Development of cranial flexure and Rathke's pouch in the chick embryo

Experiments were done to investigate the cause of the cranial (mesencephalic) flexure of the chick brain during stages 10 to 14. Measurements of the length and thickness of the roof and floor of the mesencephalon gave values similar to the values obtained previously by others. The labeling index was determined in the roof and floor of the prosencephalon, mesencephalon, and rhombencephalon as a preliminary measure of cell division. The labeling index was about the same in all regions, and was high enough to suggest that most of the cells were dividing. The labeling indices did not suggest that differential growth was caused by differential rates of cell division in the roof and floor of the mesencephalon. It was found through time lapse photography that the foregut and heart remained stationary along the rostrocaudal axis, whereas the prosencephalon moved rostrally and the mesencephalon underwent flexure. Measurements suggested that the neural tube cranial to the otic primordium grew in volume exponentially at a rate consistent with the labeling index. The rostral tip of the neural tube was observed to be linked to the rostral tip of the foregut by the ectoderm that formed Rathke's pouch at the neural tube and the pharyngeal membrane (prospective stomodeum) at the foregut. As the neural tube grew in length, the link between the neural tube and the foregut did not. We suggest that because of this link, the growing neural tube had to bend around the foregut, forming the cranial flexure, and the ectoderm folded where it attached to the prosencephalon, forming Rathke's pouch. © 1994 Wiley-Liss, Inc.     

The role of extracardiac factors in normal and abnormal development of the chick embryo heart: cranial flexure and ventral thoracic wall

The present study was designed to investigate whether the formation of the cranial flexure is involved in the normal positional changes of the embryonic heart tube that occur during its transformation from the c- to s-shaped loop. For this purpose, the formation of the cranial flexure was locally suppressed in chick embryos by introducing a straight hair into the neural canal. In the experimental embryos, prevention of cranial flexure did not suppress the normal positional changes of the heart tube. However, other anomalies in the looping of the heart tube were frequently observed. These anomalies were caused by alterations in the formation of the ventral thoracic wall, which in turn seemed to be related not to the prevention of the cranial flexure but rather to accidental injuries during the implantation of the hair. In the embryos with abnormal looping of the heart tube, the incidence of delayed/defective septation of the heart was significantly higher than in embryos with normal looping. These results show that in the chick embryo: (1) cranial flexure is not involved in normal positional changes of the heart loop; (2) manipulations at the head region of the embryo can unintentionally result in developmental disorders of the ventral thoracic wall; (3) such disorders can result in congenital heart defects through mechanical interference with normal looping of the embryonic heart. The possible significance of these findings for the evaluation of experimental studies of chick embryos is discussed in the context of anomalies observed after surgical ablation of the premigratory cranial neural crest.     

The onset of myotome formation in the chick

Summary The onset of myotome formation in somites of chick embryos was studied by use of a polyclonal antidesmin antibody and by histochemical demonstration of acetylcholine esterase activity. The myotome cells originate from the dermatome only; sclerotome cells do not contribute to the myotome. The formation of the myotome starts in the craniomedial corner of the dermatome. From there the myotome formation continues simultaneously along the medial and the cranial edge of the dermatome. It was found that only the already longitudinally oriented cells of the cranial dermatome edge give rise to the myotome; the cells of the dorsomedial dermatome edge do not contribute to the myotome. Myotome cells do not originate directly from the surface of the overlying dermatome by delamination.Dedicated to Professor Dr. T.H. Schiebler, Würzburg, on the occasion of his 65th birthday     

Experimental study of the development of the truncus arteriosus of the chick embryo heart. I. Time of appearance

The time of appearance of the truncus arteriosus was studied in the chick embryo using an in ovo labeling technique. Three hundred embryos at stages 13–18 of Hamburger and Hamilton were selectively labeled at the distal end of the heart tube, using gelatine-india ink label; 122 of these embryos were reincubated and 111 of them reached stages 25–28. In these stages the final location of the label was determined. Only 95 of these embryos showed both a normal heart and a label located in it. The remaining embryos were discarded due to abnormal cardiac morphology or because the label was not found. Embryos labeled at stages 13–14 had label in the conus in 42.8% of the cases and in the boundary between the conus and the truncus arteriosus in 57.1% of the cases. Label placed at stages 15–16 was located in the conus in 6.1% of the cases, in the boundary between the conus and the truncus arteriosus in 44.8% of the cases, and in the truncus arteriosus in 48.9% of the cases. Finally, label placed at stages 17–18 was located in the boundary between the conus and the truncus arteriosus in 18.7% of the cases and in the truncus arteriosus in 81.2% of the cases. Our results permit us to conclude that the truncus arteriosus appears in the chick embryo as early as stages 15–16 of Hamburger and Hamilton (50–56 hours of incubation). © 1993 Wiley-Liss, Inc.     

Changes in the arrangement of actin bundles during heart looping in the chick embryo

Summary We assessed the arrangement of actin bundles in the looping chick heart. Actin filaments were stained with rhodamine-labeled phalloidin, and their total arrangement was observed in whole mount specimens. Before the straight heart tube was formed, actin bundles were in a net-like arrangement as if to indicate the cell borders. With progress of the heart tube formation, actin bundles were gradually arranged in a circumferential direction. In the looped heart, regional differences in actin arrangements were observed. In the truncus arteriosus, actin bundles ran in a net-like arrangement. In the bulbus cordis, actin bundles ran in random directions. In the ventricle, actin bundles were roughly arranged in a circumferential direction. Between these three regions, actin bundles ran in a circumferential direction especially on the concave side. Near the right contour on the ventral face, some actin bundles ran in a longitudinal direction along the axis of the tubular heart. In the bulbus cordis and the ventricle at the looped stage, there was another group of actin bundles in the inner layer of the myocardium which ran in a circumferential direction. We presume that the arrangement of actin bundles is related to heart looping.     

Analysis of duodenojejunal flexure formation in mice: implications for understanding the genetic basis for gastrointestinal morphology in mammals

The mammalian gut undergoes morphological changes during development. We studied the developing mouse duodenojejunal flexure (DJF) to elucidate the mechanism of formation. During embryonic days 10.75–13.75, DJF formation was morphologically classified into three stages: the expansion stage, flexure formation stage, and flexure elongation stage. From the expansion to the flexure formation stages, the DJF wall showed asymmetric morphology and proliferation along the left‐right intestinal axis. From the flexure formation to the flexure elongation stage, the DJF started to bend dorsally with counterclockwise rotation along the antero‐caudal intestinal axis, indicating that the original right side of the duodenum was rotated towards the dorsal body wall during development of the DJF. The direction of attachment of the dorsal mesentery to the DJF did not correspond to the bending direction of the DJF during flexure formation, and this finding indicated that the dorsal mesentery contributed very little to DJF formation. During DJF formation, Aldh1a2 and hedgehog mRNAs were detected at the DJF, and their expression levels differed along the bending axis. In conclusion, DJF formation might be triggered by asymmetric morphology and proliferation along the left‐right intestinal axis under the control of retinoic acid and hedgehog signaling.     

Fibronectin distribution in the chick embryo during formation of the blastula

Summary The distribution of the fibronectin-rich extracellular matrix (ECM) in the chick embryo during formation of the blástula has been evaluated semiquantitatively using an electron microscopical immunogold staining technique. During the first 10 h of postlaying development, fibronectin was found in both embryonic area pellucida and extra-embryonic area opaca of the blastoderm. In the area pellucida, the fibronectin was (1) associated with the basal lamina of the epiblast, (2) present between epiblastic and hypoblastic cells and (3) occasionally internalized in hypoblastic cells. Along the embryonic axis, a transient and high density of ECM was associated with the front of the anteriorly and rapidly expanding hypoblast. Very high density of fibronectin was observed in the marginal zone of the area pellucida, where the epiblastic and deeper cell layers show contacts and intense re-arrangements. In the area opaca, fibronectin was at first found only sporadically between contacting cells, but its density increased steadily and markedly during the first day of development. These rapid and significant changes in the regional distribution of fibronectin-rich ECM are discussed with respect to the early morphogenesis of the chick embryo.     

Experimental study on the formation of the epicardium in chick embryos

In chick embryos, the formation of the epicardium proceeds from the attachment of a secondary sinuventricular mesocardium. This mesocardium is formed by the adhesion of pericardial villi with the dorsal surface of the heart. It was the aim of this study to clarify the role of the pericardial villi in the formation of the epicardium. For this purpose, the contact between the pericardial villi and the heart was prevented by placement of a piece of the shell membrane between them. After re-incubation, the hearts of the experimental embryos could be assigned to one of two different groups: hearts completely lacking a secondary mesocardium (Group A), and hearts without the sinu-ventricular but with a dystopic secondary mesocardium (Group B). In Group A, the formation of the epicardium and subepicardial mesenchyme was found to be severely disturbed. In Group B, the formation of the epicardium proceeded from the point of attachment of a dystopic secondary mesocardium; defective development of the subepicardial mesenchyme was not encountered. These results support the view that the epicardium is derived from the pericardial epithelium.     

Actin bundles on the right side in the caudal part of the heart tube play a role in dextro-looping in the embryonic chick heart

Summary The role of actin bundles on the heart looping of chick embryos was examined by using cytochalasin B, which binds to the barbed end of actin filaments and inhibits association of the subunits. It was applied to embryos cultured according to New's method. Looping did not occur when cytochalasin B was applied diffusely in the medium. Further, we disorganized actin bundles in a limited part of the heart tube to examine the role of actin bundles in each part in asymmetry formation. A small crystal of cytochalasin B was applied to the caudal part of the heart tube on either the left or right side. The disorganization of actin bundles on the left side resulted in the right-bending of the heart, an initial sign of dextro-looping (normal pattern), and right side disorganization resulted in left-bending. We suggest that actin bundles on the right side of the caudal part of a heart tube generate tension and cause dextro-looping. Embryos whose hearts bent to the right rotated their heads to the right, and embryos with left-bent-hearts rotated their heads to the left. The rotation of the heart tube may therefore decide in which direction the body axis rotates.     

Scanning and light microscope studies of the development of the chick embryo semilunar heart valves

Summary The development of the semilunar valves of the great arteries was studied by light and scanning electron microscopy in the chick embryo. The results show that three distinct developmental periods can be distinguished. The formation of the anlage of the valves takes place in the first period (stages 26–29). These early anlage consist of three pyramidal shaped cusps formed by a core of loosely packed mesenchymal cells covered by a flattened endothelium. In the second period (stages 30–35) the cusps undergo excavation on their distal face. Morphological evidence is reported suggesting that this excavation process is produced by an initial solid ingrowth of the endothelium of the arterial face of the cusps which is immediately luminated by detachment of cells towards the bloodstream and by cell death. The histogenesis of the valves takes place in the third period (from stage 36 until hatching). It was observed that during this period some myocardial cells of the outflow tracts of the ventricles invade the valvular tissue and that in the upper part of the cusps a prominent fibrous layer is formed.     

Correlation between the embryonic head flexures and cardiac development

The aim of the present study is to examine whether the formation of the cranial and cervical flexures is involved in the process of cardiac looping, and whether looping anomalies are causally involved in the development of cardiac malformations. For this purpose, the formation of the cranial and cervical flexures was experimentally suppressed in chick embryos by introducing a straight human hair into the neural tube. In the experimental embryos, the absence of the cervical flexure, alone or in combination with a reduced cranial flexure, was always associated with anomalies in the looping of the tubular heart. The convergence of the primary distant venous and arterial ends of the heart, as well as the normal movement of the ventricular region from its original position, cranial and ventral from the cardiac inflow, to its final position caudal to the presumptive atria, was suppressed to an extent related to the degree to which the formation of the flexures was prevented. Positional immaturity of the heart loop (increased distance between its inflow and outflow, and cranio-ventral position of the ventricular region) was associated with incomplete deformations (reduced angulations) of the cardiac wall at the atrioventricular or conoventricular junctional areas. Reduced angulations were associated with the hypoplasia of the anlagen of the cardiac septa at the level of the angulation (av-cushions, conal ridges). Hypoplasia of these anlagen was followed by incomplete or absent fusion of their opposite free edges, which finally resulted in atrioventricular or ventricular septal defects. These results show that the convergence of the venous and arterial ends of the tubular heart and the caudo-dorsal movement of its ventricular region are related to the formation of the cervical flexure, and that the mesenchymal septa of the heart seem to develop in response to deformations of the embryonic heart, which are generated by the process of cardiac looping. Therefore, the positional and morphological changes of the looping heart are regarded as playing a key role in the process of normal and abnormal morphogenesis of the heart.     

The development of cholinergic ganglia in the chick embryo heart

Summary Cholinesterase activity was investigated in the heart of the developing chick from the 6th to 20th day of incubation. The earliest cholinesterase-positive nerve cells and fibers could be demonstrated between the 7th and 9th day. On the 13th day the nervous structure attained full development comparable with that seen in the hatched chicken.The number of ganglia increases up to the 15th day, and remains constant thereafter. The right ventricle is associated with the largest number of ganglia.     

Developmental expression of connexins in the chick embryo myocardium and other tissues

Background: Connexins are cell surface proteins that form specialized regions of cell-cell communication called gap junctions. These allow impulse conduction in involuntary muscle tissue such as the heart, but also allow the formation of communities of like cells during development of organs. Methods: We used an antipeptide antibody to connexin 43 in immunolocalization studies and an anti-peptide antibody to an external loop domain common to most connexins in Western blotting of total heart protein to measure the accumulation of connexins in the heart as it develops from 33 hours to 21 days (hatching), and in the adult. Results: Immunolocalization revealed that connexin 43 is widely distributed in the earliest organ rudiments. It is especially prominent in the neural tube and its derivatives, in the lens and nasal placodes, in the foregut and its derivatives, in the somites, in the mesonephric tubules, and in the heart and major arteries. Heart tissue staining grew more intense with development through day 8. However, at day 11 and day 15, and in the adult, heart staining diminished. Endocardium and valve tissue did not stain. Western blotting of heart homogenates with the antibody directed against the external loop domain peptide showed 26, 32, 43, 45, and 56 kilodalton connexins, which changed in relative abundance, displaying unique patterns during development. Conclusions: Our results show patterns of connexin immunolocalization in early germ layers and organ rudiments that are similar to those known in the mouse, but with certain differences. Our results show a distinctive pattern of multiple connexin gene expression in the developing heart from days 2–21. © 1995 Wiley-Liss, Inc.     

Formation of extra-digits induced by surgical removal of the apical ectodermal ridge of the chick embryo leg bud in the stages previous to the onset of interdigital cell death

Summary In the present work we have studied the mechanism of formation and the possible morphogenetic significance of the process of ectopic chondrogenesis induced by surgical removal of AER of the interdigital spaces of the chick leg bud at stage 28–30 (Hurle and Gañan 1986). Our results show that ridge removal causes condensation and rounding of the underlaying mesenchymal cells followed by chondrogensis. The long-term study of the fate of these ectopic cartilages shows that in a high percentage of the cases the cartilages undergo morphogenesis taking by day 10 of incubation the appearance of the two distal phalanges of an extra-digit. These extra-digits lack tendons and are joined by thin interdigital membranes to the neighboring digits.     

Natural wound formation: Endodermal responses in experimental primary neural induction in the chick embryo

Natural wound formation in experimental primary neural induction has been studied by SEM and in paraffin wax sections in embryos from 0 minutes to 10 hours of re-incubation. Stage 4 host and graft embryos were removed from hen's eggs and mounted as for New culture. Graft Hensen's nodes were transplanted into “pockets” created in the host area pellucida and re-incubated for up to 10 hours. Initially the cut edges of the graft establish contact with the host ectoderm layer. After 4 hours the cut edges of the graft move from the host ectoderm to the host endoderm layer. Several small openings form in the host endoderm over the graft tissue. By 6 hours, these openings join to form a single natural wound through which the underlying graft is exposed to the external environment. At 8 hours the graft forms a head-fold and neural folds are evident. During 8 to 10 hours of re-incubation the edges of the graft which attach to the edges of the host edoderm meet in the midline and close the opening in the host endoderm; simultaneously, the graft forms a neural tube. The endodermal wounds form by cell re-arrangement and by a minor contribution from cell loss. © 1993 Wiley-Liss, Inc.     

Formation of the chick mesonephros

Summary Using colour microinjections the course of mesonephric nephrons was visualized in embryos of White Leghorn chicks on days 5–18. From the stage of the S-shaped body until their full maturity, the nephrons generally retain the S-shaped form, with two main flexures. The first flexure is formed at the point where the tubule runs closest to the Wolffian duct, the second, more distal flexure, at the site of juxtaposition to Bowman's capsule.The cranial, narrow part of the mesonephros contains rudimentary nephrons with mostly obliterated tubules and short nephrons often exhibiting an irregular course. Nephrons of the caudal part of the mesonephros grow rapidly in length forming secondary and tertiary infoldings. The nephrons situated ventrally are segmentally arranged in planes perpendicular to the long axis of the organ, whereas the dorsal nephrons are situated in various planes and their tubules are folded within narrow spaces of approximately ovoid form.Among the rae anomalies two-headed nephrons and nephrons lacking the main flexures are described.     

Pathway formation and the terminal distribution pattern of the spinocerebellar projection in the chick embryo

Summary Pathway formation and the terminal distribution pattern of spinocerebellar fibers in the chick embryo were examined by means of an anterograde labelling technique with wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP).Spinocerebellar fibers, which originate in the lumbar spinal cord and are located in the marginal layer of the spinal cord, reach the corsal part of the cerebellar plate on embryonic day (E)8. On the way to the cerebellum the fibers form one distinct bundle, that suggests that gross projection errors probably do not occur during the formation of the spinocerebellar pathway.On E10, labelled fibers are located mostly in the medullary zone of the anterior lobe. By E12, the number of labelled fibers increases greatly in the inner granular and molecular layers. In transverse sections labelling was distributed throughout the mediolateral extent of the medullary zone. By E14, sagittal strips of labelling were clearly recognized in lobules II–IV; however, labelled terminals were present throughout lobule I. Although the adult pattern of terminal distribution is attained by E14, the mossy fiber terminals are still quite immature. The density of labelling decreased greatly by E16, and small terminal varicosities were first recognized. Structural differentiation of mossy fiber terminals continues to the end of the embryonic or the newly posthatched period.     

Chondroitin sulphate proteoglycan and embryonic brain enlargement in the chick

Previous studies of the early development of the neural tube have shown the existence of an intraneural fluid, which causes a positive pressure inside this primordium, and seems to play a key role in the early development of the central nervous system. In the present study we investigated the composition and synthesis of this intraneural fluid. By using a sequential method, which includes fixation with glutaraldehyde plus cetylpyridinium chloride, opening the neural cavity after critical point drying and scanning electron microscopy analysis, we found a water-soluble extracellular matrix that filled up the brain vesicles of chick embryos at the earliest stages of the neural tube. An ultrastructural study of the neural epithelium during these stages revealed the existence of a secretion process in the neural cells toward the apical side, the future neural cavity. An immunocytochemical study to asses the nature of the secreted material has shown that the intraneural matrix contains chondroitin sulphate proteoglycan, which appeared homogenously distributed throughout the neural cavity. Our findings demonstrate that the intraneural liquid is a fluid of complex composition and includes chondroitin sulphate proteoglycan as an osmotically active molecule. This suggests a morphogenetic role for the proteoglycan during early brain enlargement. The neural ectoderm is a polarized epithelium from early developmental stages and secretes the intraneural matrix.     

Formation of alternating tiers in the optic chiasm of the chick embryo

Background: When the fibers of the two optic nerves of the chick cross to the contralateral side at the prospective chiasmatic region, they segregate into clearly defined bundles. These bundles form horizontally oriented tiers which alternate between the right and the left optic nerve. Methods: We have analyzed the development of these tiers qualitatively and quantitatively using light and electron microscopy between embryonic days (E) 4 and E19. Results: The formation of the chiasm begins on E4. In the course of E4, tiers become visible for the first time. Their number increases rapidly until E7. Then the increase is slowed down and the final value (32 ± 1) is approximated by E18/19. Growing axons allow one to distinguish three different segments: the growth cone, the distal, and the proximal segment. The latter originates in the perikaryon. Growth cones and distal segments are found predominantly in the ventralmost tiers. Their frequency decreases from ventral to dorsal. Proximal segments which indicate the presence of older axons appear first in the dorsal tiers and later also in more ventrally located tiers. Conclusion: Based on these criteria it is concluded that newly formed axons contribute primarily but not exclusively to the ventral tiers. There is a gradient of maturity of axons from ventral to dorsal whose slope becomes steeper with age until the last growth cones have arrived by E18. Thus, the formation of the chiasm corresponds to the spatiotemporal pattern of ganglion cell formation in the retina. The process of cell death of retinal ganglion cells is also seen in the chiasm but probably does not lead to a transitory diminution in the number of tiers. © 1994 Wiley-Liss, Inc.     

The development of pericardial villi in the chick embryo

Summary The development of pericardial villi and their relation to the development of the cardiac surface was studied in chick embryos from the 3rd to 10th day of incubation by scanning electron microscopy. During the 3rd day of incubation (stage 14–17 HH) the coelomic epithelium covering the ventral wall of the sinus venosus forms villous protrusions. By the end of the 3rd day (stage 17 HH) these protrusions contact the dorsal wall of the heart, so that a secondary dorsal mesocardium is formed. This bridges the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles. This sinu-ventricular mesocardium exists only temporarily, as on the 8th day of incubation it becomes a part of the cardiac wall due to fusion with the epicardium of the coronary sulcus. During the 4th and 5th day of incubation (stage 17 – 25 HH), the formation of the epicardium proceeds from the point of attachment of the sinu-ventricular mesocardium. Although these findings suggest that the epithelium of the villous protrusions spreads over the surface of the embryonic heart, one cannot exclude other hypotheses on epicardial origin. The impression of a spreading epicardium could also be created if epicardial cells were to delaminate from a local epithelium in a temporally and spatially organized pattern.