Muscular dysgenesis in fowl: ultrastructural study of skeletal muscles in the crooked neck dwarf (cn/cn) mutant

In vivo evolutive aspects of muscular dysgenesis were studied in normal and crooked neck dwarf (cn/cn) 7.5- to 20-day chick embryos. Wing, leg and breast muscles were processed for electron microscopy. It appears that the effects of the gene cn are expressed in the multinucleated cells as fine structural aberrations. Dilatation of the sarcotubular system, partial loss of the contractile elements and malorganization of the myofibrils are the major anomalies observed from day 7.5 to 18 of incubation. These changes do not constitute an abrupt phenomenon. Normal and diseased multinucleated cells always coexist in the same muscle specimen; however the frequency of the pathological cells augments with time. At the end of the incubation period, the poorly organized muscle tissue contains only morbid muscle cells. Phagocytosis or autolysis are absent.     

Histogenetic pleiotropism in the crooked neck dwarf chick embryo: degeneration of the trachea and thymus

Retrogressive analysis of the cn gene effect has been performed on crooked neck dwarf chick embryos between stages 28–38 (5–12 days). The phenocritical stage of mutant embryos studied is stage 29. Histolytic degeneration of neck tissues is first recognized by the appearance of localized degenerate nuclei in the tracheal mesenchyme. Pleiotropic autolysis of the embryonic thymus, loose mesenchyme and the ventral neck tissue is also observed. Histolysis occurs in a caudocephalic gradient in all cn-affected embryos. The degenerative effects in crooked neck dwarf embryos vary in their intensity, but the pattern of autolysis seems constant. Histological observations provide some explanation for “escapers,” homozygous lethal embryos known to survive until hatching. A mechanism for surviving developmental crises in cn embryos is proposed.     

Immunofluorescent localization of extracellular matrix components during muscle morphogenesis. II. In chick embryos with hereditary muscular dysgenesis (cn/cn)

The immunofluorescent distribution of types I and III collagen, fibronectin, and laminin during muscle morphogenesis of the crooked neck dwarf mutant chick embryo differs from that of the normal chick. The drastic difference is related to the inability of the mutant embryo to maintain a harmonious muscle pattern. The first sign of the defect is the disaggregation of type I collagen fibers of the tendons and the disorganization of the intermuscular spaces. The organization of the connective tissue never proceeds beyond the appearance of an epimysial envelope, rich in types I and III collagen, which becomes disorganized shortly after. No perimysial envelopes displaying types I and III collagen fibers and fibronectin, nor endomysial sheaths develop. Only large spaces filled with types I and III collagen fibers subdivide groups of muscle cells irregularly. On the whole, type III collagen is less abundant than type I collagen. Fibronectin disappears from the periphery of the muscle cell. Laminin is more thickly deposited in the basal lamina around irregularly sized muscle cells than around the normal muscle cell. The results are discussed in terms of morphogenetic interactions between connective tissue cells and muscle cells, and in terms of fibrosis, which characterizes some muscle diseases.     

Ontogeny of NADPH-d expression in the thymic microenvironment of the chick embryo

Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry was used to demonstrate the presence of nitric oxide in the developing chicken thymus. NADPH-d was first expressed in the epithelial cells located at the corticomedullary junction of the thymic rudiment on day 13 of incubation. The number of labelled cells gradually increased from day 13 to day 21. Ultrastructural evidence showed that the labelling was localized in a heterogeneous population of cells in the medulla near the corticomedullary junction, comprising the cystic, undifferentiated, myoid, lymphoid and epithelial reticular cells. At this age, the vascular endothelium was NADPH-d positive. Labelling was also detected in some macrophages. The reaction product primarily labelled profiles of rough endoplasmic reticulum and to a lesser extent the outer membranes of mitochondria, portions of the nuclear envelope and the Golgi apparatus. By day 18/19, NADPH-d-labelled nerve fibres were occasionally observed in the interlobular connective tissue. By day 21, these fibres formed perivascular plexuses. Labelled nerve fibres were occasionally observed in the medullary parenchyma. Possible functions of nitric oxide in the embryonic thymus are discussed.     

Ontogeny of N-acetyltransferase activity rhythm in pineal gland of chick embryo

1. N-acetyltransferase was present in pineal glands of 14-day-old chick embryos though no rhythm either in LL, DD or LD 12:12 was observed in this age. 2. Daily rhythm in pineal NAT activity was found in 18-day-old embryos incubated under LD 12:12 and LD 16:8 but no NAT rhythm was detected in DD or LL. 3. NAT rhythm persists for 2 days in constant darkness and it may be circadian in nature. 4. Presence of melatonin (85 +/- 8 pg/mg tissue) was detected in pineals of 18-day-old chick embryos.     

Origin of the iris sphincter muscle in chick embryo

The origin of the iridial sphincter muscle in chick embryo was investigated by means of immunohistochemistry. Desmin immunoreactive cells are shown in the mesenchymal stroma overlying the anterior epithelial layer of the iris in 4 1/2-day chick embryos. In 9-11-day chick embryos also some cells of the posterior epithelium near the pupillary margin, and of the iridial lamella show a slighter desmin-immunoreactivity. This finding agrees with a double origin of the iridial sphincter muscle: an early mesenchymal one and a later epithelial other.     

Tracking morphogenetic tissue deformations in the early chick embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to study the physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.     

Uptake of (3H)catecholamines by chick embryo sympathetic ganglia in tissue culture

Abstract— Chick embryo sympathetic chains were grown in tissue culture and pulse labelled with tritiated catecholamines. The uptake was restricted to sympathetic nerve cells. The capability of these cells to take up radioactive dopamine and norepinephrine from the culture media was retained after one month in tissue culture. The uptakes of both [³H]norepinephrine and [³H]dopamine were inhibited when nonradioactive DOPA, dopamine, norepinephrine or epinephrine were present in the pulse media.     

Enzymes of purine metabolism in the nuchal muscle (M. complexus) of chick embryo]

The developmental patterns of enzyme activities related to GMP metabolism have been investigated in chick embryo musculus complexus (m. Complexus). Guanylate phosphatase activity increases conspicuously from 18th to 21st day, guanosine phosphorylase increases on the 21st day and the guanase shows a very low activity during the whole period considered. Xanthine oxidase was always found absent. The results suggest that during the first period of incubation GMP breakdown in chick embryo m. complexus might follow a catabolic pathway, while starting from the 18th day some guanine might be converted to GMP originating a new metabolic pathway as previously suggested for AMP metabolism.     

Effect of the sex-linked dwarf gene on thyrotrophic and somatotrophic axes in the chick embryo

Plasma concentrations of thyroxine (T4), triiodothyronine (T3), reversed triiodothyronine (rT3), and insulin-like growth factors I and II (IGF-I, IGF-II) together with peripheral 5'-monodeiodination activity were measured in both normal and sex-linked dwarf embryos between day 14 of incubation and day 1 posthatch. Plasma T4 levels increased gradually during embryonic development while T3 concentrations remained low until day 20, when a sharp increase was observed. rT3 levels also increased from day 14 and dropped on day 20 when T3 levels started to increase. 5'-monodeiodination activity was high on day 14 of incubation, decreased thereafter, and showed an increase at the time of air sac penetration together with increased T3 levels. At this stage, differences between normal and dwarf embryos were observed; the latter had lower nonsignificant 5'-Monodeiodination activity and lower (P less than 0.01) plasma T3 levels. Plasma IGF-II levels were high during the whole embryonic period studied. Dwarf embryos had lower (P less than 0.05) IGF-II levels at the time of hatching. IGF-I levels were high on days 14 and 16, declined afterwards, and started to increase again around hatching. With the exception of T3 and IGF-II levels, introduction of the dwarf gene did not cause major changes in the hormonal parameters studied. This may explain the identical body weight at hatching.     

Spatial analysis of limb bud myogenesis: A proximodistal gradient of muscle colony-forming cells in chick embryo leg buds

The regional distribution of myogenic cells in developing chick leg buds has been investigated using an in vitro clonal assay. Leg buds were embedded in gelatin and sectioned at intervals of 100–300 μm utilizing a vibratome, and cells dissected from prospective myogenic areas were analyzed for their ability to form colonies containing multinucleated myotubes. The results show that muscle colony-forming (MCF) cells from stage 23 ( to 4-day incubation) are exclusively of the early morphological type, and are found in the proximal two-thirds of the bud. Late-type MCF cells are first obtained from the proximal sections of stage 24–25 (4- to day) buds; in succeeding stages (26–29), late MCF cells supercede the early MCF cell type in the proximal regions, and extend into progressively more distal sections in a graded fashion. Results from sequential sections suggest that early and late MCF cells are located within the same muscle groups. The proportion of late MCF cells continues to increase throughout this period, until by stage 31 (7 days) only the most distal myogenic regions (the toe muscle regions) have an appreciable proportion of early MCF cells. Clonal plating efficiencies increase throughout the period of analysis, and by stage 31 precisely dissected myogenic regions yield plating efficiencies as high as 36% with greater than 95% of these colonies differentiating as muscle.     

The crooked neck dwarf muscular dysgenesis in fowl is due to a selective alteration of the somitic myogenic cell line

Summary Muscular dysgenesis in trunk and limb regions of the crooked neck dwarf (cn/cn) fowl is characterized by a complete disorganization of the muscles, starting at 7.5 days of incubation and resulting, at the end of the incubation period, in a profound muscular atrophy. It has previously been attributed to progressively extending defects of the myotubes. In this paper, embryonic cn/cn head and neck muscles were subjected to histological and ultrastructural analysis. The mononucleated myoblasts of the skeletal muscles are not diseased. Pathology is only expressed in the multinucleated cells, mainly by impaired sarcomerogenesis and distension of the sarcoplasmic reticulum. In the non-skeletal (cardiac or smooth) muscles, the connective tissue scaffolding and the ultrastructural features are similar to those of normal muscles at the same age. The present report confirms that the cn defect is confined to the skeletal muscle cells. All of them belong to the same lineage, which is contained in the somitic mesoderm, whether the latter becomes segmented or not during embryogenesis.