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Biomedical subjects

J R Hinchliffe

Publications and source records attributed to J R Hinchliffe.

At least 19 recordsLinked to original sources
PubMed1995

Immunohistological and ultrastructural study of the developing tendons of the avian foot.

The aim of the present report is to provide a detailed description of the morphogenesis and initial differentiation of the long tendons of the chick foot, the long autopodial tendons (LAT), from day 6 to day 11 of development. The fine structure of the developing LAT was studied by light and transmission electron microscopy. The characterization by immunofluorescent techniques of the extracellular matrix was performed using laser scanning confocal (tenascin, elastin, fibrillin, emilin, collagen type I, II, III, IV and VI) or routine fluorescence (tenascin, 13F4) microscopy. In addition, cell proliferation in pretendinous blastemas was analyzed by the detection of BrdU incorporation by immunofluorescence. The light microscopic analysis permitted the identification of different stages during LAT morphogenesis. The first stage is the formation of a thick ectoderm-mesenchyme interface along the digital rays, followed by the differentiation of the "mesenchyme lamina", an extracellular matrix tendon precursor, and ending with the formation and differentiation of the cellular condensation that forms the tendon blastema around this lamina. The immunofluorescence study revealed the presence and arrangement of the different molecules analyzed. Tenascin and collagen type VI are precocious markers of the developing tendons and remain present during the whole process of tendon formation. Collagen type I becomes mainly restricted to the developing tendons from day 7.5. Collagens type II and IV are never detected in the developing tendons, while a faint labeling for collagen type III is first detected at day 7. The analysis of the distribution of the elastic matrix components in the developing tendons is a major contribution of our study. Elastin was detected in the periphery of the tendons from day 8 and also in fibrils anchoring the tendons to the skeletal elements. At the same stage, emilin strongly stains the core of the tendon rods, while fibrillin is detected a little later. Our study indicates the existence of an ectoderm-mesoderm interaction at the first stage of the tendon formation. In addition, our results show the different spatial and temporal pattern of distribution of extracellular matrix molecules in developing tendons. Of special importance are the findings concerning the tendinous elastic matrix and its possible role in tendon maturation and stabilization.

Animals↗
PubMed1994

Immunolocalization of basement membrane components and beta 1 integrin in the chick wing bud identifies specialized properties of the apical ectodermal ridge.

To examine whether the extracellular matrix (ECM) plays a role in mediating interactions between the apical ectodermal ridge (AER) and the subjacent mesoderm in the chick wing bud, we used immunohistochemistry to locate the following tissue components during wing morphogenesis: types I and IV collagens, fibronectin, the basal lamina form of heparan sulphate proteoglycan (HSPG), laminin, and the beta 1 integrin subunit. The notch region at the base of the AER exhibits particularly strong labelling for type IV collagen, fibronectin, laminin, and beta 1 integrin. This suggests that the ridge cells are firmly anchored to their underlying basement membrane. In nonridge ectoderm, the beta 1 integrin subunit is present only at the basal cell surface, whereas in the AER it has a pericellular distribution. The localization of beta 1 integrin receptors at the lateral ridge cell surfaces, in the apparent absence of fibronectin, collagens I and IV, and laminin, suggests that they may function in cell-cell adhesion in the AER. The normal AER-mesenchyme interface was compared to an experimental situation in which the AER flattens. This was induced in the anterior region of the wing bud by inserting an impermeable barrier at intersomite level 17/18, at stage 21. At 12 hr (stage 23) and 24 hr (stage 25) after the operation, each of the ECM components listed above is uniformly distributed along the experimental epithelial-mesenchymal interface. By 24 hr postoperation, the beta 1 integrin subunit is restricted to the basal surface of the flattened apical ectoderm. Similar changes occur in the AER as it flattens during later stages of normal development. These results point to a possible role for the ECM and integrin receptors in maintaining the thickened structure of the AER.

Animals↗
PubMed1994

Evolutionary developmental biology of the tetrapod limb.

New insights into the origin of the tetrapod limb, and its early development and patterning, are emerging from a variety of fields. A wide diversity of approaches was reported at the BSDB Spring Symposium on 'The Evolution of Developmental Mechanisms' (Edinburgh, 1994); here I review the contributions these various approaches have made to understanding the evolutionary developmental biology of the tetrapod limb. The fields covered included palaeontology, descriptive embryology, experimental embryological analysis of interactions within developing limbs plus description and manipulation of homeobox gene expression in early limb buds. Concepts are equally varied, sometimes conflicting, sometimes overlapping. Some concern the limb 'archetype' (can the palaeontologists and morphologists still define this with precision? how far is there a limb developmental bauplan?); others are based on identification of epigenetic factors (eg secondary inductions), as generating pattern; while yet others assume a direct gene-morphology relationship. But all the contributors ask the same compelling question: can we explain both the similarity (homology) and variety of tetrapod limbs (and the fins of the Crossopterygians) in terms of developmental mechanisms?

Animals↗
PubMed1990

Experimental analysis of the role of ECM in the patterning of the distal tendons of the developing limb bud.

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.

Animals↗
PubMed1989

Posterior half amputation of the chick wing bud: the response of the developing vasculature, and subsequent wound healing.

Experimental analyses examining pattern formation in the developing chick limb have concentrated on the skeleton, muscles and nerves, and have rarely considered blood vessels. To investigate the relationship between the vasculature and limb development, posterior amputations were performed on 3.5-4 day chick limb-buds. It has been shown that the removal of the posterior half alters the developmental fate of the anterior tissue: it becomes necrotic and fails to differentiate into the complement of skeletal parts predicted by fate maps. The possibility that this developmental failure results from interference with the future arterial supply was examined by Indian ink injection between 3-48 h after operation. Scanning electron microscopy (SEM) and resin histology were used to examine the wound repair at similar post-operative intervals. Results from the Indian ink injections showed that within 6 h of operation a collateral circulation was established by means of a branch from the truncated primary subclavian artery. The capillary density in the operated limbs appeared normal when compared to the contralateral limb. The results support the view that the poor developmental performance of the anterior half is due to removal of the zone of polarizing activity (ZPA) rather than to experimentally-induced alteration to the vascular supply. Histological and SEM examination of the wound healing process showed that epithelialization of the cut surface occurred within 24 h, and that the peridermal cells of the bilayered ectoderm appeared to initiate the regrowth. The wound site was not visible 48 h after operation, showing that wound healing at these developmental ages occurs quickly, with no scar tissue formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amputation, Surgical↗
PubMed1989

The fate map of the chick forelimb-bud and its bearing on hypothesized developmental control mechanisms.

Carbon particles and isotopic quail grafts were used as markers to study the salient features of the fate map of the chick forelimb between stages 20 and 27. The grafting technique confirmed the reliability of the carbon method: they both revealed striking asymmetries in which apical mesodermal tissue was progressively displaced in a proximal direction (as would be expected on the basis of growth by net apical addition of tissue) but also in a preaxial direction, while postaxial tissue became elongated in the direction of limb outgrowth. Ectoderm showed a similar preaxial-postaxial asymmetry but became displaced from initially underlying mesoderm. In marked contrast to mesoderm, distal ectoderm remained at a constant distance from the apical ectodermal ridge (or became incorporated into it), thus implying that the ectodermal sheet is anchored distally and grows by uniform stretching proximally. Within the ectoderm itself, the outer peridermal layer is displaced distally relative to the underlying epidermal basal layer. Peripheral mesoderm showed patterns of displacement which were intermediate between those of ectoderm and chondrogenic core mesoderm. It is argued that such morphogenetic phenomena are integral components of developmental mechanisms of significance in the control of pattern generation. Implications of the interpretation and use of the fate map in relation to theories of limb development, particularly those based on mechanisms defined in terms of limb axes, are reviewed.

Animals↗
PubMed1989

The extracellular matrix architecture relating to myotendinous pattern formation in the distal part of the developing chick limb: an ultrastructural, histochemical and immunocytochemical analysis.

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.

Animals↗
PubMed1988

Spatial and temporal changes in the pattern of glycosylation of the developing chick limb tissue components as revealed by fluorescent conjugated lectin probes.

The changing pattern of expression of glycoconjugates during the differentiation of the chick leg bud between stages 17 to 34 (days 3 to 8 of incubation) was studied using fluorochrome-labelled plant lectins. Limb buds were fixed in cold acetic-alcohol and wax-embedded. Agglutinins of peanut (PNA), soybean (SBA) and succinylated wheat germ (WGAs) revealed a specific binding pattern in the apical ectodermal ridge (AER) between Hamburger and Hamilton stages 19-32. These stages coincide with the period of elevation of the AER. This specific binding pattern was absent from the adjacent dorsal and ventral ectoderm. Prechondrogenic cells were positive for WGA and for PNA, and the PNA-binding capacity was intensified after neuraminidase treatment. Premyogenic cells at stage 23 can be identified as negative to PNA after neuraminidase, while the blood vessels became positive. PNA, SBA, WGA, WGAs and, in addition, Ricinus communis (RCA-I) lectins stained the basal membrane. Strands of extracellular matrix which connect with the basal membrane and cross the limb transversely between dorsal and ventral ectoderm were stained by RCA-I, SBA and PNA after neuraminidase.

Animals↗
PubMed1987

The effect of the zone of polarizing activity (ZPA) on the anterior half of the chick wing bud.

Removal of the posterior half of the chick wing bud between stages 17-22 results in failure of the anterior distal tissue to survive and differentiate. This observation has been interpreted in terms of a requirement by the anterior half of a factor supplied by the posterior half of the limb containing the zone of polarizing activity (ZPA). This relationship has been tested by grafting ZPA tissue to the posterior surface of the anterior half after posterior half removal. Grafts made proximally on the cut surface did not significantly improve survival and development, nor did the ZPA prevent the expansion of the cell death in the ANZ beyond its normal boundaries into the distal mesenchyme. However, when grafted distally the ZPA inhibited cell death in the apical mesenchyme and caused the anterior mesenchyme to change its normal prospective fate (radius and digit 2). In all these cases, in addition to digit 2, digit 3 and frequently also digit 4 differentiated. The anterior half went on to develop a full set of digits and zeugopod parts in almost 50% of cases, although no skeleton resulting from this regulation of the anterior half had totally size regulated. These results demonstrate a developmental 'rescue' effect by the ZPA, and further support the view that the ZPA has a central and unique function in normal limb bud development, controlling survival and differentiation of the mesenchyme along the anteroposterior axis.

Animals↗
PubMed1985

Experimental analysis of the role of the ZPA in the development of the wing buds of wingless (ws) mutant embryos.

Phenotypically wingless (ws) chick embryos have wing buds characterized by the spreading of mesenchymal cell death in an anterior-to-posterior direction beginning at stage 19. It has been argued that this may reflect the absence of a functional polarizing zone (ZPA). When tested by preaxial grafting into normal wing buds (stages 20-21), wingless ZPAs (stage 18-19) had duplicating properties identical with those of normal ZPAs. Equally, normal chick or quail ZPA (stages 20-22) grafted into the posterior margin of wingless wing buds (stages 18-20) failed to inhibit the pattern of cell death or to evoke any improvement in their developmental performance. The wingless (ws) condition is not, therefore, due to a ZPA deficiency. Possible explanations are the prior programming for cell death of the wingless mesenchyme, or somitic deficiency, but it appears more likely that the mutant limb mesenchyme fails to transmit or respond to factor(s) produced by the ZPA.

Animals↗
PubMed1985

The distribution of the polarizing zone (ZPA) in the legbud of the chick embryo.

The stage-21 to 22 legbud polarizing zone (ZPA) was mapped by transplanting small blocks of posterior marginal mesenchyme preaxially into stage-20 to -22 chick wing buds and assessing the degree of duplication of the wing digital skeleton produced in the host. Blocks taken from the posterior flank, from the angle between posterior flank and the proximal base of the limb bud, and from the most anterior distal position chosen (under the AER), all had very low activity. Blocks taken from the posterior margin of the legbud, plus the next distal block under the posterior part of the AER, all had high activity. We consider that barrier and amputation results on wing and legbud, when interpreted in the light of maps of the ZPA in both limb buds, are consistent with the hypothesis that both leg and wing have their growth and anteroposterior axis of pattern formation controlled by the ZPA.

Animals↗