Four and a half domain 2 (FHL2) scaffolding protein is a marker of connective tissues of developing digits and regulates fibrogenic differentiation of limb mesodermal progenitors.

Four and a half LIM domain 2 (FHL2) is a multifunctional scaffolding protein of well-known function regulating cell signalling cascades and gene transcription in cancer tissues. However, its function in embryonic systems is poorly characterized. Here, we show that Fhl2 is involved in the differentiation of connective tissues of developing limb autopod. We show that Fhl2 exhibits spatially restricted and temporally dynamic expression around the tendons of developing digits, interphalangeal joint capsules, and fibrous peridigital tissue. Immunolabelling analysis of the skeletal progenitors identified a predominant, but not exclusive, cytoplasmic distribution of FHL2 being associated with focal adhesions and actin cytoskeleton. In the course of chondrogenic differentiation of cultures of limb skeletal progenitors, the expression of Fhl2 is down-regulated. Furthermore, cultures of skeletal progenitors overexpressing Fhl2 take on a predominant fibrogenic appearance. Both gain-of-function and loss-of-function experiments in the micromass culture assays revealed a positive transcriptional influence of Fhl2 in the expression of fibrogenic markers including Scleraxis, Tenomodulin, Tenascin C, ßig-h3, and Tgif1. We further show that the expression of Fhl2 is positively regulated by profibrogenic signals including Tgfß2, all-trans-retinoic acid, and canonical Wnt signalling molecules and negatively regulated by prochondrogenic factors of the bone morphogenetic protein family. Expression of Fhl2 is also regulated negatively in immobilized limbs, but this influence appears to be mediated by other connective tissue markers, such as Tgfßs and Scleraxis.

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

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

Cathepsin D gene expression outlines the areas of physiological cell death during embryonic development.

The implication of lysosomes in the activation of physiological cell death (PCD) was proposed some decades ago. In this work, we show that the expression of the lysosomal enzyme cathepsin D is up-regulated in developing tissues undergoing apoptosis. By comparing vital and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) labeling patterns with in situ hybridization for this gene in a variety of tissues and organs, we show that this procedure constitutes a reliable technique to map the regions of PCD in the embryo. Using this methodological approach, we report the occurrence of two new areas of PCD in the developing limb.

[Janus' brain]. / Le cerveau de Janus.

In this first lesson, the author uses a neuro-physiological and philosophical approach to describe the functioning of the two cerebral hemispheres, emphasizing their asymmetry and the consequences of this differentiation.

Expression of Sox8, Sox9 and Sox10 in the developing valves and autonomic nerves of the embryonic heart.

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.

Comparative analysis of the expression and regulation of Wnt5a, Fz4, and Frzb1 during digit formation and in micromass cultures.

Previous studies have shown that three members of the Wnt signaling pathway, the ligand WNT5A, the receptor FZ4, and the Wnt antagonist FRZB1, are implicated in the formation and differentiation of the digits. In this study, we have attempted to establish a functional correlation between them by comparing their expression patterns and their regulation by the signals controlling proliferation and differentiation of the limb mesoderm during formation of the avian digits in vivo and in micromass cultures. In vivo Wnt5a and Fz4 are expressed in the undifferentiated mesoderm of the autopod and in the differentiating digit cartilages. In the undifferentiated mesoderm, the expression of both genes is regulated positively by FGFs and negatively by bone morphogenetic proteins (BMPs). As chondrogenic differentiation starts, Fz4 becomes intensely up-regulated in the aggregate and in the developing perichondrium, whereas transcripts of Wnt5a are excluded from the core of the aggregate but maintained in the surrounding mesenchyme and perichondrium. In addition, at this stage, the expression of both genes become positively regulated by BMPs. These changes in expression and regulation are coincident with the induction of Frzb1 in the chondrogenic aggregate, which is expressed under the positive control of BMPs. Our findings fit with a role of Wnt5a/Fz4 negatively regulating in vivo the initiation and progression of cartilage differentiation. In vitro, only Frzb1 is expressed and regulated in a manner resembling that observed in vivo. Wnt5a and Fz4 are both expressed in the differentiating mesenchyme of micromass cultures, but their expression is not significantly regulated by the addition of FGF-2 or BMP-7 to the culture medium.

Stem cell emergence and hemopoietic activity are incompatible in mouse intraembryonic sites.

In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/ aorta-gonad-mesonephros (AGM) region. Here, we address the following question: can the P-Sp/AGM environment support hemopoietic differentiation as well as generate stem cells, and, conversely, are other sites where hemopoietic differentiation occurs capable of generating stem cells? Although P-Sp/AGM generates de novo hemopoietic stem cells between 9.5 and 12.5 days post coitus (dpc), we show here that it does not support hemopoietic differentiation. Among mesoderm-derived sites, spleen and omentum were shown to be colonized by exogenous cells in the same fashion as the fetal liver. Cells colonizing the spleen were multipotent and pursued their evolution to committed progenitors in this organ. In contrast, the omentum, which was colonized by lymphoid-committed progenitors that did not expand, cannot be considered as a hemopoietic organ. From these data, stem cell generation appears incompatible with hemopoietic activity. At the peak of hemopoietic progenitor production in the P-Sp/AGM, between 10.5 and 11.5 dpc, multipotent cells were found at the exceptional frequency of 1 out of 12 total cells and 1 out of 4 AA4.1+ cells. Thus, progenitors within this region constitute a pool of undifferentiated hemopoietic cells readily accessible for characterization.

Antigenic profiles of endothelial and hemopoietic lineages in murine intraembryonic hemogenic sites.

Two hemogenic sites are present in mouse embryos before the onset of fetal liver hemopoiesis. While the yolk sac provides for immediate erythropoiesis, an intraembryonic region encompassing the dorsal aorta produces definitive hematopoietic stem cells, as shown experimentally. At early developmental stages this region, that we named paraaortic splanchnopleura, produces multipotent progenitors. At the time of fetal liver colonisation, the paraaortic splanchnopleura further evolves into aorta, gonads and mesonephros (AGM) and contains progenitors capable of long term multilineage reconstitution. Only then are cytologically identifiable collections of early hemopoietic cells present in various arteries and in the mesentery. The present report focuses on the antigenic characterisation of immature hemopoietic progenitors in order to trace back the intraembryonic precursors at earlier developmental stages. CD34, an antigen expressed by immature progenitors and endothelial cells, labels all potential hemopoietic sites. Markers, supposed to counterstain endothelial cells and spare CD34+ hemopoietic cells, also stain various hemopoietic cells. The meaning of these shared antigenic expressions between cells of the endothelial and hemopoietic lineages in the early embryo is discussed.

A1 demonstrates restricted tissue distribution during embryonic development and functions to protect against cell death.

Members of the bcl-2 gene family are essential regulators of cell survival in a wide range of biological processes. A1, a member of the family, is known to be expressed in certain adult tissues. However, the precise tissue distribution and function of A1 remains poorly understood. We show here that A1 is expressed in multiple tissues during murine embryonic development. In the embryo, A1 was detected first at embryonic day 11.5 in liver, brain, and limbs. At day 13.5 of gestation, A1 expression was observed in the central nervous system, liver, perichondrium, and digital zones of developing limbs in a pattern different from that of bcl-X. In the central nervous system of 15.5-day embryos, A1 was expressed at high levels in the ventricular zone and cortical plate of brain cortex. Significantly, the interdigital zones of limbs and the intermediate region of the developing brain cortex, two sites associated with extensive cell death, were devoid of A1 and bcl-X. The expression of A1 was retained in many adult tissues. To assess the ability of A1 to modulate cell death, stable transfectants expressing different amounts of A1 protein were generated in K562 cells. Expression of A1 was associated with retardation of apoptotic cell death induced by actinomycin D and cycloheximide as well as by okadaic acid. Confocal microscopy showed that the A1 protein was localized to the cytoplasm in a pattern similar to that of Bcl-2. These results demonstrate that the expression of A1 is wider than previously reported in adult tissues. Furthermore, its distribution in multiple tissues of the embryo suggests that A1 plays a role in the regulation of physiological cell death during embryonic development.

Potential intraembryonic hemogenic sites at pre-liver stages in the mouse.

In the course of a previous experimental study on the early development of the mouse embryo hemopoietic system, we found that, at the 10-25 pairs of somite stages, the para-aortic splanchnopleure contains hemopoietic progenitors. Trying to discover a structural basis for this potentiality, we have looked for cytological signs of hemopoiesis in the embryo proper between 8.5 and 12 days post-coitum, i.e. prior to full-blown fetal liver hemopoiesis. Two suggestive findings are reported: (1) intra-arterial hemopoietic cells aggregates are present in the omphalomesenteric and umbilical arteries and to a lesser degree in the dorsal aorta; (2) cells groups resembling yolk sac blood islands are observed in the mesentery. The intra-arterial aggregates are strikingly similar to the intra-aortic clusters of avian embryos. These cytological aspects provide the anatomical basis underlying recent functional data that revealed the hemogenic capacity of the para-aortic splanchnopleure.

Para-aortic splanchnopleura from early mouse embryos contains B1a cell progenitors.

Definitive erythropoiesis in birds originates from stem cells that emerge in the splanchnopleural mesoderm near the embryonic aorta. The yolk sac is still generally held to be the unique provider of haematopoietic stem cells during mammalian ontogeny, although there may be an alternative intraembryonic source of stem cells in the mouse fetus. Here we search for a possible non-yolk-sac source of stem cells by grafting intraembryonic splanchnopleura from 10- to 18-somite mouse embryos into adult immunodeficient SCID mice. We find significant amounts of donor-derived serum IgM, normal numbers of IgM-secreting plasma cells, and the B1a (IgM(a)brightB220dullCD5+) cell subset to be fully reconstituted by donor progenitors 3 to 6 months after engraftment. The haematogenic capacity revealed in our experiments is present in a previously unrecognized site, the earliest described in the embryo, 12 hours before fetal liver colonization.

Basement membrane alterations during development and regression of tubular cysts.

Tubular cysts consisting of dilatation of the collecting ducts at the level of the subcapsular zone of the kidney were induced in newborn rabbits by a single injection of methylprednisolone acetate. We describe here the structural and compositional modifications of the tubular basement membrane (BM) during the formation, growth, and regression of the tubular cysts. During development of the tubular cysts the cystic BM appeared thickened and multilayered, with numerous matrix vesicles. Alcian blue- (AB) and ruthenium red- (RR) positive material distributed differently along the BM of control and cystic tubuli. While the amount of RR-positive material appeared increased in the cystic BM, no differences in the intensity of the AB staining could be discerned between normal and cystic tubuli. Immunofluorescent staining for laminin and type IV collagen appeared to be slightly decreased in the cystic tubuli. However, the amount of fibronectin appeared clearly increased. These changes in the cystic BM appear at the beginning of the tubular dilatation and are not observed in other renal BM. We suggest that there is a causal relationship between the modifications of the BM and the development of the tubular cysts. Glucocorticoids appear to modify the synthesis and/or secretion of the BM components. An abnormal BM should modify the spatial and chemical signals encoded within the BM that, in turn, could lead to abnormal behavior of the tubular cells. This may result in a loss of the normal developmental constraints imposed upon the tubular epithelium, which then undergoes cystic dilatation. During the regression of the cysts, the abnormalities of the BM progressively disappear. The sharp increase in the number of interstitial cells, which show close relationships with the components of the BM, suggests that these cells may be involved in the removal of the cyst BM.

Structural and morphometric characteristics of the basement membrane of rabbit parietal podocytes induced by corticoids.

This paper presents a structural and morphometric study of the basement membrane underlying the parietal epithelium of cysts developed in the rabbit kidney after a single postnatal injection of methylprednisolone acetate. This epithelium consists of podocyte-like cells named parietal podocytes. Our results show that the parietal podocytes synthesize their own basement membrane in vivo. However, the different laminae of this membrane present differences in structure and thickness when compared to the analogous layers of both the normal glomerular and parietal basement membranes. Differences between the different zones of the parietal podocytic basement membrane are also observed, depending upon the structure that surrounds the cyst (capillaries, interstitial cells or loose connective tissue). A lamina lucida, analogous to the lamina lucida interna of the glomerular basement membrane, is formed only in segments of the parietal podocytic basement membrane in close contact with endothelial or interstitial cells. The thickness of the lamina lucida externa of the parietal podocytic basement membrane appears regulated by the presence of interstitial cells and capillaries. In the zones in contact with capillaries, the lamina densa of the parietal prodocytic basement membrane appears thinner than in other segments. This fact, together with structural observations, suggests that the formation of a single basement membrane, interposed between the parietal podocytes and the endothelium, does not take place by fusion of the two basement membranes but by previous removal of the capillary basement membrane. In areas where the parietal podocytic basement membrane is in contact with loose connective tissue, a lamina fibroreticularis is observed. All these data indicate that the formation of the basement membrane by the parietal podocytes is influenced by nonpodocytic cells. Around the glomerular cysts, a tardy formation of capillaries is observed. Parietal podoytes can be hypothesized to synthesize an angiogenic factor that may be implicated in the process of angiogenesis.

Histochemical characteristics of the basement membranes of the parietal podocytes in the rabbit kidney. A light- and electron-microscopic study.

The patterns of silver affinity and following treatment with guanidine were studied in the basement membrane produced by pareital podocytes induced by corticoids in newborn rabbits. The goal of this study was to analyze the role of the different cell types of the renal corpuscle in the determination of the histochemical characteristics of the glomerular basement membrane (GBM). Jones' method shows that while the GBM exhibited silver affinity only after periodic-acid oxidation, the basement membrane of parietal podocytes exhibits the same histochemical characteristics as the normal parietal basement membrane, appearing deep black both after periodic-acid or permanganate oxidation, and after elastase or lysozyme digestions. Since the treatment with guanidine shows that the basement membrane of the parietal podocytes lacks the endothelial component typical of the GBM, it may be suggested that the special resistance to silver impregnation exhibited by the basement membrane after permanganate oxidation or after different enzymatic digestions is due to its endothelial component.

Arterial segmentation and subsegmentation in the human spleen.

The segmental nature of the arterial tree of the human spleen was analyzed in 181 subjects of both sexes, who had died of various accidental causes. Based on the observation of the pattern of the terminal and polar splenic branches, selective arteriographs and corrosion casts, and taking account of the ideas reported in the literature, we proposed that the spleen is divided in arterial segments and subsegments. Segments are the territories corresponding to both the primary branches of the splenic artery (primary segments) and the polar arteries (polar segments). In 92.82% of the cases there are two primary segments and in 7.18% three primary segments. Associated to these, in 29.28% there is a superior polar segment, in 44.75% an inferior polar segment, and in 10.49% both superior and inferior polar segments are present. Thus, the number of segments varies from two to five. Occasionally, two or three inferior polar segments can be present. Subsegments are the territories corresponding to the extrasplenic subdivisions of the primary branches and the polar arteries. According to the number of arterial subdivisions, the subsegments can be of second, third, fourth, fifth or sixth order. The last branches of the splenic artery (penetrating arteries) are all subsegmentary in nature and supply hilar or polar subsegments. Anastomoses between extrasplenic branches of the splenic artery were observed in 19.89% of the cases. Sometimes, thin anastomotic bridges could be observed between arterial splenic compartments.

Retinal cell death occurs in the absence of retinal disc invagination: experimental evidence in papaverine-treated chicken embryos.

In an attempt to clarify the relationship between the presence of retinal cell death and the invagination of the optic vesicle, we have tested the occurrence and cytological characteristics of the retinal necrotic areas in the embryonic chicken after the administration in ovo of papaverine. Papaverine, a Ca2+ antagonist, was found to prevent the invagination of the optic vesicle. All embryonic retinae presented two distinct necrotic areas. However, these areas of cell death appeared abnormally located in the experimental, uninvaginated retina. One area was located at the transition between the retinal disc and the ventral wall of the optic vesicle; a second area was located in the dorsal wall of the optic vesicle, close to the optic stalk. We suggest that these necrotic areas represent the normal necrotic areas, should the invagination of the retinal disc have taken place. Retinal cell death appears to be programmed; it occurs whether the retinal disc invaginates or not. Cell death appears, in this experimental model, as a natural marker giving evidence that the embryonic retinal cells move from the optic stalk into the invagination retinal disc during normal eye cup formation. In addition to the uninvaginated optic vesicle the lens placode failed to invaginate in 45% of the cases, forming a lens vesicle in 55% of the remaining cases. This suggests that the two processes of invagination are governed by a different set of factors.

Duplication of slit diaphragms in the cystic glomeruli of rabbits treated with methylprednisolone acetate.

Duplication of the slit diaphragms between the pedicels of the visceral and parietal podocytes of glomerular cysts induced by a single intramuscular injection of methylprednisolone acetate is reported here. These changes are not accompanied by modifications of the cell coat of the podocytes.