Tropomyosin expression and dynamics in developing avian embryonic muscles

The expression of striated muscle proteins occurs early in the developing embryo in the somites and forming heart. A major component of the assembling myofibrils is the actin-binding protein tropomyosin. In vertebrates, there are four genes for tropomyosin (TM), each of which can be alternatively spliced. TPM1 can generate at least 10 different isoforms including the striated muscle-specific TPM1alpha and TPM1kappa. We have undertaken a detailed study of the expression of various TM isoforms in 2-day-old (stage HH 10-12; 33 h) heart and somites, the progenitor of future skeletal muscles. Both TPM1alpha and TPM1kappa are expressed transiently in embryonic heart while TPM1alpha is expressed in somites. Both RT-PCR and in situ hybridization data suggest that TPM1kappa is expressed in embryonic heart whereas TPM1alpha is expressed in embryonic heart, and also in the branchial arch region of somites, and in the somites. Photobleaching studies of Yellow Fluorescent Protein-TPM1alpha and -TPM1kappa expressed in cultured avian cardiomyocytes revealed that the dynamics of the two probes was the same in both premyofibrils and in mature myofibrils. This was in sharp contrast to skeletal muscle cells in which the fluorescent proteins were more dynamic in premyofibrils. We speculate that the differences in the two muscles is due to the appearance of nebulin in the skeletal myocytes premyofibrils transform into mature myofibrils.     

The control of avian cephalic neural crest cytodifferentiation. II. Neural tissues.

A series of neural crest transplantations has been performed to (1) analyze whether avian premigratory cranial neural crest cells are pluripotential or restricted to specific developmental pathways and (2) examine the ability of trunk neural crest cells to develop in an environment usually occupied by cranial crest cells. Quail embryos, the cells of which have a unique nuclear marker, were used as donors and chick embryos as hosts. Hindbrain crest cells grafted in the place of diencephalic crest cells failed to form neurons in all but one case, in which a small ectopic ganglion was found. In the reciprocal transplants, neural crest cells emigrating from a segment of forebrain crest tissue grafted in the place of metencephalic crest cells produced trigeminal and ciliary ganglia which were completely normal. Thus, crest cells which normally never form ganglionic neurons will do so if placed in a suitable neurogenic environment. These results prove that premigratory avian cranial crest cells are not restricted to specific developmental pathways, but are initially pluripotential. Trunk crest cells grafted in the place of metencephalic crest cells form neuronal ganglia along the proximal trigeminal motor roots but do not form normal trigeminal ganglia. These root ganglia do not display normal peripheral projections, and placode cells, a normal component of the trigeminal ganglion, form ganglia in ectopic locations. Thus, while trunk crest cells respond to the metencephalic environment and form neurons, their response is different from that of cranial crest cells in the same location. Whether this is due to differences in developmental potential or in initial population size is not known.     

The MADS-domain protein AGAMOUS-like 15 accumulates in embryonic tissues with diverse origins.

AGL15 (AGAMOUS-like 15), a member of the MADS-domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.     

Kindlin-2 expression in adult tissues correlates with their embryonic origins

Kindlin-2 functions in the maintenance of homeostasis and in human diseases. This study investigated the interrelationship between Kindlin-2 expression in tissues and the corresponding germ layers from which these tissues originated. Kindlin-2 expression was examined in normal adult human organs and human cancer tissues by immunohistochemical analyses. Analysis of Kindlin-2 mRNA levels in adult human organs in the Oncomine dataset revealed Kindlin-2 is highly expressed in mesoderm-derived organs. However, Kindlin-2 was negative or weakly expressed in endoderm/ectoderm-derived organs. Interestingly, the abnormal expression of Kindlin-2 was observed in a variety of human cancers. In agreement with its expression profile in humans, Kindlin-2 was also highly expressed in mesoderm-derived organs in mouse embryos with the exception of strong Kindlin-2 expression in ectoderm-derived spinal cord and ganglia, tissues that are highly mobile during embryonic development. Importantly, we demonstrated the expression level of Kindlin-2 in adult organs correlated with their embryonic dermal origins and deregulation of Kindlin-2 in tissues is associated with tumor progression. This finding will help us understand the dual role of Kindlin-2 in the regulation of tumor progression and embryonic development.     

Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian skeletal muscles

Myotubes were isolated from enzymically disaggregated embryonic muscles and examined with light microscopy. Primary myotubes were seen as classic myotubes with chains of central nuclei within a tube of myofilaments, whereas secondary myotubes had a smaller diameter and more widely spaced nuclei. Primary myotubes could also be distinguished from secondary myotubes by their specific reaction with two monoclonal antibodies (MAbs) against adult slow myosin heavy chain (MHC). Myonuclei were birth dated with [3H]thymidine autoradiography or with 2-bromo-5'-deoxyuridine (BrdU) detected with a commercial monoclonal antibody. After a single pulse of label during the 1-2 day period when primary myotubes were forming, some primary myotubes had many myonuclei labelled, usually in adjacent groups, while in others no nuclei were labelled. If a pulse of label was administered after this time labelled myonuclei appeared in most secondary myotubes, while primary myotubes received few new nuclei. Labelled and unlabelled myonuclei were not grouped in the secondary myotubes, but were randomly interspersed. We conclude that primary myotubes form by a nearly synchronous fusion of myoblasts with similar birthdates. In contrast, secondary myotubes form in a progressive fashion, myoblasts with asynchronous birthdates fusing laterally with secondary myotubes at random positions along their length. These later-differentiating myoblasts do not fuse with primary myotubes, despite being closely apposed to their surface. Furthermore, they do not generally fuse with each other, as secondary myotube formation is initiated only in the region of the primary myotube endplate.     

Oxytocin and cervical connective tissue

The effect of oxytocin on collagen synthesis in the pregnant human cervix and lower uterine segment was studied in incubation experiments by measuring the incorporation of 3H-proline. Oxytocin had a concentration related inhibitory effect on the labelling with 3H-proline. Vasopressin in the corresponding concentrations had only a weak effect on the incorporation of 3H-proline. Addition of indomethacin did not influence the response to oxytocin indicating that the effect was probably not mediated by prostaglandins. These results suggest that oxytocin under in vitro experimental conditions influences cervical connective tissue metabolism which is in contrast to current clinical experience.     

Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.     

Oxygen and the connective tissues.

Avascular connective tissues (cartilage, discs, cornea) change with maturation and aging, particularly in large animals, where diffusion paths are longest. It is suggested that the changes in such tissues are responses to increasing difficulties in obtaining oxygen. Two almost identical structural polymers are made in these tissues: chondroitin sulphate, which requires large amounts of oxygen for biosynthesis and keratan sulphate, which requires relatively little. The observed balance of these polymers in the tissue is proposed to depend on the control of biosynthesis by the ambient oxygen tension, and/or selective breakdown.     

The embryonic development of larval muscles in Drosophila

Each of the abdominal hemisegments A2-A7 in the Drosophila larva has a stereotyped pattern of 30 muscles. The pattern is complete by 13 h after egg laying, but the development of individual muscles has begun with the definition of precursors at least by the onset of germ band shortening, some 5.5 h earlier. The earliest signs of muscle differentiation are cell fusions, which occur in the ventralmost mesoderm overlying the CNS and at stereotyped positions in the rest of the mesoderm as the germ band shortens. At the end of shortening, the pattern of muscle precursors produced by these fusions is complete. Precursors filled with dye reveal extensive fine processes probably involved initially in cell fusion and, subsequently, in navigation over the epidermis to form attachment points. The muscle pattern is formed before innervation and without cell death. Thus, neither of these processes is involved in determining the distribution of precursors. Evidence is presented for the view that the development of the larval muscle pattern in Drosophila depends on a prior segregation of founder cells at appropriate locations in the mesoderm with which other cells fuse to form the precursors.     

Rheology of embryonic avian blood

Shear stress, a mechanical force created by blood flow, is known to affect the developing cardiovascular system. Shear stress is a function of both shear rate and viscosity. While established techniques for measuring shear rate in embryos have been developed, the viscosity of embryonic blood has never been known but always assumed to be like adult blood. Blood is a non-Newtonian fluid, where the relationship between shear rate and shear stress is nonlinear. In this work, we analyzed the non-Newtonian behavior of embryonic chicken blood using a microviscometer and present the apparent viscosity at different hematocrits, different shear rates, and at different stages during development from 4 days (Hamburger-Hamilton stage 22) to 8 days (about Hamburger-Hamilton stage 34) of incubation. We chose the chicken embryo since it has become a common animal model for studying hemodynamics in the developing cardiovascular system. We found that the hematocrit increases with the stage of development. The viscosity of embryonic avian blood in all developmental stages studied was shear rate dependent and behaved in a non-Newtonian manner similar to that of adult blood. The range of shear rates and hematocrits at which non-Newtonian behavior was observed is, however, outside the physiological range for the larger vessels of the embryo. Under low shear stress conditions, the spherical nucleated blood cells that make up embryonic blood formed into small aggregates of cells. We found that the apparent blood viscosity decreases at a given hematocrit during embryonic development, not due to changes in protein composition of the plasma but possibly due to the changes in cellular composition of embryonic blood. This decrease in apparent viscosity was only visible at high hematocrit. At physiological values of hematocrit, embryonic blood viscosity did not change significantly with the stage of development.     

Ontogeny of avian extrinsic ocular muscles

Summary Light- and electron-microscopic studies were performed on those tissues that are supposed to deliver the anlagen of the extrinsic ocular muscles. Since the blastemata of the ocular muscles can be traced back into the prechordal mesoderm, it can be concluded that this tissue is the source of these muscles. In embryos from stage 8–10 according to Hamburger and Hamilton (HH) cells are found to detach from the lateral border of the prechordal mesoderm. These cells are assumed to give rise to the trochlearis and abducens musculature. In stage-14 embryos the paired premandibular cavity arises within the lateral wings of the prechordal mesenchyme. In 4-day embryos the lateral wall of each premandibular cavity becomes denser forming a premuscular mass, which is subdivided into the anlagen of the oculomotorius muscles in 5-day embryos. The head cavities are not homologous to somites because their structures, origins and sites are very different.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (CH 44/6-1).This paper is dedicated to Prof. Dr. med. Dr. h.c. Hermann Voss on the occasion of his 90th birthday.     

Species-common antigen of connective tissues.

Collagen-free extracts were prepared from bovine, porcine and canine hyaline, elastic and fibrous cartilages, articular capsule, tendon, aorta, cortical bone and regenerating articular surfaces. The extracts were investigated with antisera to bovine nasal septal cartilage, dog articular cartilage and non-collagenous protein fraction of bovine cortical bone. Immunodiffusion, immunoelectrophoresis, and immunohistochemical methods were used. In the different supporting tissues of the three animal species a common antigen, probably of proteoglycan origin, was demonstrated. The finer differences in antigenicity between the different tissues are probably due to the variations in proteoglycan composition of the given supporting tissues. Owing to the wide-spread occurrence of the antigen, the authors suggest the term "species-common connective tissue antigen" instead of the "species-common cartilage antigen" used so far.     

Histochemistry of sialo-mucins in connective tissues

Summary The present investigation was undertaken in an effort to differentiate histochemically between sialo-mucins and acid mucopolysaccharides in connective tissues.On the basis of previous studies regarding the histochemical identification of sialic acid in sections of animal salivary glands the author applied different technical procedures on cartilage and tooth germs of newborn rats.The results obtained indicated that in the tissues examined sialic acid is a normal component of the polysaccharide fraction of the connective tissue ground substance.On leave of absence at the University of Rome, Medical School, Viale Regina Elena 287-A, Rome, Italy.     

Distribution and origins of VIP-immunoreactive nerves in the cephalic circulation of the cat

VIP-immunoreactive (IR) nerves were visualized in whole mounts and sections of cephalic arteries and cranial nerves of cats with indirect immunofluorescence. Perivascular VIP-IR nerves were very widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-IR nerves were most abundant in the Circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brain stem and ventral areas of the cerebral cortex. VIP-IR nerves were absent from arterial branches supplying the posterior brain stem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya within microganglia found in the cavernous plexus and external rete. Extracerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya in microganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. It seems likely, therefore, that each major segment of the cephalic circulation is supplied by local VIP-IR neurons.