On the histogenetic potency of the tailhud mesoderm

Summary The caudalmost part of the tailbud mesoderm (terminal paraxial tailbud mesoderm) does not develop into somites. It is not clear whether this terminal paraxial tailbud mesoderm can be considered to be a part of the segmental plate. To elucidate the nature of the tailbud mesoderm, grafts containing caudal somites, caudal prospective somitic mesoderm and the terminal paraxial tailbud mesoderm were grafted from quail embryos into the wing bud mesoderm of chick embryos. The distinct nuclear difference between quail and chick allows the identification of the grafts on a cellular level. The grafts containing caudalmost somites and the prospective somitic mesoderm differentiate into muscle and cartilage. The terminal paraxial tailbud mesoderm, on the other hand, did not give rise to either of these tissues. From this it can be concluded that the terminal paraxial tailbud mesoderm cannot be considered to be a part of the segmental plate.     

Positional control of mesoderm movement and fate during avian gastrulation and neurulation.

Segments of primitive streak from donor quail embryos at stages of gastrulation and neurulation were transplanted heterotopically and isochronically to primitive streaks of host chick embryos. The subsequent movement and fate of grafted cells was determined using the quail nucleolar marker to define grafted cells. The pattern of movement of grafted cells depended on their new position within the primitive streak, not on their original position. When cells of cranial regions were placed more caudally, they moved to mesodermal subdivisions that were located lateral to those they would have populated if left in their original position. When caudal segments were placed more cranially, they moved to more medial mesodermal subdivisions. Whether the fate of grafted cells corresponded to their original location or their new location depended on both their level of origin and their new position. Cells from heterotopically transplanted Hensen's nodes, which migrated to the somitic and more lateral mesoderm, self-differentiated notochords. Similarly, in some cases, heterotopically transplanted prospective somitic cells, which migrated to lateral plate mesoderm, formed ectopic somites. In other cases, however, grafted cells contributed to the host's somites, intermediate mesoderm, and lateral plate mesoderm. Moreover, prospective somitic cells, which migrated to the extraembryonic lateral plate mesoderm, changed their fate and formed extraembryonic lateral plate mesoderm; and prospective lateral plate mesoderm cells, which migrated to the somitic mesoderm, formed somites as well as intermediate mesoderm and lateral plate mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early mesoderm differentiation in the chick embryo

Summary Two groups of experiments were carried out. In the first group, grafts of quail mesoderm whose presumptive fate was to form somites or heart tissues, were taken from quail embryos (stage 4–5 of Hamburger and Hamilton 1951) and inserted beneath the ectoderm of chick embryos of stage 3–4 immediately lateral to the primitive streak. Whilst most grafts contributed to the somites and/or the heart, 22 out of a total of 46 were found to have contributed also to the pharyngeal endoderm. Although three of these grafts were known to have included some quail endoderm cells, the remainder were considered to consist of mesoderm alone. It is concluded that mesoderm at the primitive streak stages is still capable of forming endoderm.In the second group of experiments, grafts of quail somites (stage 10–14) were inserted beneath the ectoderm of chick embryos of stage 3–4. In 18 out of 23 cases the graft cells were found in somitic tissue, but they were also found in the endoderm (4 specimens), lateral plate (3 specimens) and endothelium (4 specimens). It is concluded that even at stages 10–14, the somite-derived cells are still not completely determined to form somite derivatives. In those cases where the grafted somites differentiated further, sclerotome cells which migrated from them did not necessarily move towards the host notochord.     

The lymphatic endothelium of the avian wing is of somitic origin.

It has recently been shown that there are lymphangioblasts in the early avian wing bud, but fate map studies on the origin of these cells have not yet been performed. The lymphatics in the wings of 10-day-old chick and quail embryos are characterized by both the position along with all major blood vascular routes and by the Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) expression. In the quail, the endothelium of both blood vessels and lymphatics can be marked with the QH1 antibody. We have grafted the dorsal halves of epithelial somites of 2-day-old quail embryos homotopically into chick embryos. The grafting was performed at the wing level and the host embryos were reincubated until day 10. The chimeric wings were studied with the QH1 antibody alone and with double staining consisting of VEGFR-3 in situ hybridization and QH1 immunofluorescence. Our results show that in the wing the endothelium of both the blood vessels and the lymphatics is derived from the somites. QH1-positive endothelial cells form the vasculature of the chimeric wings. Chimeric lymphatics of the wing can be identified because of their typical position and their VEGFR-3 and QH1 double-positivity. This shows that not only the blood vascular cells but also the lymphatic endothelial cells of the avian wing are born in the paraxial/somitic mesoderm.     

The relationship between limb muscle and endothelial cells migrating from single somite

Somites contribute myogenic and endothelial precursor cells to the limb bud. Transplantations of single somites have shown the pattern of muscle cell distribution from individual somites to individual limb muscles. However, the pattern of the endothelial cell distribution from individual somites to the limb has not been characterized. We have mapped quail muscle and endothelial cell distribution in the distal part of the chick limb after single somite transplantation to determine if there is a spatial relationship between muscle and endothelial cells originating from the same somite. Single brachial somites from quail donor embryos were transplanted into chick embryos, and, following incubation, serial sections were stained with a quail-endothelial cell-specific monoclonal antibody (QH-1), an anti-quail antibody (QCPN) and an anti-desmin antibody to distinguish the quail endothelial and muscle cells from chick cells. Our results show that transplants of somite 16-21 each gave rise to quail endothelial cells in the wing. The anterioposterior position of the blood vessels formed by somitic endothelial cells corresponded to the craniocaudal position of the somite from which they have originated. Endothelial cells were located not only in the peri- and endomysium but also in the subcutaneous, intermuscular, perineural and periost tissues. There was no strict correlation between the distribution of muscle and endothelial cell from a single transplanted somite. Blood vessels formed by grafted quail endothelial cells could invade the muscle that did not contain any quail muscle cells, and conversely a muscle composed of numerous quail muscle cells was lacking any endothelial cells of quail origin. Furthermore, a chimeric limb with very little quail muscle cells was found to contain numerous quail endothelial cells and vice versa. These results suggest that muscle and endothelial cells derived from the same somite migrate on different routes in the developing limb bud.     

Grafting experiments on determination and migratory behaviour of presomitic, somitic and somatopleural cells in avian embryos

Summary The state of determination of somites, parts of somites, unsegmented paraxial mesoderm and of somatopleure was investigated by grafting these tissues from quail embryos to the wing buds of chicken embryos. It was found that muscular and chondrogenic determination occur before the formation of somites. Muscular determination takes place earlier than previously assumed and ahead of chondrogenic determination. Somatopleure yields cartilage, but no skeletal muscle. Prospective sclerotomes are primarily capable of differentiating into muscle and loose this potency in the course of development. Myogenic cells extensively migrate within the wing bud in a proximo-distal direction, whereas chondrogenic cells both of somitic and somatopleural origin show no overt migratory tendency.This work was supported by a grant of the Deutsche Forschungsgemeinschaft     

Intraspecific chick/chick chimaeras: dystrophic somitic mesoderm transplanted to a normal host forms muscles with a dystrophic phenotype

Intraspecific chick/chick chimaeras were prepared by transplanting thoracic somitic mesoderm from donor chick embryos with hereditary muscular dystrophy to replace extirpated brachial somites of normal host embryos at stage 13 (48-52 h in ovo). Since the wings of unoperated dystrophic embryos exhibit significantly reduced motility between day-10 in ovo (day-10E) to day-15E, this parameter was used as a marker both to verify the viability of the transplant and to assess if the dystrophic phenotype of impaired functional activity is preserved in the mutant wing muscles innervated by brachial nerves of normal embryos. Our motility analyses of the chimaeras confirmed that transplanted thoracic somitic mesoderm gives rise to brachial musculature and that the experimental muscles maintained the inherent dystrophic phenotype.     

An autoradiography study of myogenic cell movement in avian limb buds following heterospecific and homospecific transplantation

Summary Species specificity and the use of quail cells as a marker in the study of myogenic cell movement in the developing avian limb was investigated. In order to establish whether or not observed myogenic cell movement in quail/chick limb transplantation experiments might be an artefact produced by cellular interaction between these cell types a series of homospecific and heterospecific transplantations was performed. Chick wing fragments (staged 20–25 H.H.) were labelled with tritiated thymidine and inserted into unlabelled chick wing bud (homospecific) in ovo. In addition, quail wing fragments were also labelled with tritiated thymidine and transplanted in the same manner into chick (heterospecific), so that the effectiveness of tritium as a marker could be assessed. After 4 days post-incubation, myogenic cell movement was detected in eight out of the ten homospecific transplantions performed. Myogenic cell movement in avian limbs is therefore not produced by interaction between chick and quail cells, as migration was also detected in the chick/chick transplants. Nonetheless, heterospecific transplantation results revealed that autoradiographic methods failed to reveal completely the true extent to which myogenic cell movement occurred, because tritiated thymidine was subject to dilution.     

Participation of individual brachial somites in skeletal muscles of the avian distal wing

In this paper we investigate the somitic origin of the individual muscles of the forearm and hand using quail-chick chimeras. Our results show that only somites 16–21 give rise to wing muscle, but they take part in muscle formation to different extents. Somite 21 does not always participate in the formation of muscle of the forearm and hand. The most cranial somite (16) takes part in the radial muscles and the most caudal somites (20, 21) in the ulnar muscles, reflecting their position with respect to the limb bud. The centrally located somites (17, 18, 19) are involved in all (18) or most (17, 19) muscle primordia. This pattern of distribution is clearest in the forearm, whereas the participation of somites in particular muscle groups is not so distinct in the hand. Hand muscles are mainly made up of cells from somites 18–20. All brachial somites participate in dorsal (extensor) as well as ventral (flexor) muscles of the forearm and hand. Each somite takes part in more than three muscle primordia in a reproducible fashion, and every muscle primordium is derived from at least three somites. Especially the M. ulnimetacarpalis ventralis takes origin from all somites involved in limb muscle formation (16–21). Apart from muscle cells, endothelial cells also and a few fibroblasts of quail origin are found in the limb bud after somite grafting.     

Dynamic Id2 expression in the medial and lateral domains of avian dermamyotome.

Id2 cDNA was isolated from a subtractive screen of stage-12 quail caudal somites. In situ hybridisation analysis identified the previously un-described expression of Id2 mRNA in distinct medial and lateral domains of the somitic dermamyotome in both quail and chick embryos. Id2 expression in somites was highly dynamic being first initiated in the lateral domain of the dermamyotome of stage-8-10 embryos, followed by expression in a separate medial domain. Id2 mRNA during subsequent embryonic development could be detected in both medial and lateral domains in the anterior to mid regions while the posterior, recently segmented somites, showed expression only in the lateral domain, which was eventually down regulated in the anterior-most somites. Tissue manipulation studies revealed that Id2 expression in somites required positive signalling from not only axial structures and lateral plate mesoderm but also surface ectoderm. In addition, Id2 expression was also observed in anterior and posterior domains of developing avian limb buds and interdigital tissue.     

Angioblast differentiation is influenced by the local environment: FGF-2 induces angioblasts and patterns vessel formation in the quail embryo.

The embryonic vasculature forms by the segregation, migration, and assembly of angioblasts from mesoderm, a process termed vasculogenesis. The initial role of fibroblast growth factor 2 (FGF-2) in vascular development appears to be in the induction of endothelial precursors, angioblasts. Quail somites transplanted into chick embryos will give rise to angioblasts of quail origin. The number of angioblasts present within the chimera is dependent on the host environment. Angioblast induction can be demonstrated in vitro by the addition of FGF-2 to cultures of dissociated somitic mesoderm, as assessed by QH-1 epitope expression. Manipulation of FGF-2 concentration in the quail/chick chimeras by FGF-2 peptide or neutralizing antibody injections increases or decreases angioblast induction in the predicted manner. To better control growth factor release in vivo we have implanted beads that release FGF-2 into the embryonic environment. FGF-2 beads implanted into the somite induce angioblast differentiation in the epithelial somite; whereas, beads lateral to the somitic mesoderm induce the formation of ectopic vessels. These studies suggest that FGF-2 is important for both the induction of angioblasts and the assembly of angioblasts into the initial vasculature pattern.     

Origin of spinal cord meninges, sheaths of peripheral nerves, and cutaneous receptors including Merkel cells

Summary The origin of cells covering the nervous system and the cutaneous receptors was studied using the quail-chick marking technique and light and electron microscopy. In the first experimental series the brachial neural tube of the quail was grafted in place of a corresponding neural tube segment of the chick embryo at HH-stages 10 to 14. In the second series the leg bud of quail embryos at HH-stages 18–20 was grafted in place of the leg bud of the chick embryos of the same stages and vice versa. It was found that all meningeal layers of the spinal cord, the perineurium and the endoneurium of peripheral nerves, as well as the capsular and inner space cells of Herbst sensory corpuscles, develop from the local mesenchymal cells. Schwann cells and cells of the inner core of sensory corpuscles are of neural crest origin. The precursors of Merkel cells migrate similarly to the Schwann cells into the limb bud where they later differentiate. This means that in addition to the Schwann cells and the melanocytes a further neural crest-derived subpopulation of cells enters the limb.     

Rib development in chick embryos analyzed by means of tantalum foil blocks

Rib development in chick embryos, with special emphasis on the origin of ribforming cells, has been analyzed by inserting tantalum foil blocks parallel to certain somites of the prospective rib-forming level. Intact edges of bent pieces of foil the width of 4–6 somites were inserted through the somatopleure at stage 14–16 either (1) immediately lateral to the nephrotome, (2) between proximal and distal portions of the lateral plate or (3) within the distal lateral plate. Edges of similar pieces of foil containing a triangular notch the width of 2 somites were likewise inserted through the somatopleure of stage 14–16 embryos (4) immediately lateral to the nephrotome or (5) between proximal and distal portions of the lateral plate. On the basis of the modified rib pattersn studied in toto after 11 days of incubation it was concluded that the (1) original somatic mesoderm does not participate in formation of vertebral rib components, (2) original somatic mesoderm plays some role in formation of sternal rib components (ribs 3–7) but probably does not contribute chondroblasts for this purpose, and (3) distalmost somatic mesoderm is not involved in rib formation. Consistent types of modified wings are described and also modifications of the body wall, breast and ventral feather tracts of the right side.     

On the origin of cells determined to form skeletal muscle in avian embryos

Summary Pieces of quail embryos from various developmental stages ranging from unincubated blastoderms (before the appearance of a primitive streak) to embryos having formed somites were grafted to the wing buds or into the coelomic cavity of chicken embryos. The grafts, which can be identified on a cellular level by virtue of the prominent nucleolus-associated chromatin, present in the quail and absent in the chicken, were screened after suitable periods of reincubation for the presence or absence of skeletal myotubes containing quail nuclei. Grafts having contributed to such skeletal myotubes were considered as having contained determined myogenic cells at the time of the grafting procedure. Determined myogenic cell appeared first in the primitive streak and in the mesodermal cells formed by the invagination (gastrulation) of epiblastic cells through the primitive streak. This is true for both the head process and the paraxial mesoderm. Epiblastic cells never gave rise to skeletal myotubes. Therefore it can be said, that the onset of myogenic determination coincides with gastrulation. It remains, however, to be established, whether these two events are causally related to one another.     

On the differentiation and origin of myoid cells in the avian thymus

Summary The avian thymus and its myoid cells were investigated paying special attention to the developmental and morphological differences between chick and quail.By means of light- and electron microscopy, and immunofluorescence technique using an anti-myosin antibody, the myoid cells were found to express characteristics corresponding to those of skeletal muscle cells. They change their appearance during embryonic development. In the chick the myoid cells become located singly and rounded, and their cross-striation disappears. In the quail they remain small, elongated, cross-striated, and become arranged in long cords.The origin of myoid cells was studied using the quailchick marking technique: Cranial somites and the prechordal mesoderm were grafted from quail into chick embryos. After somite transplantation the host thymus does not contain graft-derived cells. The myoid cells are exclusively derived from the chick. After implantation of prechordal mesoderm, graft-derived quail cells are found in the central cores of all visceral arches and also within the early epithelial anlage of chimeric thymus. These findings indicate that the thymus myoid cells are derived from the axially located prechordal head mesoderm.Supported by the Deutsche Forschungsgemeinschaft (Ch 44/8-1)     

Study of the origin of connective tissue sheaths of peripheral nerves in the limb of avian embryos

Summary Quail-chick and chick-quail chimeras were constructed by grafting, isotopically, the limb bud of quail embryos into a chick of the same developmental stage and vice versa, prior to the entry of nerve fibres into the limb.After 5–14 days reincubation of the embryos, the components of the connective tissue sheaths of the peripheral nerves were observed by using Feulgen-Rossenbeck staining and light microscopy, in order to distinguish quail cells and chick cells.In all the chimeras studied, the connective tissue sheaths of peripheral nerves (the epineurium, perineurium, perineural septa and endoneural fibroblasts) were formed from the mesenchyme of the limb bud, while Schwann cells were of host origin. Also the outer and inner capsule of muscle spindles originated from the limb bud mesenchyme.These experiments suggest that the connective tissue sheaths of peripheral nerves (at least in the limb region of avian embryos) are not of neural crest origin, but are formed from limb bud mesenchyme.     

From the primitive streak to the somitic mesoderm: labeling the early stages of chick embryos using <Emphasis Type="Italic">EGFP</Emphasis> transfection

Mesoderm is derived from the primitive streak. The rostral region of the primitive streak forms the somitic mesoderm. We have previously shown the developmental origin of each level of the somitic mesoderm using DiI fluorescence labeling of the primitive streak. We found that the more caudal segments were derived from the primitive streak during the later developmental stages. DiI labeled several pairs of somites and showed the distinct rostral boundary; however, the fluorescence gradually disappeared in the caudal region. This finding can be explained in two ways: the primitive streak at a specific developmental stage is primordial of only a certain number of pairs of somites, or the DiI fluorescent dye was gradually diluted within the primitive streak by cell division. Here, we traced the development of the primitive streak cells using enhanced green fluorescent protein (EGFP) transfection. We confirmed that, the later the EGFP transfection stage, the more caudal the somites labeled. Different from DiI labeling, EGFP transfection performed at any developmental stage labeled the entire somitic mesoderm from the anterior boundary to the tail bud in 4.5-day-old embryos. Furthermore, the secondary neural tube was also labeled, suggesting that not only the somite precursor cells but also the axial stem cells were labeled.     

Cardiac looping in the chick embryo: the role of the posterior precardiac mesoderm

Summary Grafts of mesoderm taken from the precardiac region of quail embryos of stages 5–7 were inserted into the precardiac mesoderm of chick embryos of stages 5–7. The experiments were of four types and were codenamed to indicate the origin and the destination of the graft. QACP: tissue from the anterior end of the quail precardiac area was inserted into the posterior end of the chick precardiac mesoderm; QPCA: tissue from the posterior end of the quail precardiac area was inserted into the anterior end of the chick precardiac mesoderm; QACA: tissue from the anterior end of the quail precardiac area was inserted into the anterior end of the chick precardiac mesoderm; QPCP: tissue from the posterior end of the quail precardiac area was inserted into the posterior end of the chick precardiac mesoderm. In no case was precardiac tissue removed from the host. Three main types of anomaly were obtained: inverted hearts, in which looping took place to the left rather than to the right; compact hearts, in which no looping occurred, and hearts in which extra tissues or regions were apparent. The incidence of compact hearts was significantly greater with QPCA than with any other category of experiment. When older donors were used (stages 8–9), the incidence of compact hearts fell. No variations in the origin of the graft, nor in its ultimate destination in the host, were found to affect the frequency of any of the anomalies. Sections showed that quail hearts tended to have thicker walls than chick hearts; although quail tissues were often incorporated into the host chick hearts, they retained the histological characteristics of the donors. The fact that no compact hearts resulted from the experiment QACA, or from the mock operations, leads us to conclude that failure to loop in the compact hearts was not due to mechanical trauma caused by the operation, but to some specific difference between grafts taken from the anterior and posterior precardiac mesoderm. The fact that compact hearts were obtained when chick donors were used instead of quails, shows that the effect is not species-specific. We propose that a morphogen is secreted by the posterior end of the precardiac mesoderm and this plays a role in controlling the cessation of looping.     

On the migration of myogenic stem cells into the prospective wing region of chick embryos. A scanning and transmission electron microscope study

In chick embryos undifferentiated myogenic stem cells migrate from the ventrolateral somite respectively dermatome edge into the prospective wing region after the second day of incubation. At first, single cells that are elongated in mediolateral direction, later also small groups of cells, are found in the space between somites and somatopleura at the wing bud level. The leading ends of the migrating cells are formed like finger-shaped lobopodia as well as flattened lamellipodia from which thin filopodia arise. The main structural features of the cell processes are microtubules and microfilaments predominantly oriented parallel to the long axis of the cells. The filopodia are found to be in close connection with the surrounding network of collagen fibrils. Since the main strands of the fibrils show a mediolateral orientation, it may be assumed that the direction of cell migration depends on the arrangement of the collagen fibrils.     

Somitogenesis in cultured embryos of the Japanese quail,Coturnix coturnix japonica

The ability of unsegmented paraxial mesoderm from Japanese quail embryos to form somites was studied by culturing pieces of embryos, containing the segmental plates, on an agar medium. In the first experiments, two explants were prepared from each donor embryo. Both explants contained a segmental plate and neural tube, but only one contained notochord. The explants containing notochord formed 11.4 ± 2.1 somites, while the explants without notochord formed 11.1 ± 1.3 somites. It was concluded that explants containing Japanese quail segmental plates readily form somites in culture and that the continued presence of the notochord is not required for these somites to form. In a second series of experiments, one explant from each donor embryo contained neural tube and notochord along with the segmental plate, while the corresponding explant did not contain axial structures. The results, which were similar to those obtained in the first experiments, indicated that neither neural tube nor notochord is required for somitogenesis in vitro. Additional experiments demonstrated that bilateral symmetry extends to the unsegmented somite mesoderm, where there was a strong tendency for each segmental plate of a given embryo to form the same number of somites. It was also shown that over a three-fold range of segmental plate length, there was only a slight tendency for shorter segmental plates to make fewer somites. It was estimated that Japanese quail embryos having five to 21 pairs of somites have segmental plates that represent 11.3 ± 2.9 prospective somites each.