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BOR - Papers in Press, published online ahead of print November 9, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.046904
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BIOLOGY OF REPRODUCTION 74, 463–472 (2006)
DOI: 10.1095/biolreprod.105.046904
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Expression of a Dominant Negative FGF Receptor in Developing GNRH1 Neurons Disrupts Axon Outgrowth and Targeting to the Median Eminence1

John C. Gill 2 , and Pei-San Tsai 

Department of Integrative Physiology and the Center for Neuroscience, University of Colorado, Boulder, Colorado 80309-0354

ABSTRACT

During development, neurons that synthesize and release gonadotropin-releasing hormone (GNRH1) extend their axons to the median eminence (ME) to establish neurosecretory contacts necessary for hormone secretion. Signals that coordinate this process are not known, but could involve the activation of fibroblast growth factor receptors (FGFRs) expressed on developing GNRH1 neurons. Using both whole-animal and cell culture approaches, this study examines the direct role of FGFR signaling in the extension and guidance of GNRH1 axons to the ME. In vivo retrograde labeling with fluorogold (FG) first showed a significant reduction in the projections of GNRH1 axons to the circumventricular organs (including the ME) in transgenic mice expressing a dominant negative FGF receptor (dnFGFR) in GNRH1 neurons. Using a primary GNRH1 neuronal culture system, we examined if compromised axon extension and directional growth led to the reduced axon targeting efficiency seen in vivo. Primary cultures of GNRH1 neurons were established from Embryonic Day 15.5 embryos, an age when GNRH1 neurons are actively targeting the ME. Cultured GNRH1 neurons expressing dnFGFR (dnFGFR neurons) exhibited attenuated activation of signaling pathways and reduced neurite outgrowth in response to FGF2. Further, dnFGFR neurons failed to preferentially target neurites toward cocultured ME explant and FGF2-coated beads, suggesting a defect in axon pathfinding. Together, these findings describe a direct role of FGFR signaling in the elongation and guidance of GNRH1 axons to the ME.

developmental biology, embryo, gonadotropin-releasing hormone, kinases, neuroendocrinology

INTRODUCTION

The secretion of gonadotropin-releasing hormone (GNRH1) at the median eminence (ME) maintains vertebrate reproduction. In most vertebrates, the ME is vascularized by capillaries of the portal plexus that carry GNRH1 to the pituitary [1, 2]. During development, axons from GNRH1 neurons extend ventral caudally to contact the perivascular space of the ME to ensure the pulsatile release of GNRH1 into circulation. Developmental defects that compromise the ability of GNRH1 axons to reach the ME could lead to a deficiency in GNRH1 secretion, resulting in the failure to initiate sexual maturation and sterility.

The targeting of GNRH1 axons to the ME occurs after the migration of GNRH1 neurons from the peripheral nasal compartment into the medial preoptic area (POA) [3, 4]. Upon the arrival of GNRH1 neurons in the forebrain, diffusible signals emanating from the ME appear to provide chemical cues that attract the growing GNRH1 axons [5, 6]. This chemoattractive concept is supported by the ability of conditioned medium from the medial basal hypothalamus (containing ME) to attract GNRH1 axons in culture [6] and the ability of GNRH1 neurons transplanted into the brains of the hypogonadal (hpg) mice to properly target their axons to the ME of the recipient mice [7]. At present, the identity of chemoattractive factors from the ME remains largely unknown.

Fibroblast growth factors (FGFs) signal through a family of receptor tyrosine kinases, FGF receptors (FGFRs 1–4), by an interaction stabilized by the cofactors, heparin or heparin sulfate proteoglycans. The range of developmental roles mediated by FGFR signaling includes proliferation, differentiation, and survival of multiple cell types [8]. FGFR signaling contributes to neuronal migration and axonal pathfinding in various systems [9] and has previously been shown to be critical for several aspects of GNRH1 neuron development [10, 11].

Recently, a study suggested FGFR signaling might be involved in the targeting of GNRH1 axons to the ME. This study examined cultured GNRH1 axon outgrowth from embryonic POA explants and demonstrated that GNRH1 axons extended toward FGF2-soaked heparin-coated beads [12]. However, these results demonstrated only the effects of exogenous FGFs; the contribution of endogenous FGF signals in the regulation of this process was not determined. Thus, the goal of the present study is to investigate if endogenous signals acting through FGFRs contribute to the successful targeting of GNRH1 axons to the ME during development by stimulating axon elongation and directional growth.

To accomplish this goal, we used transgenic mice that were engineered to express a dominant negative FGF receptor (dnFGFR) specifically in GNRH1 neurons to disrupt the function of endogenous FGF receptors [11]. The expression of this transgene was detected in 79% of GNRH1 neurons and led to a 30% reduction in the GNRH1 neuron population, delayed puberty, and shortened reproductive life span [11]. Reproduction in these transgenic mice may also be compromised by a reduction in GNRH1 axonal fibers that innervate the ME. To test this hypothesis, we first performed a retrograde labeling experiment to determine if in vivo targeting of GNRH1 axons to the ME is compromised in these transgenic mice. Because the dynamics of axon extension were difficult to study in vivo, primary cultures of embryonic GNRH1 neurons were established to examine if neurite outgrowth and directional extension to cultured ME explant were disrupted in GNRH1 neurons expressing dnFGFR. The presence of more than 20 classical and unconventional FGFR ligands precluded the determination of the exact FGFR ligands involved, which is a goal beyond the scope of this study. However, the current approach enables us to clearly determine if FGFR signaling contributes directly to the elongation and preferential extension of GNRH1 axons to the ME.

MATERIALS AND METHODS

Animal Husbandry

All mice were housed in the animal facility under a 12L:12D cycle and fed water and rodent chow ad libitum. Embryos were generated through timed breeding of 2- to 6-mo-old females, with Embryonic Day (E) 0.5 being the morning that copulatory plugs were detected. All animal procedures complied with protocols approved by the Institutional Animal Care and Use Committee at the University of Colorado.

Transgenic Mice

Mice expressing a dnFGFR specifically in GNRH1 neurons, officially designated as Tg(Gnrh1-Fgfr1)1Tsai (abbreviated FGFRm mice) were previously described [11]. In these mice, the expression of a single transgene that codes for a truncated murine FGFR1 lacking the tyrosine kinase domain was targeted to GNRH1 neurons. These mice were derived from the mating of C57BL/6J x DBA/2J hybrid mice and their offspring.

To facilitate the live imaging of GNRH1 neurons in culture, all in vitro studies were conducted on GNRH1 neurons isolated from transgenic mice engineered to express enhanced-green fluorescent protein (GFP) specifically in GNRH1 neurons. Transgenic mice with Gnrh1 promoter-driven expression of enhanced GFP (abbreviated control mice), derived from the mating of C57BL/6J and CBA/J mice, were generated at the University of Virginia [13]. Control mice were used as a source of GNRH1 neurons with normal FGFR function. The expression of GFP in these animals has proven useful in the identification of live GNRH1 neurons in dissociated cultures [10].

To generate mice with the targeted expression of both GFP and dnFGFR in GNRH1 neurons, homozygous FGFRm mice were bred with homozygous control mice to generated F1 mice heterozygous for both Gfp and Tg(Gnrh1-Fgfr1)1Tsai. F1 mice were intercrossed, and the resulting F2 generation was screened for the presence of both Gfp and Tg(Gnrh1-Fgfr1)1Tsai transgenes by polymerase chain reaction (PCR) of genomic tail DNA. To ensure homozygosity, F2 mice positive for both transgenes were mated with nontransgenic mice. F2 mice were considered homozygous for both transgenes when 100% of their offspring from two consecutive litters (8–10 pups per litter) were positive for both transgenes. The resulting mice, homozygous for both Gfp and Tg(Gnrh1-Fgfr1)1Tsai, were designated as dnFGFR mice. A homozygous breeding pair was generated in this fashion and used as founders to generate all subsequent dnFGFR mouse embryos for this study.

Wild-Type Mice

Wild-type mice (abbreviated WT mice) were used as nontransgenic controls for the fluorogold (FG) retrograde labeling experiment and as the source for target explants in coculture experiments. All WT mice have the same genetic background as the FGFRm mice and were derived from the mating of C57BL/6J and DBA/2J mice and their offspring.

Imaging of GNRH1 Fibers in the ME of Control and dnFGFR Mice

The brains of one control male and one dnFGFR male (2–3 mo old) were removed, placed in a Petri dish, and its ventral surfaced viewed on an Olympus IX70 inverted microscope. GFP fluorescence of GNRH1-GFP fibers projecting to the surface of the ME was imaged using the appropriate fluorescent filter with a Retiga 2000R camera (Qimaging, Burnaby, BC, Canada). Identical exposure settings were used for both control and dnFGFR brains. Brain tissues were then trimmed and fixed with 4% paraformaldehyde, cryoprotected in 20% sucrose, and 60-µm coronal sections cut with a cryostat. Sections containing the ME were mounted, coverslipped, and immediately imaged as described above.

Retrograde Labeling of GNRH1 Neurons with FG

Four FGFRm males and four WT males (2–3 mo old) were lightly sedated by Metafane vapor (Shering-Plow, Union, NJ) and then given a single intraperitoneal injection of 40 mg/kg FG (Molecular Imaging Products, Ann Arbor, MI) dissolved in saline [14]. FG is a fluorescent retrograde tracer (excitation maxima = 365 nm, emission maxima = 565 nm) incorporated primarily by axon terminals in the circumventricular organs (CVOs), where the blood-brain barrier is absent. Five days after the FG injection, animals were killed by pentobarbital overdose and perfused intracardially with 50 ml of 4% paraformaldehyde. Brains were removed and postfixed for 1–2 h in the same fixative and cryoprotected in 20% sucrose. Cryostat sections (40 µm thickness) containing the medial septal region to the anterior paraventricular nucleus were collected, and alternating sections were processed for GNRH1 immunocytochemistry (ICC).

For GNRH1 ICC, sections were washed with 0.5% hydrogen peroxide in 0.1 M phosphate-buffered saline (PBS) containing 0.4% Triton X 100 (PBST) for 10 min to quench the endogenous peroxidase activity, rinsed five times with PBST, and incubated for 48 h at 4°C in PBST containing an anti-GNRH1 antibody (LR1, 1:20,000; a gift of Dr. Robert Benoit, Montreal General Hospital) and 4% normal donkey serum. After incubation, sections were washed with PBST and incubated with a biotinylated donkey-anti-rabbit IgG (1:400; Jackson Laboratory, West Grove, PA), washed, and incubated with Cy3-conjugated streptavidin (1:1500; Jackson Immunoresearch). Following extensive washes in PBS, sections were mounted on slides and coverslipped with an aqueous antifade medium (2% Tris-buffered DABCO, pH 8.5; Sigma).

On slides coded to conceal their identity, GNRH1 and FG signals were visualized using the standard rhodamine and long-pass ultraviolet filter sets, respectively, under a Leica SM RE microscope (Leica Microsystems, Exton, PA). The total number of GNRH1 neurons was scored first by counting Cy3-positive neurons with distinct cellular morphology and visible nuclei. Colocalization of FG in GNRH1 neurons was then determined by first focusing on a GNRH1 neuron under the rhodamine filter set (excitation BP 515–560 nm, emission LP 590 nm) and then switching to the ultraviolet filter set (excitation BP 340–380 nm, emission LP 425 nm) to visualize FG without readjusting the focus. GNRH1 neurons containing FG signals within the same focal plane were scored positive for colocalization.

Dissociated GNRH1 Neuron Cultures and Measurement of Neurite Length and Branching

GNRH1 neurons from E15.5 brains were isolated from control and dnFGFR embryos and dissociated to investigate the role of FGFR signaling in neurite extension. This age (E15.5) represents a period of heightened axon targeting and thus is an age suitable for this study. From each embryo, brain tissue approximately 1 x 2 mm in size was excised from the ventromedial area between the olfactory bulbs and the ME. This tissue was dissociated and the recovered cells were maintained in a serum-free medium (SFM) as previously described [10]. Both control and dnFGFR cultures were treated with various doses of recombinant human FGF2 (Promega Inc., Madison, WI) for 1 day in vitro (DIV). FGF2 was used because it is a prototypic FGF commonly used in FGFR signaling studies due to its high affinity for all FGFRs. Neurite length and branching in cultured GNRH1 neurons were measured after 1 DIV according to the method described previously [10]. Each dissociation experiment included brain tissues from an entire E15.5 litter (6–10 embryos each). After 1 DIV, the typical recovery of visible GFP-expressing GNRH1 neurons in culture was approximately 5% of the estimated GNRH1 population found in the entire brain. Because there were noticeable variations in their morphology and initial neurite length, we assumed the cultured population was heterogeneous. However, it is not clear how well the population in culture represents the original E15.5 ventromedial population in the brain. In all treatment groups, 30–40 GNRH1 neurons obtained from three separate dissociation experiments were measured.

ICC of FGFRs

To ensure dissociated GNRH1 neurons retain the ability to respond to FGFs, the presence of FGFRs in these cells was verified by FGFR ICC. For this, dissociated control and dnFGFR neurons were cultured for 1 DIV in SFM on glass coverslips coated with poly-D,L-lysine (5 µg/ml) and mouse laminin (5 µg/ml). Cells were fixed for 30 min in 4% paraformaldehyde and examined for the presence of FGFR proteins by ICC as previously reported [10]. The following anti-FGFR antisera (Santa Cruz Biotechnology, Santa Cruz, CA) were used: anti-FGFR1 (SC-121G), anti-FGFR2 (SC-122), and anti-FGFR3 (SC-123). The presence of FGFR4 was not examined because previous ICC of E15.5 brain sections failed to detect FGFR4 in GNRH1 neurons [10]. FGFR signals were visualized by the Cy3-tyramide signal amplification (TSA; Molecular Probes, Eugene, OR), whereas GNRH1 neurons were identified by the presence of the GFP signal. After ICC, all cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma). Images were collected using a Leica TCS SP2 AOBS laser scanning confocal microscope and presented as 2- to 4-µm-thick z-stack projections.

Signal Transduction Marker Bioassay

To ensure FGFR responsiveness is blunted in GNRH1 neurons expressing dnFGFR, the induction of extracellular signal-regulated protein kinases (ERKs), cyclic AMP-responsive element binding protein (CREB1), and FOS following FGF2 addition was compared between control and dnFGFR neurons. Dissociated control and dnFGFR neurons were plated at a density of 5 x 105 cells/cm2 in uncoated 12-well culture plates and cultured for a 2-h pretreatment period in unsupplemented DME/F12 (Invitrogen, Carlsbad, CA). Cultures were then treated with 0, 1, 10, or 50 ng/ml recombinant human FGF2 (Promega), a dose range previously shown to include stimulatory doses of FGF2 [10]. At 10, 30, or 60 min after the FGF2 treatment, cells were fixed in 4% paraformaldehyde and immunostained for phosphorylated ERKs (anti-pERKs; 1:500; Promega), phosphorylated CREB1 (anti-pCREB1; 1:1000; Upstate Biotech, Charlottesville, VA), or FOS (anti-FOS; 1:750; EMD Bioscience, San Diego, CA). Primary antibody incubation was conducted overnight at 4°C in the presence of 4% normal donkey serum. Following three 5-min washes, cultures were incubated with a biotinylated donkey-anti-rabbit IgG (1:400; Jackson Immunoresearch) for 60 min, washed, and incubated with streptavidin-conjugated Cy3 (1:1500; Jackson Immunoresearch) for 60 min.

The activation of the three signaling markers was measured by a semiquantitative method. Using this method, levels of marker immunofluorescence in randomly identified GNRH1 neurons were measured. GNRH1 neurons were first identified by the GFP signal under an Olympus IX70 inverted microscope. For each GNRH1 neuron, images were captured with a SPOT digital camera (Diagnostic Industries, Inc., Sterling Heights, MI) using first the fluorescein (for GFP) and then the rhodamine (for Cy3) filter set. Image analysis was conducted in Adobe Photoshop by first tracing with a mouse the perimeter of the GFP neurons with the program's Lasso tool. This delineates the soma of the GNRH1 neuron to be measured. The entire soma was measured because activated markers could be found in both the cytoplasm and the nucleus. Within the traced area, Cy3 brightness levels were measured with the histogram function to obtain mean pixel intensity of the red channel. Net pixel intensity (NPI) was calculated by subtracting the background Cy3 intensity of the surrounding cell-free area from Cy intensity within the traced soma area. Final data, presented as percent control, were derived by dividing the values of NPI of FGF2-treated cells by the NPI of control cells (no FGF2 treatment). These final data reflect FGF2-induced changes in the intensity of the marker immunofluorescence (and thus marker protein levels) in randomly sampled GNRH1 neurons.

Neurite Extension and Directional Outgrowth in Cocultures with Test Targets

A coculture system was employed to study the effects of test targets on GNRH1 neurite extension and directional growth. Dissociated GNRH1 neurons were harvested from E15.5 control or dnFGFR embryos and cocultured with the test target in SFM. Dissociated cells were adjusted to 7.5 x 105 cells per ml and plated uniformly on poly-D,L-lysine- and laminin-coated 12-well plates. The estimated cell density in wells was 1.4 x 105 cells/cm2. Test targets (see below) or heparin beads soaked with recombinant human FGF2 or FGF8 isoform b (FGF8B; R&D Systems, Minneapolis, MN) were adhered to the center of the wells with 10 µl growth factor-reduced Matrigel (Becton Dickson, Franklin Lakes, NJ). FGF2 and FGF8B were chosen for this study because the former represents a prototypic FGF capable of binding to all FGFRs, and the latter is a secretable splice variant of FGF8 produced by the ME [15] and has been shown to play a role in axon targeting [16]. FGF2- and FGF8B-soaked heparin beads were prepared as described [12]. Test targets included ME or cerebellum (CB) explants, each approximately 2 x 2 mm in size, isolated from Postnatal Day (P) 21 WT mice. Negative controls included Matrigel alone (blank) or beads prepared with no growth factors (blank beads). After 1 DIV, GNRH1 neurons were identified by GFP fluorescence and imaged with a SPOT digital camera. The position of each GNRH1 neuron, assessed by the positions of the X- and Y-axes of the microscope stage, was recorded.

Based on the recorded coordinates, a photomontage was reconstructed to represent the relative positions of the GNRH1 neuron, its neurites, and the test target. All neurons scored were randomly distributed in the culture wells, and there were no differences in the average distance to target between control and dnFGFR cultures. Figure 6A illustrates the method used to score the direction of neurites. GNRH1 neurites were given a directional score depending on if their terminals extended into the zone with the target (score = toward) or the zone without the target (score = away; Fig. 6A). For data analysis, the number of neurites growing toward a test target was divided by the total number of neurites scored (toward + away). This percentage value was designated as the chemoattractive index. Chemoattractive indices of cultures containing explants or FGF beads were normalized to those of the appropriate experimental controls in which only a blank target or uncoated beads were present. In addition to neurite direction, neurite lengths were also measured. GNRH1 neurons (n = 40–80 for explant experiments and n = 20–30 for bead experiments) from at least three separate dissociation experiments were scored in this fashion for each target.


Figure 6
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  		FIG. 6. A) Diagrammatic demonstration of the coculture system used to assess GNRH1 neurite growth and targeting to the test target. The shortest distance from the cell body to the target was indicated with line 1, and another line perpendicular to it (line 2) defines neurite growth toward (T) or away (A) from the target. B) GNRH1 neurite length in dissociated control or dnFGFR neurons cocultured with one of the target explants for 1 DIV. * P < 0.05 between control and dnFGFR neurons within the same test target group (Mann-Whitney U-test). Each bar represents mean ± SEM, n = 30–50. Two-way ANOVA indicated GNRH1 neurite length was significantly affected by the type of target (P = 0.0009) and genotype (P = 0.0401). There is no interaction between target and genotype

Statistics

For all analyses, parametric statistics were used with data that satisfied Bartlett tests for equal variances. Otherwise, data were square-root transformed or nonparametric statistics were used. Differences in percent FG-positive GNRH1 neurons were analyzed by Mann-Whitney U-test and two-way ANOVA. Signal transduction assay values were analyzed by one-way ANOVA and Student-Newmann-Keul post hoc tests; differences between genotypes within the same treatments were compared by the Student t-test. Differences in neurite lengths and branching were analyzed by one-way ANOVA followed by Student-Newmann-Keul or two-way ANOVA. Mann-Whitney U-test was used to analyze differences in neurite length in explant cocultures between genotypes. Differences in normalized chemoattractive indices between genotypes were analyzed by Student t-test. Differences were considered significant when P < 0.05.

RESULTS

Reduced GNRH1 Axon Terminals in the ME of dnFGFR Mice

The loss of GNRH1 fibers in the ME of FGFRm mice has been documented previously [11]. In this experiment, we confirmed this loss and showed, by GFP signal, that the defect was also present in dnFGFR mice. In unfixed whole brains, the ME is positive for GFP signal and clearly visible at the medial aspect of the ventral surface of both control (Fig. 1A) and dnFGFR (Fig. 1B) mice. A visible reduction in GFP intensity was observed in the ME of dnFGFR mouse compared with control mouse. Following fixation, frozen sections cut at the level of the ME clearly showed fewer GFP-positive fibers in the ME of dnFGFR (Fig. 1D) mouse compared with control mouse (Fig. 1C). The reduction of GFP-expressing GNRH1 neurosecretory terminals was consistently observed through all sections of the dnFGFR ME (data not shown).


Figure 1
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  		FIG. 1. Transgenic mice expressing dnFGFR in GNRH1 neurons exhibited reduced innervation by GNRH1 neurosecretory fibers at the ME. GFP signals, indicative of GNRH1 fibers, were imaged from the ventral surface of the whole brains isolated from control (A) and dnFGFR (B) mice. Arrowheads indicate the ME and the plane of section of images C and D. C and D) Coronal sections through the ME of the brains in A and B following fixation and cryosectioning. A marked difference was observed in the GFP fluorescence at the medial aspect of the ME (arrow) between control (C) and dnFGFR ME (D). R, Rostral; C, caudal; L, lateral; 3V, third ventricle. Bars = 250 µm (A, B) and 100 µm (C, D)

Reduced GNRH1 Axon Targeting in FGFRm Mice

An in vivo FG uptake study was conducted to examine if GNRH1 neurons from FGFRm mice showed reduced targeting to the ME. FG is a retrograde tracer taken up selectively by brain regions lacking the blood-brain barrier, including the ME. To ensure effective FG labeling, two brain regions, the paraventricular nuclei (PVN) and the organum vasculosum of the lamina terminalis (OVLT), were examined as positive controls. The magnocellular and parvocellular neurons of the PVN project to the neurohypophysis and the ME, respectively, and both targets lie outside the blood-brain barrier. Similarly, no blood-brain barrier exists in the OVLT, a CVO. The selective FG labeling of the PVN (Fig. 2A) and cells around the OVLT (Fig. 2, B and C), a secondary target of GNRH1 axons, confirmed the uptake in neurosecretory nuclei or regions lacking a blood-brain barrier. Here, and as previously reported, the color of FG appeared bright yellow to orange when examined at neutral to high pH [17]. Other brain areas protected by the blood-brain barrier, such as the striatum and cortex, were devoid of any FG labeling.


Figure 2
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  		FIG. 2. Peripherally injected FG retrogradely labels neurons and axons in contact with vasculature outside the blood-brain barrier. A) Specific FG labeling (orange) of the paraventricular nuclei in a coronal brain section of a WT mouse; asterisk indicates third ventricle. Fibers in OVLT (arrow) of a WT mouse are positive for both GNRH1 (red, B) and FG (C). A single GNRH1 neuron from a WT mouse showing overlapping signals for GNRH1 (D) and FG (E). Bars = 200 µm (A); 100 µm (B, C); and 8 µm (D, E). F) When quantified, the percentage of FG-positive GNRH1 neurons was significantly lower in FGFRm compared with WT mice (* P = 0.0002, n = 4, Mann-Whitney U-test). G) The reduction in FG-positive GNRH1 neurons in FGFRm mice was not brain-region specific. The X-axis, from left to right, represents the rostral to caudal forebrain sequence. OVLT was used as a landmark and denoted as 0 µm. Negative numbers on the X-axis indicate distance rostral to the OVLT, and positive numbers indicate distance caudal to the OVLT. Each error bar represents ± SEM

GNRH1 neurons in both WT and FGFRm mice were visualized by Cy3 immunostaining. No visible differences in cellular morphology were observed in GNRH1 neurons between the genotypes. FG labeling in GNRH1 neurons varied from smooth cytoplasmic staining that extended into GNRH1 neuronal processes to sparse puncta within GNRH1 neurons of both WT (Fig. 2, D and E) and FGFRm mice (not shown). The proportion of FG-labeled GNRH1 neurons was consistent with the 65–90% efficiency reported previously [14, 18, 19]. Percentage of GNRH1 neurons labeled with FG was significantly lower in FGFRm mice than WT (Fig. 2F). The reduction in FG-positive GNRH1 neurons occurred in most sections examined and was not specific to any particular brain region in the FGFRm mice (Fig. 2G). Confirming these observations, two-way ANOVA revealed the number of FG-positive GNRH1 neurons was significantly affected by the genotype (WT vs. FGFRm; P < 0.0001) but not by the location of neurons (P = 0.1459). No significant interaction was observed between the genotype and neuron location (P = 0.4237; two-way ANOVA). A GNRH1 neuronal count conducted on alternating sections between the medial septal nucleus and anteroventral periventricular nucleus showed a 30% reduction in FGFRm mice (182.8 ± 10.75 GNRH1 neurons) compared with WT mice (250 ± 17.75 GNRH1 neurons), a finding consistent with the previous report [11].

FGFRs Are Expressed in Primary Cultures of E15.5 GNRH1-GFP Neurons

To investigate if developing GNRH1 neurons have the ability to respond directly to FGFR-mediated signals, primary cultures of E15.5 GNRH1 were examined for the presence of FGFRs by ICC. This information is important in ascertaining if biological effects induced by FGFR signaling can be direct upon cultured GNRH1 neurons. In dissociated control GNRH1 neurons, approximately 60%, 10%, and 70% were positive for FGFRs 1, 2, and 3, respectively (Fig. 3, A–C). As described previously from our laboratory and other laboratories, FGFR1 was observed in the cytoplasm [10] (Fig. 3A), and FGFR3 immunoreactivity was restricted to the nucleus [20] (Fig. 3C). FGFR2 was present in only a very small fraction of cultured GNRH1 neurons and appeared as localized punctate staining in the cytoplasm (Fig. 3B). In dissociated dnFGFR GNRH1 neurons, approximately 70%, 50%, and 75% were positive for FGFRs 1, 2, and 3, respectively (Fig. 3, D–F). Because all FGFR antibodies were directed to the C-terminal, they do not detect dnFGFR, which lacks the C-terminal domain. There were no visible differences in the subcellular localization of FGFRs between control and dnFGFR GNRH1 neurons. These experiments showed, for the first time, dissociated GNRH1 neurons in culture expressed FGFRs and thus retained the ability to respond directly to FGFR-mediated signals.


Figure 3
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  		FIG. 3. Colocalization of FGFRs (red) in dissociated control and dnFGFR neurons (identified by GFP, green) by ICC. DAPI (blue) was used as a nuclear counterstain. Representative control neurons (A–C) and dnFGFR neurons (D–F) positive for FGFR1 (A, D), FGFR2 (B, E), and FGFR3 (C, F) are shown. Arrowheads indicate FGFR immunoreactivity. Each image is a 1.5-µm projection of a z-stack captured by a confocal microscope. Bars = 8 µm

Attenuation of FGFR Signaling in dnFGFR Neurons

To ensure that the expression of a dnFGFR in GNRH1 neurons attenuates FGFR responsiveness in dissociated GNRH1 neurons in culture, several signal transduction markers associated with FGFR activation were examined in control and dnFGFR. This is a critical control experiment because the attenuation of FGFR signaling in these dnFGFR neurons has never been directly demonstrated. Control neurons responded to FGF2 exposure for 30 min (1, 10, and 50 ng/ml) with a significant activation of ERKs and CREB1 (Fig. 4, A and B). Interestingly, dnFGFR neurons responded differently under the same treatment condition (Fig. 4, A and B). Whereas CREB1 was activated by both 10 and 50 ng/ml of FGF2, only 1 ng/ml FGF2 was effective in activating ERKs in dnFGFR neurons. Under the 30-min FGF2 treatment, control neurons consistently had higher levels of ERK and CREB1 activation compared with dnFGFR neurons (Fig. 4, A and B), indicating attenuated FGFR responsiveness in dnFGFR neurons. FGF2 exposure for 10 min did not activate ERKs in either control or dnFGFR neurons (Fig. 4A) but activated CREB1 at the highest dose (Fig. 4B). Representative photomicrographs of control neurons showed the increased accumulation of pCREB1 after 10 and 30 min of 50 ng/ml FGF2 treatment (Fig. 4, C–E).


Figure 4
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  		FIG. 4. FGF2-induced activation of ERKs (A), CREB1 (B–E), and FOS (F) in dissociated E15.5 control and dnFGFR neurons. ERKs (A) and CREB1 (B) activation were assessed in control (white bars) and dnFGFR (black bars) at 10 and 30 min after FGF2 addition. Dissimilar letters indicate P < 0.05 between treatment groups for control (upper case) and dnFGFR (lower case) neurons. * P < 0.05 between control and dnFGFR neurons within the same treatment. Representative confocal images of GFP-expressing GNRH1 control neurons (green) showing low level of pCREB1 in the absence of FGF2 (C) and increased accumulation of pCREB1 (red) after 10 (D) and 30 (E) min of FGF2 (50 ng/ml) treatment. Bars = 8 µm. F) FOS, activation was assessed in control (white bars) and dnFGFR (black bars) at 60 min after FGF2 addition. * P < 0.05 between control and FGF2-treated groups. In all graphs, each bar represents mean ± SEM, n = 15

Last, the induction of FOS was compared between control and dnFGFR neurons following a 60-min incubation with or without FGF2 (50 ng/ml). Only control neurons responded to FGF2 treatment with significantly elevated levels of FOS. FGF2 failed to induce the appearance of FOS immunoreactivity in dnFGFR GNRH1 neurons (Fig. 4F). Together, these results confirmed a significantly reduced responsiveness of dnFGFR neurons to FGFR stimulation.

Reduced Neurite Outgrowth in Primary Cultures of dnFGFR Neurons

After confirming the blunted FGF responsiveness in dnFGFR neurons, the next step is to ascertain if this attenuated signaling pathway interferes with an important component of GNRH1 axon targeting—the elongation and branching of axons. For this, we investigated if the length and number of neurites in dnFGFR neurons were reduced compared with control neurons.

Quantification of neurite lengths confirmed dnFGFR neurons were defective in neurite extension (Fig. 5A). Whereas FGF2 at 10 and 50 ng/ml significantly stimulated neurite outgrowth in control neurons (Fig. 5A), FGF2 stimulated neurite outgrowth in dnFGFR neurons only at the highest dose (50 ng/ml; Fig. 5A). In all treatment groups, dnFGFR neurons had markedly shorter neurites compared with control neurons (Fig. 5A). Two-way ANOVA confirmed a significant inhibitory effect of the dnFGFR transgene on neurite length (P < 0.0001). To assess the degree of neurite branching in control and dnFGFR neurons, the number of neurites per GNRH1 neuron was scored (Fig. 5B). Although FGF2 treatment did not significantly alter the number of neurites in control or dnFGFR neurons, the number of neurites was significantly affected by the presence of the dnFGFR transgene (two-way ANOVA; P = 0.016). However, because many dnFGFR neurons had no neurites, this observation likely reflected the reduced ability of dnFGFR neurons to sprout neurites rather than a difference in branching.


Figure 5
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  		FIG. 5. Neurite outgrowth in dissociated control and dnFGFR GNRH1 neurons after 1 DIV. Quantification of neurite length (A) and number of branches (B) in control (white bars) and dnFGFR neurons (black bars). Dissimilar letters indicate P < 0.05 between FGF treatments within the control (lowercase) or the dnFGFR group (uppercase). Each bar represents mean ± SEM, n = 30–40. C) An image of a single dissociated control GNRH1 neurons treated with FGF2 (50 ng/ml) for 1 DIV. Bar = 20 µm

Diffusible Factors from Median Eminence Influence Neurite Outgrowth

The previous experiment confirmed exogenous FGF2 stimulated GNRH1 neurite outgrowth and attenuating FGFR activity blunted this stimulation. However, it was not clear if endogenous FGF signals emanating from the ME provide a physiological cue to promote GNRH1 axon extension. To test this, a method that employed the coculture of dissociated GNRH1 neurons and a test target was used to examine the ability of the target to influence the length and directional outgrowth of GNRH1 neurites (Fig. 6A). After 1 DIV, total neurite length was significantly affected by the type of target explant in the coculture (Fig. 6B; two-way ANOVA, P = 0.0009). Both control and dnFGFR neurites were longest when cocultured with ME compared with CB or blank. However, neurite length was also significantly affected by the presence of the dnFGFR transgene (two-way ANOVA, P = 0.0401). For GNRH1 neurons cocultured with blank or CB, shorter neurites were measured in dnFGFR neurons compared with control neurons (Fig. 6B). In ME cocultures, large variability in neurite length was observed in control neurons. Consequently, no statistically significant difference between control (n = 51) and dnFGFR neurons (n = 41) was seen (Fig. 6B). This experiment suggests that higher levels of FGF signals emanate from the ME than blank and CB. However, because control and dnFGFR neurons do not differ significantly in neurite length when cocultured with the ME, FGF signals from the ME were either insufficient or unlikely the sole contributors to the lengthening of GNRH1 axons.

GNRH1 Neurites Target Median Eminence Explants and FGF2 Beads

In order for GNRH1 axons to successfully target the ME, the axons must not only grow but also grow in a directional manner toward the target. The previous experiments confirmed an effect of FGFR signaling on the former. In this experiment, we examined if FGF signals emanating from the ME are important for the process of directional extension.

Using the same coculture method described above (see Fig. 6A), we examined if cultured GNRH1 neurons extend neurites preferentially toward a physiological target (ME) or a nonphysiological target (CB; Fig. 7A). Neurites growing randomly without the directional influence of target would have a chemoattractive index of 50%; that is, 50% of neurites would be growing toward target and 50% growing away. Consistent with this, chemoattractive indices of cultures with Matrigel-only negative control (blank) were 46.5% ± 6.24% for control neurons and 50.51% ± 6.74% for dnFGFR neurons. Coculturing with ME explant elevated the chemoattractive index of control neurites to approximately 150% of blank cultures (Fig. 7A), demonstrating a growth preference toward this target. Interestingly, this preferential growth of the neurites toward ME was not observed in dnFGFR neurons (Fig. 7A). Neither control nor dnFGFR neurons preferentially targeted CB (Fig. 7A). To determine if neurite length and directionality are correlated, e.g., longer neurites are better able to find their targets, or chemoattractants from the targets stimulate neurite outgrowth, we compared the length of neurites according to their direction of growth. The lengths of neurites growing toward explant targets were not different from the lengths of neurites growing away from any explant target for either control or dnFGFR GNRH1 neurons (data not shown).


Figure 7
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  		FIG. 7. Control neurons, but not dnFGFR neurons, preferentially targeted to the ME and FGF2 beads. A) Dissociated E15.5 control and dnFGFR neurons were cultured with tissue explants (ME or CB). The ME had a greater attractive effect on control neurites than dnFGFR neurites (* P < 0.05), but no difference was observed between the genotypes in cultures with CB. B) FGF2-soaked heparin beads had a greater attractive effect on control neurites compared with dnFGFR (* P < 0.05), but no difference was observed between the genotypes in cultures with FGF8B-soaked heparin beads. Dotted line though 100% value indicates the level of neurite attraction to the blank or uncoated beads. Data represent chemoattractive indices normalized against blank (A) or blank beads (B). Error bars represent ± SEM

To ascertain that the loss of FGFR responsiveness contributes, at least in part, to the inability of dnFGFR neurons to identify secretable cues, FGF2- and FGF8B-soaked heparin-coated beads were tested. Chemoattractive indices of neurites grown with uncoated beads again indicated random neurite growth (46.25% ± 10.3% for control and 57.99% ± 6.41% for dnFGFR neurons). As expected, FGF2-soaked beads attracted control neurites (Fig. 7B). In contrast, the preference for FGF2 beads was not observed in dnFGFR neurons (Fig. 7B). Neither control nor dnFGFR neurons preferentially targeted the FGF8B-coated beads.

DISCUSSION

In this study, we demonstrated that FGFR signaling is involved in the extension and targeting of GNRH1 axons to the ME, a developmental process essential for GNRH1 secretion and ultimately fertility. Retrograde labeling in adult FGFRm mice showed compromised ability of GNRH1 axons to reach their targets. Analyses with primary cultures of embryonic GNRH1 neurons confirmed dnFGFR neurons exhibited blunted FGFR signaling and markedly reduced neurite outgrowth. Importantly, cultured embryonic dnFGFR neurons, unlike control neurons, were incapable of targeting their neurites preferentially toward the cocultured ME explant. These observations strongly suggest signals emanating from the ME guide the directional growth of GNRH1 axons by activating functional FGFRs expressed in GNRH1 neurons.

The reduced FG labeling efficiency in FGFRm mice likely resulted from the failure of GNRH1 axons to properly reach the ME during development. To our knowledge, postnatal alterations in GNRH1 axons that could reduce FG labeling, such as axonal pruning or rerouting, have not been previously observed. Also, in a previous study, we showed postnatal GNRH1 neurons were less likely to elaborate neurites than embryonic GNRH1 neurons [10]. This diminished capacity to remodel axons further implies the disruption shown by FG labeling occurred embryonically. However, attenuated FGFR signaling might interfere with the physical access of GNRH1 axons to portal capillaries in adult animals. In adults, dynamic alterations of such physical access can result from plastic changes in tanycytic end feet and basal lamina within the ME [21, 22]. However, the tissue-specific expression of the transgene should minimize disruption in non-GNRH1 cells, such as in the tanycytes, that may occlude GNRH1 axons from the vasculature or the vascular flow through the portal capillaries [11]. That said, we cannot exclude the possibility that dnFGFR might also interfere with plastic changes within GNRH1 neurons that are required for the postnatal access to the portal vessels. Regardless of the mechanisms, we believed that our results offer the first evidence that GNRH1 axon targeting is compromised when FGFR signaling has been compromised. The underlying cause of this defect requires further investigation.

Because FG labels all neurons with contacts outside the blood-brain barrier, this method cannot distinguish between GNRH1 neurons that project to the ME and those that project to other CVOs. Although the ME is the primary site innervated by GNRH1 axon terminals, GNRH1 axons also target the OVLT [23, 24] and, to a lesser extent, the subfornical organ [25, 26]. Because whole-animal FG experiments provided only circumstantial evidence of an ME-targeting defect, a series of cell culture experiments were performed to determine if attenuated FGFR signaling affected the outgrowth and directional targeting of GNRH1 neurites specifically to the ME.

The observation that FGFRs 1 and 3 are present in dissociated GNRH1 neurons confirmed these neurons remained competent to respond to FGFR signaling for up to 1 DIV. However, in contrast with a previous ICC study using tissue sections [10], a small fraction of control neurons also expressed FGFR2. This could reflect increased sensitivity of ICC detection in cultured cells [27] or dissociation-induced FGFR2 expression. Interestingly, more dnFGFR neurons were positive for FGFR2 compared with control GNRH1 neurons, suggesting a compensatory upregulation of FGFR2 in response to attenuated FGFR function.

The signal transduction markers, pERKs, pCREB1, and FOS, were rapidly activated in cultures of control GNRH1 neurons in response to FGF2 treatment. All three markers have been associated with the induction of neurite outgrowth by neurotrophic factors [28–31]. Phosphorylation of CREB1 has been shown in other cell types to occur downstream of activated ERKs [32], and the induction of FOS likely occurred downstream of CREB1 activation [33–35]. However, our results demonstrated significant CREB1 phosphorylation was achieved before ERK activation (Fig. 3A), perhaps suggesting the activation of a separate pathway that led to the phosphorylation of CREB1. The robust differences between control and dnFGFR in all three markers measured clearly indicated a significant reduction in dnFGFR cells' response to FGF2. Importantly, although dnFGFR neurons exhibited attenuated responsiveness to FGF2, they still retained partial responsiveness to FGFs and could be stimulated with higher doses or longer exposure to FGF2. This observation could explain, in part, why the GNRH1 system in FGFRm mice was only partially disrupted and not completely abolished.

Under the basal culture condition, control neurons consistently extended longer neurites than dnFGFR neurons. This observation was similar to the previous report that dnFGFR-transfected GT1 cells were largely devoid of neurites [11] and suggests FGFs locally produced in the cultures were responsible for maintaining the basal levels of neurite length. The ability of the highest dose (50 ng/ml) of FGF2 to stimulate neurite outgrowth in dnFGFR neurons again demonstrates that FGF responsiveness in these cells was only reduced, not eliminated.

Both control and dnFGFR neurons were cocultured with the ME explant to examine if FGFs emanating from the ME represent endogenous signals responsible for stimulating GNRH1 neurite extension and directional outgrowth. Surprisingly, the difference in neurite length between control and dnFGFR neurons was no longer significant when these neurons were cocultured with ME. These results suggest, in addition to FGFR ligands, other factors emanating from the ME were capable of supporting the extension of GNRH1 neurites. In general, the compromised neurite extension in dnFGFR neurons may result from the dominant negative interference with normal FGFR-mediated growth and differentiation in vivo in the transgenic mice before harvesting of the cells. Thus, the impairment of FGFR-dependent physiology in GNRH1 neurons may render them less capable of extending long neurites. This would explain the diminished magnitude of neurite extension that is exhibited by these cells in the presence of multiple targets and why additional signals from the ME may only partially override the deficit. Of interest was the finding that brain-derived neurotrophic factor, a factor present at high levels in the ME [36], was also a potent stimulator of neurite outgrowth in primary cultures of GNRH1 neurons [37]. These results represent an excellent example of the redundant regulatory mechanisms that exist to ensure the proper formation of an important neuroendocrine system.

In contrast with the less significant involvement in neurite extension, FGFR signaling appears to play a greater role in the directional guidance of neurites toward the ME. This conclusion was based on the observation that dnFGFR neurons lack the ability to preferentially direct their neurites toward cocultured ME explants. Interestingly, beads coated with FGF2, but not FGF8B, mimicked the chemoattractive effect of the ME. In vitro bioassays showed that both FGF2 and FGF8B were activated by isoforms of FGFRs 2–4, but only FGF2 possessed the affinity to activate FGFR1 [38, 39]. Collectively, these results suggest that FGFR1 might be the primary receptor subtype mediating the directional targeting of GNRH1 neurites toward the ME.

FGFR1 could mediate the directional guidance of GNRH1 axons via several mechanisms. The simplest mechanism involves the activation of FGFR1 by one or more unspecified FGFR ligands emanating from the ME to serve as chemoattractants for GNRH1 axons. In support of this notion, FGFs have been shown to act as chemoattractants that guide the migration of neural crest cells [40] and trochlear axons [16]. Our results that FGF2-coated beads attract GNRH1 neurites provide further support for this possibility. However, because FGFs bind heparan sulfate proteoglycans in the extracellular matrix and diffuse poorly from their source [41], their ability to form a long-range chemoattractive gradient in vivo is questionable. Another mechanism involves alternative factors acting as the primary modulator. For instance, target-derived factors other than FGFs could modulate the expression levels of FGFR1 in GNRH1 neurons and FGF ligands on the axon-targeting pathway, enhancing target-directed axon extension. Evidence for this was the observation that nerve growth factor could modulate FGFRs1, 2 and FGF2 expression levels in PC12 cells to induce neuritogenesis [42]. Last, other soluble forms of nontraditional FGFR1 ligands, such as anosmin and cell adhesion molecules, may mediate directional neurite outgrowth [43, 44]. Anosmin, for instance, has been found at high levels in the ME [45]. Anosmin has been implicated as a soluble short-range chemoattractant [46] and could represent a target-derived signal needed to activate FGFR1. It is important to note that homologues of anosmin, a molecule indispensable for human GNRH1 neuronal development, have not been found in mice [47]. Thus, the implication of anosmin or similar molecules in mouse GNRH1 neuronal development requires caution and additional verification.

In humans, anosmin-1 (or Kal1) was first characterized as the gene underlying the X-linked form of Kallmann syndrome (KS), a disorder characterized by hypogonadotropic hypogonadism (HH) and associated anosmia [48, 49]. The clinical relevance of our current results is evident from the recent finding that mutations in human FGFR1 gene (or Kal2) underlie an autosomal dominant form of KS [50]. KS is a phenotypically heterogeneous disorder that likely involves other unidentified genes, but HH in KS has always been attributed to a migrational defect in GNRH1 neurons. This was based on a single case of X-linked KS that described the arrest of GNRH1 neuronal migration during development, a defect likely secondary to the incomplete formation of the olfactory system [51]. Our current results suggest FGFR signaling is also involved in establishing the neurosecretory contacts between GNRH1 neurons and the ME. These data should therefore expand our view of potential causes of HH in KS to include mechanisms beyond the disruption of GNRH1 migration.

In conclusion, FGFRm mice exhibited a noticeable reduction in the GNRH1 fiber density in the ME [11], but this was previously interpreted as a defect secondary to the reduced number of GNRH1 neurons. Using both in vivo and in vitro models, the current study demonstrated that a defect in axon targeting contributed to this phenotype. That a large number of GNRH1 axons still targeted to the ME in FGFR mice likely reflected the incomplete inhibition of FGFR function by dnFGFR and/or the presence of redundant factors capable of supporting axon extension in vivo. Regardless, these results provide clear evidence that signaling through FGFRs contributes significantly to the establishment of contacts between the GNRH1 axons and the vasculature of the ME, a process essential for fertility.

ACKNOWLEDGMENTS

The authors thank Dr. Sue Moenter for the gift of the control mice (GFP-expressing transgenic mice) and editorial comments on the manuscripts and Dr. Richard Weiner (University of California at San Francisco) for sharing with us the FGFRm mice. We also thank Mai Hashimoto and Jeremy Jones for breeding and genotyping of the double homozygous transgenic mice, and Dr. Ned Friedman for the assistance in fluorescence imaging.

FOOTNOTES

2 Correspondence. FAX: 303 492 0811; John.Gill{at}colorado.edu Back

1 Supported by NIH RO1 HD042634 to P.-S.T. and NIH NRSA Predoctoral Fellowship 1 F31 HD47991–01 to J.C.G. Back

Received: 31 August 2005.

First decision: 28 September 2005.

Accepted: 4 November 2005.

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