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Micromass cultures of embryonic limb skeletal progenitors replicate the tissue remodelling processes observed during digit morphogenesis. Here, we have employed micromass cultures in an in vitro assay to study the nature of cell degeneration events associated with skeletogenesis. In the assay, "naive" progenitors obtained from the autopod aggregate to form chondrogenic nodules and those occupying the internodular spaces exhibit intense apoptosis and progressive accumulation of larger cells, showing intense SA-β-Gal histochemical labelling that strictly overlaps with the distribution of neutral red vital staining. qPCR analysis detected intense upregulation of the p21 gene, but P21 immunolabelling showed cytoplasmic rather than the nuclear distribution expected in senescent cells. Semithin sections and transmission electron microscopy confirmed the presence of canonical apoptotic cells, degenerated cell fragments in the process of phagocytic internalization by the neighbouring cells, and large vacuolated cells containing phagosomes. The immunohistochemical distribution of active caspase 3, cathepsin D, and β-galactosidase together with the reduction in cell death by chemical inhibition of caspases (Q-VAD) and lysosomal cathepsin D (Pepstatin A) supported a redundant implication of both pathways in the dying process. Chemical inhibition of P21 (UC2288) revealed a complementary role of this factor in the dying process. In contrast, treatment with the senolytic drug Navitoclax increased cell death without changing the number of cells positive for SA-β-Gal. We propose that this model of tissue remodelling involves the cooperative activation of multiple degradation routes and, most importantly, that positivity for SA-β-Gal reflects the occurrence of phagocytosis, supporting the rejection of cell senescence as a defining component of developmental tissue remodelling., (© 2023. The Author(s).)

Considering the importance of programmed cell death in the formation of the skeleton during embryonic development, the aim of the present study was to analyze whether regulated cell degeneration also accompanies the differentiation of embryonic limb skeletal progenitors in high-density tridimensional cultures (micromass cultures). Our results show that the formation of primary cartilage nodules in the micromass culture assay involves a patterned process of cell death and cell senescence, complementary to the pattern of chondrogenesis. As occurs in vivo, the degenerative events were preceded by DNA damage detectable by γH2AX immunolabeling and proceeded via apoptosis and cell senescence. Combined treatments of the cultures with growth factors active during limb skeletogenesis, including FGF, BMP, and WNT revealed that FGF signaling modulates the response of progenitors to signaling pathways implicated in cell death. Transcriptional changes induced by FGF treatments suggested that this function is mediated by the positive regulation of the genetic machinery responsible for apoptosis and cell senescence together with hypomethylation of the Sox9 gene promoter. We propose that FGF signaling exerts a primordial function in the embryonic limb conferring chondroprogenitors with their biological properties.

The present paper proposes a new level of regulation of programmed cell death (PCD) in developing systems based on epigenetics. We argue against the traditional view of PCD as an altruistic "cell suicide" activated by specific gene-encoded signals with the function of favoring the development of their neighboring progenitors to properly form embryonic organs. In contrast, we propose that signals and local tissue interactions responsible for growth and differentiation of the embryonic tissues generate domains where cells retain an epigenetic profile sensitive to DNA damage that results in its subsequent elimination in a fashion reminiscent of what happens with scaffolding at the end of the construction of a building. Canonical death genes, including Bcl-2 family members, caspases, and lysosomal proteases, would reflect the downstream molecular machinery that executes the dying process rather than being master cell death regulatory signals.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marín-Llera, Lorda-Diez, Hurle and Chimal-Monroy.)

During limb formation in vertebrates with free digits, the interdigital mesoderm is eliminated by a massive degeneration process that involves apoptosis and cell senescence. The degradation process is preceded by intense DNA damage in zones located close to methylated DNA, accompanied by the activation of the DNA repair response. In this study, we show that trimethylated histone 3 (H3K4me3, H3K9me3, and H3K27me3) overlaps with zones positive for 5mC in the nuclei of interdigital cells. This pattern contrasts with the widespread distribution of acetylated histones (H3K9ac and H4ac) and the histone variant H3.3 throughout the nucleoplasm. Consistent with the intense labeling of acetylated histones, the histone deacetylase genes Hdac1 , Hdac2 , Hdac3 , and Hdac8 , and at a more reduced level, Hdac10 , are expressed in the interdigits. Furthermore, local treatments with the histone deacetylase inhibitor trichostatin A, which promotes an open chromatin state, induces massive cell death and transcriptional changes reminiscent of, but preceding, the physiological process of interdigit remodeling. Together, these findings suggest that the epigenetic profile of the interdigital mesoderm contributes to the sensitivity to DNA damage that precedes apoptosis during tissue regression.

Digits develop in the distal part of the embryonic limb primordium as radial prechondrogenic condensations separated by undifferentiated mesoderm. In a short time interval the interdigital mesoderm undergoes massive degeneration to determine the formation of free digits. This fascinating process has often been considered as an altruistic cell suicide that is evolutionarily-regulated in species with different degrees of digit webbing. Initial descriptions of interdigit remodeling considered lysosomes as the primary cause of the degenerative process. However, the functional significance of lysosomes lost interest among researcher and was displaced to a secondary role because the introduction of the term apoptosis. Accumulating evidence in recent decades has revealed that, far from being a unique method of embryonic cell death, apoptosis is only one among several redundant dying mechanisms accounting for the elimination of tissues during embryonic development. Developmental cell senescence has emerged in the last decade as a primary factor implicated in interdigit remodeling. Our review proposes that cell senescence is the biological process identified by vital staining in embryonic models and implicates lysosomes in programmed cell death. We review major structural changes associated with interdigit remodeling that may be driven by cell senescence. Furthermore, the identification of cell senescence lacking tissue degeneration, associated with the maturation of the digit tendons at the same stages of interdigital remodeling, allowed us to distinguish between two functionally distinct types of embryonic cell senescence, "constructive" and "destructive.", (Copyright © 2020 Montero, Lorda-Diez and Hurle.)

During embryonic development, organ morphogenesis requires major tissue rearrangements that are tightly regulated at the genetic level. A large number of studies performed in recent decades assigned a central role to programmed cell death for such morphogenetic tissue rearrangements that often sculpt the shape of embryonic organs. However, accumulating evidence indicates that far from being the only factor responsible for sculpting organ morphology, programmed cell death is accompanied by other tissue remodeling events that ensure the outcome of morphogenesis. In this regard, cell senescence has been recently associated with morphogenetic degenerative embryonic processes as an early tissue remodeling event in development of the limbs, kidney and inner ear. Here, we have explored cell senescence by monitoring β-galactosidase activity during embryonic heart development where programmed cell death is believed to exert an important morphogenetic function. We report the occurrence of extensive cell senescence foci during heart morphogenesis. These foci overlap spatially and temporally with the areas of programmed cell death that are associated with remodeling of the outflow tract to build the roots of the great arteries and with the septation of cardiac cavities. qPCR analysis allowed us to identify a gene expression profile characteristic of the so-called senescence secretory associated phenotype in the remodeling outflow tract of the embryonic heart. In addition, we confirmed local upregulation of numerous tumor suppressor genes including p21, p53, p63, p73 and Btg2. Interestingly, the areas of cell senescence were also accompanied by intense lysosomal activation and non-apoptotic DNA damage revealed by γH2AX immunolabeling. Considering the importance of sustained DNA damage as a triggering factor for cell senescence and apoptosis, we propose the coordinated contribution of DNA damage, senescence and apoptotic cell death to assure tissue remodeling in the developing vertebrate heart., (© 2019 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Embryonic development requires the coordinated regulation of apoptosis, survival, autophagy, proliferation and differentiation programs. Senescence has recently joined the cellular processes required to master development, in addition to its well-described roles in cancer and ageing. Here, we show that senescent cells are present in a highly regulated temporal pattern in the developing vertebrate inner ear, first, surrounding the otic pore and, later, in the otocyst at the endolymphatic duct. Cellular senescence is associated with areas of increased apoptosis and reduced proliferation consistent with the induction of the process when the endolymphatic duct is being formed. Modulation of senescence disrupts otic vesicle morphology. Transforming growth factor beta (TGFβ) signaling interacts with signaling pathways elicited by insulin-like growth factor type 1 (IGF-1) to jointly coordinate cellular dynamics required for morphogenesis and differentiation. Taken together, these results show that senescence is a natural occurring process essential for early inner ear development.

Next-generation sequencing in combination with quantitative polymerase chain reaction analysis revealed a dynamic miRNA signature in the interdigital mesoderm of the chick embryonic hinlimb in the course of interdigit remodelling. During this period, 612 previously known chicken miRNAs (gga-miRNAs) and 401 non-identified sequences were expressed in the interdigital mesoderm. Thirty-six microRNAs, represented by more than 750 reads per million, displayed differential expression between stages HH29 (6 id) and HH32 (7.5 id), which correspond to the onset and the peak of interdigital cell death. Twenty miRNAs were upregulated by at least 1.5-fold, and sixteen were downregulated by at least 0.5-fold. Upregulated miRNAs included miRNAs with recognized proapoptotic functions in other systems (miR-181 family, miR-451 and miR-148a), miRNAs associated with inflammation and cell senescence (miR-21 and miR-146) and miRNAs able to induce changes in the extracellular matrix (miR-30c). In contrast, miRNAs with known antiapoptotic effects in other systems, such as miR-222 and miR-205, became downregulated. In addition, miR-92, an important positive regulator of cell proliferation, was also downregulated. Together, these findings indicate a role for miRNAs in the control of tissue regression and cell death in a characteristic morphogenetic embryonic process based on massive apoptosis., (© 2017 Anatomical Society.)

In tetrapods the digit pattern has evolved to adapt to distinct locomotive strategies. The number of digits varies between species or even between hindlimb and forelimb within the same species. These facts illustrate the plasticity of embryonic limb autopods. Sox9 is a precocious marker of skeletal differentiation of limb mesenchymal cells. Its pattern of expression in the developing limb has been widely studied and reflects the activity of signaling cascades responsible for skeletogenesis. In this assay we stress previously overlooked differences in the pattern of expression of Sox9 in limbs of avian, mouse and turtle embryos which may reflect signaling differences associated with distinct limb skeletal morphologies observed in these species. Furthermore, we show that Sox9 gene expression is higher and maintained in the interdigital region in species with webbed digits in comparison with free digit animals.

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 γH2AX 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 γH2AX-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 γH2AX-only cells increases after caspase inhibition while the relative number of TUNEL-only 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.

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.

Bone morphogenetic proteins (BMPs) are key molecules in the differentiation of skeletal tissues. We have investigated whether differentiation of limb embryonic mesodermal progenitors into different connective tissue lineages depends on specific stimulation of distinct BMP ligands or on the differential response of target cells to a common BMP stimulus. We show that Bmp2,4,5,7 and Gdf5 exhibit differential expression domains during the formation of tendons, cartilages, and joint tissues in digit development, but their respective effects on digit progenitors cell cultures cannot sustain the divergent differentiation of these cells into tendons, joints, and cartilage. However, the influence of BMPs differs based on the culture length. Early cultures respond to any of the BMPs by inducing chondrogenic factors and inhibiting fibrogenic and osteogenic markers. Later, a second phase of the culture occurs when BMPs attenuate their prochondrogenic influence and promote the fibrogenic marker Scleraxis. At advanced culture stages, BMPs inhibit prochondrogenic and profibrogenic markers and promote osteogenic markers. The switch from the prochondrogenic to the profibrogenic response appears critically dependent on the basal expression of Noggin. Thus, the differential regulation of Scleraxis at these stages was abrogated by treatments with a BMP-analogous compound (AB204) that escapes NOGGIN antagonism. Gene regulation experiments in absence of protein synthesis during the first period of culture indicate that BMPs activate at the same time master chondrogenic and fibrogenic genes together with cofactors responsible for driving the signaling cascade toward chondrogenesis or fibrogenesis. Gene-silencing experiments indicate that Id2 is one of the factors limiting the profibrogenic influence of BMPs. We propose that connective tissues are dynamic structures composed of cartilage, fibrous tissue, and bone that form in successive steps from the differentiation of common progenitors. This sequential differentiation is regulated by BMPs through a process that is dependent on the basal expression of BMP cofactors or signaling modulators., (© 2014 American Society for Bone and Mineral Research.)

The purpose of this study is to gain insight into the role of BMP signaling in the diversification of the embryonic limb mesodermal progenitors destined to form cartilage, joints, and tendons. Given the importance of extracellular BMP modulators in in vivo systems, we performed a systematic search of those expressed in the developing autopod during the formation of the digits. Here, we monitored the expression of extracellular BMP modulators including: Noggin, Chordin, Chordin-like 1, Chordin-like 2, Twisted gastrulation, Dan, BMPER, Sost, Sostdc1, Follistatin, Follistatin-like 1, Follistatin-like 5 and Tolloid. These factors show differential expression domains in cartilage, joints and tendons. Furthermore, they are induced in specific temporal patterns during the formation of an ectopic extra digit, preceding the appearance of changes that are identifiable by conventional histology. The analysis of gene regulation, cell proliferation and cell death that are induced by these factors in high density cultures of digit progenitors provides evidence of functional specialization in the control of mesodermal differentiation but not in cell proliferation or apoptosis. We further show that the expression of these factors is differentially controlled by the distinct signaling pathways acting in the developing limb at the stages covered by this study. In addition, our results provide evidence suggesting that TWISTED GASTRULATION cooperates with CHORDINS, BMPER, and NOGGIN in the establishment of tendons or cartilage in a fashion that is dependent on the presence or absence of TOLLOID.

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.

Background: Transforming growth factor beta proteins (Tgfbetas) are secreted cytokines with well-defined functions in the differentiation of the musculoskeletal system of the developing limb. Here we have studied in chicken embryos, whether these cytokines are implicated in the development of the embryonic limb bud at stages preceding tissue differentiation., Results: Immunohistochemical detection of phosphorylated Smad2 and Smad3 indicates that signaling by this pathway is active in the undifferentiated mesoderm and AER. Gene expression analysis shows that transcripts of tgfbeta2 and tgfbeta3 but not tgfbeta1 are abundant in the growing undifferentiated limb mesoderm. Transcripts of tgfbeta2 are also found in the AER, which is the signaling center responsible for limb outgrowth. Furthermore, we show that Latent Tgfbeta Binding protein 1 (LTBP1), which is a key extracellular modulator of Tgfbeta ligand bioavailability, is coexpressed with Tgfbetas in the early limb bud. Administration of exogenous Tgfbetas to limb buds growing in explant cultures provides evidence of these cytokines playing a role in the regulation of mesodermal limb proliferation. In addition, analysis of gene regulation in these experiments revealed that Tgfbeta signaling has no effect on the expression of master genes of musculoskeletal tissue differentiation but negatively regulates the expression of the BMP-antagonist Gremlin., Conclusion: We propose the occurrence of an interplay between Tgfbeta and BMP signaling functionally associated with the regulation of early limb outgrowth by modulating limb mesenchymal cell proliferation.

Transforming growth factor beta (TGFbeta) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGFbeta signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGFbetas shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved down-regulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGFbeta signaling up-regulates Sox9 in the in vivo experimental model system in which TGFbeta treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGFbeta signaling, we found that TGFbetas appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGFbeta signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGFbeta signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGFbeta treatments.

The progress zone (PZ) is a specialized area at the distal margin of the developing limb where mesodermal cells are kept in proliferation and undifferentiated, allowing limb outgrowth. At stages of digit morphogenesis the PZ cells can undergo two possible fates, either aggregate initiating chondrogenic differentiation to configure the digit blastemas, or to die by apoptosis if they are incorporated in the interdigital mesenchyme. While both processes are controlled by bone morphogenetic proteins (BMPs) the molecular basis for such contrasting differential behavior of the autopodial mesoderm remains unknown. Here we show that a well-defined crescent domain of high BMP activity located at the tip of the forming digits, which we termed the digit crescent (DC), directs incorporation and differentiation of the PZ mesenchymal cells into the digit aggregates. The presence of this domain does not correlate with an exclusive expression domain of BMP receptors and its abrogation by surgical approaches or by local application of BMP antagonists is followed by digit truncation and cell death. We further show that establishment of the DC is directed by Activin/TGFbeta signaling, which inhibits Smad 6 and Bambi, two specific BMP antagonists expressed in the interdigits and progress zone mesoderm. The interaction between Activin/TGFbeta and BMP pathways at the level of DC promotes the expression of the chondrogenic factor SOX9 accompanied by a local decrease in cell proliferation. Characteristically, the DC domain is asymmetric, it being extended towards the posterior interdigit. The presence of the DC is transitorily dependent of the adjacent posterior interdigit and its maintenance requires also the integrity of the AER.

Here we report a new role for the small GTPase RhoC in the control of limb chondrogenesis. Expression of rhoC is a precocious marker of the zeugopodial and digit blastemas and is induced by treatments with TGFbetas preceding the formation of ectopic digits. As development progresses, expression of rhoC outlines the growing distal tip of the digits, and marks the regions of interphalangeal joint formation. Functional experiments show that RhoC is a negative regulator of chondrogenesis, which controls digit outgrowth and joint segmentation. These functions appear to be mediated by reorganization of the actin cytoskeleton and modification of the adhesive properties of the mesenchymal cells.

Vertebrate muscle morphogenesis is a complex developmental process, which remains quite yet unexplored at cellular and molecular level. In this work, we have found that sculpturing programmed cell death is a key morphogenetic process responsible for the formation of individual foot muscles in the developing avian limb. Muscle fibers are produced in excess in the precursor dorsal and ventral muscle masses of the limb bud and myofibers lacking junctions with digital tendons are eliminated via apoptosis. Microsurgical experiments to isolate the developing muscles from their specific tendons are consistent with a role for tendons in regulating survival of myogenic cells. Analysis of the expression of Raldh2 and local treatments with retinoic acid indicate that this signaling pathway mediates apoptosis in myogenic cells, appearing also involved in tendon maturation. Retinoic acid inhibition experiments led to defects in muscle belly segmentation and myotendinous junction formation. It is proposed that heterogeneous local distribution of retinoids controlled through Raldh2 and Cyp26A1 is responsible for matching the fleshy and the tendinous components of each muscle belly.

During limb development, expression of cathepsin D and B genes prefigure the pattern of interdigital apoptosis including the differences between the chick and the webbed digits of the duck. Expression of cathepsin L is associated with advanced stages of degeneration. Analysis of Gremlin-/- and Dkk-/- mouse mutants and local treatments with BMP proteins reveal that the expression of cathepsin B and D genes is regulated by BMP signaling, a pathway responsible for triggering cell death. Further cathepsin D protein is upregulated in the preapoptotic mesenchyme before being released into the cytosol, and overexpression of cathepsin D induces cell death in embryonic tissues by a mechanism including mitochondrial permeabilization and nuclear translocation of AIF. Combined inhibition of cathepsin and caspases suggests a redundancy in the apoptotic molecular machinery, providing evidence for compensatory activation mechanisms in the cathepsin pathway when caspases are blocked. It is concluded that lysosomal enzymes are functionally implicated in embryonic programmed cell death.

Here, we have studied how Sox genes and BMP signaling are functionally coupled during limb chondrogenesis. Using the experimental model of TGFbeta1-induced interdigital digits, we dissect the sequence of morphological and molecular events during in vivo chondrogenesis. Our results show that Sox8 and Sox9 are the most precocious markers of limb cartilage, and their induction is independent and precedes the activation of BMP signaling. Sox10 appears also to cooperate with Sox9 and Sox8 in the establishment of the digit cartilages. In addition, we show that experimental induction of Sox gene expression in the interdigital mesoderm is accompanied by loss of the apoptotic response to exogenous BMPs. L-Sox5 and Sox6 are respectively induced coincident and after the expression of Bmpr1b in the prechondrogenic aggregate, and their activation correlates with the induction of Type II Collagen and Aggrecan genes in the differentiating cartilages. The expression of Bmpr1b precedes the appearance of morphological changes in the prechondrogenic aggregate and establishes a landmark from which the maintenance of the expression of all Sox genes and the progress of cartilage differentiation becomes dependent on BMPs. Moreover, we show that Ventroptin precedes Noggin in the modulation of BMP activity in the developing cartilages. In summary, our findings suggest that Sox8, Sox9, and Sox10 have a cooperative function conferring chondrogenic competence to limb mesoderm in response to BMP signals. In turn, BMPs in concert with Sox9, Sox6, and L-Sox5 would be responsible for the execution and maintenance of the cartilage differentiation program.

We describe the expression pattern of Sox8, Sox9 and Sox10 during the development of the chick embryo heart. These Sox genes constitute the group E of the large Sox family of transcription factors. We show that the expression of Sox8, Sox9 and Sox10 in the developing heart correlates with heart septation and with the differentiation of the connective tissue of the valve leaflets. Sox10 appears also as a specific marker of developing heart nerves. These findings fit with the occurrence of morphological and functional anomalies of the heart reported in humans deficient for Sox9 and Sox10.

We have investigated the role of FGFs in the control of programmed cell death during limb development by analyzing the effects of increasing and blocking FGF signaling in the avian limb bud. BMPs are currently considered as the signals responsible for cell death. Here we show that FGF signaling is also necessary for apoptosis and that the establishment of the areas of cell death is regulated by the convergence of FGF- and BMP-mediated signaling pathways. As previously demonstrated, cell death is inhibited for short intervals (12 hours) after administration of FGFs. However, this initial inhibition is followed (24 hours) by a dramatic increase in cell death, which can be abolished by treatments with a BMP antagonist (Noggin or Gremlin). Conversely, blockage of FGF signaling by applying a specific FGF-inhibitor (SU5402) into the interdigital regions inhibits both physiological cell death and that mediated by exogenous BMPs. Furthermore, FGF receptors 1, 2 and 3 are expressed in the autopodial mesoderm during the regression of the interdigital tissue, and the expression of FGFR3 in the interdigital regions is regulated by FGFs and BMPs in the same fashion as apopotosis. Together our findings indicate that, in the absence of FGF signaling BMPs are not sufficient to trigger apoptosis in the developing limb. Although we provide evidence for a positive influence of FGFs on BMP gene expression, the physiological implication of FGFs in apoptosis appears to result from their requirement for the expression of genes of the apoptotic cascade. We have identified MSX2 and Snail as candidate genes associated with apoptosis the expression of which requires the combined action of FGFs and BMPs.

In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.

Major advances in the genetics of vertebrate limb development have been obtained in recent years. However, the nature of the signals which trigger differentiation of the mesoderm to form the limb skeleton remains elusive. Previously, we have obtained evidence for a role of TGFbeta2 in digit formation. Here, we show that activins A and B and/or AB are also signals involved in digit skeletogenesis. activin betaA gene expression correlates with the initiation of digit chondrogenesis while activin betaB is expressed coincidently with the formation of the last phalanx of each digit. Exogenous administration of activins A, B or AB into the interdigital regions induces the formation of extra digits. follistatin, a natural antagonist of activins, is expressed, under the control of activin, peripherally to the digit chondrogenic aggregates marking the prospective tendinous blastemas. Exogenous application of follistatin blocks physiological and activin-induced digit formation. Evidence for a close interaction between activins and other signalling molecules, such as BMPs and FGFs, operating at the distal tip of the limb at these stages is also provided. Chondrogenesis by activins is mediated by BMPs through the regulation of the BMP receptor bmpR-1b and in turn activin expression is upregulated by BMP signalling. In addition, AER hyperactivity secondary to Wnt3A misexpression or local administration of FGFs, inhibits activin expression. In correlation with the restricted expression of activins in the course of digit formation, neither activin nor follistatin treatment affects the development of the skeletal components of the stylopod or zeugopod indicating that the formation of the limb skeleton is regulated by segment-specific chondrogenic signals.

Bone morphogenetic proteins (BMPs) constitute a large family of secreted signals involved in the formation of the skeleton but the specific function of each member of this family remains elusive. GDF-5 is a member of the BMP family which has been implicated in several skeletogenic events including the induction and growth of the appendicular cartilages, the determination of joint forming regions, and the establishment of tendons. Here, we have studied the function of GDF-5 in digit skeletogenesis by analyzing the effects of its local administration in the developing autopod of embryonic chick and the regulation of its pattern of gene expression by other signals involved in digit morphogenesis. As reported in the mouse, the gdf-5 gene exhibits a precise distribution in the joint-forming regions of the developing chicken digital rays. GDF-5 beads implanted at the tip of the digits promote intense cartilage growth and fail to induce morphological or molecular signs of joint formation. Furthermore, GDF-5 beads implanted in the interdigits inhibit the formation of joints in the adjacent digits. These data suggest that the role of GDF-5 in joint formation is the control of growth and differentiation of the cartilage of the epiphyseal regions of the phalanges rather than accounting for the differentiation of the sinovial joint tissues. The interdigital mesoderm in spite of its potential to form ectopic digits with their tendinous apparatus failed to form either ectopic cartilages or ectopic tendons after the implantation of GDF-5 beads in the stages preceding cell death. At difference with other BMPs, GDF-5 exhibited only a weak cell death promoting effect. The BMP antagonist Noggin binds to GDF-5 and is able to inhibit all the observed effects of this growth factor in vivo. Potential interactions of GDF-5 with other signals involved in digits morphogenesis were also explored. BMP-7 regulates negatively the expression of gdf-5 gene in the joint forming regions and local treatment with Noggin induces the ectopic expression of gdf-5 in the interdigital mesoderm. Retroviral-induced misexpression of Indian or Sonic Hedgehog genes in the developing digits leads to the formation of digits without joints in which gdf-5 expression occurs throughout the entire perichondrial surface. In conclusion, this study indicates that GDF-5 is a signal regulated by other BMPs which controls the growth and differentiation of the epiphyses of the digital cartilages acting in close relationship with Hedgehog signaling., (Copyright 1999 Academic Press.)

In the final stages of limb morphogenesis, autopodial cells leaving the progress zone differentiate into cartilage or undergo apoptotic cell death, depending on whether they are incorporated into the digital rays or interdigital spaces. Most evidence indicates that these two opposite fates of the autopodial mesoderm are controlled by BMP signaling. However, the molecular basis for these two distinct actions of BMPs, including the receptors involved in the process, is controversial. In this study we have addressed this question by exploring the presence in the developing autopod of diffusible signals able to modulate BMP function and by analyzing the effects of their exogenous administration on the pattern of expression of BMP receptor genes. Our findings show that tgfbeta2 and noggin genes are expressed in the condensing region of the developing digital rays in addition to the well-known distribution in the autopodial tissues of FGFs (apical ectodermal ridge, AER) and BMPs (AER, progress zone mesoderm, and interdigital regions). Exogenous administration of all the factors causes changes in the expression of the bmpR-1b gene which are followed by parallel alterations of the skeletal phenotype: FGFs inhibit the expression of bmpR-1b compatible with their function in the maintenance of the progress zone mesoderm in an undifferentiated state; and TGFbetas induce the expression of bmpR-1b and promote ectopic chondrogenesis, compatible with a function in the establishment of the position of the digital rays. In addition we provide evidence for the occurrence of an interactive loop between BMPs and noggin accounting for the spatial distribution of bmpR-1b which may control the size and shape of the skeletal pieces. In contrast to the bmpR-1b gene, the bmpR-1a gene is expressed at low levels in the autopodial mesoderm and its expression is not modified by any of the tested factors regardless of their effects on chondrogenesis or cell death. Finally, the role of BMPs in programmed cell death is confirmed here by the intense inhibitory effect of noggin on apoptosis, but the lack of correlation between changes in the pattern of cell death induced by treatment with the studied factors and the expression of either bmpR-1a or bmpR-1b genes suggest that a still-unidentified BMP receptor may account for this BMP function., (Copyright 1998 Academic Press.)

The formation of the digits in amniota embryos is accompanied by apoptotic cell death of the interdigital mesoderm triggered through BMP signaling. Differences in the intensity of this apoptotic process account for the establishment of the different morphological types of feet observed in amniota (i.e., free-digits, webbed digits, lobulated digits). The molecular basis accounting for the differential pattern of interdigital cell death remains uncertain since the reduction of cell death in species with webbed digits is not accompanied by a parallel reduction in the pattern of expression of bmp genes in the interdigital regions. In this study we show that the duck interdigital web mesoderm exhibits an attenuated response to both BMP-induced apoptosis and TGFbeta-induced chondrogenesis in comparison with species with free digits. The attenuated response to these signals is accompanied by a reduced pattern of expression of msx-1 and msx-2 genes. Local application of FGF in the duck interdigit expands the domain of msx-2 expression but not the domain of msx-1 expression. This change in the expression of msx-2 is followed by a parallel increase in spontaneous and exogenous BMP-induced interdigital cell death, while the chondrogenic response to TGFbetas is unchanged. The regression of AER, as deduced by the pattern of extinction of fgf-8 expression, takes place in a similar fashion in the chick and duck regardless of the differences in interdigital cell death and msx gene expression. Implantation of BMP-beads in the distal limb mesoderm induces AER regression in both the chick and duck. This finding suggests an additional role for BMPs in the physiological regression of the AER. It is proposed that the formation of webbed vs free-digit feet in amniota results from a premature differentiation of the interdigital mesoderm into connective tissue caused by a reduced expression of msx genes in the developing autopod., (Copyright 1998 Academic Press.)

Bone Morphogenetic Protein 2 (BMP-2) and Osteogenic Protein 1 (OP-1, also termed BMP-7) are members of the transforming growth factor beta superfamily. In the present study, we have analyzed the effects of administering them locally at different stages and locations of the chick limb bud using heparin beads as carriers. Our results show that these BMPs are potent apoptotic signals for the undifferentiated limb mesoderm but not for the ectoderm or the differentiating chondrogenic cells. In addition, they promote intense radial growth of the differentiating cartilages and disturb the formation of joints accompanied by alterations in the pattern of Indian hedgehog and ck-erg expression. Interestingly, the effects of these two BMPs on joint formation were found to be different. While the predominant effect of BMP-2 is alteration in joint shape, OP-1 is a potent inhibitory factor for joint formation. In situ hybridizations to check whether this finding was indicative of specific roles for these BMPs in the formation of joints revealed a distinct and complementary pattern of expression of these genes during the formation of the skeleton of the digits. While Op-1 exhibited an intense expression in the perichondrium of the developing cartilages with characteristic interruptions in the zones of joint formation, Bmp-2 expression was a positive marker for the articular interspaces. These data suggest that, in addition to the proposed role for BMP-2 and OP-1 in the establishment of the anteroposterior axis of the limb, they may also play direct roles in limb morphogenesis: (i) in regulating the amount and spatial distribution of the undifferentiated prechondrogenic mesenchyme and (ii) in controlling the location of the joints and the diaphyses of the cartilaginous primordia of the long bones once the chondrogenic aggregates are established.

The establishment of the digital rays and the interdigital spaces in the developing limb autopod is accompanied by the occurrence of corresponding domains of expression of TGF beta s and BMPs. This study analyzes whether these coincident events are functionally correlated. The experiments consisted of local administration of TGF beta-1, TGF beta-2 or BMP-4 by means of heparin or Affi-gel blue beads to the chick limb autopod in the stages preceding the onset of interdigital cell death. When beads bearing either TGF beta-1 or -2 were implanted in the interdigits, the mesodermal cells were diverted from the death program forming ectopic cartilages or extra digits in a dose- and stage-dependent fashion. This change in the interdigital phenotype was preceded by a precocious ectopic expression of ck-erg gene around the bead accompanied by down-regulation of bmp-4, msx-1 and msx-2 gene expression. When BMP-beads were implanted in the interdigital spaces, programmed cell death and the freeing of the digits were both accelerated. Implantation of beads bearing BMP-4 at the tip of the growing digits was followed by digit bifurcation, accompanied by the formation of an ectopic area of cell death resembling an extra interdigit, both morphologically and molecularly. The death-inducing effect of the BMP beads and the chondrogenic-inducing effect of the TGF beta beads were antagonized by the implantation of an additional bead preabsorbed with FGF-2, which constitutes a signal characteristic of the progress zone. It is concluded that the spatial distribution of digital rays and interdigital spaces might be controlled by a patterned distribution of TGF beta s and BMPs in the mesoderm subjacent to the progress zone.

In this work we have analyzed the presence of elastic components in the extracellular matrices of the developing chick leg bud. The distributions of elastin and fibrillin were studied immunohistochemically in whole-mount preparations using confocal laser microscopy. The association of these constituents of the elastic matrix with other components of the extracellular matrix was also studied, using several additional antibodies. Our results reveal the transient presence of an elastin-rich scaffold of extracellular matrix fibrillar material in association with the establishment of the cartilaginous skeleton of the leg bud. The scaffold consisted of elastin-positive fibers extending from the ectodermal surface of the limb to the central cartilage-forming regions and between adjacent cartilages. Fibrillin immunolabeling was negative in this fibrillar scaffold while other components of the extracellular matrix including: tenascin, laminin and collagens type I, type III and type VI; appeared codistributed with elastin in some regions of the scaffold. Progressive changes in the spatial pattern of distribution of the elastin-positive scaffold were detected in explant cultures in which one expects a modification in the mechanical stresses of the tissues related to growth. A scaffold of elastin comparable to that found in vivo was also observed in high-density micromass cultures of isolated limb mesodermal cells. In this case the elastic fibers are observed filling the spaces located between the cartilaginous nodules. The fibers become reoriented and attach to the ectodermal basal surface when an ectodermal fragment is located at the top of the growing micromass. Our results suggest that the formation of the cartilaginous skeleton of the limb involves the segregation of the undifferentiated limb mesenchyme into chondrogenic and elastogenic cell lineages. Further, a role for the elastic fiber scaffold in coordinating the size and the spatial location of the cartilaginous skeletal elements within the limb bud is also suggested from our observations.

In this work we have attempted to characterize the programmed cell death process in the chick embryonic interdigital tissue. Interdigital cell death is a prominent phenomenon during limb development and has the role of sculpturing the digits. Morphological changes in the regressing interdigital tissue studied by light, transmission and scanning electron microscopy were correlated with the occurrence of internucleosomal DNA fragmentation, evaluated using agarose gels. Programming of the cell death process was also analyzed by testing the chondrogenic potential of the interdigital mesenchyme, in high density cultures. Our results reveal a progressive loss of the chondrogenic potential of the interdigital mesenchyme, detectable 36 hours before the onset of the degenerative process. Internucleosomal DNA fragmentation was only detected concomitant with the appearance of cells dying with the morphology of apoptosis, but unspecific DNA fragmentation was also present at the same time. This unspecific DNA fragmentation was explained by a precocious activation of the phagocytic removal of the dying cells, confirmed in the tissue sections. From our observations it is suggested that programming of cell death involves changes before endonuclease activation. Further, cell surface changes involved in the phagocytic uptake of the dying cells appear to be as precocious as endonuclease activation.

We have shown previously that from stage 27 the distal growing region of the limb exhibits a tenascin-rich sheet of extracellular matrix termed the "mesenchyme lamina" (ML), which runs from the ectodermal basement membrane in a proximal direction until it contacts the distal tip of the muscle blocks. This study reports experimental evidence that the mesenchyme lamina is a pretendinous structure that controls the spatial organization of the flexor and extensor tendons of the distal part of the chick leg. Two sets of experiments were designed to alter the ML position and examine subsequent tendon pattern formation. In a first series of experiments limbs with digits lacking phalangeal elements were induced by AER removal at stages 26 and 27. This procedure induced an abnormal arrangement of the ML around the distal tip of each terminal phalange of the truncated digit, which was followed by the development of a precisely similar pattern of abnormal extensor and flexor tendons. In the second set of experiments, an extradigit was induced to form in the interdigital mesenchyme through surgical removal of the marginal ectoderm of the third interdigit of stage 29 leg buds. By day 4 post-operation, a chondrogenic extradigit had formed, together with a ML that ran from the cartilage to the normal ventral flexor and dorsal extensor tendons. By day 6 post-operation, the experimentally induced ML had transformed into a tendinous structure connecting with the adjacent normal tendon. Both experiments show that the position of the ML defines the position of subsequent tendon development, thus supporting its role as a pretendinous structure which might be responsible for the alignment of the pretendinous condensing cells.

In the later developmental stages (Hamburger and Hamilton, 25-34) the distal part of the chick leg possesses a distinctive extracellular matrix (ECM) architecture which relates to myotendinous patterning. There are two components: firstly, a system of dorsoventrally oriented fibrils which link the two ectodermal surfaces through the undifferentiated distal mesenchyme and secondly, a 'mesenchyme lamina' originates at the basement membrane distally, but proximally runs through the mesoderm, subjacent and parallel to the basement membrane. The 'mesenchyme lamina' appears to be a precursor of developing tendons and is spatially related to the distal tips of the myogenic blocks. As developing tendons form on the inner surface of the lamina at its proximal end, it becomes less distinct and disappears. Further dorsoventral fibrils run from the 'mesenchyme lamina' into the developing condensations and chondrogenic elements of the phalanges. The architecture of the ECM was revealed by silver and lectin staining (peanut and Ricinus communis agglutinins, PNA and RCA I), by immunocytochemistry (for fibronectin, tenascin, collagen type I) and by ultrastructural analysis. Both components stain with silver, PNA following neuraminidase digestion, RCA I, tenascin and collagen type I. However, the dorsoventral fibrils are positive for fibronectin and negative for PNA, while conversely the mesenchyme lamina is positive for PNA but much less so for fibronectin. Tenascin has been shown to be a specialized mesenchyme component of tendons and myotendinous junctions (Chiquet and Fambrough, 1984). Such a basement membrane forming a 'mesenchyme lamina' appears to be unique in epithelial-mesenchymal developing systems and points to an ectodermal role in tendon pattern formation within the mesenchyme. We discuss the possible role of mechanical force in converting the dorsoventral tenascin-positive fibrils into the localized pattern of tendon insertions into the proximal parts of the phalanges. Distally the dorsoventral fibrils may shape the digital plate by pulling together the two ectodermal surfaces. A similar ECM architecture is found in corresponding stages in the developing wing.

Phagocytosis of naturally degenerating cells, by healthy myocardial cells of the bulbus cordis of 5--7-day chick embryos, was studied by electron microscopy. Myocardial cells showed cell processes surrounding dead cell fragments. The cell fragments appeared to be internalized later and digested within phagosomes. Ruthenium red was employed to ascertain whether the cell fragments were in fact internalized or located in pockets of the cell membrane. The results are discussed in the light of present knowledge of phagocytosis.

The hearts of chick embryos of stages 9-13 were subjected to SEM, TEM and histochemical studies to ascertain possible regional differences in the structure and composition of the cardiac jelly. Two distinct regions, the cardiac jelly filling the space located between the myocardium and the endocardium (MECJ) and the cardiac jelly filling the dorsal mesocardium (EECJ), were distinguished by their structural and histochemical properties. MECJ is formed by amorphous and fibrillar material arranged between the endocardial and myocardial layer. The amount of its components increases when cetylpyridinium chloride is introduced into the fixative, and it appears intensely stained by ruthenium red and alcian blue at low concentrations of MgCl2. The amount and arrangement of its components increase during the beginning of the looping process of the heart tube. The EECJ is very rich in ruthenium-red-positive basal-lamina-like material and the addition of cetylpyridinium chloride to the fixative does not modify its appearance. It also appears poorly stained by alcian blue at low concentrations of MgCl2 and its arrangment undergoes modifications closely associated with the events of endocardial fusion. The possible significance of these results in the early morphogenesis of the heart is discussed.

There is recent evidence showing that in addition to the well-known mesenchymal necrotic mechanism involved in the disappearance of the interdigital membranes, the ectodermal tissue may also play an active role in the formation of the free digits of most vertebrates. Ultrastructural study of the regressing interdigital membrane of the chick leg revealed significant changes at the epitheliomesenchymal interface. Disruptions of the ectodermal basal lamina and an intense deposition of collagenous material were the most conspicuous changes observed in the extracellular matrix. In addition the basal ectodermal cells showed prominent cell processes projected into the mesenchymal core of the membrane, and mesenchymal macrophages appeared to migrate through the epithelial tissue to be detached into the amniotic sac. It is concluded from our results that the elimination of the interdigital membranes is a complex process requiring the interaction of all the tissue components of the membrane.

In an attempt to clarify the mechanism of production of the syndactyly induced by Janus Green B (JGB) we have studied the morphology and structural modifications of the chick embryo leg bud after JGB administration by means of neutral red vital staining, whole-mount cartilage staining and light microscopy and transmission and scanning electron microscopy. The results show that the well-known inhibition of interdigital cell death is accompanied by a precocious alteration of the epithelial tissue and especially of the epithelial-mesenchymal interface. 24 h after JGB administration the cells of the AER reduce the number of junctions and the basal ectodermal cells are detached into the mesenchymal tissue in zones in which the basal lamina undergoes disruption. In addition the interdigital mesenchymal cells diverted from the dying program are able to undergo a rapid differentiation into cartilage. It is proposed that the mechanism of production of JGB-induced syndactyly might be due to an alteration of the normal epithelial-mesenchymal interactions rather than to a direct inhibitory effect of the JGB on the dying program.

The presence of cell death in the walls of the truncus and conus of the developing chick heart was investigated by a variety of light and electron microscopic techniques. Necrotic areas were observed in the myocardial layer of the truncus and conus and within the mesenchymal cells of the truncoconal ridges and aortopulmonary septum. These necrotic zones appeared first at Stage 25-26 and reached their maximum extent at Stages 29-32 undergoing later progressive disappearance. The morphological changes of the degenerating cells detectable under both transmission and scanning electron microscopy are also reported. The possible role of cell death in the morphogenesis of the truncus and conus is discussed.

Light and electron microscopical examination of the heart of the chick embryo between stages 9 and 11 (Hamburger & Hamilton) revealed degenerating and dead cells specifically localized in the midline of endocardial tubes. The morphology of cell death in this system does not differ from that described in other embryonic tissues and organs. Phagocytosing cells are rarely seen. The results of this study show that a destruction of midline cells of both endocardial tubes takes place during the fusion of heart anlages. The possible roles of this cell death in the morphogenesis of the tubular chick heart are discussed.

The pattern and structure of the blood vessels of the interdigital spaces of the leg bud have been studied by means of Indian ink injections and transmission electron microscopy in the chick and duck embryos. The results show that in the chick the interdigital necrotic process responsible for the freeing of the digits is followed by regression of the blood vessels. In the webbed foot of the duck, the interdigital necrotic processes are not followed by vascular regression. Transmission electron microscopic studies show that both in the chick and in the duck, interdigital blood vessels are immature structures lacking basal lamina. Dead cells of presumably endothelial origin were detected in the lumen of the regressing blood vessels of the chick but not in the duck. However, the intensity of this cell death process does not appear to be high enough to account by itself for the disappearance of the interdigital blood vessels. The possible relationships between interdigital mesenchymal cell death and vascular regression are discussed.

The formation of the free digits of the chick is accompanied by conspicuous surface changes of the interdigital ectoderm. These changes were much less pronounced or absent in the duck. As early as the interdigital grooves were detected in the chick, the morphological features of the ectodermal cells changed from a polygonal shape and flattened appearance to a rounded shape and bulging appearance. These changes were not present in the webbed foot of the duck. On the other hand the development of the interdigital commissures was accompanied by the formation of ectodermal ridges consisting of an accumulation of rounded cells which were in some cases in course of detachment to the amniotic cavity. These ridges were very prominent in all the interdigital commissures of the chick. In the duck they were less pronounced and were only present in the first and third commissure. From these results it is suggested that in addition to the well-known interdigital mesenchymal necrotic process (INZ) the ectodermal tissue of the interdigits might also be actively involved in the formation of free digits.

In the present work, we have analysed the possible involvement of ectodermal tissue in the control of interdigital mesenchymal cell death. Two types of experiments were performed in the stages previous to the onset of interdigital cell death: removal of the AER of the interdigit; removal of the dorsal ectoderm of the interdigit. After the operation embryos were sacrificed at 10-12 h intervals and the leg buds were studied by whole-mount cartilage staining, vital staining with neutral red and scanning electron microscopy. Between stages 27 and 30, ridge removal caused a local inhibition of the growth of the interdigit. In a high percentage of the cases, ridge removal at these stages was followed 30-40 h later by the formation of ectopic nodules of cartilage in the interdigit. The incidence of ectopic cartilage formation was maximum at stage 29 (60%). In all cases, cell death took place on schedule although the intensity and extent of necrosis appeared diminished in relation to the intensity of inhibition of interdigital growth and to the presence of interdigital cartilages. Ridge removal at stage 31 did not cause inhibition of the growth of the interdigit and ectopic chondrogenesis was only detected in 3 out of 35 operated embryos. Dorsal ectoderm removal from the proximal zone of the interdigit at stage 29 caused the chondrogenesis of the proximal interdigital mesenchyme in 6 out of 18 operated embryos. The pattern of neutral red vital staining was consistent with these results revealing a partial inhibition of interdigital cell death in the proximal zone of the interdigit. It is proposed that under the present experimental conditions the mesenchymal cells are diverted from the death programme by a primary transformation into cartilage.

Fine structural study of interdigital membranes during formation of the digits of the duck foot reveals that the interdigital necrosis is accompanied by a high deposition of collagenous material in the epithelio-mesenchymal interface, rupture of the basal lamina and detachment of ectodermal cells into the amniotic sac. These changes are similar to those observed in the regressing interdigital membrane of the chick although their intensity and temporal extension are less pronounced in the duck. It is suggested that these changes account for the disappearance of the marginal zone of the duck interdigital membranes. The possible causal relationship between the different structural changes are discussed.

The muscular wall of the bulbus cordis of the chick embryo was studied by electron microscopy during H. H. stages 27--31. Results show that the muscle cells of the bulbus undergo necrosis during these stages. The morphology of dead cells in this area is similar to that described in other embryonic myogenic tissues. Although dead myocytes appear acid-phosphatase-negative, lysosomes may well be involved in this necrotic process as there is a significant number of cells with autolytic lesions among the neighbouring healthy muscle cells. The phagocytosis of the cell detritus resulting from the degenerative process appears to be carried out by neighbouring myocytes and by macrophages. The significance of these results is discussed both from the cytological and from the morphogenetic point of view.