expression in the interdigit. Gene silencing experiments of reelin gene or its intracellular effector Dab-1 confirmed the implication of Reelin signaling as a survival factor for the limb undifferentiated mesoderm. We found that Reelin activates canonical survival pathways in the limb mesoderm involving protein kinase B and focal adhesion kinase. Our findings support that Reelin plays a role in interdigital cell death, and suggests that anoikis (apoptosis secondary to loss of cell adhesion) may be involved in this process

UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

The primordium of the limb contains a number of progenitors far superior to those necessary to form the skeletal components of this appendage. During the course of development, precursors that do not follow the skeletogenic program are removed by cell senescence and apoptosis. The formation of the digits provides the most representative example of embryonic remodeling via cell degeneration. In the hand/foot regions of the embryonic vertebrate limb (autopod), the interdigital tissue and the zones of interphalangeal joint formation undergo massive degeneration that accounts for jointed and free digit morphology. Developmental senescence and caspase-dependent apoptosis are considered responsible for these remodeling processes. Our study uncovers a new upstream level of regulation of remodeling by the epigenetic regulators Uhrf1 and Uhrf2 genes. These genes are spatially and temporally expressed in the pre-apoptotic regions. UHRF1 and UHRF2 showed a nuclear localization associated with foci of methylated cytosine. Interestingly, nuclear labeling increased in cells progressing through the stages of degeneration prior to TUNEL positivity. Functional analysis in cultured limb skeletal progenitors via the overexpression of either UHRF1 or UHRF2 inhibited chondrogenesis and induced cell senescence and apoptosis accompanied with changes in global and regional DNA methylation. Uhrfs modulated canonical cell differentiation factors, such as Sox9 and Scleraxis, promoted apoptosis via up-regulation of Bak1, and induced cell senescence, by arresting progenitors at the S phase and upregulating the expression of p21. Expression of Uhrf genes in vivo was positively modulated by FGF signaling. In the micromass culture assay Uhrf1 was down-regulated as the progenitors lost stemness and differentiated into cartilage. Together, our findings emphasize the importance of tuning the balance between cell differentiation and cell stemness as a central step in the initiation of the so-called ?embryonic programmed cell death? and suggest that the structural organization of the chromatin, via epigenetic modifications, may be a precocious and critical factor in these regulatory events.Funding: We thank Montse Fernandez Calderon, Susana Dawalibi, and Sonia Perez Mantecon, for excellent technical assistance. This work was supported by a Grant (BFU2017-84046-P) from the Spanish Science and Innovation Ministry to J.A.M

Association of a functional microsatellite within intron 1 of the BMP5 gene with susceptibility to osteoarthritis

<p>Abstract</p> <p>Background</p> <p>In a previous study carried out by our group, the genotyping of 36 microsatellite markers from within a narrow interval of chromosome 6p12.3-q13 generated evidence for linkage and for association to female hip osteoarthritis (OA), with the most compelling association found for a marker within intron 1 of the bone morphogenetic protein 5 gene (<it>BMP5</it>). In this study, we aimed to further categorize the association of variants within intron 1 of <it>BMP5 </it>with OA through an expanded genetic association study of the intron and subsequent functional analysis of associated polymorphisms.</p> <p>Methods</p> <p>We genotyped 18 common polymorphisms including 8 microsatellites and 9 single nucleotide polymorphisms (SNPs) and 1 insertion/deletion (INDEL) from within highly conserved regions between human and mouse within intron 1 of <it>BMP5</it>. These markers were then tested for association to OA by a two-stage approach in which the polymorphisms were initially genotyped in a case-control cohort comprising 361 individuals with associated polymorphisms (<it>P </it>≤ 0.05) then genotyped in a second case-control cohort comprising 1185 individuals.</p> <p>Results</p> <p>Two <it>BMP5 </it>intron 1 polymorphisms demonstrated association in the combined case-control cohort of 1546 individuals (765 cases and 781 controls): microsatellite D6S1276 (<it>P </it>= 0.018) and SNP rs921126 (<it>P </it>= 0.013). Functional analyses in osteoblastic, chondrocytic, and adipocytic cell lines indicated that allelic variants of D6S1276 have significant effects on the transcriptional activity of the <it>BMP5 </it>promoter <it>in vitro</it>.</p> <p>Conclusion</p> <p>Variability in gene expression of <it>BMP5 </it>may be an important contributor to OA genetic susceptibility.</p

Negative Smad Expression and Regulation in the Developing Chick Limb

The inhibitory or negative Smads, Smad6 and Smad7, block TGFβ superfamily signals of both the BMP and TGFβ classes by antagonizing the intracellular signal transduction machinery. We report the cloning of one Smad6 and two Smad7 (Smad7a and Smad7b) chick homologs and their expression and regulation in the developing limb. Smad6 and Smad7a are expressed in dynamic patterns reflecting the domains of BMP gene expression in the limb. Activation and inhibition of the BMP signaling pathway in limb mesenchyme indicates that negative Smad gene expression is regulated, at least in part, by BMP family signals

The methylation status of the embryonic limb skeletal progenitors determines their cell fate in chicken

Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms.We thank Prof. Miguel Lafarga for helpful comments and advice. We thank Dr Jose E Gomez-Arozamena for helping us with the irradiation experiments. We are grateful to Montse Fernandez Calderon, Susana Dawalibi, and Sonia Perez Mantecon, for excellent technical assistance. This work was supported by a Grant (BFU2017–84046-P) from the Spanish Science and Innovation Ministry to JAM. C.S.F is recipient of a FPI grant (BES-2015–074267)

Defining the Earliest Transcriptional Steps of Chondrogenic Progenitor Specification during the Formation of the Digits in the Embryonic Limb

The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The “pre-condensation stage”, occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the “condensation stage” is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the “pre-cartilage period”, precedes the formation of molecularly identifiable cartilage by 2–3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo

Knock-Down of Cathepsin D Affects the Retinal Pigment Epithelium, Impairs Swim-Bladder Ontogenesis and Causes Premature Death in Zebrafish

The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates

Construction and analysis of a modular model of caspase activation in apoptosis

<p>Abstract</p> <p>Background</p> <p>A key physiological mechanism employed by multicellular organisms is apoptosis, or programmed cell death. Apoptosis is triggered by the activation of caspases in response to both extracellular (extrinsic) and intracellular (intrinsic) signals. The extrinsic and intrinsic pathways are characterized by the formation of the death-inducing signaling complex (DISC) and the apoptosome, respectively; both the DISC and the apoptosome are oligomers with complex formation dynamics. Additionally, the extrinsic and intrinsic pathways are coupled through the mitochondrial apoptosis-induced channel via the Bcl-2 family of proteins.</p> <p>Results</p> <p>A model of caspase activation is constructed and analyzed. The apoptosis signaling network is simplified through modularization methodologies and equilibrium abstractions for three functional modules. The mathematical model is composed of a system of ordinary differential equations which is numerically solved. Multiple linear regression analysis investigates the role of each module and reduced models are constructed to identify key contributions of the extrinsic and intrinsic pathways in triggering apoptosis for different cell lines.</p> <p>Conclusion</p> <p>Through linear regression techniques, we identified the feedbacks, dissociation of complexes, and negative regulators as the key components in apoptosis. The analysis and reduced models for our model formulation reveal that the chosen cell lines predominately exhibit strong extrinsic caspase, typical of type I cell, behavior. Furthermore, under the simplified model framework, the selected cells lines exhibit different modes by which caspase activation may occur. Finally the proposed modularized model of apoptosis may generalize behavior for additional cells and tissues, specifically identifying and predicting components responsible for the transition from type I to type II cell behavior.</p