Influence of DNA-methylation at multiple stages of limb chondrogenesis.

Precise regulation of gene expression is of utmost importance during cell fate specification. DNA methylation is a key epigenetic mechanism that plays a significant role in the regulation of cell fate by recruiting repression proteins or inhibiting the binding of transcription factors to DNA to regulate gene expression. Limb development is a well-established model for understanding cell fate decisions, and the formation of skeletal elements is coordinated through a sequence of events that control chondrogenesis spatiotemporally. It has been established that epigenetic control participates in cartilage maturation. However, further investigation is required to determine its role in the earliest stages of chondrocyte differentiation. This study investigates how the DNA methylation environment affects cell fate divergence during the early chondrogenic events. Our research has shown for the first time that inhibiting DNA methylation in interdigital tissue with 5-azacytidine results in the formation of an ectopic digit. This discovery suggested that DNA methylation dynamics could regulate the fate of cells between chondrogenesis and cell death during autopod development. Our in vitro findings indicate that DNA methylation at the early stages of chondrogenesis is integral in regulating condensation by controlling cell adhesion and proapoptotic genes. As a result, the dynamics of methylation and demethylation are crucial in governing chondrogenesis and cell death during different stages of limb chondrogenesis.

WNT5A-Ca2+-CaN-NFAT signalling plays a permissive role during cartilage differentiation in embryonic chick digit development.

During digit development, the correct balance of chondrogenic signals ensures the recruitment of undifferentiated cells into the cartilage lineage or the maintenance of cells at the undifferentiated stage. WNT/ß catenin maintains the pool of progenitor cells, whereas TGFß signalling promotes cartilage differentiation by inducing Sox9 expression. Moreover, WNT5A promotes the degradation of ß catenin during mouse limb development. Although these mechanisms are well established, it is still unknown whether the signalling pathway downstream WNT5A is also involved in early chondrogenesis during digit formation. Thus, the aim of this study was to determine the role of WNT5A during the recruitment of progenitor cells during digit development. Our results showed that WNT5A activated calcium (Ca2+) release in the undifferentiated region during digit development. Further, the blockade of Ca2+ release or calcineurin (CaN) or nuclear factor of activated T-cells (NFAT) functions resulted in an inhibition of cartilage differentiation. Together, our results demonstrate that non canonical WNT5A-Ca2+-CaN-NFAT signalling plays a key role during embryonic digit development in vivo promoting the competence for chondrogenic signals and also acts as a permissive factor for chondrogenesis independently of cell death mechanisms.

Multi-organ transcriptomic landscape of Ambystoma velasci metamorphosis.

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. In this process, these organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages of Ambystoma velasci to investigate its metamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4064 differentially expressed genes in the heart, 4107 in the lung and 8265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways modulation involved in the transformation of the facultative metamorphic salamander A. velasci in an organ-specific manner.

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues.

Full regeneration of the tribasal Polypterus fin.

Full limb regeneration is a property that seems to be restricted to urodele amphibians. Here we found that Polypterus, the most basal living ray-finned fish, regenerates its pectoral lobed fins with a remarkable accuracy. Pectoral Polypterus fins are complex, formed by a well-organized endoskeleton to which the exoskeleton rays are connected. Regeneration initiates with the formation of a blastema similar to that observed in regenerating amphibian limbs. Retinoic acid induces dose-dependent phenotypes ranging from inhibition of regeneration to apparent anterior-posterior duplications. As in all developing tetrapod limbs and regenerating amphibian blastema, Sonic hedgehog is expressed in the posterior mesenchyme during fin regeneration. Hedgehog signaling plays a role in the regeneration and patterning processes: an increase or reduction of fin bony elements results when this signaling is activated or disrupted, respectively. The tail fin also regenerates but, in contrast with pectoral fins, regeneration can resume after release from the arrest caused by hedgehog inhibition. A comparative analysis of fin phenotypes obtained after retinoic acid treatment or altering the hedgehog signaling levels during regeneration allowed us to assign a limb tetrapod equivalent segment to Polypterus fin skeletal structures, thus providing clues to the origin of the autopod. We propose that appendage regeneration was a common property of vertebrates during the fin to limb transition.

Changes in receptivity epithelial cell markers of endometrium after ovarian stimulation treatments: its role during implantation window.

BACKGROUND: To compare the expression of receptivity markers in epithelial and stromal cells in the endometrium of ovulatory women and infertile with hypothalamic pituitary dysfunction (HPD), untreated or treated with clomiphene citrate (CC), or with recombinant follicle stimulating hormone (rFSH). METHODS: Twelve control ovulatory and 32 anovulatory women, 22 of whom received ovulation induction with CC (n = 12) or rFSH (n = 10). Endometrial biopsies were obtained during the mid-secretory phase. Hormonal secretion was measured by chemiluminescence immunoassay, endometrial dating and cellular expression and distribution of receptivity proteins were evaluated by quantitative immunohistochemistry. RESULTS: CC or rFSH treatments, modified the expression of epithelial receptivity markers, such as Glycodelin A, beta-catenin, CD166/ALCAM and IGF-1R, but not in stromal markers. Also, a change in their cell distribution was observed. CONCLUSIONS: Treatment of infertile women with HPD modified the expression and distribution of receptivity markers in the mid-secretory phase of the endometrium in epithelial but not stromal cells, which can help to explain changes in the receptivity of the endometrium during treatments and suggest an important role of these cells in the receptivity window.

Expression pattern of mRNA A and mRNA B of alpha sarcoglycan gene during mouse embryonic development and regulation of their expression by myogenic and cardiogenic transcription factors.

BACKGROUND: Type 2D limb-girdle muscular dystrophy (LGM2D) is a progressive disorder caused by mutations in the alpha sarcoglycan (α-SG) gene. In mice, the α-SG gene contains two promoters that regulate the expression of two different mRNAs (A and B). However, their gene expression pattern during embryonic development has not been explored and their regulation by myogenic and cardiogenic transcription factors has been only partially studied. RESULTS: During embryonic development, mRNA A and B of α-SG gene were initially detected in hypaxial muscles, heart, stomach, tongue, and mesenchymal cells, which surround the dorsal region of the somites. Moreover, mRNA B was exclusively expressed in the floor plate and notochord and in the interdigits of limbs. In vitro, MyoD and myogenin positively regulated the transcription of mRNA B during skeletal myogenesis, whereas mRNA A was activated only for MyoD in differentiated skeletal muscle. In addition, Gata-4 together with Mef2c may regulate the expression of mRNA B in heart development, whereas Nkx2.5 and myocardin may activate expression of mRNA A in the differentiated cardiomyocyte. CONCLUSIONS: The differential expression of α-SG mRNAs during mouse embryonic development may be a consequence of the differential regulation of both promoters by myogenic and cardiogenic factors.

Commitment of human mesenchymal stromal cells to skeletal lineages is independent of their morphogenetic capacity.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent cell populations obtained from fetal and adult tissues. They share some characteristics with limb bud mesodermal cells such as differentiation potential into osteogenic, chondrogenic, and tenogenic lineages and an embryonic mesodermal origin. Although MSCs differentiate into skeletal-related lineages in vitro, they have not been shown to self-organize into complex skeletal structures or connective tissues, as in the limb. In this work, we demonstrate that the expression of molecular markers to commit MSCs to skeletal lineages is not sufficient to generate skeletal elements in vivo. AIM: To evaluate the potential of MSCs to differentiate into skeletal lineages and generate complex skeletal structures using the recombinant limb (RL) system. METHODS: We used the experimental system of RLs from dissociated-reaggregated human placenta (PL) and umbilical cord blood (UCB) MSCs. After being harvested and reaggregated in a pellet, cultured cells were introduced into an ectodermal cover obtained from an early chicken limb bud. Next, this filled ectoderm was grafted into the back of a donor chick embryo. Under these conditions, the cells received and responded to the ectoderm's embryonic signals in a spatiotemporal manner to differentiate and pattern into skeletal elements. Their response to differentiation and morphogenetic signals was evaluated by quantitative polymerase chain reaction, histology, immunofluorescence, scanning electron microscopy, and in situ hybridization. RESULTS: We found that human PL-MSCs and UCB-MSCs constituting the RLs expressed chondrogenic, osteogenic, and tenogenic molecular markers while differentially committing into limb lineages but could not generate complex structures in vivo. MSCs-RL from PL or UCB were committed early to chondrogenic lineage. Nevertheless, the UCB-RL osteogenic commitment was favored, although preferentially to a tenogenic cell fate. These findings suggest that the commitment of MSCs to differentiate into skeletal lineages differs according to the source and is independent of their capacity to generate skeletal elements or connective tissue in vivo. Our results suggest that the failure to form skeletal structures may be due to the intrinsic characteristics of MSCs. Thus, it is necessary to thoroughly evaluate the biological aspects of MSCs and how they respond to morphogenetic signals in an in vivo context. CONCLUSION: PL-MSCs and UCB-MSCs express molecular markers of differentiation into skeletal lineages, but they are not sufficient to generate complex skeletal structures in vivo.

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay.

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation.

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Recombinant Limb Assay as in Vivo Organoid Model.

Organ formation initiates once cells become committed to one of the three embryonic germ layers. In the early stages of embryogenesis, different gene transcription networks regulate cell fate after each germ layer is established, thereby directing the formation of complex tissues and functional organs. These events can be modeled in vitro by creating organoids from induced pluripotent, embryonic, or adult stem cells to study organ formation. Under these conditions, the induced cells are guided down the developmental pathways as in embryonic development, resulting in an organ of a smaller size that possesses the essential functions of the organ of interest. Although organoids are widely studied, the formation of skeletal elements in an organoid model has not yet been possible. Therefore, we suggest that the formation of skeletal elements using the recombinant limb (RL) assay system can serve as an in vivo organoid model. RLs are formed from undissociated or dissociated-reaggregated undifferentiated mesodermal cells introduced into an ectodermal cover obtained from an early limb bud. Next, this filled ectoderm is grafted into the back of a donor chick embryo. Under these conditions, the cells can receive the nascent embryonic signals and develop complex skeletal elements. We propose that the formation of skeletal elements induced through the RL system may occur from stem cells or other types of progenitors, thus enabling the study of morphogenetic properties in vivo from these cells for the first time.

Molecular control of cell differentiation and programmed cell death during digit development.

During the hand plate development, the processes of cell differentiation and control of cell death are relevant to ensure a correct shape of the limb. The progenitor cell pool that later will differentiate into cartilage to form the digits arises from undifferentiated mesenchymal cells beneath the apical ectodermal ridge (AER). Once these cells abandon the area of influence of signals from AER and ectoderm, some cells are committed to chondrocyte lineage forming the digital rays. However, if the cells are not committed to chondrocyte lineage, they will form the prospective interdigits that in species with free digits will subsequently die. In this work, we provide the overview of the molecular interactions between different signaling pathways responsible for the formation of digit and interdigit regions. In addition, we briefly describe some experiments concerning the most important signals responsible for promoting cell death. Finally, on the basis that the interdigital tissue has chondrogenic potential, we discuss the hypothesis that apoptotic-promoting signals might also act as antichondrogenic factors and chondrogenic factors might operate as anti-apoptotic factors.

Smad8 is expressed in the anterior necrotic zone: evidence for a role of bone morphogenetic proteins/SMAD signaling in the activation of a molecular cascade that culminates in cell death.

Bone morphogenetic proteins (BMPs) play a crucial role in programmed cell death (PCD), a biological process required for the sculpturing of the embryonic limbs. However, it is unknown if BMP signaling directly promotes cell death, or if it induces a molecular cascade that culminates in cell death. Given that Smad8, which encodes one component of BMP signaling, is expressed during the regression of interdigital tissue and responds to BMPs, we presumed that it may be expressed in other cell death areas during chick limb development such as the anterior and posterior necrotic zones (ANZ and PNZ). The present study found that the Smad8 expression pattern in the anterior mesoderm of the hindlimb is very similar to that observed in limbs stained to detect cell death. Also, BMPs and retinoic acid, which act as apoptosis-promoting factors, induced expression of Smad8 before the onset of cell death, while sonic hedgehog protein, acting as a survival factor, inhibited Smad8 expression in the ANZ. However, although there was correlation between Smad8 expression patterns and PCD in the ANZ, phosphorylated forms of SMAD1/5/8 and TUNEL staining did not co-localize in dying cells. Interestingly, a short pulse of BMP was sufficient to trigger cell death. On the other hand, most dying cells were located in the avascular region, while many cells expressing Smad8 were located in the vascular region of the ANZ. These results suggest that BMPs mediated by SMAD signaling activate a molecular cascade that culminates in PCD.

Epigenetic control of cell fate - an interview with Maria-Elena Torres-Padilla.

Maria-Elena Torres-Padilla's research is focused on how cell fate arises from a single-cell embryo, the fertilized egg or zygote. After the initial divisions, cell potency becomes restricted, originating the first cell lineage fates. She studies how epigenetic information controls transitions in cell identity and cellular reprogramming during embryonic development. Currently, she is the founding Director of the Institute of Epigenetics and Stem Cells, Helmholtz Centre, and Professor of Stem Cell Biology at the Ludwigs Maximilians University in Munich. In this interview, Maria-Elena Torres-Padilla talks to us about her beginnings in the biology field in Mexico. She also tells us about how she became interested in the control of genome regulation within the nucleus during the transition from totipotency to pluripotency and how the control of gene regulation and chromatin organization during the early stages of cell fate decision in the one-cell embryo occurs. She considers that science has no borders; visiting Mexico gives her the possibility to discuss her work with colleagues and the new generation of students trained in Mexico.

Cell fusion and fusogens - an interview with Benjamin Podbilewicz.

Cell fusion is a process in which cells unite their membranes and cytoplasm. It is fundamental for sexual reproduction and embryonic development. Among the best-known cell fusion processes during animal development are fertilization, myoblast fusion, osteoclast generation, and vulva formation in Caenorhabditis elegans. Although it is involved in many other functions in unicellular and multicellular organisms, little is known about the mechanisms of cell fusion and the genes that code for the proteins participating in this process. Benjamin Podbilewicz has dedicated many years to understanding the processes and mechanisms of cell fusion. In this interview, he spoke to us about how he began his studies of this process, his contributions to this exciting field, his scientific ties with Ibero-America and his strategies for a well-balanced scientific/personal life.

Insights into the mechanism of adult neurogenesis - an interview with Arturo Álvarez-Buylla.

Neurogenesis is the process by which new neurons are formed from progenitor cells. The adult nervous system was long considered unable to generate new neurons, especially in mammals. It was not until the 1960s that Joseph Altman and Gopal Das, using H3-thymidine autoradiography to trace newly formed cells, that the first suggestions of new neurons added to the olfactory bulb and the dentate gyrus of the rat hippocampus came about. These observations remained controversial for many years as they went against the dogmatic view that the structure of the adult brain precluded processes of neurogenesis. It was not until two decades later that work in songbirds and then in mammals, not only confirmed that new neurons could be produced in the adult brain, but revealed basic processes of how young neurons are produced, how they could migrate long distances and become incorporated into adult brain circuits. Arturo Álvarez-Buylla has made important contributions to the understanding of the mechanism of adult neurogenesis, including the identification of adult neural stem cells. Here we summarize a discussion with him related to the field of adult neurogenesis, the root of his interest in neural development and the ramifications of some of his laboratory findings.

Inhibition of WNT/ß-catenin is necessary and sufficient to induce Scx expression in developing tendons of chicken limb.

The cell differentiation of the musculoskeletal system is highly coordinated during limb development. In the distal-most region of the limb, WNT and FGF released from the apical ectodermal ridge maintain mesenchymal cells in the undifferentiated stage. Once the cells stop receiving WNT and FGF, they respond to differentiation signals. Particularly during tendon development, mesenchymal cells enter the cell differentiation program once Scleraxis (Scx) gene expression occurs. Among the signals that trigger the cell differentiation programs, TGFß signaling has been found to be closely involved in tendon differentiation. However, whether Scx gene expression depends merely on TGFß signaling or other signals is still not fully understood. In the present study, considering that WNT/ß-catenin is an inhibitory signal of cell differentiation, we speculated possible antagonistic or additive effects between canonical Wnt/ß-catenin and TGFß/SMAD signaling pathways to control Scx gene expression. We found that the blockade of WNT/ß-catenin promoted Scx gene expression. In contrast, the inhibition of TGFß/SMAD signaling did not maintain Scx gene expression. Interestingly, the blockade of both WNT/ß-catenin and TGFß/SMAD signaling at the same time promoted Scx gene expression. Thus the inhibition of WNT/ß-catenin signaling appears to be necessary and sufficient to induce Scx gene expression.

SCA-1/Ly6A Mesodermal Skeletal Progenitor Subpopulations Reveal Differential Commitment of Early Limb Bud Cells.

At early developmental stages, limb bud mesodermal undifferentiated cells are morphologically indistinguishable. Although the identification of several mesodermal skeletal progenitor cell populations has been recognized, in advanced stages of limb development here we identified and characterized the differentiation hierarchy of two new early limb bud subpopulations of skeletal progenitors defined by the differential expression of the SCA-1 marker. Based on tissue localization of the mesenchymal stromal cell-associated markers (MSC-am) CD29, Sca-1, CD44, CD105, CD90, and CD73, we identified, by multiparametric analysis, the presence of cell subpopulations in the limb bud capable of responding to inductive signals differentially, namely, sSca+ and sSca- cells. In concordance with its gene expression profile, cell cultures of the sSca+ subpopulation showed higher osteogenic but lower chondrogenic capacity than those of sSca-. Interestingly, under high-density conditions, fibroblast-like cells in the sSca+ subpopulation were abundant. Gain-of-function employing micromass cultures and the recombinant limb assay showed that SCA-1 expression promoted tenogenic differentiation, whereas chondrogenesis is delayed. This model represents a system to determine cell differentiation and morphogenesis of different cell subpopulations in similar conditions like in vivo. Our results suggest that the limb bud is composed of a heterogeneous population of progenitors that respond differently to local differentiation inductive signals in the early stages of development, where SCA-1 expression may play a permissive role during cell fate.

Activation of the WNT-BMP-FGF Regulatory Network Induces the Onset of Cell Death in Anterior Mesodermal Cells to Establish the ANZ.

The spatiotemporal control of programmed cell death (PCD) plays a significant role in sculpting the limb. In the early avian limb bud, the anterior necrotic zone (ANZ) and the posterior necrotic zone are two cell death regions associated with digit number reduction. In this study, we evaluated the first events triggered by the FGF, BMP, and WNT signaling interactions to initiate cell death in the anterior margin of the limb to establish the ANZ. This study demonstrates that in a period of two to 8 h after the inhibition of WNT or FGF signaling or the activation of BMP signaling, cell death was induced in the anterior margin of the limb concomitantly with the regulation of Dkk, Fgf8, and Bmp4 expression. Comparing the gene expression profile between the ANZ and the undifferentiated zone at 22HH and 25HH and between the ANZ of 22HH and 25HH stages correlates with functional programs controlled by the regulatory network FGF, BMP, and WNT signaling in the anterior margin of the limb. This work provides novel insights to recognize a negative feedback loop between FGF8, BMP4, and DKK to control the onset of cell death in the anterior margin of the limb to the establishment of the ANZ.

The spatiotemporal expression patterns of MSC-associated markers contribute to the identification of progenitor subpopulations in developing limbs.

During limb development, skeletal tissues differentiate from their progenitor cells in an orchestrated manner. Mesenchymal stromal cells (MSCs), which are considered to be adult undifferentiated/progenitor cells, have traditionally been identified by the expression of MSC-associated markers (MSC-am) and their differentiation capacities. However, although MSCs have been isolated from bone marrow and a variety of adult tissues, their developmental origin is poorly understood. Remarkably, adult MSCs share similar differentiation characteristics with limb progenitors. Here, we determined the expression patterns of common MSC-am throughout mouse hindlimb development. Our results demonstrate that MSC-am expression is not restricted to undifferentiated cells in vivo. Results from the analysis of MSC-am spatiotemporal expression in the embryonic hindlimb allowed us to propose five subpopulations which represent all limb tissues that potentially correspond to progenitor cells for each lineage. This work contributes to the understanding of MSC-am expression dynamics throughout development and underlines the importance of considering their expression patterns in future MSC studies of the limb.