Slug, a zinc finger gene previously implicated in the early patterning of the mesoderm and the neural crest, is also involved in chick limb development

The great advances made over the last few years in the identification of signalling molecules that pattern the limb bud along the three axes make the limb an excellent model system with which to study developmental mechanisms in vertebrates. The understanding of the signalling networks and their mutual interactions during limb development requires the characterisation of the corresponding downstream genes. In this study we report the expression pattern of Slug, a zinc-finger-containing gene of the snail family, during the development of the limb, and its regulation by distinct axial signalling systems. Slug expression is highly dynamic, and at different stages of limb development can be correlated with the zone of polarizing activity, the progress zone and the interdigital areas. We show that the maintenance of its expression is dependent on signals from the apical ectodermal ridge and independent of Sonic Hedgehog. We also report that, in the interdigit, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at stages of tissue differentiation is consistent with its role in early development, in maintaining the mesenchymal phenotype and repressing differentiation processes. We suggest that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone.     

Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo

After gastrulation, progenitor cells of the cardiac, visceral and body wall musculature arise at defined positions within the mesodermal layer of the Drosophila embryo. The regulatory mechanisms underlying this process of pattern formation are largely unknown, although ablation experiments carried out in other insects indicate that inductive influences from ectodermal cells have major roles in embryonic mesoderm differentiation. An early and important event in the regional subdivision of the mesoderm is the restriction of tinman expression to dorsal mesodermal cells. Genetic analysis has shown that this homeobox gene controls the formation of the visceral musculature and the heart from dorsal portions of the mesoderm. We now show that an inductive signal from dorsal ectodermal cells is required for activation of tinman in the underlying mesoderm and present evidence that Decapentaplegic (Dpp), a member of the transforming growth factor-beta superfamily, serves as a signalling molecule in this process. This demonstrates that the spatial expression of dpp in the ectoderm determines which cells of the mesoderm become competent to develop into visceral mesoderm and the heart.     

Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain

The Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutation in the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of both the paired domain and the homeodomain, suggesting that they may functionally interact. To investigate this possibility further, we have analyzed the DNA-binding properties of additional point mutants in the Pax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H, G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53G in the homeodomain), the Pax-1 un mutation (G15A) and a substitution associated with Peters' anomaly in the PAX-6 gene (R23G). Within the paired domain, seven of 10 mutations were found to abrogate DNA-binding by the paired domain. Remarkably, these seven mutations also affected DNA binding by the homeodomain, causing either a complete loss (P17L and G66D), a reduction (R23G, R23L, G15S and G15A) or an increase in DNA-binding activity (N14H). In addition, the effect of paired domain mutations occurred at the level of monomer formation by the homeodomain, while the dimerization potential of this domain seemed unaffected in mutants where it could be analyzed. Furthermore, while both homeodomain mutations were found to abolish DNA binding by this domain, the R53G mutation also abrogated DNA binding by the paired domain. The important observation that independent mutations in either domain can affect DNA binding by the other in the intact Pax-3 protein strongly suggests that the two domains are not functionally independent but bind DNA through cooperative interactions. Modeling the deleterlous mutations on the three-dimensional structure of the paired domain of Drosophila Prd shows that these mutations cluster at the DNA interface, thus suggesting that a series of DNA contacts are essential for DNA binding by both the paired domain and the homeodomain of Pax-3.     

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.     

Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.     

BMP1-related metalloproteinases promote the development of ventral mesoderm in early Xenopus embryos

Bone morphogenetic protein 1 (BMP1) is a metalloproteinase closely related to Drosophila Tolloid (Tld). Tld regulates dorsoventral patterning in early Drosophila embryos by enhancing the activity of Dpp, a member of the TGF-beta family most closely related to BMP2 and BMP4. In Xenopus BMP4 appears to play an essential role in dorsoventral patterning, promoting the development of ventral fates during gastrula stages. To determine if BMP1 has a role in regulating the activity of BMP4, we have isolated cDNAs for Xenopus BMP1 and a novel closely related gene that we have called xolloid (xld). Whereas xbmp1 is uniformly expressed at all stages tested, the initial uniform expression of xld becomes localized to two posterior ectodermal patches flanking the neural plate and later to the inner ectoderm of the developing tailbud. xld is also expressed in dorsal regions of the brain during tailbud stages and is especially abundant in the ventricular layer of the dorsal hindbrain caudal to the otic vesicle. Overexpression of either gene inhibits the development of dorsoanterior structures in whole embryos and ventralizes activin-induced dorsal mesoderm in animal caps. Since ventralization of activin-induced animal caps can be blocked by coinjecting a dominant-inhibitory receptor for BMP2 and BMP4, we suggest a role for BMP1 and Xld in regulating the ventralizing activity of these molecules.     

Induction of primitive streak and Hensen's node by the posterior marginal zone in the early chick embryo

In the preprimitive streak chick embryo, the search for a region capable of inducing the organizer, equivalent to the Nieuwkoop Center of the amphibian embryo, has focused on Koller's sickle, the hypoblast and the posterior marginal zone. However, no clear evidence for induction of an organizer without contribution from the inducing tissue has been provided for any of these structures. We have used DiI/DiO labeling to establish the fate of midline cells in and around Koller's sickle in the normal embryo. In the epiblast, the boundary between cells that contribute to the streak and those that do not lies at the posterior edge of Koller's sickle, except at stage X when it lies slightly more posteriorly in the epiblast. Hypoblast and endoblast (a second lower layer formed under the streak) have distinct origins in the lower layer, and goosecoid expression distinguishes between them. We then used anterior halves of chick prestreak embryos as recipients for grafts of quail posterior marginal zone; quail cells can be identified subsequently with a quail-specific antibody. Anterior halves alone usually formed a streak, most often from the posterior edge. Quail posterior marginal zones without Koller's sickle were grafted to the anterior side of anterior halves. These grafts were able to increase significantly the frequency of streaks arising from the anterior pole of stage X-XI anterior halves without contributing to the streak or node. Stage XII anterior halves no longer responded. A goosecoid-expressing hypoblast did not form under the induced streak, indicating that it is not required for streak formation. We conclude that the marginal zone posterior to Koller's sickle can induce a streak and node, without contributing cells to the induced streak.     

Cross talk between adhesion molecules: control of N-cadherin activity by intracellular signals elicited by beta1 and beta3 integrins in migrating neural crest cells

During embryonic development, cell migration and cell differentiation are associated with dynamic modulations both in time and space of the repertoire and function of adhesion receptors, but the nature of the mechanisms responsible for their coordinated occurrence remains to be elucidated. Thus, migrating neural crest cells adhere to fibronectin in an integrin-dependent manner while maintaining reduced N-cadherin-mediated intercellular contacts. In the present study we provide evidence that, in these cells, the control of N-cadherin may rely directly on the activity of integrins involved in the process of cell motion. Prevention of neural crest cell migration using RGD peptides or antibodies to fibronectin and to beta1 and beta3 integrins caused rapid N-cadherin-mediated cell clustering. Restoration of stable intercellular contacts resulted essentially from the recruitment of an intracellular pool of N-cadherin molecules that accumulated into adherens junctions in tight association with the cytoskeleton and not from the redistribution of a preexisting pool of surface N-cadherin molecules. In addition, agents that cause elevation of intracellular Ca2+ after entry across the plasma membrane were potent inhibitors of cell aggregation and reduced the N-cadherin- mediated junctions in the cells. Finally, elevated serine/ threonine phosphorylation of catenins associated with N-cadherin accompanied the restoration of intercellular contacts. These results indicate that, in migrating neural crest cells, beta1 and beta3 integrins are at the origin of a cascade of signaling events that involve transmembrane Ca2+ fluxes, followed by activation of phosphatases and kinases, and that ultimately control the surface distribution and activity of N-cadherin. Such a direct coupling between adhesion receptors by means of intracellular signals may be significant for the coordinated interplay between cell-cell and cell-substratum adhesion that occurs during embryonic development, in wound healing, and during tumor invasion and metastasis.     

Role of nonrandomly oriented cell division in shaping and bending of the neural plate

Neural plate shaping and bending are crucial to the process of neural tube formation. Shaping requires intrinsic forces generated by alterations in neuroepithelial cell behavior, whereas bending requires similar intrinsic forces and extrinsic forces generated by alterations in cell behaviors outside the neural plate. This study evaluates the role of nonrandomly oriented cell division in neural plate shaping and epidermal ectoderm expansion during bending by examining mitotic spindle orientation in the neuroepithelium and epidermal ectoderm throughout neurulation. Neuroepithelial mitotic spindles are oriented preferentially in the rostrocaudal plane, suggesting a role for nonrandomly oriented (i.e., rostrocaudal) neuroepithelial cell division in longitudinal lengthening of the neural plate during shaping. Epidermal ectoderm mitotic spindles are oriented preferentially in both rostrocaudal and mediolateral planes, suggesting a role for nonrandomly oriented cell division in epidermal ectoderm elongation and expansion. In neural plate and epidermal ectoderm isolates separated prior to shaping and bending, mitotic spindles continued to be oriented preferentially in the rostrocaudal plane; however, a preferential mediolateral mitotic spindle orientation could not be demonstrated in the epidermal ectoderm isolates. We conclude that the nonrandom rostrocaudal orientation of cell division in the neuroepithelium and epidermal ectoderm is an autonomous process, occurring in the absence of forces from adjacent tissues, whereas the nonrandom mediolateral orientation of cell division in the epidermal ectoderm is dependent upon interactions with the neural plate.     

Negative regulation by interleukin-3 (IL-3) of mouse early B-cell progenitors and stem cells in culture: transduction of the negative signals by betac and betaIL-3 proteins of IL-3 receptor and absence of negative regulation by granulocyte-macrophage colony-stimulating factor

The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit betac. However, in the mouse, there is an additional IL-3 signaling protein, betaIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from betac- and betaIL-3-knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin-) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin-Sca-1-positive, c-kit-positive marrow cells from 5-FU-treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with "compromised" marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors.     

A functional soluble form of the murine mannose receptor is produced by macrophages in vitro and is present in mouse serum

A soluble form of the mannose receptor (sMR) has been found in conditioned medium of primary macrophages in vitro and in mouse serum. sMR was released as a single species, had a smaller size than the cell-associated form, and accumulated in macrophage-conditioned medium, in a cytokine-regulated manner, to levels comparable with those found for cell-associated mannose receptor. Pulse-chase experiments showed that sMR production in culture occurred by constitutive cleavage of pre-existing full-length protein. A binding assay was developed to determine the sugar specificity of sMR and its ability to interact with pathogens and particulate antigens (i.e. Candida albicans and zymosan). Protease inhibitor studies suggested that sMR was produced by cleavage of an intact mannose receptor by a matrix metalloprotease or ADAM metalloprotease. A role for sMR in the immune response is proposed based on its binding properties, regulation by cytokines, and the previous discovery of putative ligands for the cysteine-rich domain of the mannose receptor in lymph nodes and spleen.     

The hyperinsulinemia produced by concanavalin A in rats is opioid-dependent and hormonally regulated

BACKGROUND: Peroxidatively modified low-density lipoprotein (LDL) may contribute to the atherosclerotic process; therefore, protecting LDL against peroxidation may reduce or retard the progression of atherosclerosis. We evaluated the effect of alpha-tocopherol on copper-catalyzed LDL peroxidative modification. METHODS: The protective effects of alpha-tocopherol on copper-catalyzed LDL peroxidative modification were examined by measurement of the concentration of lipid hydroperoxides in LDL and by the provision of LDL cholesterol to lymphocytes via the LDL receptor-mediated pathway. RESULTS: The measurement of concentration of lipid hydroperoxides in LDL showed that alpha-tocopherol inhibited the peroxidative modification of LDL. Also, alpha-tocopherol preserved the ability of LDL to be recognized by LDL receptors in peripheral blood lymphocytes to the same extent as native LDL. CONCLUSION: These findings indicate that alpha-tocopherol may protect LDL against peroxidative modification, maintaining its ability to act as a ligand for LDL receptors in vivo.     

Expression of protein kinase C gene family members is temporally and spatially regulated during neural development in vitro

The dog mastocytoma BR cell line provides us with a permanent source of canine mast cells, allowing a characterization of secretory mediators that exert important effects in canine allergic and nonallergic diseases and in physiological processes. We studied the ultrastructural characteristics and histamine releasing activity after immunological and non-immunological stimuli of the dog mastocytoma BR cell line, and compared the cell line to normal skin mast cells enzymatically isolated from healthy dogs. The histamine content of BR cells was 0.04 +/- 0.002 pg/cell, approximately 100-fold less than that found in canine skin mast cells. Non-immunologic stimuli induced similar concentration-dependent histamine release from skin mast cells and BR cells: 29.3 +/- 0.9% vs. 12.7 +/- 0.7% (calcium ionophore A23187), 23.3 +/- 0.7% vs. 18.8 +/- 0.7% (substance P) and 12.5 +/- 0.3% vs. 12.1 +/- 0.9% (compound 48/80), respectively. Immunologic stimulation, however, was only effective on canine skin mast cells, causing 30.9 +/- 1.7%, 27.7 +/- 0.6% and 12.2 +/- 0.9% histamine release in response to anti-canine IgE, concanavalin A, and antigen Asc S 1, respectively. The absence of functional IgE receptors in BR cells was confirmed by the lack of response to anti-IgE and antigen Asc S 1 following passive sensitization with dog atopic serum and dog antigen sensitized serum. We conclude that BR cells are able to release histamine after non-immunologic stimulation in a similar manner to canine skin mast cells, but that there are morphological and functional differences possibly due to different states of maturity or differentiation. For this reason the study of the highly homogeneous BR cells could offer insights into dog mast cell biology in contexts where freshly isolated cells cannot be used because of low purity and recovery.     

Expression of the POU transcription factor Brn-5 is an early event in the terminal differentiation of CNS neurons

OBJECTIVE: To evaluate transesophageal echocardiography (TEE) as an intraoperative monitoring modality and to assess its safety, reliability, and overall utility in real-time use during cardiac surgery. DESIGN: Prospective, observational cohort study performed from January 1993 to June 1997. SETTING: Operating room of a tertiary care hospital for cardiology and cardiovascular surgery. PARTICIPANTS: Five thousand and sixteen adult patients with acquired heart disease, who underwent 1,356 valve procedures and 3,660 coronary artery bypass graftings (CABGs). INTERVENTIONS: All patients were monitored with radial artery and pulmonary artery catheters, along with continuous TEE monitoring with a multiplane transducer. MEASUREMENTS AND MAIN RESULTS: Prebypass imaging yielded unsuspected findings that either helped or modified the surgical plan in 158 of 1,356 valve procedures (11.65%) and in 993 of 3,660 CABGs (27.13%). There were 3,217 TEE-guided hemodynamic interventions in 944 patients (25.79%) in the CABG group and 629 in 142 patients (10.47%) in the valve group. TEE was the sole guiding factor in initiating therapy in 23.53% of events, whereas it was supportive to other monitoring modalities in 76.46% of events. Postbypass TEE identified the need for graft revision in 29 patients (0.8%), intra-aortic balloon pump (IABP) requirement in 29 patients (0.8%), and inadequate valve repair in 28 patients (2.08%). For the entire series, 38.78% of patients benefited from prebypass and 39.16% from postbypass use of TEE. There were no complications attributable to the use of TEE in the entire series. There was 87% concordance between online interpretation by a trained anesthesiologist and offline analysis by a cardiologist. CONCLUSION: Intraoperative TEE is useful in formulating the surgical plan, guiding various hemodynamic interventions, and assessing the immediate results of surgery. It is safe and the results are reliable in the hands of trained anesthesiologists.