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Fishberg Center for Neurobiology and Division of Endocrinology, Mount Sinai School of Medicine (M.C.R., M.J.G.), New York, New York 10029; and the Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons (A.-J.S.), New York, New York 10032
Address all correspondence and requests for reprints to: Dr. Marie J. Gibson, Mount Sinai School of Medicine, Box 1055, 1 Gustave Levy Place, New York, New York 10029. E-mail: MGIBSON{at}SMTPLINK.MSSM.EDU
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Introduction |
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GnRH cells within embryonic or neonatal septal/POA grafts placed in the third ventricle of adult hypogonadal mice (hpg) also innervate the median eminence. The hpg mouse has a deletion of two exons in the GnRH gene and does not synthesize the peptide (7). Its reproductive organs, therefore, remain undeveloped, and the mice are infertile. Only when GnRH cells in the grafts innervate the host median eminence do the mice undergo reproductive development (8), including ovulation and pregnancy (9, 10).
Several lines of evidence using the model of a normal POA graft into the central nervous system of the adult hpg mouse suggest that the MBH or the median eminence itself plays a critical role in GnRH axonal targeting to the portal capillaries. When the graft is placed within the third ventricle, GnRH axons ramify within the graft and exit into the central nervous system of the host when they reach the MBH or the median eminence (11). If grafts are placed into the lateral ventricle, GnRH neurons survive, but the axons that enter the host brain, following nearby fiber tracts, do not reach the median eminence (12). The location of the graft in the proximity of the MBH seems to be critical for proper targeting. The importance of this region in providing cues to GnRH axons was emphasized by experiments in which embryonic MBH was cografted with POA in the third ventricle. GnRH terminals robustly innervated the median eminence within the cograft as well as the adult host median eminence (13). These latter experiments also suggested that the cues for inducing axonal targeting were either similar between the adult and embryonic median eminence or had overlapping functions.
A cellular compartment within the MBH may form an attractive surface for GnRH axons. Many of the GnRH axons exiting from the graft arch through the arcuate nucleus to terminate in the median eminence, mimicking a normal pathway (11). However, despite selective destruction of the majority of arcuate nucleus neurons of the hpg host by neonatal treatment with monosodium glutamate, grafted GnRH neurons formed functional connections with the host median eminence, suggesting a limited role for this neuronal population (14). Graft-derived GnRH axons were often associated with glial channels and/or glial processes in this region, but the degree of reactive gliosis induced by the graft procedure might make this association fortuitous rather than instructive (15). It thus appears that direct surface interactions of GnRH axons with cells in the MBH are unlikely to be the only factor involved in targeting.
In recent years it has become clear that chemoattractants participate in axonal guidance in many systems (16). To test the hypothesis that GnRH axonal outgrowth to the median eminence is regulated by diffusible substances from the target region, we established cocultures of embryonic POA, containing GnRH neurons, with embryonic MBH, containing the median eminence. The explants grew on the porous membrane surface of insert chambers. This organotypic culture system is used for studies of axonal targeting in other systems and is suitable for the study of chemotropic diffusible factors (17, 18, 19).
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Materials and Methods |
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The tissue explants were fed through the porous membrane by the underlying medium. In all experiments the medium used was DMEM and Ham’s nutrient mix F-12 with L-glutamine and 15 mM HEPES without phenol, supplemented with putrescine (10-4 M), sodium selenite (0.02 µg/ml), and apotransferrin (100 µg/ml; all medium ingredients obtained from Sigma Chemical Co., St. Louis, MO). The coated insert chamber membrane was washed with 200 µl medium before use.
Dissections
Embryos of gestational age E15 or newborn pups of either sex
were used in these experiments as indicated in the text. In previous
in vivo studies, the sex of the donor had no effect on
successful targeting of GnRH neurons in recipients (11). For embryos,
time-mated normal female mice (C3H/HeH x 101H) were killed by
cervical dislocation on gestational day 15. The day the vaginal plug
was found was designated E0, and staging was confirmed (22). The uterus
was removed and placed immediately into a sterile petri dish on ice.
Newborn pups were collected less than 24 h after delivery (this
stage was defined as postnatal day 1, or P1) and placed on ice for
anesthesia. The embryo (or the pup) was decapitated, and the head was
placed on a sterile stage under a binocular dissection microscope. When
P1 pups were used, the head was rinsed with a solution of 70% ethanol
before dissection to assure sterility. The skull was removed, and the
brain inverted on the stage so that the ventral surface of the brain
faced up. The POA was dissected under sterile conditions as described
previously (8). Using a separate set of instruments, the MBH, including
the median eminence, was dissected as a midline strip of tissue from
the ventral surface of the brain caudal to the POA. We cannot exclude
the possibility that remnants of the pars tuberalis were present in MBH
explants. However, our earlier in vivo studies with
hypophysectomized hypogonadal host mice showed that the presence of the
pituitary is not necessary for targeting of grafted GnRH cell outgrowth
to the median eminence (13). Cervical spinal cord (SC) or cerebellar
cortex (CE) were also dissected in some experiments. All procedures
were approved by the institutional animal care and use committee.
Tissue explants were placed immediately on the collagen- and laminin-coated surface of the insert chamber membrane. In the case of the POA explant, the block of tissue was sectioned at the midline after placement in the chamber and applied flat on the surface of the membrane. All explants were kept moist by the addition of 10–20 µl medium above the membrane. The chambers were then placed in wells of 12-well plates containing 1 ml of the same medium. In all experiments, the medium was not changed, and care was taken to leave the cultures undisturbed until the time of fixation.
Experimental design of single POA cultures and cocultures
The POA explant was either cultivated alone or with other brain
regions placed on the membrane adjacent to the POA. Using E15 tissue,
the following explant partners were established: POA alone or POA with
MBH, SC, or CE. In experiments using P1 tissue, the POA explant was
cultivated only with MBH. In the majority of experiments, explants were
cultured for 7 days at 37 C in 5% CO2 and 100% humidity
and then fixed by immersion in 4% paraformaldehyde for 3 h. For a
time-course analysis, E15 coexplants of POA and MBH were fixed after 1,
4, 7, or 10 days of culture. All fixed cultures were kept at 4 C in
phosphate buffer (PB; 0.1 M; pH 7.3) with 0.1% sodium
azide (Sigma Chemical Co., St. Louis, MO) until they were processed by
immunocytochemistry.
Some cultures were counterstained with a solution of 1% of cresyl violet in 20% ethanol solution. The staining was differentiated with a rinse in water, followed by a brief rinse in a solution of acidified 90% ethanol.
Immunocytochemistry
Immunocytochemistry was performed directly on membranes after
cutting them out of the chamber. To detect GnRH neurons and processes,
SW1 antiserum (23) was used at a dilution of 1:2500 in PB containing
0.1% Triton X-100 (TX100; Sigma), 1% normal goat serum (Life
Technologies) or 3% normal donkey serum (Jackson ImmunoResearch
Laboratories, West Grove, PA) for 4 days at 4 C. The biotinylated
antirabbit secondary antiserum [1:200, made in goat (Vector
Laboratories, Burlingame, CA), or 1:200, made in donkey (Jackson
ImmunoResearch Laboratories)] in the same diluent was applied
overnight for increased penetration of the explants. Explants were then
incubated in an avidin-biotin solution (Vector Laboratories) conjugated
with horseradish peroxidase for 2 h. The chromogen was 1 mg/ml
3,3'-diaminobenzidine (Sigma), and H2O2 was
generated by the oxidation of glucose by glucose oxidase (Sigma). There
were no differences in GnRH labeling or background staining with the
two different secondary antibodies.
In a group of cultures double label immunocytochemistry was performed to visualize GnRH and growth-associated protein-43 (GAP-43). The visualization of GnRH in this case was performed using SW1 with a secondary antirabbit antiserum directly conjugated with fluorescein isothiocyanate (1:200, made in donkey; Jackson ImmunoResearch Laboratories). The cultures were then rinsed overnight, treated with 3% normal donkey serum in PB with 0.1% TX100, incubated in the GAP-43 antibody at a dilution of 1:1000 (mouse monoclonal antibody, clone 91E12; Boehringer Mannheim) in PB containing 3% normal donkey serum and 0.1% TX100 for 4–6 days. The cultures were incubated subsequently in antimouse biotinylated secondary antiserum (1:200, made in donkey; Jackson ImmunoResearch Laboratories), and the sites of antibody binding were visualized with Texas Red-conjugated avidin D (Vector Laboratories).
Double labeling was also performed to characterize the possible interactions between GnRH and glial processes or cells. Glial cells were identified by using antiserum against glial fibrillary acidic protein (GFAP), vimentin, or S-100. GnRH was visualized with the anti-GnRH mouse monoclonal antibody (no. 19304, QED Advanced Research Technologies, San Diego, CA) at a dilution of 1:5000 in 0.2% TX100 and 3% normal donkey serum in PB for 4 days at 4 C. The cultures were incubated overnight in biotinylated antimouse secondary antiserum (1:200, made in donkey; Jackson ImmunoResearch Laboratories) followed by Texas Red-streptavidin. The labeling of glial markers was performed with rabbit anti-GFAP (24) at a dilution of 1:1000, rabbit antivimentin (gift from Dr. Wang) at a dilution of 1:1000, or rabbit anti-S100 (Dako, Carpenteria, CA) at a dilution of 1:2000. Visualization of the glial marker labeling was carried out with the use of an antirabbit antiserum conjugated with fluorescein isothiocyanate. All glial marker antisera were prepared in 0.1% TX100 and 3% normal donkey serum.
After immunocytochemistry, the tissue was washed, mounted directly onto glass slides, and immediately coverslipped with Gelmount (Biomedia, Foster City, CA). The cultures were observed and photographed under brightfield or fluorescent optics as appropriate. In certain cases, observation of double labeled cultures was performed first with epifluorescence, then with a Leica inverted confocal laser scanning microscope (Leica Lasertechnik GmbH, Heidelberg, Germany), using an argon/krypton laser as its light source. For these studies the objective x40 was used with a pinhole of 40 that allowed confocal sections of 0.5 µm.
Quantitative analysis of the cultures
GnRH axonal outgrowth was assessed in the cocultures of POA-MBH,
POA-SC, and POA-CE and in cultures of POA alone. Cultures were not
considered for quantitative analysis if the explants were fused or
torn, if the location of the MBH or control tissue could not be
determined, or if there were less than 50 GnRH-immunoreactive (GnRH-ir)
cell bodies in the POA. Only GnRH-ir fibers terminating in a growth
cone were considered.
GnRH axons extending on the surface of the membrane from the POA
explant were counted in two sectors. Sector I is the region of membrane
facing the cocultivated explant, and sector II is the region of
membrane away from the cocultivated explant (Fig. 1
). A few GnRH cell bodies were detected
within the MBH coexplants, similar to previous findings in
vivo (Wu, T. J., and A.-J. Silverman, unpublished
observations). The occasional GnRH axons that extended out of the MBH
were not included in the quantitative analyses. In cultures of POA
alone, GnRH axons extending from the entire circumference of the POA
were counted. All values were expressed as the mean ±
SEM. When homogeneity of variances was not achieved, the
appropriate transformation of the data were applied (25). The numbers
of GnRH axons in sectors I and II were then compared between POA-MBH
cocultures and control cultures, and similarly at each time point of
the time course, with a two-way ANOVA. When P < 0.05,
Newman-Keuls t test with Duncan’s correction was used for
post-hoc comparisons.
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For cell counts the POA was divided into two analogous regions, so that
counts could be compared between the half facing the coexplant (side I)
and the half facing away (side II; Fig. 1
).
Analysis for trophic effects on GnRH cell survival was performed by counting the number of GnRH cell bodies in the POA in the presence or absence of the MBH coexplant and in the presence of control tissue coexplants. Possible trophic effects on GnRH axonal outgrowth were assessed by adding the GnRH axonal outgrowth in sectors I and II (which corresponds to the total outgrowth) in the presence and absence of the coexplants. Comparisons among groups were performed using one-way ANOVA or t test as appropriate.
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), and GnRH fibers, occasionally
extending over very long distances (up to 1800 µm), could be followed
on the surface of the chamber membrane as they exited the explants
(Fig. 2B). Most of these terminated in growth cones (Fig. 2, B and C).
Preabsorption of the antiserum with 10 µg GnRH peptide eliminated
reaction product in cells and fibers. All quantitative analyses were
performed using the SW1 antibody.
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). GnRH processes ramified extensively within the
entire POA explant, and numerous growth cones were visible therein.
Survival of the GnRH neurons in culture
After 1 day of culture, the E15-derived POA region (n = 13)
contained 281 ± 24 GnRH cells. During the first 7 days in
vitro this number declined moderately [day 4, 240 ± 16
cells (n = 21); day 7, 197 ± 14 cells (n = 21)] and
then stabilized after an additional 3 days (day 10, 216 ± 11;
n = 32). In some cultures, as many as 410 GnRH neurons could still
be detected at 7 days. The normal mouse brain contains approximately
800 GnRH neurons scattered in the basal forebrain, primarily in the
region of the diagonal band, POA, and hypothalamus (2).
For the 7-day cultures used in most experiments, the presence of a
cograft did not alter the number of surviving GnRH neurons (see Table 1). In addition, there was no difference
among the cultures in the number of GnRH cell bodies within the POA in
the region facing toward or away from any of the cocultures under any
condition tested. MBH-derived trophic factors were implicated in
studies by Daikoku using both organ cultures and transplantation
in vivo (27). The number of GnRH neurons derived from rat
early embryonic olfactory placode or vomeronasal organ was higher in
the presence of the MBH in culture or in animals with transplants. In
contrast, in our study the presence of the cocultivated MBH explant did
not influence either the survival or distribution of GnRH cell bodies
in the POA.
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). This effect was not seen with SC or
CE coexplants. GnRH axonal outgrowth toward the MBH was significantly
greater than that toward the CE (P < 0.05; Fig. 3A).
GnRH axons also extended significantly farther from the POA on the
membrane in the sector facing the MBH (P < 0.001; Fig. 3B) than opposite it. This effect was not observed with the control
tissues. Indeed, the GnRH axons extended farther in the sector away
from the CE (P < 0.02; Fig. 3B). For P1 tissue (n
= 28), the number of GnRH axons growing in sector I (64.8 ± 6.8)
was also significantly greater than that in sector II (38.3 ±
5.2) in POA-MBH cocultures (P < 0.01).
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To assess whether the MBH was providing trophic support for GnRH axons, we counted the total number of GnRH axons extending out of the POA explants grown alone or with the MBH in E15 cultures. The total number of GnRH axons on the membrane did not differ between the groups [POA alone, 42.2 ± 8.9 (n = 12); POA-MBH, 50.4 ± 4.5 (n = 48)].
Effect of time on GnRH axonal outgrowth
One possible explanation for differential GnRH axonal outgrowth
toward the MBH after 7-day cultures is that the target provides support
for the axons that project nearby. Under such conditions, GnRH axons
would initially grow equally in all directions, but only those
extending toward the target would be maintained. To assess this
possibility, we performed a time-course analysis of GnRH axonal
outgrowth in the presence of a MBH coexplant using E15 tissue.
The total number of GnRH fibers increased steadily between days 1–10
of culture (Fig. 4A). The preferential
location of GnRH fibers in sector I reached significance on day 4
(P < 0.01) and was maintained until day 10
(P < 0.05). The extent of GnRH axonal outgrowth on the
membrane from the POA explant was significantly longer facing the MBH
explant than opposite from it as early as day 4 and at every later time
point (P < 0.001; Fig. 4B).
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, A and B).
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), with numerous growth cones. GnRH axons grew over comparatively
shorter distances from the POA onto the membrane and were invariably
found in the company of GAP-43 axons. Observations using confocal
microscopy confirmed that when associations between GnRH and GAP-43
axons were observed, GnRH axons traveled on top of GAP-43 axons (Fig. 6A), such that some GnRH fibers followed
a bundle of GAP-43 axons (Fig. 6B), and others traveled on the surface
of a network of GAP-43 fibers (Fig. 6C). Every GnRH axon observed with
confocal microscopy was consistently found in the same confocal plane
(estimated to be 0.5 µm) as GAP-43 axons.
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), such that GnRH axons extending onto
the membrane were not found in close proximity to GFAP-ir cellular
elements (Fig. 6E). Vimentin-ir cells and processes extended further
onto the membrane (Fig. 6F), but GnRH axons were not associated with
them either and did not align along vimentin-ir elements. Occasionally,
a GnRH axon would cross over a vimentin-ir process or cell, but such an
occurrence seemed fortuitous. Most GnRH axons grew past the extent of
the vimentin processes on the membrane and were then found
unaccompanied (Fig. 6G). S-100 antibody-labeled small astrocytes
sparsely scattered on the membrane around the explants, with processes
confined to the immediate surroundings of the cell bodies. GnRH axons
did not contact S-100-labeled cells (Fig. 6, H and I).
Presence of other cellular elements on the membrane surrounding the
explants
A cresyl violet counterstain as well as direct observation with
phase contrast microscopy revealed the presence of cells of globular or
spindle-like shape on the membrane. These cells did not extend on the
entire surface of the membrane, but remained confined in the region of
membrane surrounding the explants. Only erratic associations of GnRH
axons with such elements were observed (results not shown).
Presence of the median eminence within the MBH explant
The median eminence was visible in most of the MBH explants
dissected from P1-derived tissue after 7 days in culture. In single
labeled experiments, GnRH axons concentrated in one region within the
MBH (not shown). In cultures double labeled for GnRH and glial-specific
antibodies, tanycytes, the specialized ependymal cells of the MBH were
identified by their intermediate filaments, vimentin or GFAP. Tanycytes
formed the characteristic arched palisade arrangement seen in
vivo. GnRH axons terminated among them (Fig. 6J).
Intrinsic effects of the POA region on GnRH axonal outgrowth
Within the majority of POA explants, regardless of the coexplant,
GnRH fibers tended to form dense, highly branched plexi. The ependymal
lining derived from the third ventricle was often visible, and GnRH
fibers were abundant in this region. The morphological appearance of
this area resembled the organosum vasculosum of the lamina terminalis
(OVLT), a circumventricular organ located at the anterior tip of the
third ventricle that receives an abundant GnRH innervation in the
normal animal (30). Within the POA explant, tanycytes, immunoreactive
for vimentin or GFAP, were detected (Fig. 6K), and GnRH axons were seen
closely associated with these processes. Occasionally some POA explants
separated into two pieces of tissue. When this happened, numerous GnRH
terminals were seen crossing between the two segments.
It is possible that the POA explant itself possesses some attractive properties, influencing GnRH outgrowth. To assess this hypothesis, cultures were performed in which the POA was intentionally dissected into two halves and placed separately on the surface of the membrane. The number of GnRH axons extending between the two pieces of POA was significantly higher than the number of GnRH terminals extending away from the pieces of POA (49.7 ± 5.8 and 31.8 ± 4.3; n = 24; P < 0.05).
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Discussion |
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The MBH, dissected to contain the median eminence, exerted an action on GnRH axons that resulted in both longer and greater numbers of GnRH axons growing on the membrane region facing the MBH explant. This result was not the consequence of an uneven distribution of GnRH cell bodies within the explant, nor was it a nonspecific effect exerted on all axonal outgrowth, as axons labeled with GAP-43 did not show such directionality. This preference for GnRH outgrowth toward an explant was specific to the MBH, in that neither spinal cord nor cerebellum attracted GnRH axons.
Diffusible factors have been previously described to have chemoattractant actions involved in the guidance of axons (16, 31). For example, cortical axons, which form collaterals that grow toward the basilar pons, follow a diffusible signal originating from their target, as demonstrated with organotypic cultures in three-dimensional collagen gel matrexes (32). Similarly, visual cortical axons project directly in culture to their appropriate thalamic target (33). Trigeminal neurons project to peripheral targets using a similar mechanism, although the molecular nature of the signal is not yet known (26, 34). The floor plate of the spinal cord secretes diffusible factors that orient the outgrowth of commissural axons within collagen gel matrexes (35). This latter observation has led to the identification and isolation of netrins, expressed by floor plate cells. Netrins form a family of factors involved in the guidance of commissural axons of the spinal cord (36). They are homologous to a protein related to laminin involved in similar action in the nematode Caenorhabditis elegans (37). Studies of UNC-6, the nematode netrin, show that it is structurally closely related to laminin and might interact with it to promote the establishment of diffusible gradients involved in axonal guidance (38). However, netrin is unlikely to be involved in the guidance of GnRH axons, as there was an absence of directional outgrowth of GnRH axons toward the spinal cord in our control experiments. In addition, the expression of netrin is not confined to the region of the median eminence, but extends along the ventral midline of the embryo.
The fact that more and longer axons were present in sector I vs. sector II from days 4–10 suggests that a concentration gradient between the MBH and the POA was established early and was maintained (39). In studies attempting to demonstrate that outgrowth follows a concentration gradient established by the release of a diffusible target-derived substance, one consideration is the distance separating the target from the innervating source. In our culture conditions, the distance separating the POA and the coexplants did not affect GnRH axonal outgrowth. In a study of sensory fiber targeting, Lumsden and Davies (26) showed that trigeminal ganglion explants could be placed one behind the other in collagen gel cushions, and that both would still extend axons in the direction of the target. The researchers interpreted this to indicate the maintenance of a gradient of diffusible factor(s) past the boundaries of the first ganglion. This gradient directed axonal outgrowth from the most distal ganglion regardless of the distance. Similar to Lumsden’s experiment, GnRH axons in the present study adopted their pattern of preferential outgrowth whether they were close to or distant from the MBH explant, suggesting that a concentration gradient was the important factor influencing GnRH outgrowth and challenging the purely trophic hypothesis, where the more distant the target the less effective the outgrowth.
Unfortunately, we cannot perform an experiment similar to that conducted by Lumsden and Davies, as the POA itself has attractive properties. In vivo, there are abundant GnRH axonal projections to the OVLT, which is located close to the majority of the GnRH cell bodies (30). The presence of an intrinsic target such as the OVLT would account for the abundant projection of GnRH terminals within the explant and in between two separated pieces of POA. As the POA possesses a target for GnRH projections in culture, it is less surprising that so few GnRH axons succeeded in extending out of the explant in the direction of the MBH. This observation emphasizes the power of the median eminence as a target, as axons exiting the POA have to overcome an intrinsic attraction.
The persistence of a mediobasal hypothalamic effect on GnRH outgrowth after 10 days of culture suggests that the factors involved were synthesized by this brain region rather than simply associated with the tissue at the time of explantation. Blood-borne factors present at the time of dissection would probably be destroyed over this extensive time course. In turn, this supports our hypothesis that the diffusible signal is at least partly acting via a chemotaxic mechanism. One would expect that a neurotrophic substance diffusing throughout the undisturbed culture dish over 10 days would lead to a decline in the ratio of sector I vs. sector II and disappearance of significant differences between the two sectors. Further, the directionality rather than the total amount of GnRH axons growing out on the membrane was influenced by the presence of the MBH. The progressive increase in GnRH outgrowth facing the MBH in time-course experiments, with the outgrowth in the opposite sector always remaining lower, as well as the absence of influence of the MBH on the total number of axons exiting the POA suggest that mechanisms other than preferential maintenance of axons close to the target are involved.
Although there appear to be chemoattractant factors for GnRH axons from
the median eminence/MBH, little else is yet known about these
materials. Circumventricular organs, such as the median eminence and
the OVLT, may be attractive for GnRH axons because of their special
nature, which includes the presence of tanycytes, specialized ependymal
cells lining the walls of the third ventricle, and fenestrated
endothelial cells (40, 41). The secretion(s) of these endothelial cells
might reach the nervous tissue to direct axonal outgrowth. Endothelial
cells have been shown to release many substances, including
endothelins, which seem to have an important physiological role in
regulating neuropeptide secretion (42). Tanycytes may also release
diffusible trophic or tropic factors, including insulin-like growth
factor I (43) and transforming growth factor-
(44).
Alternative possibilities have been suggested to explain the specificity of an axonal projection to its target. On-going communication between axons and their target was hypothesized to regulate the secretion of chemotropic factors in the developing spinal cord. For example, substance P, secreted by some commissural terminals, increases the release of chemoattractants from the floor plate (45). However, the secretion of the GnRH peptide by GnRH axons is not necessary for proper targeting to the median eminence; axons originating from cell bodies containing the defective GnRH gene of hypogonadal mice, unable to synthesize the peptide, nevertheless reach the median eminence (46).
The extent of GnRH outgrowth from the POA explant was longer on the side away from the cerebellum explant. These findings suggest that the cerebellum may have some repulsive activity on GnRH outgrowth. Although diffusible substances such as netrins have been shown to have chemoattractive actions on developing commissural axons in the embryonic spinal cord (36), it was recently demonstrated that the same proteins can have a chemorepellent activity toward other sets of axons, such as the trochlear motor axons (47). Other examples suggesting the presence of chemorepulsive factors include the repulsion of olfactory bulb axons by the septum (48), the inhibitory effect of connectin, expressed by muscles, on motoneuron growth cones (49), and the family of semaphorins, which may be either transmembrane or secreted repellent molecules (50). Chemoattraction and chemorepulsion appear to act in concert to assure the proper guidance of axons to their target over long distances by creating inhibitory and attractive territories along axonal projection pathways.
In addition to following a concentration gradient of diffusible signals, GnRH axons may also need a specific substrate for proper guidance. The absence of association between GnRH axons extending on the membrane with glial elements suggests that the signals guiding GnRH axonal outgrowth toward the MBH are not provided by a direct cell to cell interaction with the surrounding glial environment. The interactions of neurons with glial elements during development to provide substrate-bound guidance to specific axonal projections have been widely documented (51, 52, 53). In our study, despite the presence of tanycytes in the preparation, we did not find consistent interactions of growing GnRH axons with these glial elements on the membrane. In contrast, a close association existed between GnRH- and GAP-43-positive axons extending on the membrane. GAP-43 labels most if not all growing axons, and this association may provide a facilitative substrate. Our experiments, which require fixing and immunocytochemistry staining, do not allow us to study whether GnRH axons dynamically follow some other elongating axons.
The precise nature of the guidance mechanism of the GnRH axons is still unknown. The present experiments were designed to test the hypothesis that the MBH provides signals to guide the GnRH axons to their target, the median eminence. These in vitro studies as well as the prior in vivo transplantation experiments clearly show that the MBH produces a diffusible factor whose most potent effect is to act as a chemoattractant.
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Acknowledgments |
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Footnotes |
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Received April 15, 1997.
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M. S. Deiner and D. W. Sretavan Altered Midline Axon Pathways and Ectopic Neurons in the Developing Hypothalamus of Netrin-1- and DCC-Deficient Mice J. Neurosci., November 15, 1999; 19(22): 9900 - 9912. [Abstract] [Full Text] [PDF]
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,
P < 0.05). The number of axons in sector I was
significantly greater than that in sector II on days 4 (••,
P < 0.01), 7 (•, P < 0.05),
and 10 (•, P < 0.05). B, The extent of GnRH
axonal outgrowth from the POA on the membrane was longer in sector I,
facing the MBH, than in sector II, facing away from the MBH, on days 4,
7, and 10 of culture (••, P < 0.001).
