Scoliosis in chickens: responsiveness of severity and incidence to dietary copper.

The severity and incidence of spinal lesions were manipulated in a line of chickens susceptible to scoliosis by varying their dietary intake of copper. A decrease in expression of the lesion was related to increased intake of copper. The change in expression, however, appeared to be related only indirectly to the defects in collagen cross-linking, maturation, and deposition known to be associated with dietary copper deficiency. Thus, a dietary constituent in the range of normal intakes may act as an environmental factor in the expression of scoliosis.

Inductive interactions between human dermis and chick chorionic epithelium.

In a study of specificity in mesenchymal-epithelial interactions, human embryonic dermis has been recombined with chick chorionic epithelium and cultured for 7 days on a host chick chorioallantoic membrane. Dermis from the sole of the foot or palm of the hand induces chick chorionic epithelium to form an epidermis that resembles chick rather than human epidermis. Chick epithelium, though it has the capacity to respond to a human dermal stimulus, is limited to forming chick-type tissue. The human dermis was modified in its turn by culture in combination with chick epithelium.

Collagen, proteoglycan and hyaluronidase activity in cultures from normal and scoliotic chicken fibroblasts.

Connective tissue matrix components were investigated using skin fibroblasts from normal or inbred scoliotic lines of chickens. Specifically, the fibroblasts were obtained from either an isogenic line or a backcross, derived by crossing the isogenic line with a pure line of scoliotic birds. From the backcross, both affected (35-45%) and non-affected (55-65%) progeny were produced. The affected birds had spinal curves greater than 20 degrees. Several abnormalities of connective tissue were observed when cells from scoliotic chicks were grown in culture: increased collagen extractability, decreased aggregatability of proteoglycans under associative conditions and lower than normal levels of hyaluronic acid. There was also less collagen deposited in the cell layer with proportionately increased amounts of collagen secreted into the culture media by cells from scoliotic versus normal chick fibroblasts. Values for collagen matrix stability, as estimated by extractability and net deposition, were intermediate for cells from the backcrossed, but non-affected, birds. Moreover, hyaluronidase, an enzyme that degrades hyaluronic acid, was abnormally elevated in the fibroblast cultures from scoliotic chicks. It is proposed that the increase in hyaluronidase contributes to the abnormalities observed in extracellular matrix components and may be a factor in the expression of scoliosis in susceptible birds.

Collagen crosslinking and cartilage glycosaminoglycan composition in normal and scoliotic chickens.

The amounts of lysine-derived crosslinks in collagens from tendon, cartilage, intervertebral disc, and bone and changes in the composition of sternal cartilage glycosaminoglycans were estimated in two lines of chickens, a control-isogenic line and a line that develops scoliosis. In the scoliotic line, scoliosis first appears at 3-4 weeks and progressively increases in severity and incidence so that 90% of the birds express the lesion by week 10. We have reported previously that cartilage, tendon, and bone collagens from scoliotic birds are more soluble than corresponding collagens from normal birds. Herein, collagen crosslinking and altered proteoglycan metabolism are examined as possible mechanisms for the differences in collagen solubility. At 1 week of age there were fewer reducible crosslinking amino acids (hydroxylysinonorleucine, dihydroxylysinonorleucine, and lysinonorleucine) in collagens from sternal cartilage and tendon in the scoliotic line than in the isogenic line. However, by week 3 and at weeks 5 or 7 values were similar in both groups. The amounts of hydroxypyridinium in vertebral bone and intervertebral disc collagen were also similar in both groups of birds. Consequently, differences in collagen crosslinking do not appear to be a persistent developmental defect underlying the expression of scoliosis in the model. However, differences were observed in cartilage proteoglycans and glycosaminoglycans from the scoliotic line that were not present in cartilage from the isogenic line. The average molecular weight of the uronide-containing glycosaminoglycans was 30% less in the scoliotic line than in the isogenic line, i.e., 12,000 compared to 18,000. The size distribution of cartilage proteoglycans from the scoliotic line also differed from that of proteoglycans from the isogenic line.(ABSTRACT TRUNCATED AT 250 WORDS)

Scoliosis in chickens.

Scoliosis developed in 55 per cent of sexually mature birds (68 per cent of male and 46 per cent of female birds) in a highly inbred line of chickens originally produced from white Leghorns. The curve could first be detected at five to six weeks of age and progressed until spontaneous fusion of the thoracic vertebrae occurred. Studies of these chickens indicated that abnormalities of growth and development of the spine are not the primary cause of the scoliosis. Preliminary studies of the paravertebral musculature also indicated that simple muscle imbalance is not responsible for the curve. Initial studies of collagen extracted from the scoliotic line of chickens showed it to be more soluble than similar collagen extracted from white Leghorn controls.

Neuromorphometric changes in the ventral spinal roots in a scoliotic animal.

The morphologic changes were investigated in the ventral and dorsal spinal nerve roots in a strain of white leghorn chickens that genetically was predisposed to have idiopathic scoliosis. In the ventral spinal roots in chickens with scoliotic curves, small myelinated fibers were increased in number and appeared in clusters. The number of small fibers did not correlate with the degree or the duration of the deformity. In 8 of the 15 chickens of the genetic scoliotic strain that did not have spinal curves, changes in the myelinated fibers, similar to those in chickens with scoliotic curves, were seen. These findings suggest that abnormalities in the myelinated fibers of the ventral spinal nerve roots may be the primary genetic lesion that, with variable penetrance, influences the development of scoliosis.

Adult and embryo responses to organophosphate pesticides: azodrin.

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.

Tissue densities in developing avian embryos.

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.

Making the connection: exploring classical concepts in normal and abnormal limb development using contemporary approaches.

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.

Facial development in normal and mutant chick embryos. I. Scanning electron microscopy of primary palate formation.

Early facial development in normal chick embryos was studied by scanning electron microscopy, and compared to the abnormal facial development of a mutant in which primary palate formation does not occur, thus resulting in bilateral cleft lip. In both normal and "cleft primary palate" mutant embryos, subsequent to the appearance of the nasal placodes, the surrounding tissues elevate to give rise to the presumptive facial primordia. As the facial primordia grow forward, they gradually assume the configuration of a square which is most pronounced at five days development. In normal embryos, the square configuration is then lost as the facial primordia become aligned in preparation for primary palate formation. The primary palate is formed at six days development by fusion of the "free-ended" medial nasal processes with the combined lateral nasal and maxillary processes across the nasal grooves. Just prior to fusion, long, slender filaments extend from the apposing surfaces of the facial primordia in the regions of prefusion contact. It is speculated that these "prefusion filaments" may function in alignment or adhesion of the facial primordia. In "cleft primary palate" embryos, facial morphogenesis appears to arrest at five days development, so that the square configuration persists. The medial nasal processes never contact the lateral nasal and maxillary processes, but instead remain separated from them by wide nasal grooves. Furthermore, facial primordia of mutant embryos do not exhibit the "prefusion filaments" characteristic of normal embryos.

An avian model for comparative studies of insulin teratogenicity.

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.

Temporal, morphological, and genetic responses of avian embryos to Azodrin, an organophosphate insecticide.

The effect of Azodrin on avian development was studied using a bobwhite quail line and two chicken lines--a single comb White Leghorn (SCWL) and an Australorp line. The bobwhite quail embryos did not respond to injections of Azodrin until stages 22 to 23 (6 days of incubation); the SCWL embryos, not until stage 18 (3 days of incubation) with increasing susceptibility through stage 20. The threshold concentration, at stage 19, for the SCWL was 0.4 mg/kg, for the Australorp, less. Within one hour of treatment stage-20 embryo shape was altered. There was, however, no immediate response from embryos treated earlier. The effect appeared as a reduced growth rate in the cervical flexure, and may be mediated by interference with the normal energy balance. The response, once initiated, was continuous through 10 days of incubation.

Gene expression in the limbless mutant: polarized gene expression in the absence of Shh and an AER.

Limb buds in the limbless chick begin to form normally but fail to form an AER and ultimately degenerate. Wnt7a and LMX-1, which are restricted to the dorsal half of a normal limb bud, are expressed throughout the ectoderm and mesenchyme of the mutant buds, respectively. Engrailed-1, normally expressed in ventral limb ectoderm, is not expressed in the limbless bud. This defect precedes the normal period of AER formation and no localized expression of genes normally found in the AER is observed in limbless buds. Consistent with the lack of molecular specialization of an AER, Shh and BMP-2 are not expressed in the ZPA of the mutant bud. Despite the lack of Shh, FGF-4, or BMP-2 expression, the hoxd genes are expressed at low levels in the posterior mesenchyme of the bud. Forced expression of Shh fails to rescue the positive feedback loop between the AER and the ZPA and does not lead to distal outgrowth. However, Shh does maintain the part of the bud formed during pre-AER stages. These results support the importance of the dorsal/ventral boundary in the initiation of AER formation and imply that Shh is not required for the initial activation of polarized hoxd gene expression during limb development.

Distribution of extracellular matrix in the migratory pathway of avian primordial germ cells.

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.

Effects of the sex-linked dwarf gene (dw) on the expression of the muscular dystrophy gene (am) in chicken.

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.

Limb development in diplopodia4: a polydactylous mutation in the chicken.

In a study of the polydactylous mutation of the domestic chicken, diplopodia4, we have found that the genetic lesion affects primarily the mesoderm and only secondarily the ectoderm. The effect of this mutant mesenchyme on overlying ectodermal ridge, either mutant or normal, is to thicken the ridge preaxially, leading to increased outgrowth and preaxial polydactylism. A "zone of polarizing activity" in the normal limb-bud seems to have a role in the control of its anteroposterior polarity. We have examined diplopodia4 limb-buds for polarizing activity and found it to be normal in its activity and distribution. These results suggest that the supernumerary outgrowth in the mutant limbs result from increased ridge mainon of the polarizing zone.