- Email: [email protected]
Formation and malformation of the enteric nervous system in mice: An organ culture study
Formation and Malformation of the Enteric Nervous System in Mice: An Organ Culture Study By Eiji Nishijima,
J.H. Care1 Meijers,
Dick Tibboel,
Arthur W.M.
Theo M. Luider, Marjo M.J.
van der Kamp,
Peters-van
der Sanden,
and Jan C. Molenaar
Rotterdam, The Netherlands and Kobe, Japan 0 Despite some progress in the treatment of congenital malformations of the enteric nervous system, there is no knowledge about the pathogenesis. The study of the normal formation of the entaric nervous system is hampered by the difficulty of manipulating and culturing mammalian embryos and their organs. Three methods to culture bowel explants of murine embryos, (chorioallantoic membrane grafting, organotypic tissue culture, and renal subcapsular space grafting) were compared. The three-dimensional cytoarchitecture of the bowel developed almost normally in the renal subcapsular space cultures. Using this culture system, it was found that neural crest cells colonize the murine bowel in distinct phases. The distal bowel was colonized at the 13th day of development. In a spontaneous mouse mutant model for intestinal aganglionosis. the lethal spotted mouse, the colonixation of the distal 2 mm of the bowel did not occur at E13. 0 1990 by W.B. Saunders INDEX WORDS:
ing neural crest cells, such as monoclonal antibody HNK-1 .14 The culture environment most suited for the growth and differentiation of embryonic murine bowel was determined. Bowel that was cultured (1) on the chorioallantoic membrane of chicken embryos; (2) in a organotypic tissue culture; and (3) in the murine renal subcapsular space, were compared. The renal subcapsular space of adult mice turned out to be the environment of choice for the culture of embryonic murine bowel. Using this culture environment, it was determined when neural crest cells colonize the distal segments of the bowel of control mice and of lethal spotted (ls/ls) mice, in which a recessive gene leads to intestinal aganglionosis.
Cotnpany. MATERIALS
Enteric nervous system; Hirschsprung’s
METHODS
Animals
disease.
C
ONGENITAL malformations of the enteric nervous system (ENS), such as Hirschsprung’s disease, usually present with disturbances in intestinal motility. During the past two decades, several pathological abnormalities of the ENS have been described.’ Although progress has been made in the treatment of these disorders, there is little knowledge about the pathogenesis. Most knowledge about normal ENS formation concerns the avian species. It is generally agreed that precursor cells of avian enteric neurons derive from the vagal neural crest. 24 Vagal neural crest cells migrate from their site of origin along defined pathways to the developing bowel,6 proliferate,7 aggregate at the sites of the future enteric ganglia,8 and differentiate into various neuronal phenotypes.’ Although an experimental model for intestinal aganglionosis has been obtained in the chicken embryo,” there are no spontaneous mutant chicken strains with intestinal aganglionosis. However, in mammals a number of spontaneous mutations lead to congenital intestinal aganglionosis. Consequently, mammals are more suitable experimental animal models than birds.‘1-‘3 The lack of knowledge about the formation of the mammalian ENS is due to the difficulty of manipulating and culturing mammalian embryos and their organs, and the lack of an early marker for migratJournalof Pediatric Surgery, Vol
AND
25, No 6 (June). 1990: pp 627-631
Normal C57BL.BLl and Is/is mice were bred in the Laboratory Animal Center at Erasmus University Medical School. Mouse embryos of several gestational ages were obtained after overnight matings. The day on which a vaginal plug was observed was called day EO. Pregnant female mice were killed by cervical dislocation. Embryos were staged according to the number of gestational days. The bowel was dissected under sterile conditions using fine tungsten needles and a dissecting microscope (Nikon, Zeist, The Netherlands). Chorioallantoic
Membrane
Grafting
Bowel segments were grafted onto the chorioallantoic membrane of ‘I-day-old chicken embryos. The technique is described in detail elsewhere.” Briefly, after abrasion of the superficial layer, the bowel segment was put at the bifurcation of two great vessels, and fixed in position with a piece of cellophane (sterilized in 70% ethanol and dried in air). The egg was sealed with cellophane tape and incubated for 1 week.
From the Departments of PediatricSurgery and Cell Biology and Genetics, Erasmus University Medical School, Rotterdam. The Netherlands. and the Department of Surgery, Kobe University School of Medicine, Kobe Children’s Hospital, Kobe. Japan. Supported by Grants No. 85-52 and No. 88-84 from the Sophia Foundation for MedicaI Research, Rotterdam. The Netherlands. Date accepted: October 11.1989. Address reprint requests to J.H.C. Meijers. MD, PhD. Department of Cell Biology and Genetics. Erasmus University Medical School, Dr Molewaterplein JO. PO Box 1738, 3000 DR Rotterdam, The Netherlands. o 1990 by W.B. Saunders Company. 0022-3468/90/2506~11$03.00j0 627
NISHIJIMA ET AL
Organotypic Tissue Culture Bowel segments were cultured according to a modification of the method of Rothman et al.‘6-‘8Briefly, bowel explants were grown on plastic Costar tissue culture dishes in a mixture of Ham’s FlO, (Flow Labs, Zwanenburg, The Netherlands) cultured medium and Dulbecco’s modification of EagIe medium. The culture medium was changed every other day.
Renal Subcapsular
Grafting
Dissected bowel segments of different embryonic ages were transplanted to heterozygous (Is/ +) or control adult male mice that were anesthetized by intraperitoneal injection of 0.3 mL of 2.5% avertine per 20 g body weight. Bowel explants were inserted in the subcapsular space using a 20-gauge mandrin needle. The recipients were killed 10 days after surgery.
Histology The grafts were embedded in OCT Compound (Tissue Tek 2; Miles Inc, Elkhart, IN) and snap frozen in liquid-nitrogen-cooled isopentane. Cryosections (10 pm) were mounted on slides coated with chrome alum and gelatin and stored at -20% The sections were routinely stained using hematoxylin and eosin. The presence of neural crest cells in explants was determined using immunoperoxidase staining of neurofilaments (NF). Polyclonal rabbit antibodies raised against 68, 160, and 200 kd purified NF triplet proteins” were kindly provided by Dr Y. Nakazato (Gunma University, Gunma, Japan). Peroxidase-conjugated swine antirabbit immunoglobulins (Dakopatts, Glostrup, Denmark) were used as second step antibodies in a dilution of 1 to 80. The grafts were considered to contain neural crest cells when positive staining was observed in at least two serial sections. RESULTS
Three different culture environments for the growth and differentiation of embryonic murine bowel were compared. Table 1 summarizes the characteristics of the three different culture environments. Culture of Embryonic Murine Bowel on the Chorioallantoic Membrane and in the Coelomic Cavity of Chicken Embryos It was found that when murine bowel is cultured on the chorioallantoic membrane of chicken embryos (E7), almost all grafts showed severe loss of normal architecture and hypoplasia of capillaries in the bowel wall, as well as in the bowel lumen, after l-week culture, whereas some grafts showed a delayed differentiation of the layers of the bowel wall and accumulations of cells in the bowel lumen.
Table 1. Culture Methods
Chorioallantoic
No. Environment Growth Differentiation
Organotypic
Renal
Tissue
Subcapsular Space
Membrane
Culture
32 in viva
56 in vitro
in viva
POW poor
moderate
good
poor
good
148
It was argued that the failure of growth of the bowel segments was related to the age of the acceptor embryo and its chorioallantoic membrane. In younger embryos (E4), the chorioallantoic membrane is not sufficiently developed. Therefore, embryonic murine bowel was inserted into the coelomic cavity of younger chicken embryos (E4). After l-week culture, we observed a more differentiated aspect of the bowel compared with the chorioallantoic cultures, but there were still abnormal accumulations of cells in the bowel lumen, and the differentiation of the layers of the bowel wall was delayed compared with normal in vivo development. Culture of Embryonic Bowel in Organotypic Tissue Culture
To avoid possible immunological reactions and differences in nutrient/oxygen requirements in interspecies culture systems, attempts to culture bowel segments in organotypic tissue culture were made. It was found that the differentiation of the bowel explants that were cultured using the described method was poor. The three-dimensional organization of the tissue architecture of the layers of the bowel wall had not been initiated. Rothman et ali6 reported that precursor cells for enteric neurons, present in the bowel segment at the time of explantation, differentiated into enteric neurons. The three-dimensional organization of a tissue plays an important role in the differentiation of neurons.” Therefore, organotyp ic tissue culture does not seem to be suitable for study of the differentiation of enteric neurons. Culture of Embryonic Murine Bowel in the Renal Subcapsular Space
One hundred forty-eight bowel segments of murine embryos of various ages were transplanted to the renal subcapsular space of adult control mice. The success rate was approximately 71%. The success rate of bowel explants of E9 and El0 embryos was significantly lower than the success rate of bowel explants of older embryos (33% and 44% for E9 and El0 embryos, and over 7 1% for older embryos [E 11 through El 51). Table 2 gives the success rates of the culture of bowel segments from embryos of various ages. Cystic dilatation was observed in 25 of the 148 grafts. Microscopical analysis showed an undulating undifferentiated epithelium and a retardation of smooth muscle differentiation. The renal subcapsular space was the method of choice from the three tested culture techniques. Using the renal subcapsular space culture method, the presence of neuronal precursor cells in bowel explants of control embryos was determined. The grafts that showed cystic dilatation were excluded from the series
ENS DEVELOPMENT
629
IN MICE
Table 2. Growth and Differentiation of Murine Bowel Explants Cultured in the Renal Subcapsular
No.
E9
El0
El1
El2
El3
Space El4
El5
Total
148
7
36
39
15
16
15
20
Death of host
1
2
0
0
3
3
6
15
Growth
2
17
37
15
13
12
14
110
Cystic dilatation
0
1
9
4
7
2
2
25
Differentiation
2
15
32
14
11
11
11
96
33
44
82
93
85
92
71
71
of grafts
Success rate (%)
in which the presence of precursor cells was screened for enteric neurons. The Presence of Neuronal Precursor Cells in Bowel Explants of Murine Embryos
Using neurofilament immunoperoxidase staining, enteric neurons in cultured bowel explants of E9 embryos were observed (Fig 1). These neurons were not noted at the time of explantation. Apparently, the neurons had developed from neural crest cells that were present in the bowel segment at the time of explantation. This indicates that neural crest cell colonization of murine bowel starts at least as early as E9. The entire bowel measured 1.5 mm at E9.
Fig 1. Cryostat section of an explant of the distal bowel of an El3 embryo that was cultured in the renal subcapsular space. Neurofilament staining shows the presence of enteric neurons.
Enteric neurons were not observed in cultures of the distal 2 mm of the bowel from ElO, El 1, and El2 embryos (Fig 2). The consistent absence of enteric neurons in these cultured explants indicates that neural crest cells do not colonize the entire bowel at E9. Enteric neurons were observed in cultured explants of the distal 2 mm of the bowel of El3 embryos. This indicates that the distal 2 mm of the bowel is colonized at E13. The Presence of Neuronal Precursor Cells in Bowel Explants of 1~11sEmbryos
The pattern of neural crest cell colonization of normal bowel was compared with that in Is/s bowel. We found that neural crest cell colonization of ls/ls bowel proceeded analogous to that observed in control mice up until E12. However, in ls/ls mice the colonization of the distal 2 mm of the bowel did not occur after E13. DISCUSSION
Comparing the three culture environments, it was determined that the three-dimensional architecture of the layered bowel came closest to the natural occurring pattern when embryonic murine bowel was cultured in the renal subcapsular space. Extrapolation of these culture results indicates that neural crest cells are present in the bowel of E9 embryos, and in the foregut and a part of the midgut of older embryos (El0 through E12). The distal 2 mm of the
Fig 2. Cryostat section of explant of the normal differentiated distal bowel of an El0 embryo that was cultured for 10 days in the renal subcapsular space. lmmunoperoxidase staining using antineurofilament antibodies shows the absence of enteric neurons. K, kidney.
630
NISHIJIMA ET AL
bowel remains devoid of neural crest cells until E13. Neural crest cells colonize this bowel segment at E13. Contrasting with our observations, Rothman and Gershon” stated that the entire bowel is colonized at E9. They demonstrated acetylcholine-positive cells in bowel explants that were grown in organotypic tissue culture. The cultured explants lacked the layered structure of the normal bowel wall. Furthermore, the number of explants that contained the acetylcholine positive cells was low (l/5). They could not rule out that the culture environment had elicited an abnormal phenotype in otherwise nonneuronal cells. Compared with the culture of embryonic avian bowel, the culture of embryonic murine bowel remains difficult. For instance, although suited for the culture of single bowel segments, the renal subcapsular space culture method is not suitable to induce neural crest cell colonization, by coculturering aneuronal bowel and the neural primordium (data not shown). Malformation
of the Is/Is ENS
It was found that neural crest cell colonization of the bowel proceeded identical in ls/ls and control mice until El 3. In Is/s mice the final phase of colonization of the distal 2 mm of the bowel does not take place.
These culture results do not support the hypothesis that the speed of enteric neural crest cell migration is lower in ls/ls mice.2’ It might well be that the intestinal aganglionosis in ls/ls mice is due to a localized abnormality in the mesenchyme of the distal 2 mm of the bowel. This hypothesis is supported by the findings of JacobsCohen et a1,22who reported that the distal bowel of Is/s mice is not colonized by neural crest cells from various sources (neural primordium of quail embryos, innervated bowel). In addition, differences in the composition of the extracellular matrix in the distal bowel of adult and embryonic ls/ls mice have been reported.23*24It is of interest to investigate whether the reported differences in the composition of the extracellular matrix in the caudal part of Is/s embryos is related to the inhibition of neural crest cell colonization of the bowel. It might well be that in Hirschsprung’s disease or other congenital malformations of the ENS, a local defect in the embryonic rectosigmoid colon prevents the homing of neural crest cells. Cloning of the Is/s gene, which is located on chromosome 2 of the mouse, will provide a direct insight into the cause of the intestinal aganglionosis.
REFERENCES 1. Holschneider AM: Hirschsprung’s Disease. Hippokrates, Stuttgart, West Germany, 1982 2. Yntema CL, Hammond WS: The origin of intrinsic ganglia of trunk viscera from vagal neural crest in the chick embryo. J Comp Nemo1 101515-541, 1954 3. Le Douarin NM, Teillet MA: The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryo1 Exp Morph01 30~31-48, 1973 4. Allan IJ, Newgreen DF: The origin and differentiation of enteric neurons of the intestine of the fowl embryo. Am J Anat 157:137-154.1980 5. Le Douarin NM: The Neural Crest. New York, NY, Cambridge, 1982 6. Tucker GC, Ciment G, Thiery JP: Pathways of avian neural crest cell migration in the developing gut. Dev Biol 116:439-450, 1986 7. Meijers JHC, Tibboel D, van der Kamp AWM, et al: Cell division in migratory and aggregated neural crest cells in the developing gut: An experimental approach to innervation-related motility disorders of the gut. J Pediatr Surg 22:243-245, 1987 8. Thiery JP, Duband J.-L, Rutishauser U, et al: Cell adhesion molecules in early chicken embryogenesis. Proc Nat1 Acad Sci USA 79:6737-6741,1982 9. Saffrey MJ, Polak JM, Burnstock G: Distribution of vasoactive intestinal polypeptide-, substance P-, enkephalin-, and neurotensinlike immunoreactive nerves in the chicken gut during development. Neuroscience 7:279-293,1982 10. Meijers JHC, Tibboel D, van der Kamp AWM, et al: A model for aganglionosis in the chicken embryo. J Pediatr Surg 24:282-285, 1989 11. Lane PW: Association of megacolon with two recessive spotting genes in the mouse. J Hered 57:18 l- 183,1966
12. Ikadai H, Fujita H, Agematsu Y, et al: Observation of congenital aganglionosis rat (Hirschsprung’s disease rat) and its genetical analysis. Congen Anom 19:3 l-36, 1979 13. Lane PW, Liu HM: Association of megacolon with a new dominant spotting gene (Dom) in the mouse. J Hered 75:435-439, 1984 14. Tucker GC, Aoyama H, Tursz T, et al: Identical reactivity of monoclonal antibodies HNK-1 and NCl: Conservation in vertebrates on cells derived from the neural primordium and on some leukocytes. Cell Differ 14:223-230,1984 15. Meijers JHC, Tibboel D, van der Kamp AWM, et al: The influence of the stage of differentiation of the gut on the migration of neural cells: An experimental study of Hirschsprung’s disease. Pediatr Res 21:466-470, 1987 16. Rothman TP, Nilaver G, Gershon MD: Colonization of the developing murine enteric nervous system and subsequent phenotypic expression by the precursors of peptidergic neurons. J Comp Neurol225:13-23,1984 17. Nakazato Y: Immunohistological localization of neurofilament protein in neuronal degenerations. Acta Neuropathol 64:3036,1984 18. Ito Y, Donahue PK, Hendren WH: Differentiation of intramural ganglia in the dissociated rectosigmoid of the rat: An organ culture study. J Pediatr Surg 20:969-974, 198 19. Bronner-Fraser M, Fraser SE: Cell lineage analysis reveals multipotency of some avian neural crest cells. Nature 335: 16 1-164, 1988 20. Rothman TP, Gershon MD: Phenotypic expression in the developing murine enteric nervous system. J Neurosci 2:381-393, 1982
ENS DEVELOPMENT
IN MICE
2 1. Webster W: Embryogenesis of the enteric ganglia in normal mice and in mice that develop congenital aganglionic megacolon. J Embryo1 Exp Morph01 30573-585.1973 22. Jacobs-Cohen RJ, Payette RF, Gershon MD, et al: Inability of neural crest cells to colonize, the presumptive aganglionic bowel of Is/Is mutant mice: Requirement for a permissive microenvironment. J Comp Neurol255:425-438,1987 23. Tennyson VM, Pham TD, Rothman TP, et al: Abnormalities
631
of smooth muscle, basal laminae, and nerves in the aganglionic segments of the bowel of lethal spotted mutant mice. Anat Ret 215:267-281, 1986 24. Payette RF, Tennyson VM, Pomeranz HD, et al: Accumulation of components of basal laminae: Association with the failure of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice. Dev Biol 125:341-360, 1988
J.H. Care1 Meijers,
Dick Tibboel,
Arthur W.M.
Theo M. Luider, Marjo M.J.
van der Kamp,
Peters-van
der Sanden,
and Jan C. Molenaar
Rotterdam, The Netherlands and Kobe, Japan 0 Despite some progress in the treatment of congenital malformations of the enteric nervous system, there is no knowledge about the pathogenesis. The study of the normal formation of the entaric nervous system is hampered by the difficulty of manipulating and culturing mammalian embryos and their organs. Three methods to culture bowel explants of murine embryos, (chorioallantoic membrane grafting, organotypic tissue culture, and renal subcapsular space grafting) were compared. The three-dimensional cytoarchitecture of the bowel developed almost normally in the renal subcapsular space cultures. Using this culture system, it was found that neural crest cells colonize the murine bowel in distinct phases. The distal bowel was colonized at the 13th day of development. In a spontaneous mouse mutant model for intestinal aganglionosis. the lethal spotted mouse, the colonixation of the distal 2 mm of the bowel did not occur at E13. 0 1990 by W.B. Saunders INDEX WORDS:
ing neural crest cells, such as monoclonal antibody HNK-1 .14 The culture environment most suited for the growth and differentiation of embryonic murine bowel was determined. Bowel that was cultured (1) on the chorioallantoic membrane of chicken embryos; (2) in a organotypic tissue culture; and (3) in the murine renal subcapsular space, were compared. The renal subcapsular space of adult mice turned out to be the environment of choice for the culture of embryonic murine bowel. Using this culture environment, it was determined when neural crest cells colonize the distal segments of the bowel of control mice and of lethal spotted (ls/ls) mice, in which a recessive gene leads to intestinal aganglionosis.
Cotnpany. MATERIALS
Enteric nervous system; Hirschsprung’s
METHODS
Animals
disease.
C
ONGENITAL malformations of the enteric nervous system (ENS), such as Hirschsprung’s disease, usually present with disturbances in intestinal motility. During the past two decades, several pathological abnormalities of the ENS have been described.’ Although progress has been made in the treatment of these disorders, there is little knowledge about the pathogenesis. Most knowledge about normal ENS formation concerns the avian species. It is generally agreed that precursor cells of avian enteric neurons derive from the vagal neural crest. 24 Vagal neural crest cells migrate from their site of origin along defined pathways to the developing bowel,6 proliferate,7 aggregate at the sites of the future enteric ganglia,8 and differentiate into various neuronal phenotypes.’ Although an experimental model for intestinal aganglionosis has been obtained in the chicken embryo,” there are no spontaneous mutant chicken strains with intestinal aganglionosis. However, in mammals a number of spontaneous mutations lead to congenital intestinal aganglionosis. Consequently, mammals are more suitable experimental animal models than birds.‘1-‘3 The lack of knowledge about the formation of the mammalian ENS is due to the difficulty of manipulating and culturing mammalian embryos and their organs, and the lack of an early marker for migratJournalof Pediatric Surgery, Vol
AND
25, No 6 (June). 1990: pp 627-631
Normal C57BL.BLl and Is/is mice were bred in the Laboratory Animal Center at Erasmus University Medical School. Mouse embryos of several gestational ages were obtained after overnight matings. The day on which a vaginal plug was observed was called day EO. Pregnant female mice were killed by cervical dislocation. Embryos were staged according to the number of gestational days. The bowel was dissected under sterile conditions using fine tungsten needles and a dissecting microscope (Nikon, Zeist, The Netherlands). Chorioallantoic
Membrane
Grafting
Bowel segments were grafted onto the chorioallantoic membrane of ‘I-day-old chicken embryos. The technique is described in detail elsewhere.” Briefly, after abrasion of the superficial layer, the bowel segment was put at the bifurcation of two great vessels, and fixed in position with a piece of cellophane (sterilized in 70% ethanol and dried in air). The egg was sealed with cellophane tape and incubated for 1 week.
From the Departments of PediatricSurgery and Cell Biology and Genetics, Erasmus University Medical School, Rotterdam. The Netherlands. and the Department of Surgery, Kobe University School of Medicine, Kobe Children’s Hospital, Kobe. Japan. Supported by Grants No. 85-52 and No. 88-84 from the Sophia Foundation for MedicaI Research, Rotterdam. The Netherlands. Date accepted: October 11.1989. Address reprint requests to J.H.C. Meijers. MD, PhD. Department of Cell Biology and Genetics. Erasmus University Medical School, Dr Molewaterplein JO. PO Box 1738, 3000 DR Rotterdam, The Netherlands. o 1990 by W.B. Saunders Company. 0022-3468/90/2506~11$03.00j0 627
NISHIJIMA ET AL
Organotypic Tissue Culture Bowel segments were cultured according to a modification of the method of Rothman et al.‘6-‘8Briefly, bowel explants were grown on plastic Costar tissue culture dishes in a mixture of Ham’s FlO, (Flow Labs, Zwanenburg, The Netherlands) cultured medium and Dulbecco’s modification of EagIe medium. The culture medium was changed every other day.
Renal Subcapsular
Grafting
Dissected bowel segments of different embryonic ages were transplanted to heterozygous (Is/ +) or control adult male mice that were anesthetized by intraperitoneal injection of 0.3 mL of 2.5% avertine per 20 g body weight. Bowel explants were inserted in the subcapsular space using a 20-gauge mandrin needle. The recipients were killed 10 days after surgery.
Histology The grafts were embedded in OCT Compound (Tissue Tek 2; Miles Inc, Elkhart, IN) and snap frozen in liquid-nitrogen-cooled isopentane. Cryosections (10 pm) were mounted on slides coated with chrome alum and gelatin and stored at -20% The sections were routinely stained using hematoxylin and eosin. The presence of neural crest cells in explants was determined using immunoperoxidase staining of neurofilaments (NF). Polyclonal rabbit antibodies raised against 68, 160, and 200 kd purified NF triplet proteins” were kindly provided by Dr Y. Nakazato (Gunma University, Gunma, Japan). Peroxidase-conjugated swine antirabbit immunoglobulins (Dakopatts, Glostrup, Denmark) were used as second step antibodies in a dilution of 1 to 80. The grafts were considered to contain neural crest cells when positive staining was observed in at least two serial sections. RESULTS
Three different culture environments for the growth and differentiation of embryonic murine bowel were compared. Table 1 summarizes the characteristics of the three different culture environments. Culture of Embryonic Murine Bowel on the Chorioallantoic Membrane and in the Coelomic Cavity of Chicken Embryos It was found that when murine bowel is cultured on the chorioallantoic membrane of chicken embryos (E7), almost all grafts showed severe loss of normal architecture and hypoplasia of capillaries in the bowel wall, as well as in the bowel lumen, after l-week culture, whereas some grafts showed a delayed differentiation of the layers of the bowel wall and accumulations of cells in the bowel lumen.
Table 1. Culture Methods
Chorioallantoic
No. Environment Growth Differentiation
Organotypic
Renal
Tissue
Subcapsular Space
Membrane
Culture
32 in viva
56 in vitro
in viva
POW poor
moderate
good
poor
good
148
It was argued that the failure of growth of the bowel segments was related to the age of the acceptor embryo and its chorioallantoic membrane. In younger embryos (E4), the chorioallantoic membrane is not sufficiently developed. Therefore, embryonic murine bowel was inserted into the coelomic cavity of younger chicken embryos (E4). After l-week culture, we observed a more differentiated aspect of the bowel compared with the chorioallantoic cultures, but there were still abnormal accumulations of cells in the bowel lumen, and the differentiation of the layers of the bowel wall was delayed compared with normal in vivo development. Culture of Embryonic Bowel in Organotypic Tissue Culture
To avoid possible immunological reactions and differences in nutrient/oxygen requirements in interspecies culture systems, attempts to culture bowel segments in organotypic tissue culture were made. It was found that the differentiation of the bowel explants that were cultured using the described method was poor. The three-dimensional organization of the tissue architecture of the layers of the bowel wall had not been initiated. Rothman et ali6 reported that precursor cells for enteric neurons, present in the bowel segment at the time of explantation, differentiated into enteric neurons. The three-dimensional organization of a tissue plays an important role in the differentiation of neurons.” Therefore, organotyp ic tissue culture does not seem to be suitable for study of the differentiation of enteric neurons. Culture of Embryonic Murine Bowel in the Renal Subcapsular Space
One hundred forty-eight bowel segments of murine embryos of various ages were transplanted to the renal subcapsular space of adult control mice. The success rate was approximately 71%. The success rate of bowel explants of E9 and El0 embryos was significantly lower than the success rate of bowel explants of older embryos (33% and 44% for E9 and El0 embryos, and over 7 1% for older embryos [E 11 through El 51). Table 2 gives the success rates of the culture of bowel segments from embryos of various ages. Cystic dilatation was observed in 25 of the 148 grafts. Microscopical analysis showed an undulating undifferentiated epithelium and a retardation of smooth muscle differentiation. The renal subcapsular space was the method of choice from the three tested culture techniques. Using the renal subcapsular space culture method, the presence of neuronal precursor cells in bowel explants of control embryos was determined. The grafts that showed cystic dilatation were excluded from the series
ENS DEVELOPMENT
629
IN MICE
Table 2. Growth and Differentiation of Murine Bowel Explants Cultured in the Renal Subcapsular
No.
E9
El0
El1
El2
El3
Space El4
El5
Total
148
7
36
39
15
16
15
20
Death of host
1
2
0
0
3
3
6
15
Growth
2
17
37
15
13
12
14
110
Cystic dilatation
0
1
9
4
7
2
2
25
Differentiation
2
15
32
14
11
11
11
96
33
44
82
93
85
92
71
71
of grafts
Success rate (%)
in which the presence of precursor cells was screened for enteric neurons. The Presence of Neuronal Precursor Cells in Bowel Explants of Murine Embryos
Using neurofilament immunoperoxidase staining, enteric neurons in cultured bowel explants of E9 embryos were observed (Fig 1). These neurons were not noted at the time of explantation. Apparently, the neurons had developed from neural crest cells that were present in the bowel segment at the time of explantation. This indicates that neural crest cell colonization of murine bowel starts at least as early as E9. The entire bowel measured 1.5 mm at E9.
Fig 1. Cryostat section of an explant of the distal bowel of an El3 embryo that was cultured in the renal subcapsular space. Neurofilament staining shows the presence of enteric neurons.
Enteric neurons were not observed in cultures of the distal 2 mm of the bowel from ElO, El 1, and El2 embryos (Fig 2). The consistent absence of enteric neurons in these cultured explants indicates that neural crest cells do not colonize the entire bowel at E9. Enteric neurons were observed in cultured explants of the distal 2 mm of the bowel of El3 embryos. This indicates that the distal 2 mm of the bowel is colonized at E13. The Presence of Neuronal Precursor Cells in Bowel Explants of 1~11sEmbryos
The pattern of neural crest cell colonization of normal bowel was compared with that in Is/s bowel. We found that neural crest cell colonization of ls/ls bowel proceeded analogous to that observed in control mice up until E12. However, in ls/ls mice the colonization of the distal 2 mm of the bowel did not occur after E13. DISCUSSION
Comparing the three culture environments, it was determined that the three-dimensional architecture of the layered bowel came closest to the natural occurring pattern when embryonic murine bowel was cultured in the renal subcapsular space. Extrapolation of these culture results indicates that neural crest cells are present in the bowel of E9 embryos, and in the foregut and a part of the midgut of older embryos (El0 through E12). The distal 2 mm of the
Fig 2. Cryostat section of explant of the normal differentiated distal bowel of an El0 embryo that was cultured for 10 days in the renal subcapsular space. lmmunoperoxidase staining using antineurofilament antibodies shows the absence of enteric neurons. K, kidney.
630
NISHIJIMA ET AL
bowel remains devoid of neural crest cells until E13. Neural crest cells colonize this bowel segment at E13. Contrasting with our observations, Rothman and Gershon” stated that the entire bowel is colonized at E9. They demonstrated acetylcholine-positive cells in bowel explants that were grown in organotypic tissue culture. The cultured explants lacked the layered structure of the normal bowel wall. Furthermore, the number of explants that contained the acetylcholine positive cells was low (l/5). They could not rule out that the culture environment had elicited an abnormal phenotype in otherwise nonneuronal cells. Compared with the culture of embryonic avian bowel, the culture of embryonic murine bowel remains difficult. For instance, although suited for the culture of single bowel segments, the renal subcapsular space culture method is not suitable to induce neural crest cell colonization, by coculturering aneuronal bowel and the neural primordium (data not shown). Malformation
of the Is/Is ENS
It was found that neural crest cell colonization of the bowel proceeded identical in ls/ls and control mice until El 3. In Is/s mice the final phase of colonization of the distal 2 mm of the bowel does not take place.
These culture results do not support the hypothesis that the speed of enteric neural crest cell migration is lower in ls/ls mice.2’ It might well be that the intestinal aganglionosis in ls/ls mice is due to a localized abnormality in the mesenchyme of the distal 2 mm of the bowel. This hypothesis is supported by the findings of JacobsCohen et a1,22who reported that the distal bowel of Is/s mice is not colonized by neural crest cells from various sources (neural primordium of quail embryos, innervated bowel). In addition, differences in the composition of the extracellular matrix in the distal bowel of adult and embryonic ls/ls mice have been reported.23*24It is of interest to investigate whether the reported differences in the composition of the extracellular matrix in the caudal part of Is/s embryos is related to the inhibition of neural crest cell colonization of the bowel. It might well be that in Hirschsprung’s disease or other congenital malformations of the ENS, a local defect in the embryonic rectosigmoid colon prevents the homing of neural crest cells. Cloning of the Is/s gene, which is located on chromosome 2 of the mouse, will provide a direct insight into the cause of the intestinal aganglionosis.
REFERENCES 1. Holschneider AM: Hirschsprung’s Disease. Hippokrates, Stuttgart, West Germany, 1982 2. Yntema CL, Hammond WS: The origin of intrinsic ganglia of trunk viscera from vagal neural crest in the chick embryo. J Comp Nemo1 101515-541, 1954 3. Le Douarin NM, Teillet MA: The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryo1 Exp Morph01 30~31-48, 1973 4. Allan IJ, Newgreen DF: The origin and differentiation of enteric neurons of the intestine of the fowl embryo. Am J Anat 157:137-154.1980 5. Le Douarin NM: The Neural Crest. New York, NY, Cambridge, 1982 6. Tucker GC, Ciment G, Thiery JP: Pathways of avian neural crest cell migration in the developing gut. Dev Biol 116:439-450, 1986 7. Meijers JHC, Tibboel D, van der Kamp AWM, et al: Cell division in migratory and aggregated neural crest cells in the developing gut: An experimental approach to innervation-related motility disorders of the gut. J Pediatr Surg 22:243-245, 1987 8. Thiery JP, Duband J.-L, Rutishauser U, et al: Cell adhesion molecules in early chicken embryogenesis. Proc Nat1 Acad Sci USA 79:6737-6741,1982 9. Saffrey MJ, Polak JM, Burnstock G: Distribution of vasoactive intestinal polypeptide-, substance P-, enkephalin-, and neurotensinlike immunoreactive nerves in the chicken gut during development. Neuroscience 7:279-293,1982 10. Meijers JHC, Tibboel D, van der Kamp AWM, et al: A model for aganglionosis in the chicken embryo. J Pediatr Surg 24:282-285, 1989 11. Lane PW: Association of megacolon with two recessive spotting genes in the mouse. J Hered 57:18 l- 183,1966
12. Ikadai H, Fujita H, Agematsu Y, et al: Observation of congenital aganglionosis rat (Hirschsprung’s disease rat) and its genetical analysis. Congen Anom 19:3 l-36, 1979 13. Lane PW, Liu HM: Association of megacolon with a new dominant spotting gene (Dom) in the mouse. J Hered 75:435-439, 1984 14. Tucker GC, Aoyama H, Tursz T, et al: Identical reactivity of monoclonal antibodies HNK-1 and NCl: Conservation in vertebrates on cells derived from the neural primordium and on some leukocytes. Cell Differ 14:223-230,1984 15. Meijers JHC, Tibboel D, van der Kamp AWM, et al: The influence of the stage of differentiation of the gut on the migration of neural cells: An experimental study of Hirschsprung’s disease. Pediatr Res 21:466-470, 1987 16. Rothman TP, Nilaver G, Gershon MD: Colonization of the developing murine enteric nervous system and subsequent phenotypic expression by the precursors of peptidergic neurons. J Comp Neurol225:13-23,1984 17. Nakazato Y: Immunohistological localization of neurofilament protein in neuronal degenerations. Acta Neuropathol 64:3036,1984 18. Ito Y, Donahue PK, Hendren WH: Differentiation of intramural ganglia in the dissociated rectosigmoid of the rat: An organ culture study. J Pediatr Surg 20:969-974, 198 19. Bronner-Fraser M, Fraser SE: Cell lineage analysis reveals multipotency of some avian neural crest cells. Nature 335: 16 1-164, 1988 20. Rothman TP, Gershon MD: Phenotypic expression in the developing murine enteric nervous system. J Neurosci 2:381-393, 1982
ENS DEVELOPMENT
IN MICE
2 1. Webster W: Embryogenesis of the enteric ganglia in normal mice and in mice that develop congenital aganglionic megacolon. J Embryo1 Exp Morph01 30573-585.1973 22. Jacobs-Cohen RJ, Payette RF, Gershon MD, et al: Inability of neural crest cells to colonize, the presumptive aganglionic bowel of Is/Is mutant mice: Requirement for a permissive microenvironment. J Comp Neurol255:425-438,1987 23. Tennyson VM, Pham TD, Rothman TP, et al: Abnormalities
631
of smooth muscle, basal laminae, and nerves in the aganglionic segments of the bowel of lethal spotted mutant mice. Anat Ret 215:267-281, 1986 24. Payette RF, Tennyson VM, Pomeranz HD, et al: Accumulation of components of basal laminae: Association with the failure of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice. Dev Biol 125:341-360, 1988