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Species specificity in the responsiveness of chick embryo neural tube explants to target-conditioned medium
lx?
Developmental
Brain Research,
3X
(1988)m- 1% Elsevicr
Species speci#icity in the rmsi neural tub8 sto Marieta B. Heaton Department
of’ Neuroscience.
University
of Florida
(Accepted Key words:
Neurogenesis;
Neural
College
I5 September
tube explant;
ofMedicine,
Gainesville,
FL 32610
(1I/.S.A, I
1987)
Species specificity;
Target-conditioned
medium
Explants from the metencephalic region of 40-h chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured in appropriate target muscle-conditioned media (MCM) derived from chick, quail and rat embryos. Enhanced neuritic outgrowth was found only in the presence of chick MCM. indicating that this early. initial responsiveness to target-released materials within this system is species-specific.
Growth factors which influence developing neuronal populations have been shown to both enhance early nerve fiber outgrowth and to guide it. Targetreleased factors have received particular attention in recent years, with one hypothesis being that such substances might play a vital role in vivo in directing outgrowing fibers to their proper points of termination. If such a peripheral-central interactive relationship does exist, then it seems essential that some degree of specificity be present, such that a given neuronal population may respond selectively to agents associated only with its own target tissue. Such regional specificity has recently been demonstrated within the developing trigeminal (V) system, both with respect to the eariy V motor nucleus population’ and the early trigeminal ganglion”. In the present study. the question of species specificity in the responsiveness of the early trigeminal motor population to target-released materials was addressed. Some earlier studies had suggested that growth factors tend to be conserved during evolution (nerve growth factor (NGF) being the best example). with similar influences demonstrated across a variety of species’.‘. Explants from Hamburger and Hamilton’ stage I1
(40 h) chick embryos were taken from the region of the metencephalic basal plate which gives rise to the V motor nucleus. The explants were grown on coliagen-polyornithine-coated tissue culture dishes, using F-12 medium, with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.7% fungizone, and 1.6% glutamine. The experimental explants were supplemented with muscle-conditioned media (MCM) derived from the normal target tissue of motor V (the mandibular process of the first visceral arch) from stage 22 (3.5-4 days) chick embryos (Gallus gulfus domesticus), from comparably staged bobwhite quail embryos (Colinus virginianus). and from El4 rat embryos (Ra~us norvegicus a&mu). This point in development represents the time that motor V peripheral innervation first begins’. The MCM was produced as previously described’, using medium without FBS. After 3 days, the medium was removed from the underlying cell layer, assayed for relative protein content using the Bradford’ dye-binding assay, and added in 0.5mi aliquots to experimental cultures on the day after explantation. Protein levels were equated to the average found in chick MCM (approximately 35 @g/ml). In control cultures. 0.5 ml of medium without FBS was
__ (‘orrespondenct~:
nesville.
FL.32610.
M.B. Hcaton. L1.S.A.
0165-3806/X8/$03.50@
Department
1988 Elsevier
of Neuroscience,
Science Publishers
University
B.V. (Biomedical
of Florida
Division)
C‘ollepe of Medicine.
Bar J-244. JHMHC‘.
Gal-
153
added at the same time, so that all elements
within
the medium were equivalent, except for substances present in the MCM. After approximately 36 h in vitro, ah cultures were supplemented with 2-pglml cytosine arabinoside (ara-C), to inhibit excessive proliferation of non-neuronal cells and to eliminate lategenerated neurons. At this point in vivo (i.e. 76 h), the motor V population has just completed its proliferation and is by far the predominant postmitotic population in this region of the metencephalon’. Thus, these preparations are enriched for motor V neuron&‘. After 5 days in vitro, the neuritic
outgrowth
from
the explants was quantified by placing a transparent template consisting of concentric circles over photomicrographs or camera lucida drawings of the explants, and counting neuritic intersections with the circles. This procedure produces a quantitative ‘neuritic growth index’, reflecting both the number of neurites present and the length of such processes’. In the presence of chick target MCM, neuritic outgrowth from the neural tube explants was significantly enhanced (P < 0.0001, Mann-Whitney UTest. 2-tailed), as we had found previously’. The neuritic growth index in this experimental group averaged 387.2, compared to 193.6 in their control group (each count represents one intersection with the circles of the template described above). In the presence of quail target MCM, the mean growth index was 151.8, compared to 224.4 for their controls. This difference was not significant (P > 0.10). In the presence of rat target MCM, the growth index was 205.2, compared to 164.9 in their control group. Again, this difference was not significant (P > 0.30). These comparisons are presented in Fig. 1. In a previous study’, we found that explants of the motor V region of the metencephalon respond preferentially to MCM derived from target tissue specific to this population (from the mandibular process). No enhancement of neuritic outgrowth was found when these explants were grown in the presence of limb bud-derived MCM (of the same ontogenetic stage as the mandibular process tissue). We also found that this growth enhancement effect is developmentally regulated. Mid- and late-incubation target MCM inhibit neurite outgrowth from this early neuronal population’. The present data demonstrate that in addition to this regional and stage specificity, the respon-
Fig. 1. Comparison of mean neuritic growth indices from chick embryo metencephalic explants grown in the presence of target muscle-conditioned media (MCM) derived from chick embryos
(CMCM), from bobwhite quail embryos (QMCM), and from rat embryos (RMCM). CON, control. The neuritic growth in the CMCM group (n = 36) significantly exceeded that of its control group (n = 33; P < 0.0001). The growth in the QMCM group (n = 25) and in the RMCM group (n = 21) did not differ from their controls (n = 28, n = 22 respectively). Vertical bars = S.E.M.
siveness of these preparations
to appropriate
target-
released substances is also species-specific. Even target MCM derived from a species within the avian class, but of a different genus and species, is not competent to elicit the outgrowth promoting effects seen in the presence of chick-derived MCM. A high degree of selective responsiveness has also been seen in other aspects of the trigeminal system. Lumsden and Davies’.” have elegantly characterized interactions between the early V ganglion and its target tissues in vitro: V ganglion fibers direct their neuritic outgrowth toward their own target field (either the maxillary or mandibular process), but not toward the adjoining field (the hyoid process of the second visceral arch, normally innervated by the geniculate ganglion). This precision with which the early trigeminal system apparently recognizes or responds to appropriate elements may be somewhat exceptional, however. Lumsden and Davies”, for example, also found that unlike the V ganglion, the geniculate (VII) gan-
154 glion showed no orientation of neuritic growth toward its normal target tissue. Similarly, Dribin” cultured spinal cord explants
from embryonic
chick with MCM derived from mouse. embryos.
In this study.
hance neurite origin. cord
outgrowth
In fact, neurite explants
showed
the MCM appeared regardless outgrowth the
when grown in the presence
rat and
rat and chick
noted
whether
the protein
content
of the various
MCM were equated in this study, so the basis of these differences is not clear, and cannot bc directly compared to the data from the present study.
to en-
of the species of from chick spinal
greatest
enhancement
of mouse and rat MCM.
rather than in chick MCM. Unfortunately.
it is not
1 Bradford, M.M.. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 24% 254. 2 Collins, F., Electrophoretic similarity of the ciliary ganglion survival factors from different tissues and species. Del’. Biol., 109 (1985) 255-258. 3 Dribin, L.B., On the species and substrate specificity of conditioned medium enhancement of neuritic outgrowth from spinal cord explants. Dev. Bruin Res., 3 ( 1982) 300-304. 4 Hamburger. V. and Hamilton. H.L., A series of normal stages in the chick embryo, J. Morphol., 88 (1951) 49-92. 5 Heaton, M.B. and Moody, S.A., Early development and migration of the trigeminal motor nucleus in the chick embryo,
This research was supported
by Nattonal
Institutes
of Health Grant NS-20387. I thank Michael Paiva for his excellent
assistance,
and Dr. Paul Reier and Dr.
John Houle for their generous
gift of the rat embryos.
J. C’omp. Neural.. 1X’)(1980) hl-YY. 6 Heaton, M.B. and Paiva. M.. The inlluence of target tissue age on neurite outgrowth from chick embryo trigeminal motor nucleus exptants, Dev. Rid.. 116 (1%X5) 314-318. 7 Heaton, M.B. and Wayne. D.B., Specific responsiveness ol chick trigeminal motor nucleus explants to target-conditioned media, J. Camp. Neural., 14.3(1+1X6)3Xl-387. 8 Lumsden. A.G.S. and Davies, A.M., Earliest sensory nerve fibers are guided to peripheral target\ hy attractants other than nerve growth factor. ,Vafure c1.orrd.j. 306 (19X3) 786-788. Y Lumsden. A.G.S. and Davies, A.M.. (‘hemotropic effect 01 specific target epithelium in the developing mammatian ncrvous system, Nature (Land.), 323 (19X6) 53X-539.
Developmental
Brain Research,
3X
(1988)m- 1% Elsevicr
Species speci#icity in the rmsi neural tub8 sto Marieta B. Heaton Department
of’ Neuroscience.
University
of Florida
(Accepted Key words:
Neurogenesis;
Neural
College
I5 September
tube explant;
ofMedicine,
Gainesville,
FL 32610
(1I/.S.A, I
1987)
Species specificity;
Target-conditioned
medium
Explants from the metencephalic region of 40-h chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured in appropriate target muscle-conditioned media (MCM) derived from chick, quail and rat embryos. Enhanced neuritic outgrowth was found only in the presence of chick MCM. indicating that this early. initial responsiveness to target-released materials within this system is species-specific.
Growth factors which influence developing neuronal populations have been shown to both enhance early nerve fiber outgrowth and to guide it. Targetreleased factors have received particular attention in recent years, with one hypothesis being that such substances might play a vital role in vivo in directing outgrowing fibers to their proper points of termination. If such a peripheral-central interactive relationship does exist, then it seems essential that some degree of specificity be present, such that a given neuronal population may respond selectively to agents associated only with its own target tissue. Such regional specificity has recently been demonstrated within the developing trigeminal (V) system, both with respect to the eariy V motor nucleus population’ and the early trigeminal ganglion”. In the present study. the question of species specificity in the responsiveness of the early trigeminal motor population to target-released materials was addressed. Some earlier studies had suggested that growth factors tend to be conserved during evolution (nerve growth factor (NGF) being the best example). with similar influences demonstrated across a variety of species’.‘. Explants from Hamburger and Hamilton’ stage I1
(40 h) chick embryos were taken from the region of the metencephalic basal plate which gives rise to the V motor nucleus. The explants were grown on coliagen-polyornithine-coated tissue culture dishes, using F-12 medium, with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.7% fungizone, and 1.6% glutamine. The experimental explants were supplemented with muscle-conditioned media (MCM) derived from the normal target tissue of motor V (the mandibular process of the first visceral arch) from stage 22 (3.5-4 days) chick embryos (Gallus gulfus domesticus), from comparably staged bobwhite quail embryos (Colinus virginianus). and from El4 rat embryos (Ra~us norvegicus a&mu). This point in development represents the time that motor V peripheral innervation first begins’. The MCM was produced as previously described’, using medium without FBS. After 3 days, the medium was removed from the underlying cell layer, assayed for relative protein content using the Bradford’ dye-binding assay, and added in 0.5mi aliquots to experimental cultures on the day after explantation. Protein levels were equated to the average found in chick MCM (approximately 35 @g/ml). In control cultures. 0.5 ml of medium without FBS was
__ (‘orrespondenct~:
nesville.
FL.32610.
M.B. Hcaton. L1.S.A.
0165-3806/X8/$03.50@
Department
1988 Elsevier
of Neuroscience,
Science Publishers
University
B.V. (Biomedical
of Florida
Division)
C‘ollepe of Medicine.
Bar J-244. JHMHC‘.
Gal-
153
added at the same time, so that all elements
within
the medium were equivalent, except for substances present in the MCM. After approximately 36 h in vitro, ah cultures were supplemented with 2-pglml cytosine arabinoside (ara-C), to inhibit excessive proliferation of non-neuronal cells and to eliminate lategenerated neurons. At this point in vivo (i.e. 76 h), the motor V population has just completed its proliferation and is by far the predominant postmitotic population in this region of the metencephalon’. Thus, these preparations are enriched for motor V neuron&‘. After 5 days in vitro, the neuritic
outgrowth
from
the explants was quantified by placing a transparent template consisting of concentric circles over photomicrographs or camera lucida drawings of the explants, and counting neuritic intersections with the circles. This procedure produces a quantitative ‘neuritic growth index’, reflecting both the number of neurites present and the length of such processes’. In the presence of chick target MCM, neuritic outgrowth from the neural tube explants was significantly enhanced (P < 0.0001, Mann-Whitney UTest. 2-tailed), as we had found previously’. The neuritic growth index in this experimental group averaged 387.2, compared to 193.6 in their control group (each count represents one intersection with the circles of the template described above). In the presence of quail target MCM, the mean growth index was 151.8, compared to 224.4 for their controls. This difference was not significant (P > 0.10). In the presence of rat target MCM, the growth index was 205.2, compared to 164.9 in their control group. Again, this difference was not significant (P > 0.30). These comparisons are presented in Fig. 1. In a previous study’, we found that explants of the motor V region of the metencephalon respond preferentially to MCM derived from target tissue specific to this population (from the mandibular process). No enhancement of neuritic outgrowth was found when these explants were grown in the presence of limb bud-derived MCM (of the same ontogenetic stage as the mandibular process tissue). We also found that this growth enhancement effect is developmentally regulated. Mid- and late-incubation target MCM inhibit neurite outgrowth from this early neuronal population’. The present data demonstrate that in addition to this regional and stage specificity, the respon-
Fig. 1. Comparison of mean neuritic growth indices from chick embryo metencephalic explants grown in the presence of target muscle-conditioned media (MCM) derived from chick embryos
(CMCM), from bobwhite quail embryos (QMCM), and from rat embryos (RMCM). CON, control. The neuritic growth in the CMCM group (n = 36) significantly exceeded that of its control group (n = 33; P < 0.0001). The growth in the QMCM group (n = 25) and in the RMCM group (n = 21) did not differ from their controls (n = 28, n = 22 respectively). Vertical bars = S.E.M.
siveness of these preparations
to appropriate
target-
released substances is also species-specific. Even target MCM derived from a species within the avian class, but of a different genus and species, is not competent to elicit the outgrowth promoting effects seen in the presence of chick-derived MCM. A high degree of selective responsiveness has also been seen in other aspects of the trigeminal system. Lumsden and Davies’.” have elegantly characterized interactions between the early V ganglion and its target tissues in vitro: V ganglion fibers direct their neuritic outgrowth toward their own target field (either the maxillary or mandibular process), but not toward the adjoining field (the hyoid process of the second visceral arch, normally innervated by the geniculate ganglion). This precision with which the early trigeminal system apparently recognizes or responds to appropriate elements may be somewhat exceptional, however. Lumsden and Davies”, for example, also found that unlike the V ganglion, the geniculate (VII) gan-
154 glion showed no orientation of neuritic growth toward its normal target tissue. Similarly, Dribin” cultured spinal cord explants
from embryonic
chick with MCM derived from mouse. embryos.
In this study.
hance neurite origin. cord
outgrowth
In fact, neurite explants
showed
the MCM appeared regardless outgrowth the
when grown in the presence
rat and
rat and chick
noted
whether
the protein
content
of the various
MCM were equated in this study, so the basis of these differences is not clear, and cannot bc directly compared to the data from the present study.
to en-
of the species of from chick spinal
greatest
enhancement
of mouse and rat MCM.
rather than in chick MCM. Unfortunately.
it is not
1 Bradford, M.M.. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 24% 254. 2 Collins, F., Electrophoretic similarity of the ciliary ganglion survival factors from different tissues and species. Del’. Biol., 109 (1985) 255-258. 3 Dribin, L.B., On the species and substrate specificity of conditioned medium enhancement of neuritic outgrowth from spinal cord explants. Dev. Bruin Res., 3 ( 1982) 300-304. 4 Hamburger. V. and Hamilton. H.L., A series of normal stages in the chick embryo, J. Morphol., 88 (1951) 49-92. 5 Heaton, M.B. and Moody, S.A., Early development and migration of the trigeminal motor nucleus in the chick embryo,
This research was supported
by Nattonal
Institutes
of Health Grant NS-20387. I thank Michael Paiva for his excellent
assistance,
and Dr. Paul Reier and Dr.
John Houle for their generous
gift of the rat embryos.
J. C’omp. Neural.. 1X’)(1980) hl-YY. 6 Heaton, M.B. and Paiva. M.. The inlluence of target tissue age on neurite outgrowth from chick embryo trigeminal motor nucleus exptants, Dev. Rid.. 116 (1%X5) 314-318. 7 Heaton, M.B. and Wayne. D.B., Specific responsiveness ol chick trigeminal motor nucleus explants to target-conditioned media, J. Camp. Neural., 14.3(1+1X6)3Xl-387. 8 Lumsden. A.G.S. and Davies, A.M., Earliest sensory nerve fibers are guided to peripheral target\ hy attractants other than nerve growth factor. ,Vafure c1.orrd.j. 306 (19X3) 786-788. Y Lumsden. A.G.S. and Davies, A.M.. (‘hemotropic effect 01 specific target epithelium in the developing mammatian ncrvous system, Nature (Land.), 323 (19X6) 53X-539.