Nitrergic neurons of the forepaw representation in the rat somatosensory and motor cortices: A quantitative study.

Nitrergic neurons (NNs) are inhibitory neurons capable of releasing nitric oxide (NO) that are labeled with nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. The rat primary somatosensory (S1) and motor (M1) cortices are a favorable model to investigate NN populations by comparing their morphology, since these areas share the border of forepaw representation. The distribution of the Type I NN of the forepaw representation in the S1 and M1 cortices of the rat in different laminar compartments and the morphological parameters related to the cell body and dendritic arborization were measured and compared. We observed that the neuronal density in the S1 (130 NN/mm3 ) was higher than the neuronal density in the M1 (119 NN/mm3 ). Most NN neurons were multipolar (S1 with 58%; M1 with 69%), and a minority of the NN neurons were horizontal (S1 with 6%; M1 with 12%). NN found in S1 had a higher verticality index than NN found in M1, and no significant differences were observed for the other morphological parameters. We also demonstrated significant differences in most of the morphological parameters of the NN between different cortical compartments of S1 and M1. Our results indicate that the NN of the forepaw in S1 and M1 corresponds to a neuronal population, where the functionality is independent of the different types of sensory and motor processing. However, the morphological differences found between the cortical compartments of S1 and M1, as well as the higher density of NNs found in S1, indicate that the release of NO varies between the areas.

Differences in cuticular lipid composition of the antennae of Helicoverpa zea, Heliothis virescens, and Manduca sexta.

Analyses of the hexane washes of antennae, forelegs and whole bodies of Helicoverpa zea, Heliothis virescens, and Manduca sexta revealed notable differences in the components of the cuticular coatings of each species. Most striking were the differences between the cuticular coatings of male and female antennae of both H. zea and H. virescens. Novel esters of short-chain acids (C2-C4) and long-chain secondary alcohols (C25-C32) were identified in the hexane washes of the male antenna and forelegs of H. zea and H. virescens. These compounds were found in only small amounts or were completely absent on the female antennae of both species. In H. zea, butyrates of 7- and 8-pentacosanol and 8- and 9-heptacosanol were found, whereas, in the foreleg extracts of H. virescens, acetates and propionates were detected in addition to butyrates. While cholesterol is a major component of antennal washes (10-15%), only traces were found in the foreleg extracts. Although the composition of the cuticular coating of M. sexta differed greatly from that of the other two species, the extractable coatings of the antennae of male and female M. sexta were nearly identical.

Interdigital webbing retention in bat wings illustrates genetic changes underlying amniote limb diversification.

Developmentally regulated programmed cell death sculpts the limbs and other embryonic organs in vertebrates. One intriguing example of species-specific differences in apoptotic extent is observed in the tissue between the digits. In chicks and mice, bone morphogenetic proteins (Bmps) trigger apoptosis of the interdigital mesenchyme, leading to freed digits, whereas in ducks, Bmp antagonists inhibit the apoptotic program, resulting in webbed feet. Here, we show that the phyllostomid bat Carollia perspicillata utilizes a distinct mechanism for maintaining interdigit tissue. We find that bat forelimb and hindlimb interdigital tissues express Bmp signaling components but that only bat hindlimbs undergo interdigital apoptosis. Strikingly, the retention of interdigital webbing in the bat forelimb is correlated with a unique pattern of Fgf8 expression in addition to the Bmp inhibitor Gremlin. By using a functional assay, we show that maintenance of interdigit tissue in the bat wing depends on the combined effects of high levels of Fgf signaling and inhibition of Bmp signaling. Our data also indicate that although there is not a conserved mechanism for maintaining interdigit tissue across amniotes, the expression in the bat forelimb interdigits of Gremlin and Fgf8 suggests that these key molecular changes contributed to the evolution of the bat wing.

Expression of Fgf and Tgfbeta signaling related genes during embryonic endochondral ossification.

Disturbed fibroblast growth factor (Fgf) and transforming growth factor beta (Tgfbeta) signaling lead to a variety of human skeletal disorders. To reveal the possible function and interaction of these signaling systems we have started to analyze the expression patterns of signaling factors, antagonists, receptors and transducers of these pathways in forelimbs of mouse embryos and compared them to the expression of established markers including Ihh. In addition to defining their expression domains in the developing bone, our study identified new subpopulations of chondrocytes characterized by the expression of distinct combinations of markers.

Age-related morphometry of equine calcified cartilage.

Although there are many studies in the equine literature focused on articular diseases and the aetiology of osteoarthritis, few have concentrated on normal articular structures and how they change with age. The objective of this investigation was to study the thickness and morphology of the calcified cartilage layer of the distal metacarpus over a range of ages. A parasagittal slab of bone was sectioned from the region of sesamoid contact on the medial condyle of the metacarpi from 34 horses. The slab of bone was preserved, dehydrated and embedded, undecalcified, in methylmethacrylate and then stained with toluidine blue. Six repeatable fields of interest from the distal aspect of each metacarpus were digitised and examined to determine the morphology of the calcified cartilage layer. The thickness of the calcified cartilage, range 88-426 microm, was estimated using a method of integration. The results indicate an age-related influence on the thickness of the calcified cartilage layer, generally increased in older horses. While this finding is significant, perhaps more importantly a positional relationship was also identified, indicating that pressures endured by different regions within a joint may dictate morphological development of the tissues. This study has begun to lay the groundwork to determine whether the calcified layer of the hyaline cartilage could be involved in the development of osteoarthritis.

SAGE profiling of the forelimb and hindlimb.

A recent study has used serial analysis of gene expression to compare mouse forelimb and hindlimb gene-expression profiles. The method successfully identified known regulators of limb identity and has generated a candidate set of differentially expressed genes that may regulate limb identity.

Detection of insulin receptors in newt liver and forelimb regenerates and the effects of local insulin deprivation on epimorphic regeneration.

Previous in vivo and in vitro studies indicate that insulin is required in adult newt forelimb regeneration. The objectives of the current study were 1) to detect insulin receptors in the liver (a classical target organ for insulin) and once verified, detection of insulin receptors in the adult newt forelimb regenerate; and 2) to determine whether locally implanting insulin antibody-soaked hydrolyzed polyacrylamide beads (hypa beads) into a regenerating forelimb blastema would affect its growth and/or differentiation. The results show that insulin receptors are detectable in the plasma membranes of newt liver and forelimb regenerates. Radioiodinated bovine insulin binding is time-dependent and specific; unlabeled bovine insulin competes with labeled insulin for binding to NLPM more effectively than does insulin-like growth factor-I, guinea pig insulin, and glucagon. The newt hepatic insulin receptor binds insulin with high affinity (1.1 nM-1) and low capacity (63 +/- 8 fmoles/mg). The size of the alpha subunit of the newt insulin receptor is 130 kDA and that of the beta subunit is 95 kDa. The beta subunits undergo insulin-stimulated phosphorylation in response to insulin. An autoantibody against the human insulin receptor recognizes the newt receptor protein. Insulin receptors are also detectable in 15 and 20 day newt forelimb regenerates. Specific immunogold labelling of the receptor-bound antibody appears to be restricted to the cellular processes of the regenerate. Implanting hypa beads soaked with purified insulin antibody into regenerating adult newt forelimbs results in abnormal growth and differentiation of the regenerates, confirming that insulin plays an essential role in adult newt forelimb regeneration.

Retinoic acid stimulates the synthesis of a novel heat shock protein in the regenerating forelimb of the newt.

Morphogenetic effects of retinoic acid (RA) on the urodele amphibian limb regenerate pattern have been well documented, but little is known regarding the mechanism of this action of RA at the molecular level. Since exogenous RA, at concentrations sufficient to cause proximalization, represents a significant stress to newts and has been shown previously to elicit increased synthesis of heat shock proteins (HSPs) in mouse embryo limb buds, we investigated the effects of this putative morphogen on the synthesis of members of the 70-kilodalton (70-kDa) stress protein family in amputated forelimbs of the newt Notophthalmus viridescens. Injection (i.p.) of RA in dimethyl sulfoxide (DMSO), at a dose sufficient to cause significant proximal-distal reduplication of the pattern in 50% of animals treated, resulted in increased synthesis and accumulation of a 73-kDa protein with a pI of approximately 6.75. The synthesis of this same protein is increased in limb tissues as a result of a brief 35 degrees C heat shock. This protein is electrophoretically distinct from the newt HSP 70 family members, displays a different partial peptide map, and shows no immunological cross-reactivity with an anti-human HSP 70 monoclonal antibody. It may be a member of a separate family of 70- to 73-kDa HSPs. Interestingly, the synthesis of this protein is increased and it is more abundant in control, proximal moderate-early bud stage regenerates at 6 days after i.p. injection of DMSO than in similarly treated distal regenerates. This protein is, in addition, increased in distal regenerates to proximal levels by a prior injection of RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of type I collagen, type II collagen and PNA binding glycoconjugates during chondrogenesis of three distinct embryonic cartilages.

Previous studies of chondrogenesis have been focused on limb bud cartilage, whereas little is known about chondrogenic processes of other cartilages with different developmental fates. We hypothesize that cartilages with various developmental fates might show identical characteristics of chondrogenesis. The chondrogenic processes in the nasal septum, the mandible, and the limb bud of the mouse were examined by means of PNA-binding glycoconjugate, and types I and II collagen expression. Swiss-Webster mouse embryos of 11 days (E11) to 14 days (E14) gestation were fixed and processed for immuno- and lectin histochemistry. The blastema of mesenchymal cell aggregates stained positively with anti-type I collagen, but very weakly with anti-type II collagen in all three models at E12, whereas PNA bound to the blastema in the limb bud but not in nasal septum or mandible. Types I and II collagens coexisted in cartilages at E13. Type II collagen was predominant in E14; type I collagen was confined to the peripheral region. The synchronized transitional expression of the collagen phenotypes in all three embryonic cartilages may be systemically regulated. The presence or absence of the PNA-binding glycoconjugates may be involved in characterizing the nature of the cartilages.