Appendix

Here are a few helpful notes and figures for the novice New Culture operator to facilitate easy apprehension of the most commonly applied early stages in the development of the chick embryo for this method. This summary cannot justify the diversity of nuances for all the stages that the operator will recognize with increasing awareness and experience, but serve as a guide only. The stages described here span from an unincubated blastoderm to a range of stages found after 24-30 h in culture.

Fig. 4. (A) A disk with virtually no landmarks to indicate the future polarity of the embryo. (B) Stage 1 after Hamburger and Hamilton (HH), the prestreak, incubation for 4-8 h. After several hours of incubation, the formation of the hypoblast appears as an area of higher density roughly shaped as a triangle rising from the posterior marginal wall. This is called the embryonic shield or Koller's sickle. (C) Stage 2 (HH), the

Fig. 4. (continued) initial streak (6-10 h), a short, squat, thickening of the blastoderm at the posterior marginal zone. (D) Stage 3 (HH), the intermediate streak (12-16 h), the streak is now extending to one-third of the area pellucida. Invagination of prospective mesoderm proceeds at the posterior part of the primitive streak and ingression of endoderm at more anterior levels of the primitive streak. (E,F) Stage 4- and stage 4 (HH), respectively, the definitive streak (15-20 h). The elongation of the primitive streak proceeds over several hours until it has reached its full length extending to two-thirds of the area pellucida from the posterior marginal zone. The average length of the fully extended primitive streak is about 2 mm, but varies enormously from embryo to embryo. It consists of the primitive groove flanked on either side by the primitive folds. The most anterior part of the primitive streak is broadened into a bulb, and is known as Hensen's node, the organizer region of the avian embryo, the homolog to the dorsal lip of the blastopore in amphibian embryos. Somitic mesoderm invaginates through the posterior primitive streak and heart mesoderm through the midstreak regions. The area pellucida is pear-shaped.

Fig. 4. (continued) initial streak (6-10 h), a short, squat, thickening of the blastoderm at the posterior marginal zone. (D) Stage 3 (HH), the intermediate streak (12-16 h), the streak is now extending to one-third of the area pellucida. Invagination of prospective mesoderm proceeds at the posterior part of the primitive streak and ingression of endoderm at more anterior levels of the primitive streak. (E,F) Stage 4- and stage 4 (HH), respectively, the definitive streak (15-20 h). The elongation of the primitive streak proceeds over several hours until it has reached its full length extending to two-thirds of the area pellucida from the posterior marginal zone. The average length of the fully extended primitive streak is about 2 mm, but varies enormously from embryo to embryo. It consists of the primitive groove flanked on either side by the primitive folds. The most anterior part of the primitive streak is broadened into a bulb, and is known as Hensen's node, the organizer region of the avian embryo, the homolog to the dorsal lip of the blastopore in amphibian embryos. Somitic mesoderm invaginates through the posterior primitive streak and heart mesoderm through the midstreak regions. The area pellucida is pear-shaped.

Fig. 5. (A) Stage 4+ (HH) (18-22 h) shows the first sign of the head process extending cranially from Hensen's node to give rise to the head notochord. (B,C) Stage 5-and stage 5 (HH), respectively, the head process (20-24 h), shows the condensation of mesodermal cells of the head process more clearly. (D) Stage 6 (HH), the head fold (23-26 h). The anterior rim of the medullary plate is pushed forward by the extending head process until it folds over under the tension, forming the subcephalic pocket ventrally. The ensuing fold in the endoderm is to give rise to the foregut. The duration for this stage is very short. The head fold can flip over within 1 h. Hensen's node is now beginning to regress leaving in its wake the notochord. Mesenchyme cells form isolated blood islands in the extraembryonic mesoderm.

Fig. 5. (A) Stage 4+ (HH) (18-22 h) shows the first sign of the head process extending cranially from Hensen's node to give rise to the head notochord. (B,C) Stage 5-and stage 5 (HH), respectively, the head process (20-24 h), shows the condensation of mesodermal cells of the head process more clearly. (D) Stage 6 (HH), the head fold (23-26 h). The anterior rim of the medullary plate is pushed forward by the extending head process until it folds over under the tension, forming the subcephalic pocket ventrally. The ensuing fold in the endoderm is to give rise to the foregut. The duration for this stage is very short. The head fold can flip over within 1 h. Hensen's node is now beginning to regress leaving in its wake the notochord. Mesenchyme cells form isolated blood islands in the extraembryonic mesoderm.

All stated times of incubation are very tentative. They will depend on the time an egg took to pass down from the fimbriated edge of the oviduct, where it was fertilized, to the vagina, this can take up to 22 h, depending on how long the eggs were stored before incubation commenced and the temperature at which they were stored. A further variable is that most laboratory incubators are set to slightly different temperatures, which will be reflected in the stage of

Fig. 6. Stage 5 (HH), the head process has reached its most cranial position and laid down the prospective head territory. The cells of the epiblast overlying the head process form a pseudostratified epithelium, the medullary plate, and the presumptive neural plate. Hensen's node is clearly asymmetrical. The bilateral heart primodia lie lateral and anterior to the node.

Fig. 6. Stage 5 (HH), the head process has reached its most cranial position and laid down the prospective head territory. The cells of the epiblast overlying the head process form a pseudostratified epithelium, the medullary plate, and the presumptive neural plate. Hensen's node is clearly asymmetrical. The bilateral heart primodia lie lateral and anterior to the node.

development of the embryo. Many investigators make the additional distinction between winter and summer eggs.

All embryos were fixed in 3.5% paraformaldehyde in phosphate-buffered saline (PBS) for at least 4 h and stained in saturated carmine red in 4% Borax and diluted 50/50 in 70% alcohol until they reached the required intensity, differentiated in 70% acid alcohol, dehydrated, cleared and mounted in DPX, and photographed on a Zeiss Axiophot (mag. x40) or on a Zeiss Stemi SV6 (mag. X20-30) on Fujichrome 64 T slide film. All embryos were photographed from the dorsal aspect with their antero-posterior polarity running north to south.

When the egg is laid, it has little resemblance to an embryo. The unincubated blastoderm is a flat disk made up of two layers (see Subheading 1.1.).

Fig. 7. Stage 7 (HH) (26-30 h) shows the segmentation of the first pair of somites from the para-notochrodal mesoderm. This is in fact the second pair of somites. The first somite is a phantom somite. It never forms fully and will disperse again in the next few hours of development. It is a slight mesodermal condensation anterior to the cleavage for the first somite proper. Not all embryos have a visible first somite. Subsequent pairs of somites will segment in two hourly intervals from the para-axial mesoderm. Neural folds reach to the mesencephalon. The wall of the foregut emerges close to the lateral head fold.

Fig. 7. Stage 7 (HH) (26-30 h) shows the segmentation of the first pair of somites from the para-notochrodal mesoderm. This is in fact the second pair of somites. The first somite is a phantom somite. It never forms fully and will disperse again in the next few hours of development. It is a slight mesodermal condensation anterior to the cleavage for the first somite proper. Not all embryos have a visible first somite. Subsequent pairs of somites will segment in two hourly intervals from the para-axial mesoderm. Neural folds reach to the mesencephalon. The wall of the foregut emerges close to the lateral head fold.

1 pair of somites 2 pair of somites 3 pair of somites

1 pair of somites 2 pair of somites 3 pair of somites

D

SI

F

4 pair of somites 5 pair of somites 6 pair of somites

4 pair of somites 5 pair of somites 6 pair of somites

Fig. 8. Comprises stages 7 and 8. (A) Stage 7 (HH), 1 pair of somites. (B) Stage 7+ (HH), 2 pairs of somites. (C) Stage 8- (HH), 3 pairs of somites. (D) Stage 8 (HH) 4 pairs of somites. (E) Stage 8+ (HH), 5 pairs of somites. (F) Stage 9- (HH). 6 pairs of somites, dorsally the first cleft between rhomobomere 5 and 6 can be detected.

Fig. 9. Stage 8 (HH) (28-34 h), five pairs of somites. The head is raised above the proamnion. The proamnion is a region free of mesoderm between ectoderm and endo-derm at the anterior edge of the area pellucida flanked by the lateral horns meeting cranially. Open neuropore, neural folds are beginning to close in the region of the mesencephalon (failure to close will lead to spina bifida in the adult, as seen here rostral to the first somite). Ventrally, the notochord can be seen through the open folds of the neural tube as the node regresses along the primitive streak. Bilaterally heart primodia are developing from the amnio-cardiac vesicle level with the midbrain. More blood islands are developing in the area opaca. (See color plate 10 appearing after p. 368.)

Fig. 9. Stage 8 (HH) (28-34 h), five pairs of somites. The head is raised above the proamnion. The proamnion is a region free of mesoderm between ectoderm and endo-derm at the anterior edge of the area pellucida flanked by the lateral horns meeting cranially. Open neuropore, neural folds are beginning to close in the region of the mesencephalon (failure to close will lead to spina bifida in the adult, as seen here rostral to the first somite). Ventrally, the notochord can be seen through the open folds of the neural tube as the node regresses along the primitive streak. Bilaterally heart primodia are developing from the amnio-cardiac vesicle level with the midbrain. More blood islands are developing in the area opaca. (See color plate 10 appearing after p. 368.)

Fig. 10. Comprises stages 9 and 10. (A) Stage 9 (HH), (32-38 h), seven pairs of somites, dorsally bilateral primodia of the optic vesicle appearing. Mesencephalon is clearly demarcated from rhombencephalon at the isthmus. Segmentation of rhombomeres continues with boundaries 2/3 and 3/4 appearing. Ventrally the heart primodia fusing medially into a tube. Margin of anterior intestinal portal is level with vitelline vein which is connecting with heart, and splaying out laterally to area opaca. (B) Stage 9+ (HH), 8 pairs of somites. (C) Stage10- (HH), 9 pairs of somites. (D) Stage 10 (HH) 10 pairs of somites. (E) Stage 10+ (HH) 11 pairs of somites. (F) Stage 11- (HH), 12 pairs of somites.

Fig. 10. Comprises stages 9 and 10. (A) Stage 9 (HH), (32-38 h), seven pairs of somites, dorsally bilateral primodia of the optic vesicle appearing. Mesencephalon is clearly demarcated from rhombencephalon at the isthmus. Segmentation of rhombomeres continues with boundaries 2/3 and 3/4 appearing. Ventrally the heart primodia fusing medially into a tube. Margin of anterior intestinal portal is level with vitelline vein which is connecting with heart, and splaying out laterally to area opaca. (B) Stage 9+ (HH), 8 pairs of somites. (C) Stage10- (HH), 9 pairs of somites. (D) Stage 10 (HH) 10 pairs of somites. (E) Stage 10+ (HH) 11 pairs of somites. (F) Stage 11- (HH), 12 pairs of somites.

Fig. 11. Stage 10 (HH), 10 pairs of somites (36-42 h) dorsally anterior neuropore closed, prominent optic vesicles, rhombomere boundary 4/5 formed, and neural folds are closed to almost the level of the node. Ventrally, Hensen's node has regressed almost to the end of the primitive streak (the 10th pair of somites has not fully segmented caudally in this illustration), pronephric tubules develop between somites 6 and 10, heart tube turns asymmetrical bulging out to the right and contractions can be seen, and bilateral vitelline veins fan out toward the area opaca, which shows large blood islands to establish circulation.

Fig. 11. Stage 10 (HH), 10 pairs of somites (36-42 h) dorsally anterior neuropore closed, prominent optic vesicles, rhombomere boundary 4/5 formed, and neural folds are closed to almost the level of the node. Ventrally, Hensen's node has regressed almost to the end of the primitive streak (the 10th pair of somites has not fully segmented caudally in this illustration), pronephric tubules develop between somites 6 and 10, heart tube turns asymmetrical bulging out to the right and contractions can be seen, and bilateral vitelline veins fan out toward the area opaca, which shows large blood islands to establish circulation.

Fig. 12. Stage 11 (HH), 13 pairs of somites (40-46 h), slight flexure of the head, prominent optic vesicles with slight constriction at their base form the lateral parts of the prosencephalon, mesencephalon has clear boundaries, and all rhombomeres can be distinguished. The neural tube is virtually closed along its entire length. On the ventral side, the rostral part of the heart forms the ventricle, whereas the caudal part gives rise to the atrium and the vitelline vein leading from it. The heart beat is rhythmical, but the circulation of blood is not yet fully connected up to all peripheral blood islands. Anterior somites are beginning to differentiate.

Fig. 12. Stage 11 (HH), 13 pairs of somites (40-46 h), slight flexure of the head, prominent optic vesicles with slight constriction at their base form the lateral parts of the prosencephalon, mesencephalon has clear boundaries, and all rhombomeres can be distinguished. The neural tube is virtually closed along its entire length. On the ventral side, the rostral part of the heart forms the ventricle, whereas the caudal part gives rise to the atrium and the vitelline vein leading from it. The heart beat is rhythmical, but the circulation of blood is not yet fully connected up to all peripheral blood islands. Anterior somites are beginning to differentiate.

Fig. 13. (A) Stage 11 (HH), (40-46 h), 13 pairs of somites. (B) Stage 11+ (HH), 14 pairs of somites. (C) Stage 12- (HH), 15 pairs of somites. (D) Stage 12 (HH) (48-54 h), 17 pairs of somites. Embryos in New Culture will rarely develop well beyond this stage. It is possible in exceptional circumstances to maintain embryos for up to 48 h in culture. For example, embryos on rings can be left on semipermeable membranes instead of watch glasses and given culture medium containing serum instead of albumin, but development is slowed down and abnormalities increase. The eyes are budding off from the prosencephalon, the bilateral otic vesicles are clearly visible as round indentations adjacent to rhombomere 5. Ventrally the circulation of blood from the heart to the periphery is nearly complete. Somites are segmenting off at about one every 2-3 h. The notochord has regressed to the end of the primitive streak. (See color plate 11 appearing after p. 368.)

Fig. 13. (A) Stage 11 (HH), (40-46 h), 13 pairs of somites. (B) Stage 11+ (HH), 14 pairs of somites. (C) Stage 12- (HH), 15 pairs of somites. (D) Stage 12 (HH) (48-54 h), 17 pairs of somites. Embryos in New Culture will rarely develop well beyond this stage. It is possible in exceptional circumstances to maintain embryos for up to 48 h in culture. For example, embryos on rings can be left on semipermeable membranes instead of watch glasses and given culture medium containing serum instead of albumin, but development is slowed down and abnormalities increase. The eyes are budding off from the prosencephalon, the bilateral otic vesicles are clearly visible as round indentations adjacent to rhombomere 5. Ventrally the circulation of blood from the heart to the periphery is nearly complete. Somites are segmenting off at about one every 2-3 h. The notochord has regressed to the end of the primitive streak. (See color plate 11 appearing after p. 368.)

Fig. 14. Stage 16 (HH) (about 72 h), 28 pairs of somites. Very few embryos may get to this stage in development. The vascular system is ill-equipped to function in a two-dimensional space. When the cranial flexure of the embryo reaches 90° or more the main blood vessels fold under the weight of the trunk and can no longer work efficiently. The heartbeat will increase without fullfilling a better function. The embryo fills up with fluid and development slows right down. The lens of the eye has separated from the eye cup, there is a clear demarcation between the telen- and the dien-cephalon, the isthmus divides the mesencephalon from the metencephalon, and the otic vesicle is a shallow, almost round indentation with a raised epithelial rim. All four branchial arches are clearly structured. The flexure of the body extents to the anterior intestinal portal and the body folds curve ventrally to the tail bud. The wing bud primodia can clearly be seen in a slight thickening of the flank, but there is no such indication for the leg bud yet. The posterior margin of the amnion extends to about the 20 pair of somite. Somite numbers are no longer a reliable aid to stage embryos of this stage or older. The most cranial somites are starting to be incorporated into the base of the scull, and the increase of the flexure of the body right down to the tail bud makes it difficult to give an accurate account. There are many other landmarks for accurate staging. The shape and structure of the branchial arches, the color of the eye, the angle of the head, and the shape and size of the limb buds are just some.

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