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This study was designed to explore the effects of purified insulin during early stages of chick embryo development, and to search for variations between different molecular structures of the hormone. Chicken embryos were treated in ovo with a single dose of insulin (porcine or bovine), in only one stage of development between day 0 and day 9. Two susceptible periods were found. The earliest period (day 0 to day 3), characterized by abnormalities in the caudal vertebrae and a high mortality rate, was followed by a period with a different set of malformations, a syndrome classified as achondroplasia. The rate of achondroplastic embryos was significantly higher with porcine rather than with bovine insulin. Paradoxically, insulin at physiological doses has stimulatory effects in growth and development but, in contrast, has inhibitory effects at higher doses. The precise signalling cascade of events in the target cells is still unclear. The possible interpretations of our results are discussed. The similarity between the insulin-induced abnormalities in the chicken embryos and the caudal regression syndrome, the most common malformation found in infants of diabetic women, suggests a common mechanism. This circumstance offers the chicken embryos as an excellent in vivo model for research on the mechanism of action of insulin during normal and abnormal development.

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The apical ectodermal ridge of the vertebrate limb bud lies at the junction of the dorsal and ventral ectoderm and directs patterning of the growing limb. Its formation is directed by the boundary between cells that do and cells that do not express the gene Radical fringe. This is similar to the establishment of the margin cells at the Drosophila wing dorsoventral border by fringe. Radical fringe expression in chick-limb dorsal ectoderm is established in part through repression by Engrailed-1 in the ventral ectoderm.

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In chick embryos homozygous for the limbless mutation, limb bud outgrowth is initiated, but a morphologically distinct apical ridge does not develop and limbs do not form. Here we report the results of an analysis of gene expression in limbless mutant limb buds. Fgf4, Fgf8, Bmp2 and Msx2, genes that are expressed in the apical ridge of normal limb buds, are not expressed in the mutant limb bud ectoderm, providing molecular support for the hypothesis that limb development fails in the limbless embryo because of the inability of the ectoderm to form a functional ridge. Moreover, Fgf8 expression is not detected in the ectoderm of the prospective limb territory or the early limb bud of limbless embryos. Since the early stages of limb bud outgrowth occur normally in the mutant embryos, this indicates that FGF8 is not required to promote initial limb bud outgrowth. In the absence of FGF8, Shh is also not expressed in the mutant limb buds, although its expression can be induced by application of FGF8-soaked beads. These observations support the hypothesis that Fgf8 is required for the induction of Shh expression during normal limb development. Bmp2 expression was also not detected in mutant limb mesoderm, consistent with the hypothesis that SHH induces its expression. In contrast, SHH is not required for the induction of Hoxd11 or Hoxd13 expression, since expression of both these genes was detected in the mutant limb buds. Thus, some aspects of mesoderm A-P patterning can occur in the absence of SHH and factors normally expressed in the apical ridge. Intriguingly, mutant limbs rescued by local application of FGF displayed a dorsalized feather pattern. Furthermore, the expression of Wnt7a, Lmx1 and En1, genes involved in limb D-V patterning, was found to be abnormal in mutant limb buds. These data suggest that D-V patterning and apical ridge formation are linked, since they show that the limbless mutation affects both processes. We present a model that explains the potential link between D-V positional information and apical ridge formation, and discuss the possible function of the limbless gene in terms of this model.

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Several polydactylous mutants affect the pattern of asymmetry along the anteroposterior axis of the vertebrate limb. In talpid2, diplopodia1, and diplopodia4 chick limb mutants, there is a preaxial extension that results in broader limb buds. Talpid2 shows reduction of the long bones and 9-10 syndactylous digits, none of which are specifically recognizable as members of the normal digit complement. In diplopodia1 and diplopodia4 extra digits are present preaxially in addition to the normal digits. This phenotype resembles the duplications obtained by grafting a polarizing region to the anterior margin of the limb bud. The abnormal skeletal pattern along the anteroposterior limb axis in both mutants suggests alterations in the signaling pathways that mediate growth and patterning of the limb. In situ hybridization studies reveal that whereas shh transcripts are restricted to the posterior limb margins, HoxD, Bmp-2, and Fgf-4 genes are ectopically expressed in the anterior region of the talpid2, diplopodia1, and diplopodia4 limb buds. The results obtained give insights into the molecular basis of talpid2 and diplopodia mutations and also into the possible roles of shh, Bmp-2, HoxD, and Fgf-4 genes in vertebrate limb morphogenesis.

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Classical studies of the vertebrate limb have provided a firm foundation for recent investigations into the molecular control of mechanisms governing limb patterning. The early studies revealed the importance of inductive tissue interactions in developing systems, the spatiotemporal restrictions of these interactions, and the conservation of inductive signals between different tissues and even different species. They incorporated a number of different experimental approaches, including: homologous and heterologous tissue grafting and recombination, the investigation of several limb mutations, and examination of the response of normal limb tissue to a variety of teratogenic treatments. While some of the mutations studied only affected the limbs, most were highly pleiotropic, producing complex syndromes that altered the development of several embryonic structures in addition to the limbs. Some of these syndromes could be partially or completely phenocopied (mimicked) by specific chemical or physical treatments. One such gene-phenocopy pairing that we have studied is that of the mutation wingless-2 and the syndrome produced by treatment with retinoic acid. Another aspect of abnormal pattern formation we explored is the interaction between wingless-2 and eudiplopodia.

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The sex-linked dwarf gene (dw) was introduced into companion muscular dystrophic (am) and nondystrophic (Am+) New Hampshire chicken lines to investigate influences of the dwarf gene on breast muscle weights, muscle fiber area, and the histological expression of muscular dystrophy. Dystrophic and nondystrophic chickens within dwarf or nondwarf genotypes were similar in body and carcass weights. Pectoralis and supracoracoideus muscle weights (as a percentage of adjusted carcass weight) were similar in nondystrophic dwarf and nondwarf males and females. In addition, pectoralis weight was similar in dystrophic dwarf males and dystrophic nondwarf males and females. However, pectoralis weight was significantly smaller in dystrophic dwarf females than in dystrophic nondwarf females, whereas supracoracoideus weight was significantly larger in dystrophic dwarf males than in dystrophic nondwarf males. Supracoracoideus weight was similar in dystrophic dwarf males and females and dystrophic nondwarf females. Pectoralis muscle fiber area was influenced by sex and by dwarf and dystrophy genotype. Muscle fiber area was larger in females than in males, smaller in dwarfs than in nondwarfs, and smaller in dystrophic than in nondystrophic muscles. Muscle fiber degeneration and adipose infiltration was more extensive in dystrophic than in nondystrophic females and males, and it was more advanced in dwarfs than in nondwarfs. Excessive acetylcholinesterase staining patterns were characteristic of dystrophic muscle in both dwarf and nondwarf genotypes. Nondystrophic and dystrophic dwarf male and female chickens are comparable substitutes for nondwarfs as biomedical models with respect to pectoralis histology, acetylcholinesterase staining pattern, and pectoralis muscle hypertrophy.

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A second form of hereditary chondrodystrophy (ch-2) has been discovered in a selected line of Japanese quail, Coturnix japonica. This form of chondrodystrophy is autosomal and recessive, characterized by an overall shortening and bending of the long bones of the wings and legs, slight dwarfing of the trunk, bulging of the eyes, flattening of the head, and a parrot beak. The shortened long bones vary in regard to the amount of bending from nearly straight to bends of up to 90 degrees in the midshaft region. In severe cases, the bend is evident as a protuberance of the skin. Affected embryos usually survive the 18-day incubation period. Several have hatched, but most survived no longer than 4 days after hatching. Only one female has survived long enough to lay eggs. Testcrosses indicated that this mutation is not allelic to micromelia.

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The appearance and distribution of extracellular matrix (ECM) was documented along the migratory route of chicken primordial germ cells (PGCs). The antimouse embryonal carcinoma cell antibody, EMA-1, was used to label PGCs (Urven et al.: Development 103:299-304, 1988). Antibodies against laminin, fibronectin, chondroitin sulfate proteoglycan and collagen type IV were used to label extracellular matrix components. When the PGCs emerged from the epiblast, all four ECM molecules were restricted principally to the basement membrane of the epiblast. Chondroitin sulfate was also located between hypoblast cells during this period. In late germinal crescent stages, when the PGCs entered the lumina of blood vessels, the same ECM molecules were more widespread in the mesoderm and in extracellular spaces. In addition, laminin and collagen type IV were identified on lateral surfaces of ectodermal cells at this stage. When the germ cells moved through the mesenchyme into the germinal ridge, the ECM molecules were found around mesenchymal cells, and, in the cases of laminin, fibronectin and collagen type IV, in the basement membranes of the germinal ridge epithelia. Because the appearance of these ECM components is temporally and spatially correlated with the movement of PGCs, we suggest that early PGC migration may depend on their timely appearance.

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We have found that EMA-1, a monoclonal antibody originally raised against mouse embryonal carcinoma (Nulli SCC1) cells (Hahnel & Eddy, 1982), also labels chick primordial germ cells (PGCs). We have used this antibody in immunohistological studies to follow the development of PGCs in the chick embryo from the time of their initial appearance beneath the epiblast, through their migratory phase and subsequent colonization of the germinal epithelium. During hypoblast formation, individual EMA-1-labelled cells appeared to separate from the basal surface of the epiblast and enter the blastocoel, coincident with the appearance of morphologically identifiable PGCs in this same area. EMA-1 continued to label germ cells until the initiation of gametogenesis in each sex; specifically, labelling was absent by 7-8 days of incubation in females and started to decrease at 11 days of incubation in males. There was a recurrence of the epitope on oogonia at 15 days of incubation, but not on spermatogonia during the remainder of development through hatching. These observations are consistent with an epiblast origin for the avian germ line, and are strikingly similar to those reported for the early mouse embryo using the same antibody (Hahnel & Eddy, 1986).

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Changes in the weight:mass ratio provide the physical basis for the biological responses of terrestrial organisms to alteration in the ambient acceleration field. Where organisms such as aquatic animals occupy dense media, changes in the gravitational environment produce compensating changes in the weight:mass ratios of organism and medium, such that little net load is imposed upon the organism. This relationship also applies to organs of terrestrial animals. Changes in the ambient acceleration field produce compensatory changes in surrounding tissues so that the organ may not develop a significant net load. This relationship has been investigated in the case of the vertebrate brain. However, density gradients within the organ/organism will produce a local gravitational loading, which may lead to biological responses. In fact, a significant density gradient would be an essential character for a gravity receptor. Prenatal development, both in mammals and birds, occurs characteristically in a buoyant condition. In both cases a volume of amniotic fluid develops and surrounds the embryo while it is still of microscopic size. This situation prevails until the latest stages of prenatal development. In mammals the amniotic fluid is lost immediately prior to parturition through rupture of the sac. In chick embryos the amniotic fluid is ingested, beginning on the 13th day with the process being completed by the 18th day of development, just prior to the pre-hatch reorientation of the embryo. Consequently, a net load upon the embryo/fetus is not considered to be a major factor in gravitational experiments of prenatal development. Prenatal development includes marked changes in chemical composition as well as changes in size. This is readily apparent from extensive and detailed examination of the chemical growth for the chick embryo. These chemical materials vary in density, as well as in distribution among the tissues of the developing organism. Consequently, the existence of density gradients, and changes in them may be anticipated during prenatal development.

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The genetics of an inherited form of scoliosis in chickens was studied to estimate the number of genes involved, whether they are autosomal or sex-linked, their degree of dominance and penetrance, and the heritability of this trait in this population. Expression of scoliosis and in the progeny was analyzed by radiographs of birds 12 weeks of age or older. Crosses between an inbred line selected for scoliosis expression (incidence of scoliosis - 89 percent) and a highly inbred line displaying normal spinal development provided data for genetic analyses. The incidence of expression of scoliotic parent line implicates three major autosomal, recessive genes. The significantly higher incidence of severe scoliosis found in the homogametic male sex is ascribed to a sex-influenced, on the scoliosis trait rather than to sex-linkage. Variation of expression observed in the scoliotic line is attributed to incomplete penetrance of the major genes, additive effects of minor modifying genes, and primarily to environmental effects. Because of the similarities in the expression of this disease in chickens and humans, the inheritance pattern determined for chickens may provide useful insights into that operating for so-called adolescent idiopathic scoliosis in humans.

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Azodrin was applied to adult embryo chickens, Chukar Partridge, and Bobwhite Quail. Chronic exposure of adult birds to Azodrin mixed in their feed indicated that no a priori predictions could be made about one species based on the results of another; each had a different no effect (MACT) level. The chickens were between 25 and 100 ppm, the Chukar Partridge 5 and 25 ppm, and the Bobwhite quail less than 1.25 ppm. The chicken adults were most resistant, and the quail were least resistant to chronic exposure to Azodrin. Yolk-injected Azodrin caused the embryos of all three species to develop abnormally. The chicken and Chukar embryos developed a generalized achondroplasia, the quail were amuscular, only. In general, the 3 day quail embryos were most resistant to injected Azodrin and the chicken embryo least resistant. The relationship between adult and embryo response was negative.

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