THE ANATOMICAL RECORD PART A 288A:909 –920 (2006) Staging of Intestinal Development in the Chick Embryo BRIDGET R. SOUTHWELL1–3* Gut Motility Laboratory, Murdoch Children’s Research Institute, Victoria, Australia 2 Department of Surgical Research, Royal Children’s Hospital, Melbourne, Australia 3 Department of Paediatrics, University of Melbourne, Melbourne, Australia 1 ABSTRACT Comparisons between developmental studies rely on embryonic staging systems. It is important for comparison of molecular, immunohistochemical, and physiological studies of the developing chick intestine that the developmental stage of embryos is reliably determined. Good staging systems exist for the external features of the chick embryo but not for development of internal organs. To facilitate precise comparisons of chick embryo intestine development, we prepared an intestinal staging system. Embryos were ﬁxed, other tissues dissected away, and the intestine and associated organs were then drawn to scale using a camera lucida. This produced black-and-white drawings with features of the gut clearly visible. The detailed drawings of intestine from chick embryos aged 2.5 to 10 days were correlated with age of embryos and developmental stages described in three common staging systems, Hamilton and Hamburger, Thompson and Fitzharris, and Allan and Newgreen. Descriptions of key changes in gut morphology and position are given for each stage. This staging of chick gut development will allow future studies to quote and compare development of the gut rather than external features or incubation time. This will allow much more precise reporting and comparisons in developmental studies of cell migration and gene expression. Anat Rec Part A, 288A:909 –920, 2006. © 2006 Wiley-Liss, Inc. Key words: intestinal development; fowl; growth There is considerable individual morphological variation when a daily chronology is used to stage embryonic development. Hamburger and Hamilton’s almost universally used staging system for the fowl embryo (Hamburger and Hamilton, 1951; Hamilton, 1952) describes external features characteristic at periods of embryonic development. The development of internal organs at particular stages of development has not been so well described. Detailed descriptive accounts covering the embryonic period have been reported for the changing morphology of each region of the alimentary canal (Romanoff, 1960). The primary concern of these accounts was identiﬁcation of the day when characteristic morphological features appear or change. These accounts do not allow easy consideration of morphological events occurring in a number of regions at the same time and do not allow for individual variations in time of development. Romanoff (1960) provided a summary table of previously reported data on development and length of regions of the intestine. During studies on the development of the enteric nervous system, we needed a series providing sim© 2006 WILEY-LISS, INC. ple descriptions of the key features of morphological development of the intestine to provide a basis for comparison of studies and allow the timing of cellular events to be related to morphological development (Newgreen et al., 1996). Having created this description, others have found it useful and requested we publish (McBride et al., 2003). This article provides descriptions and outline drawings of the intestine and cloaca for each Hamburger and Hamilton (1951) stage from 16 to 36 (fowl embryos 2–10 days). Also included is the morphological development of Remak’s nerve. Development of embryos is related to the two *Correspondence to: Bridget R. Southwell, Gut motility Laboratory, Murdoch Children’s Research Institute, Parkville, Victoria, 3052, Australia. Fax: 61-3-9345-6240. E-mail: firstname.lastname@example.org Received 16 December 2005; Accepted 3 February 2006 DOI 10.1002/ar.a.20349 Published online 11 July 2006 in Wiley InterScience (www.interscience.wiley.com). 910 SOUTHWELL commonly used descriptions of external features (Hamburger and Hamilton, 1951; Hamilton, 1952) and two studies on development that quote developmental age and temperatures of incubation (Thompson and Fitzharris, 1979; Allan and Newgreen, 1980). MATERIALS AND METHODS Black Australorp and White Leghorn/Black Australorp cross-breed of domestic fowl embryo were used. Both fertile and partially incubated eggs were obtained from a local research hatchery and maintained in a forced draught incubator (Multiplo, Sydney, Australia) at 38 ⫾ 1°C. Differences were not observed in rates of development between the two breeds, or for eggs received in a fertile or partially incubated state. Prior to incubation, unincubated eggs were stored at 10 –15°C. To compare to Hamburger and Hamilton (1951) staging, some eggs were incubated at 39.4°C. Embryos were staged by external features according to Hamburger and Hamilton (1951) and the precise duration of incubation was recorded. At least two embryos at each stage were examined using an Olympus dissecting microscope with continuous zoom magniﬁcation. Morphological features of the gut and closely associated tissues were traced using a camera lucida drawing tube. To limit shape changes during dissection and orientation for illustration, following careful removal from the egg, embryos were placed in 10% formalin/PBS at room temperature. The gut was drawn from both left and right aspects to illustrate features of development at each stage. Where lack of bilateral symmetry was signiﬁcant, a ventral aspect was also drawn. For stages 16 –24, the ventral and lateral body walls were removed and the gut is shown in position relative to other viscera and the dorsal body. In subsequent stages, the body dorsal to the viscera was also removed. To reduce lettering on other diagrams, features are labeled in Figure 1 only. The most common incubation times at which the identiﬁed stages were found are included with the descriptions of intestinal development at each stage and compared with the incubation times reported by Hamburger and Hamilton (1951), Hamilton (1952), Thompson and Fitzharris (1979), and Allan and Newgreen (1980). RESULTS Diagnostic Features Used for Staging The widely used Hamburger and Hamilton (1951) staging for the fowl embryo identiﬁes changes in external structural features (e.g., somites, limbs, visceral arches) or their positions (e.g., ﬂexures and rotation). A similar initial diagnostic categorization is employed here, with the displacement of the gut with respect to the embryonic axis being diagnostically useful at all stages. The structural features, which include intestinal regions and other closely associated structures, are of diagnostic signiﬁcance for shorter times. The dorsal mesentery is diagnostic early (stages 17–25) and replaced later by Remak’s nerve (stages 25–36). Three structural features, the umbilicus (stages 16 –25), the cloacal area (stages 26 –35), and bursa of Fabricius (stages 33–36), are diagnostic for periods. Other structures appear over one or more stages and are used as descriptive characteristics (e.g., the angle of the omphalomesenteric artery passing toward the umbilicus or the phallus extending as a cap to the vent in the cloaca area). Fig. 1. Key to structures in all diagrams. A, allantois; Ca, caecum; CR, colorectum; D, duodenum; G, gizzard; H, heart; K, kidney; L, liver; Lu, lung bud; M, dorsal mesentery; OA, omphalomesenteric artery; Oe, esophagus; OV, omphalomesenteric vein; P, pancreatic bud; Ph, phallus; Po, postumbilical small intestine; PoI, postumbilical intestine; Pr, preumbilical small intestine; PrI, preumbilical intestine; RN, Remak’s nerve; S, somite; St, stomach. Scale bar ⫽ 1 mm (each division). Stage aspects ⫽ L, left; R, right; V, ventral. Chronologies included in stage descriptions: H&H, Hamburger and Hamilton (1951); H, Hamilton (1952); T&F, Thompson and Fitzharris (1979); A&N, Allan and Newgreen (1980). The gut is staged differently with respect to the pre- and postumbilical regions. This arose from the clear separation by the open umbilical region of the caudal and cranial tubular primordia. At stage 16, the postumbilical gut gives rise to all regions of the cloaca, the large intestine, and the caudal part of the small intestine. As each constituent regional segment becomes an independently identiﬁable diagnostic feature, it is described under the appropriate broad gut region. In the preumbilical intestine, most of its early development was staged by ﬂexing and displacement of the gut tube, rather than according to distinct regional features. From stage 25, the whole intestine can be treated under its deﬁnitive regional segments (duodenum, preumbilical and postumbilical small intestine, caeca, colorectum). Then displacement of the gut as a INTESTINE DEVELOPMENT IN CHICK EMBRYO 911 whole is described, with ﬂexures and displacements of a particular deﬁnitive region used to characterize that region. Terminology As the primary concern of this report is a stage-by-stage characterization of the external morphology of the intestine, simple descriptive terms were used. “Preumbilical gut” refers to all tubular regions (both ectoderm-associated and endoderm-associated) lying cranial to the umbilical area, and “postumbilical gut” refers to all tubular regions caudal to this. When deﬁnitive regional primordia became clearly identiﬁable, terminology appropriate for the mid- to late-term embryo is used. There is disagreement on the appropriate regional terminology for the intestine of the bird (Hodges, 1974; McLelland, 1975; Allan and Newgreen, 1980). The mammalian distinction between jejunum and ileum is difﬁcult to identify in the avian adult and indistinguishable in the avian embryo, thus the clearly identiﬁable regions of the small intestine were referred to as duodenum, preumbilical small intestine, and postumbilical small intestine. The large intestine has two identiﬁable regions, the caeca and a combined colorectum (McLeod, 1939). The colorectum passes directly into the coprodeal compartment of the cloaca (King, 1975), the boundary being marked by an abrupt increase in the diameter of the most caudal gut. In the embryo, this boundary only becomes clearly identiﬁable at the cranial extent of the bursa of Fabricius, which is not observable until stages 33–36. The three compartments of the cloaca (coprodeum, urodeum, proctodeum) were identiﬁed according to conventional practice (Romanoff, 1960; King, 1975). The external opening from the cloaca is often identiﬁed in the avian literature as the anus (Hamilton, 1952; Romanoff, 1960). As the term “anus” has a well-established but different meaning in the mammalian literature (Romer and Parsons, 1977), the external opening from the cloaca is referred to as the “vent” (King, 1975). The term “cloaca” was applied to structures in the immediate vicinity of the vent or its primordia (e.g., cloacal area, cloacal papillae). Description of Gut at Different Stages 1 d; 2 Stage 16 [2 Hamburger and Hamilton (H&H), 51–56 hr; Hamilton (H), 51–56 hr; Thomp3 son and Fitzharris (T&F), 2 4 d; Fig. 2]. Displacement: the gut lies in the midline. Umbilicus: unformed. The gut is open from the anterior intestinal portal, immediately caudal to the sinus venosus, to the tail bud. Postumbilical gut: a wide open pocket in the tail bud. Stage 17 (2 43 d; H&H, 52– 64 hr; H, 52– 64 hr; T&F, 3 d; Fig. 2). Displacement: the preumbilical gut has moved very slightly to the left of the midline, while the postumbilical gut remains in the midline. Umbilicus: closure beginning. The gut is open from just behind the emerging liver buds to the tail bud. The anterior intestinal portal and posterior intestinal portal are present at each extremity of the open gut. Over subsequent stages, they maintain this location drawing together in the mid-umbilical intestine. Fig. 2. Intestine and embryo outline from stage 16 –18. Postumbilical gut: a short tube extending caudally into the now ventrally bending tail bud. A swelling on the ventral side of this short tube in older embryos from this stage demarcates the ﬁrst appearance of the allantoic bud. Dorsal mesentery: it is observable only cranially, extended to just caudal from the liver bud. Stage 18 (3 d; H&H, 65– 69 hr; H, 3 d; T&F, 3 41 d; Fig. 2). Displacement: little change. Umbilicus: further closure. Extends from two somites’ length caudal to the pancreatic bud to the short postumbilical gut. Postumbilical gut: a funnel-shaped tube up to four somites’ length cranial to the prominent thick walled sac of the allantois. As in the previous and subsequent stages, this tube extends into the ventral curvature of the tail. Dorsal mesentery: observable more caudally, approaching the level of the omphalomesenteric artery, which passes at right angles from the dorsal aorta to the yolk sac at the midpoint of the open gut. 912 SOUTHWELL Fig. 3. Intestine and embryo outline from stage 19 –20. Stage 19 (3 3 d; Fig. 3). 1 2 1 4 d; H&H, 68 –72 hr; H, 3–3 1 2 d; T&F, Displacement: the swollen stomach is now lying left of the midline. It constricts caudally and bends back to the midline where the intestine becomes a narrow tube passing slightly ventral, arriving at the open gut after two or three somite lengths. Umbilicus: the closed preumbilical intestine and postumbilical gut together are about equal in length to the open intestine. A ratio of open:closed intestine of about 1:1. Postumbilical gut: continued closure cranially while retaining funnel shape. Extends up to six somites’ width from the allantois, which is now a small vesicular sac. Dorsal mesentery: observable caudal to the omphalomesenteric artery but is not apparent yet at the caudal extent of the open gut. Stage 20 (3 3 d; Fig. 3). 3 4 1 3 d; H&H, 70 –72 hr; H, 3–3 1 2 d; T&F, Displacement: the intestine curves dorsally immediately after leaving the constricted caudal stomach. It then curves ventrally past the hepaticopancreatic ducts and Fig. 4. Intestine and embryo outline from stage 21–22. curves right returning to the midline. Caudal to this, the gut remains in the midline. The narrow tube for the cranial intestine extends up to four somites’ length beyond the hepaticopancreatic ducts. Umbilicus: the closed preumbilical intestine and postumbilical gut are each about equal in length to the open gut. A ratio of open:closed intestine approaching 1:2. Postumbilical gut: continues to close as a funnel-shaped tube. Now extends up to eight somites’ width from the allantois. Dorsal mesentery: now observable into the region of the closed postumbilical gut. Stage 21 (3 Fig. 4). 1 2 d; H&H, 3 1 2 d; H, 3 1 2 d; T&F, 4 d; Displacement: further development of the ﬂexures begun at stage 20. The closed tube of the intestine now INTESTINE DEVELOPMENT IN CHICK EMBRYO 913 extends up to ﬁve somites’ length from the beginning of the hepaticopancreatic ducts. Umbilicus: little change. Postumbilical gut: the allantois is attached to the cloaca by a stalk somewhat thicker than the gut. Immediately cranial to the allantoic stalk, the gut narrows for one to two somites’ length, indicating the primordia of the colorectum. Cranial to this, the postumbilical gut widens for a short distance, demarcating the point from which the caecal buds will originate. The caudal postumbilical small intestine is apparent as a result of a slight narrowing of the postumbilical gut cranial to the caecal primordia. Dorsal mesentery: observable to the level of the caecal primordia. 3 1 1 1 Stage 22 (3 4 d; H&H, 3 2 d; H, 3 2– 4 d; T&F, 4 2 d; Fig. 4). Displacement: caudal to the stomach, the intestine passes from the left to just right of the midline before returning to the midline. Caudal to this, the gut remains in the midline. The ﬁrst indication is present of the ventral curvature in the umbilical intestine, where the omphalomesenteric artery passes to the open umbilicus. Umbilicus: closed to within one or two somites’ length on either side of the omphalomesenteric artery, which passes cranial to the midpoint of the open umbilicus after leaving the aorta at an obtuse angle. Postumbilical gut: further development of stage 21, particularly an increased lengthening of the postumbilical small intestine. Dorsal mesentery: observable beyond the caecal swelling as a narrow strip attached to the dorsal colorectum. Stage 23 (4 d; H&H, 3 21– 4 d; H, 4 d; T&F, 4 Fig. 5). 1 2 d; Displacement: further development of stage 22 with the preumbilical small intestine returning to the midline at the cranial edge of the open gut. The postumbilical gut remains in the midline. A ventral U-shaped curve in the small intestine opposite the omphalomesenteric artery is now clearly apparent. Viewed from the left side, the umbilical vein passes along the cranial boarders of this ventral curvature. The region of contact between the two identiﬁes the primordia of the preumbilical small intestine from the duodenum. Umbilicus: the gut remains open in the ventral part of the U-shaped curve in the intestine. Large intestine: a short and narrow colorectal region is now clearly apparent cranial to the allantois. Viewed ventrally, the bulges of the caecal buds can be seen. While thinner than the region of the caecal pouches, the postumbilical small intestine remains thicker than the colorectum. Dorsal mesentery: observable to the caudal end of the colorectum, where it is a narrow sheet attached to the dorsal wall. Stage 24 [4 21 d; H&H, 4 d; H, 4 21 d; T&F, 4 5/8 d; Allan and Newgreen (A&N), 4 1/8 d; Fig. 5]. Displacement: caudal to the stomach, the intestine turns slightly ventrally and then dorsal, maintaining a straight line along the left side to just beyond the pancreas, where it makes a sharp bend to the right side while continuing dorsally. It then gently bends to the left and passes ventral, crossing the midline at the umbilicus. This Fig. 5. Intestine and embryo outline from stage 23–24. results in the postumbilical small intestine shifting from the midline at the caecal buds to the left side at the umbilicus. The preumbilical intestine is now clearly identiﬁable as duodenum (cranial to the completion of the sharp bend from left to right) and preumbilical small intestine (caudal to this). Umbilicus: the gut is still open at the extremity of the umbilical loop in the small intestine. This extends a little further into the postumbilical small intestine, which is a shorter, thicker tube than that of the preumbilical small intestine. The omphalomesenteric artery passes through the mesentery of the pre- and postumbilical small intestine almost parallel to the former, having arisen form the dorsal aorta at a large caudally directed obtuse angle. Large intestine: the caecal bulges have extended laterally, making a sharp demarcation from the lengthened colorectum, which is now up to three somites in length. Dorsal mesentery: widened throughout the intestine other than just cranial to the allantois, where the gut is closely attached to the dorsal body wall. Stage 25 (4 43 d; H&H, 4 21 d; H, 4 21–5 d; T&F, 4 43 d; 5 A&N, 4 8 d; Fig. 6). Displacement: the duodenal and umbilical ﬂexures of the intestine are more developed. The ventral loop of the 914 SOUTHWELL Fig. 7. Fig. 6. Outline of intestine from stage 27–28. Outline of intestine from stage 25–26. umbilical small intestine has now clearly twisted right to the left, resulting in the preumbilical region lying on the right side and the postumbilical on the left side. This shift has brought the omphalomesenteric artery into close association with the preumbilical small intestine. Umbilicus: entirely closed except for a small opening to the stalk of the yolk sac. There is little change in the subsequent stages. Small intestine: the postumbilical region is shorter and thicker than the preumbilical region. Large intestine: from the ventral aspect the caecal buds make a sharp lateral angle caudally but narrow gently cranially. The colorectum narrows in the caudal to middle half, but gently widens toward the caecal buds. Dorsal mesentery: widened slightly. Remak’s nerve present in the mesentery at its attachment to the dorsal wall of the colorectum. Stage 26 (5 d; H&H, 4 A&N, 5 d; Fig. 6). 1 2 –5 d; H, 5 d; T&F, 5 d; Displacement: the thickened loop of the duodenum crosses transversely from the left side of the body to the right, where the emerging ascending limb is directed dorsally. The preumbilical small intestine passes caudally on the right side and the postumbilical small intestine passes cranially on the left side, resulting in a sharp S-bend in the small intestine. The umbilicus arises in the midline from the apex of the ventrally looped small intestine. Small intestine: the umbilical loop is elongated ventrally with the pre- and postumbilical segments of similar length and thickness. Large intestine: the caecal buds extend laterally and slightly ventrally and are approximately symmetrical at cranial and caudal borders when viewed from the ventral aspect. The narrow segment in the middle of the colorectum is lengthened. A gradual expansion both cranially to the caecum and caudally to the allantoic stalk can be seen. Remak’s nerve: extends to the caecum. Cloacal area: on the ventral side of the tail curvature, immediately caudal to the allantois, a cranially directed, crescent-shaped depression lays transverse to the longitudinal axis of the body. Stage 27 (5 21 d; H&H, 5 d; H, 5–5 1 A&N, 5 2 d; Fig. 7). 1 2 d; T&F, 5 1 2 d; Displacement: the umbilical loop has twisted almost at right angles to the midline with the pre- and postumbilical segment of the small intestine almost parallel. The apex of the umbilical loop is displaced cranially. The omphalomesenteric artery has shifted toward the middle of the mesentery between the pre- and postumbilical segments. INTESTINE DEVELOPMENT IN CHICK EMBRYO 915 Small intestine: the duodenum retains its position well ventral to the stomach. The ascending limb is parallel with the preumbilical small intestine, both of which lie in a dorsoventral plane at right angles to the body wall. Both regions of the small intestine have increased in length. Large intestine: the cranial and caudal boarders of the caeca are not symmetrical, as the caeca extends cranially on either side of the caudal 1/3 of the postumbilical gut. The narrow segment of the colorectum has lengthened, occupying more than half the length between the caecal junction and the allantoic entry. Remak’s nerve: extends beyond the caeca to the postumbilical small intestine. Cloacal area: the crescent-shaped depression is now ﬂanked cranially and laterally by small ﬂattened papillae. Stage 28 (6 d; H&H, 5 21 d; H, 5 21 – 6 d; T&F, 5 43 d; A&N, 6 d; Fig. 7). Displacement: little change other than increased length, resulting in a larger angle between the small and large intestine. The caeca and caudal small intestine make an angle of up to 45° with the dorsal body wall and curve into the colorectum that lies parallel to the dorsal body wall. Small intestine: duodenum is similar to the previous stage, although the gizzard had moved slightly caudally and a little ventral. The descending limb is still much shorter than the ascending limb. Both pre- and postumbilical limbs have lengthened. Large intestine: due to an overall increase in length, the caeca continue to occupy the caudal 1/3 of the postumbilical small intestine. While the caeca are still connected to the postumbilical small intestine, a mesentery appears, connecting the outer thickened tubes with the inner postumbilical intestine. Further growth of the colorectum maintains the previous size relations. The enlarged cranial and caudal colorectum angle ventrally from the narrower mid-colorectum, which is against the dorsal body wall. Remak’s nerve: difﬁcult to see in the umbilical loop due to a narrow mesentery occupied almost entirely by the omphalomesenteric artery. Cloacal area: the depression evident in previous stages has deepened and the papillae are closer together, forming a cranially directed crescent shaped slit that lays transverse to the longitudinal axis of the body. Fig. 8. Outline of intestine at stage 29. the colorectum. Half or more of their ventral extent occupies the umbilicus. Large intestine: the caeca are still shorter than the colorectum and are now clearly tubular connected to the postumbilical small intestine by a thin mesentery. Together, the caeca and caudal small intestine make a sharp angle (approaching 90°) at the caecal junction with the colorectum, the mid-region of which had lengthened and descends at a 45° angle to the dorsal body wall, where the caudal enlarged region follows the ventral curve of the body. Remak’s nerve: little change. Cloacal area: the lateral papillae are raised ventrally, forming a lateral ridge. The caudal edges of the lateral papillae remain separate in the midline. The cranial papilla (the phallus) arises in the midline and is directed caudally. d; T&F, 6 d; Stage 30 (7 d; H&H, 6 21 d; H, 6 21–7 d; T&F, 6 21–7 d; A&N, 7 d; Fig. 9). Displacement: the gizzard has enlarged and extends causally to the umbilical loop. Ventrally, it extends to or beyond the duodenum. The pre- and postumbilical small intestine occupy the same level on the right and left side of the midline, respectively. Both approach a 90° angle to the dorsal body wall for two-thirds of their ventral extend before bending slightly cranial. In the apex of the umbilical loop, the postumbilical small intestine is a little caudal to the preumbilical small intestine. Small intestine: the duodenum continues to lie on the right side. The descending limb has increased in length, curving sharply caudally, and passes from left to right while the ascending limb runs ventrodorsal and slightly cranial, forming the beginning of the duodenal loop. The pancreas is present in the apex of the loop. Each limb of the pre- and postumbilical small intestine is larger than Displacement: the gizzard is enlarged and moved both caudally and ventrally. It extends to or beyond the umbilical intestine and when viewed from the left side totally obscures the duodenum and the ﬂexure between its ascending limb and the preumbilical small intestine. The caudal half of the postumbilical small intestine including the caeca begins to bend a little cranial to the preumbilical small intestine. Small intestine: the duodenal loop has formed, with the descending and ascending limbs being of equal length. The descending limb continues to pass caudally and from left to right. A midline through the apex of the loop and passing parallel to the two limbs forms an angle of about 30° with the preumbilical small intestine, which continues to pass at right angles to the body wall in a ventrodorsal plane. Each limb of the small intestine is clearly longer than the colorectum but less than twice as long. Stage 29 (6 21 d; H&H, 6 d; H, 6 – 6 1 A&N, 6 2 d; Fig. 8). 1 2 916 SOUTHWELL Fig. 10. Fig. 9. Outline of intestine at stage 30 –31. Large intestine: the caeca remain shorter than the colorectum and extend only a little over 1/3 of the caudal postumbilical small intestine. The caeca begin to shift to the caudal side of the postumbilical small intestine, almost coming together at their tips but still well separated toward the caecal junction. The cranial colorectum now descends to the dorsal body wall at an angle less than 45°. Remak’s nerve: under suitable lighting, a thin strand can be seen on the postumbilical side of the bifurcation in the omphalomesenteric artery and close to the preumbilical small intestine. Cloacal area: the phallus and lateral papillae are raised further. The caudal edges of the lateral papillae are still separated in the midline. 1 2 Outline of intestine at stage 32. 1 2 1 2 caeca have a similar ventral extent. Each limb of the small intestine is at least twice the length of the colorectum. Large intestine: the caeca maintain a similar relative extent as for previous stages. They have shifted caudolateral to the postumbilical small intestine throughout all but their most caudal extent. The colorectum is thicker than other regions of the intestine and descends from the caecal junction to the back body wall at an angle of 30° or less. Remak’s nerve: little change. Cloacal area: the papillae are raised further, forming a right angle to the body wall that is curved ventrally, resulting in the papillae being directed cranially. The caudal edges have come together in the midline, giving the papillae a short cylindrical appearance. The phallus extends caudally as a ventral cap to the vent. d; Stage 32 (7 43 d; H&H, 7 21 d; H 7 21 d; A&N, 8 d; Fig. 10). Displacement: the gizzard is now clearly ventral to all but the umbilical half of the small intestine. The postumbilical small intestine has continued to shift gradually cranial to the preumbilical small intestine. This continues over subsequent stages. Small intestine: the duodenal loop is bent ventrally with the base of each limb being approximately parallel to the preumbilical small intestine. The apex of the loop and the Displacement: little change other than increased length of all features. Small intestine: the duodenal loop has lengthened in the dorsoventral plane but is now directed a little caudally. Each limb is approximately parallel with the preumbilical small intestine and shorter than the caeca. The apex of the loop approaches or contacts the postumbilical small intestine, which continues to lie cranial to the preumbilical Stage 31 (7 Fig. 9). d; H&H, 7 d; H, 7–7 d; A&N, 7 INTESTINE DEVELOPMENT IN CHICK EMBRYO 917 intestine. Each limb of the small intestine is between two to three times longer than the caeca or colorectum. Large intestine: the caeca continue to occupy the caudal third of the postumbilical small intestine to which they are joined caudolaterally by a thin mesentery. They are now of similar length as the colorectum but are a little thinner than the small intestine. The tips of the caeca begin to separate from the intestine and recurve caudally. The colorectum is clearly thicker than the rest of the intestine, particularly the caudal quarter leading to the allantoic entry. This region will be occupied by the bursa of Fabricus in subsequent stages. The colorectum forms an open U-shape, the caudal half following the ventral curvature of the body wall and the cranial half curving at about 30° to the body wall before meeting the caecal junction. Remak’s nerve: can be seen in the ventral third of the umbilical loop as a thin ganglionated chain separating from the small intestine with which it remains in close association in the dorsal two-thirds. The chain can be seen to pass between the bifurcation of the omphalomesenteric artery. Caudally it is still a continuous, increasingly thick chord, closely applied to the colorectum. Cloacal area: the cloacal papillae have formed a cylinder (proctodeum), which has lengthened and is directed slightly caudal of a right angle to the back body wall. Caudal to the papillae, a dorsally directed depression appears. At this and subsequent stages, the phallus extends from a ventrocranial position, as a cap to the vent. Stage 33 (8 d; H&H, 7 21– 8 d; H, 7 21– 8 d; A&N, 8 d; Fig. 11). 1 3 Displacement: as for previous stages. The postumbilical small intestine is now clearly cranial to the preumbilical small intestine from the caecal junction up to 2/3 of the umbilical loop. At the most ventral 1/3 of the umbilical loop, this is reversed. Small intestine: further ventrocaudal lengthening of the duodenal loop so that the apex now overlays the postumbilical small intestine when viewed from the right side. The arms of the loop are still parallel with the preumbilical small intestine. The loop is now of a similar extent as the caeca. Each limb of the umbilical small intestine is about three times longer than the duodenal loop, the caeca, or the colorectum. Large intestine: lengthens further, maintaining the relative relationships between features identiﬁed in the previous stage. Remak’s nerve: as for the previous stage. Now ﬁne connections between the nerve and the omphalomesenteric artery can be seen. Bursa of Fabricius: can be seen as a small spherical ball both dorsal and caudal to the allantoic entry following removal of all tissue dorsal to the urogenital ducts in the cloacal area. Cloacal area: little change in the length and direction of the proctodeum. The caudal midline groove at the fused edges of the cloacal papillae descends into the deepening depression immediately caudal to the proctodeum. Stage 34 (8 21 d; H&H, 8 d; H, 8 d; A&N, 8 ⅔ d; Fig. 12). Displacement: the gizzard has shifted further caudally, now obscuring the duodenum, the caeca, and the caudal Fig. 11. Outline of intestine at stage 33. half of the postumbilical small intestine when viewed from the left side. It approaches or slightly overlays the preumbilical small intestine, which continues to lie well caudal of the postumbilical intestine. Small intestine: the duodenum has continued its ventrocaudal growth. The apex of the loop extends to the ventral quarter of the gizzard and after passing over the postumbilical small intestine, it approaches the preumbilical small intestine when viewed from the right side. Each limb of the umbilical small intestine continues to be approximately three times longer than the duodenal loop, the caeca, and the colorectum. Large intestine: lengthening and relative relationships of the caeca are maintained as in previous stages. As a result of the caudal shift of the gizzard, the cranial arm of the U-shaped bend in the colorectum is displaced dorsally. Hence the colorectum bends sharply ventral within the ﬁrst caudal quarter of its length, the remainder of which follows the curve of the body wall. Remak’s nerve: it has now separated some distance from the ventral umbilical intestine. Nerve trunks connect 918 SOUTHWELL Fig. 12. Outline of intestine at stage 34. to both the omphalmesenteric artery and the umbilical intestine. It is clearly ganglionated and observable along an increasing extent of the umbilical intestine. Little change in the region of the colorectum. Bursa of Fabricius: it has now extended cranial to the allantoic connection with urodeum. Cloacal area: the proctodeum is directed caudally following the curve of the back body wall in continuity with the colorectum. The groove in the midline of the caudal wall descends into the caudal depression, which had deepened further and is now directed cranially. Stage 35 (9 d; H&H, 8 –9 d; H, 8 –9 d; T&F, 9 d; A&N, 9 d; Fig. 13). Displacement: the gizzard extends further caudally. When viewed from the left side, it lays over the dorsal part of both limbs of the umbilical intestine and approaches the caudal colorectum. The apex of the duodenum can now be seen caudoventral to the gizzard. The relative position of the umbilical intestine had not changed. Fig. 13. Outline of intestine at stage 35. Small intestine: the duodenum runs from the midline in a ventrocaudal direction to the right body wall, where it bends caudally, reaching the postumbilical small intestine. At the caudal bend, the descending limb lays ventral to the gizzard. The relative relationships of the umbilical small intestine, identiﬁed at previous stages, are maintained. Large intestine: little relative change in the caeca. The further caudal shift of the gizzard displaces the whole of the colorectum against the dorsal body wall. The caeca and postumbilical small intestine bend sharply ventral just caudal to the caecal junction. Remak’s nerve: it is becoming ganglionated in the colorectal region with which it is still closely associated. As a result, nerve trunks to the colorectum cannot be seen. Bursa of Fabricius: extends cranially almost to the level of the clearly increased diameter of the caudal gut (coprodeum), becoming oval- rather than spherical-shaped. Cloacal area: little change other than a narrowing of the depression immediately caudal to the proctodeum. INTESTINE DEVELOPMENT IN CHICK EMBRYO 919 DISCUSSION Variations in Stage of Development With Temperature Fig. 14. Outline of intestine at stage 36. Temperature and humidity affect the development of avian embryos. A range of up to 18 hr in the times that stages appear is consistent with previously reported variability and differences in incubation temperature. A later appearance of stages 16 –36 was found also in a previous study (Allan and Newgreen, 1980) with the optimum of 38 ⫾ 1°C at 60 –70% relatively humidity for fowl embryos. Hamburger and Hamilton (1951) compiled stages 14 –35 of the commonly used fowl embryo staging from eggs incubated at higher than optimum temperature and humidity requirements (39.4°C without draft). To standardize the estimated chronology for incubation at 38°C, Hamilton (1952) made some adjustments for stages 18 –31 to the times reported by Hamburger and Hamilton (1951). Thompson and Fitzharris (1979), who incubated their eggs at 37.7 ⫾ 0.5°C at 80 –90% relative humidity, noted consistently slower developmental times than the standard times provided by Hamburger and Hamilton (1951). In this study with incubation at 38°C, stages 18 –24 were found at the times reported by Hamilton (1952). However, the common times for stages 16 and 17 were later than the slower extreme of the range given by Hamilton (1952), who did not adjust the times for these stages from those given by Hamburger and Hamilton (1951). Thompson and Fitzharris (1979) report the slower development of these stages at lower temperature (37.7°C). The times for stages ⬎ 25 occurred more often at the slower extreme of each range given by Hamilton (1952) and 12 hr slower than by Hamburger and Hamilton (1951). The times to reach stages 32–34 are slightly shorter than those in Allan and Newgreen (1980). For stages 32–34, Hamilton (1952) made no adjustments to the times originally provided by Hamburger and Hamilton (1951). In the diagrams, the time of appearance of each stage in all four studies is listed. To conﬁrm that the differences in timing were due to temperature, eggs were incubated at 39.4°C as reported by Hamburger and Hamilton (1951). Stages were reached at or before times reported by Hamburger and Hamilton (1951) (data not shown). Growth and Migration in Gut Stage 36 (9 21–10 d; H&H, 10 d; H, 10 d; Fig. 14). Displacement: a further caudal shift of the gizzard to overlay part of the caudal colorectum when viewed from the left side. The gizzard also crosses well to the right side, displacing the small intestine closer to the right body wall. Small intestine: duodenum is similar to the previous stage with a sharper caudal bend in the distal third of the loop. Little change in the relative relationships of the umbilical small intestine. Bends in the pre- and postumbilical small intestine that will form into subsidiary loops in these regions in subsequent stages can be seen. Large intestine: overall growth. Little relative change. Remak’s nerve: ganglionated throughout its length. Separation from the colorectum is beginning. Bursa of Fabricius: has reached its deﬁnitive cranial extent dorsal to the coprodeum. Clearly oval-shaped. Cloacal area: little change. The different growth rates of two closely associated primordia are important when considering how one shifts relative to the other. Thus, the displacement of one primordia relative to another may be achieved simply through differential growth. Conversely, the relationship between two apparently separate regions can be maintained by similar growth rates from earlier, more closely associated primordia. This latter point is illustrated by the growth of the caeca and caudal postumbilical small intestine. Following their early appearance as a mere swelling in the postumbilical gut (stage 21), each caecal bud extends laterally and ventrally (stage 26). In doing so, they come to lie parallel to the caudal third of the postumbilical small intestine, with which they maintain continuity (stage 27). The connection between the caeca and the caudal small intestine is maintained only by a thin mesentery during the following stages, but both regions undergo parallel growth phases. 920 SOUTHWELL Neural crest cells migrate from the neural tube into the proximal intestine and along the forming gut tube and differentiate into enteric neurons (Yntema and Hammond, 1954; Andrew, 1971; Le Douarin and Teillet, 1973; Teillet, 1978; Newgreen et al., 1980). The roles of growth (relative proliferation rate) and migration (the movement of one primordium relative to others) need to be considered carefully when dealing with such lengthy migratory pathways (Newgreen et al., 1996). In the current study, illustrations accompanying each stage description have been drawn to scale, permitting accurate estimates of length and volume measurements. In association with other data, we have used these parameters to obtain estimates of migration and proliferation rates of enteric neuron precursors (Newgreen et al., 1996). This staging system should also facilitate molecular studies (McBride et al., 2003) and comparisons between studies. ACKNOWLEDGMENTS This staging was originally prepared with Iain J. Allan, who is deceased. The author thanks Jessica Thomas for preparing the manuscript. Parts of the diagrams were used in a detailed report of the growth of gut regions (Newgreen et al., 1996). The staging was also used by McBride et al. (2003) prior to publication. LITERATURE CITED Allan I, Newgreen D. 1980. The origin and differentiation of enteric neurons of the intestine of the fowl embryo. Am J Anat 157:137– 154. Andrew A. 1971. The origin of intramural ganglia: IV, the origin of enteric ganglia—a critical review and discussion of the present state of the problem. J Anat 108:169 –184. Hamburger V, Hamilton H. 1951. 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