Scanning electron microscopy of the development of the mesoderm layer in chick embryos

Summary The distribution of mesoderm, the structure of mesoderm cells and relationship between mesoderm and ectoderm were examined by SEM in embryos at stages 3 to 5. The mesoderm was displayed by removal of the endoderm and by fracturing the embryos through mesoderm containing regions. Within the mesoderm layer four zones could be distinguished by their cell shape and arrangement — the primitive streak, a multilayered compact area around the margins of the area pellucida, multilayered and loosely arranged cells near the primitive streak and a flattened monolayer of cells around the advancing lateral and anterior edges of the mesoderm sheet. Secretion of basement membrane by the ectoderm was seen to precede the arrival of mesoderm cells. This suggests that ectoderm alone can synthesize basement membrane without mesodermal contribution.     

Experimental analysis of the origin of the wing musculature in avian embryos

Summary Interspecific grafts of somites, as well as parts of the somatic plate mesoderm, have been made between quail and chick embryos (stages 12–14 H.H.) at the level of the prospective wing bud in order to examine the relationship between somites and wing bud myogenesis. The stability of the natural quail nuclear labelling makes it possible to follow the developmental fate of grafted mesodermal cells in the host embryo. Embryos examined after subsequent incubation periods of 3–7 days show the following distribution of somatic and somitic cells within the wing bud: as soon as the three zones of different cell density within the mesoderm can be distinguished, cells of somitic origin are limited to the prospective myogenic are which is made up of a mixed population of somatic and somitic cells, whereas the prospective chondrogenic area as well as the subectodermal zone only consists of cells originated from the somatic plate mesoderm. After further incubation, single muscle blastema are present which were also seen to be a mixture of somatic and somitic cells. The cells of muscular bundles are of somitic origin, while the muscle connective tissue cells are derived from the somatic plate mesoderm. After grafting into the coelomic cavity or on the chorio-allantoic membrane, fragments of the somatic plate mesoderm previously isolated from quail embryos (stage 14 H.H.) at the level of the prospective wing bud exhibit well developed skeletal elements, but fail to differentiate any musculature. These experimental investigations support previous evidence for a somitic origin of wing bud myogenic cells. Histological and scanning electron microscopic studies of the brachial somites and the adjacent somatic plate mesoderm of chick embryo (stages 13–15 H.H.) reveal that migration of still undifferentiated somitic cells into the brachial somatic plate mesoderm begins to take place in embryos at stage 14.     

Development of the chick embryo endoderm studied by S.E.M

Summary The endoderm of a series of chick embryos from the unincubated egg to Hamburger and Hamilton stage 5 was examined by scanning electron microscopy (SEM). During this period the endoderm develops from a few scattered cells to a complete epithelial layer. Prior to the formation of the primitive streak endoderm cells can be observed delaminating from the ectoderm. These cells are round and have few processes except where they contact each other. At stage 2 cells appear in the endoderm over the primitive streak which have broad flat processes. This suggests that the cells originate directly from the streak. Away from the streak the endoderm cells are either smooth or have short microvilli. In later streak stages a mixture of smooth and some microvillous cells form a hexagonal pattern. This pattern is occasionally modified and holes are found in the endoderm with cell processes protruding from below the endoderm level. Sometimes whole cells, smaller and rounder than the majority of the endoderm cells are associated with this disturbance of the pattern. These cells are connected to the mesoderm by a long cytoplasmic process and it is suggested that they could be cells entering the endoderm from the middle layer, having accompanied the mesoderm cells through the primitive streak.     

Distribution and migration of angiogenic cells from grafted avascular intraembryonic mesoderm

Summary The hemangiogenic potencies of initially avascular intra-embryonic mesoderm were studied in chick and quail embryos and in chick-quail chimeras. The prechordal mesoderm, primitive streak and primitive node of quail embryos were heterospecifically grafted into limb buds of chick embryos. Hemangiopoietic quail cells in the host limb were detected by immunohistological staining with the monoclonal anti-MB-1 antibody after 3–6 days of re-incubation. The antibody is specifically directed against quail hemangiopoietic cells and their derivatives. Quail endothelial cells were found in pure quail and in chimeric vessels, inside as well as outside the graft. The main artery of the limb and the vessels inside the graft were connected by chimeric arteries. Proximal to the graft, quail endothelial cells were located predominantly within the lining of the main artery, while distally they were found mainly in the veins and the marginal sinus. The results show that, as early as stage 3 (according to Hamburger and Hamilton 1951, HH) all parts of the avascular intraembryonic mesoderm tested, give rise to endothelial cells. Both mechanisms, angiogenesis and vasculogenesis, contribute to the vascularization of the limb. Immunocytological and scanning electron microscopic studies indicate that centrifugal and centripetal migration of angiogenic cells occurs outside the vessels as well as on the inner surface of the endothelium.Supported by the Deutsche Forschungsgemeinschaft (CH 44/9-1)     

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)     

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 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.     

Expression of TGFβ1/β3 during early chick embryo development

We have used an antibody against a TGFβ peptide fragment to localize this growth factor in the early chick embryo from laying to the ten-somite stage of development. Western blotting showed that the antibody reacted with both mammalian TGFβ1 and chicken TGFβ3. By immunocytochemistry we find that at the earliest developmental stage (stage X of Eyal-Giladi and Kochav) immunoreactivity to this antibody is primarily located in the cells of the area opaca and marginal zone, as well as in the most peripheral edge cells of the blastoderm. The yolk is non-reactive, except in a highly localized region subjacent to the edge cells. This pattern persists at stage XII, and at both stages individual isolated cells in the epiblast and hypoblast are also reactive. By the time to gastrulation, reactivity in the epiblast is polarized to the ventral extremity of the cells, and again some isolated cells in this layer are intensely immunoreactive. At this stage also, the endoderm cells, particularly those underlying the primitive streak, are positive, as are the mesoderm cells lateral to the streak. At somite stages, the neuroepithelium is not reactive but the ectoderm lateral to it is strongly positive. At the caudal primitive streak levels of early somite embryos, the ectoderm and endoderm are immunoreactive while the mesoderm loses the reactivity it showed at the early gastrulation stages. The neuroepithelial cells later show reactivity at their apical poles, and, as at the earlier stages, individual cells show intense labelling. These results indicate that TGFβ1 and/or TGFβ3 immunoreactivity is developmentally regulated from very early stages of morphogenesis in the chick, and together with data from earlier functional studies, suggest that this factor has roles in embryonic axis formation and in blastoderm expansion. © 1994 Wiley-Liss, Inc.     

用RNA-RNA原位杂交技术研究Robo2在早期鸡胚发育中的表达

目的:探讨Robo2(roundabout homolog 2)在早期鸡胚发育中的表达。方法:利用分子生物学手段,构建Robo2/pSPT18重组质粒,并且制备地高辛标记的Robo2 RNA探针,进而通过RNA-RNA原位杂交技术检测Robo2在早期鸡胚发育中的表达。结果:原条发生前Robo2表达较弱,原条发生后主要表达在原条和神经板。体节期Robo2主要表达在脊索、神经管、体节和血岛部位。冰冻切片后观察到Robo2主要表达在外胚层,而在中胚层和内胚层只有部分表达。结论:阐明了Robo2在早期鸡胚发育中的表达,为进一步研究Robo2在正常生理和病理条件下的功能及作用机制奠定基础。     

Localization of cells of the prospective neural plate, heart and somites within the primitive streak and epiblast of avian embryos at intermediate primitive-streak stages

By constructing avian transplantation chimeras using fluorescently-labeled grafts and antibodies specific for grafted cells, we have generated a prospective fate map of the primitive streak and epiblast of the avian blastoderm at intermediate primitive-streak stages (stages 3a/3b). This high-resolution map confirms our previous study on the origin of the cardiovascular system from the primitive streak at these stages and provides new information on the epiblast origin of the neural plate, heart and somites. In addition, the origin of the rostral endoderm is now documented in more detail. The map shows that the prospective neural plate arises from the epiblast in close association with the rostral end of the primitive streak and lies within an area extending 250 microm rostral to the streak, 250 microm lateral to the streak and 125 microm caudal to the rostral border of the streak. The future floor plate of the neural tube arises within the midline just rostral to the streak, confirming our earlier study, but unlike at the late-primitive streak stages when both Hensen's node and the midline area rostral to Hensen's node contribute to the floor plate, only the area rostral to the primitive streak contributes to the floor plate at intermediate primitive-streak stages. Instead of contributing to the floor plate of the neural tube, the rostral end of the primitive streak at intermediate primitive-streak stages forms the notochord as well as the rostromedial endoderm, which lies beneath the prechordal plate mesoderm and extends caudolaterally on each side toward the cardiogenic areas. The epiblast lateral to the primitive streak and caudal to the neural plate contributes to the heart and it does so in rostrocaudal sequence (i.e., rostral grafts contribute to rostral levels of the straight heart tube, whereas progressively more caudal grafts contribute to progressively more caudal levels of the straight heart tube), and individual epiblast grafts contribute cells to both the myocardium and endocardium. The prospective somites (i.e., paraxial mesoderm) lie within the epiblast just lateral to the prospective heart mesoderm. Comparing this map with that constructed at late primitive-streak stages reveals that by the late primitive-streak stages, prospective heart mesoderm has moved from the epiblast through the primitive streak and into the mesodermal mantle, and that some of the prospective somitic mesoderm has entered the primitive streak and is undergoing ingression.     

Scanning electron microscopic study on the development of primitive blood vessels in chick embryos at the early somite-stage

Summary Primary vasculogenesis in chick embryos at the early somite stage 11–14 somites) was investigated mainly by scanning electron microscopy (SEM), with special reference to the development of primitive blood vessels such as the arteria et vena vitellina (AV, VV), aorta dorsalis (AD) and vena cardinalis (VC). After glutaraldehyde fixation, the endoderm or ectoderm was removed from the embryos to expose either the ventral (AV, VV, AD) or the dorsal (VC), vascular system. The mode of vascular formation was found to be identical in all these blood vessels, arising first in loco as isolated solid masses or cords composed of so-called angioblasts. The angioblasts at this developmental phase could be distinguished from underlying mesenchymal cells, exhibiting a relatively flat surface. The VV was recognized first on both sides of the anterior intestinal portal at the 4-somite stage, whereas the forming AD was identified on the ventral surface of the paired forming AD was identified on the ventral surface of the paired somites at the 6-somite stage, appearing almost simultaneously from the cranial to caudal somite regions. After the 8-somite stage, the AV was formed by transformation of one of the caudal plexuses spreading to the area vasculosa. In the 9-somite stage, the angioblastic cords of the VC appeared on the dorsal side of the mesoderm in the same manner as for other ventral vessels. This finding differs from the statement of a previous author that the VC is formed by longitudinal anastomosis of intersegmental diverticula of the AD.Supported in part by a Grant-in-aid for special projects in cardiovascular research from the Ministry of Education of Japan     

Emergence of endothelial and hemopoietic cells in the avian embryo

Summary During organogenesis, endothelial cells develop through two different mechanisms: differentiation of intrinsic precursors in organ rudiments constituted of mesoderm associated with endoderm, and colonization by extrinsic precursors in organs constituted of mesoderm associated with ectoderm (Pardanaud et al. 1989). On the other hand, both types of rudiment are colonized by extrinsic hemopoietic stem cells. In the present work we extend our former study by investigating the hemangioblastic (i.e. hemopoietic and angioblastic) potentialities of primordial germ layers in the area pellucida during the morphogenetic period. By means of interspecific grafts between quail and chick embryos, we show that splanchnopleural mesoderm gives rise to abundant endothelial cells, and to numerous hemopoietic cells in a permissive microenvironment, while somatopleural mesoderm produces very few cells belonging to these lineages, or none. Thus we confirm that the angioblastic capacities of the mesoderm differ radically, depending on its association with ectoderm or endoderm. Furthermore, at this embryonic period, both endothelial and hemopoietic potentialities are displayed by splanchnopleural mesoderm. However the site of emergence of intraembryonic hemopoietic stem cells appears spatially restricted by comparison to more widespread angioblastic capacities.     

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.     

Is chemotaxis a factor in the migration of precardiac mesoderm in the chick?

Summary The chick heart is formed from bilateral patches of presumptive cardiac mesoderm cells which migrate over the endoderm and fuse in the midline. We have tested the possibility that this migration is controlled, at least in part, by a chemotactic substance exuded by the anterior end of the endoderm. We have used chick/quail combinations to follow naturally marked cells during the course of their migration. Chimaeric embryos were formed by fusing together parts of chick and quail embryos of stage 5–6. Each embryo possessed two pairs of precardiac regions, the quail pair lying immediately anterior to that of the chick. These chimaeras were then explanted in embryo culture. In the event of chemotaxis, cells from the posterior end of the quail precardiac mesoderm might be expected to invade the chick area. Samples of explants and chimaeras were examined at intervals from 2 to 24 h, but in no case were cells found to have changed their direction of migration as a result of the proximity of anterior endoderm. It is concluded that this work does not provide evidence for a chemotactic attraction by the anterior end of the endoderm. Supported by the following grants: NIH HD 21048, HD 06819, and AHA 880696 (JWL); the British Heart Foundation, and Action Research (R.B.); and an SERC postgraduate studentship (HSE).     

Differentiation of gut endoderm in dependence of the notochord

Summary Presumptive intraembryonic endoderm, either isolated or together with adhering mesoderm, from 19-h chick embryos, was grafted to the coelom of 50-h host embryos. The viability of such grafts was low and endodermal differentiation was poor. In a second series the endoderm (with or without adhering mesoderm) was combined with a fragment of notochordal tissue from 48–60-h donor embryos. Then the recovery was much higher, notably after longer periods of in vivo culture. After 10 days of cultivation well-developed entero-endocrine (argyrophilic) cells were found among the regular enterocytes in both series.     

Mesoderm movement and fate during avian gastrulation and neurulation.

Quail/chick transplantation chimeras were constructed during stages of gastrulation and neurulation to follow the subsequent movement and fate of cells of the primitive streak. All grafts were placed solely within the confines of the primitive streak to prevent confusion between cells that had not yet ingressed and those that had already ingressed, and transplanted cells were distinguished from host cells on the basis of a naturally occurring cell marker. Pathways of movement of ingressing cells corresponded to their level of residence within the primitive streak. Cells residing within Hensen's node (the cranial end of the primitive streak) initially migrated mainly cranially, remaining on or near the midline, and then extended caudally along the midline as regression of Hensen's node occurred. Cells residing within the nodus posterior (the caudal end of the primitive streak) migrated caudally. Cells residing at levels of the primitive streak between Hensen's node and the nodus posterior typically migrated bilaterally, confirming that such cells had not already ingressed prior to their transplantation (in which case, they would have migrated unilaterally). Subsets of these cells residing at progressively more caudal levels of the primitive streak migrated incrementally more laterally. Hensen's node contributed cells to the gut endoderm, head mesenchyme, notochord, and median hinge-point (MHP) cells of the neural tube (future floor plate). At younger stages (i.e., stages 3a, 3b) Hensen's node contributed cells to principally the foregut endoderm and head mesenchyme, whereas at older stages (i.e., stages 3c, 3d, 4), it contributed cells to principally the notochord and MHP region. The remaining segments of the cranial half of the primitive streak contributed cells to the various mesodermal subdivisions of the embryo, and the lengths of the segments forming these subdivisions were estimated. The most cranial level of the streak (directly behind Hensen's node) contributed cells to the most medial mesodermal subdivisions (head mesenchyme, somites) and consecutively more caudal levels of the streak contributed cells to sequentially more lateral mesodermal subdivisions (intermediate mesoderm, lateral plate mesoderm). The caudal half of the primitive streak contributed cells to the extraembryonic mesoderm, with the nodus posterior contributing to the most caudal extraembryonic mesoderm, including the blood islands. Our results confirm and extend the previous avian prospective fate maps, increasing our understanding of the movement and fate of cells of the gastrula and neurula stages.     

The primitive streak

Summary The emphasis of this review is on the primitive streak of the chick embryo, collated with such information as is available on the mouse embryo. Little modern work has been published on any reptile primitive streak.The following topics are considered: — evolutionary significance; formation of the primitive streak; ingression and de-epithelialisation; the basal lamina; migration from the primitive streak of the endoderm and mesoderm; the role of the extracellular matrix; changes in cell adhesiveness; regression of the primitive streak and its role in body patterning; the primitive streak and induction.     

The formation of the embryonic mesoderm in the early post-implantation mouse embryo

Summary The formation of the mesoderm in early post-implantation mouse embryos is described and analysed. The outgrowth of the mesoderm was found to depend on the changes in the shape of the embryonic ectoderm, which lead to a relative displacement of the primitive streak in the caudal direction. The primitive streak deposits its cells laterally in the case of the lateral mesoderm, and medially in the case of the headprocess. In doing so, the primitive streak leaves a trail of mesoderm cells. This means that mesoderm cells do not migrate actively from the caudally located primitive streak towards more frontal positions in the embryo. This is confirmed by the results of scanning electron microscopy, which revealed that mesoderm cells show no polarity at all in the caudofrontal direction. In may therefore be concluded that these cells probably do not migrate.Studies on the cell-cycle parameters of the embryonic ectoderm, showed that mesoderm cells-to-be are probably recruited not only from the proliferation zone, but also from the lateral ectoderm. It is oostulated that the lateral ectoderm gives rise, via the largest part of the primitive streak, to most of the mesoderm cells, whereas the proliferation zone gives rise to the head-process mesoderm, via the anterior part of the primitive streak.     

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.