Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Feb 15.
Published in final edited form as: Dev Biol. 2007 Dec 4;314(2):317–328. doi: 10.1016/j.ydbio.2007.11.030

Shh influences cell number and the distribution of neuronal subtypes in dorsal root ganglia

Wei Guan a,*, Guoying Wang b,*, Sheryl A Scott a,b, Maureen L Condic a,b,**
PMCID: PMC2276661  NIHMSID: NIHMS40749  PMID: 18190905

Abstract

The molecular mechanisms responsible for specifying the dorsal-ventral pattern of neuronal identities in dorsal root ganglia (DRG) are unclear. Here we demonstrate that Sonic hedgehog (Shh) contributes to patterning early DRG cells. In vitro, Shh increases both proliferation and programmed cell death (PCD). Increasing Shh in vivo enhances PCD in dorsal DRG, while inducing greater proliferation ventrally. In such animals, markers characteristic of ventral sensory neurons are expanded to more dorsal positions. Conversely, reducing Shh function results in decreased proliferation of progenitors in the ventral region and decreased expression of the ventral marker trkC. Later arising trkA+ afferents make significant pathfinding errors in animals with reduced Shh function, suggesting that accurate navigation of later arising growth cones requires either Shh itself or early arising, Shh-dependent afferents. These results indicate that Shh can regulate both cell number and the distribution of cell types in DRG, thereby playing an important role in the specification, patterning and pathfinding of sensory neurons.

Keywords: dorsal root ganglia, Sonic hedgehog, proliferation, programmed cell death, pathfinding

INTRODUCTION

Shh plays a prominent role in the patterning of neuronal cell fates along the dorsal-ventral (D-V) axis of the developing neural tube (Lee and Jessell, 1999; Tanabe and Jessell, 1996). During normal spinal cord development, induction of different ventral cell types requires different concentrations of Shh derived from the notochord and floor plate (Chiang et al., 1996; Marti et al., 1995; Roelink et al., 1995). Ectopic application of Shh is sufficient to induce formation of motor neurons, a ventral phenotype, in the dorsal neural tube (Ericson et al., 1996; Ericson et al., 1995). Treatment with exogenous Shh at earlier stages results in formation of excess numbers of ventral neuronal precursors and floor plate cells, which are subsequently eliminated by increased programmed cell death (PCD) (Oppenheim et al., 1999). Conversely, blocking Shh function with neutralizing antibodies reduces progenitor cell division and subsequently prevents differentiation of ventral progenitors into motor neurons (Ericson et al., 1996). These results indicate that Shh both patterns cell fates along the D-V axis of the spinal cord and regulates cell number through its effects on proliferation and PCD.

In addition to its role in neural tube development, Shh produced by the notochord and floorplate influences the D-V patterning of the somites, mesodermal structures lateral to the neural tube. Shh induces somites to form ventral sclerotome (Capdevila et al., 1998; Fan and Tessier-Lavigne, 1994; Zeng et al., 2002), and promotes proliferation in early somite (Zeng et al., 2002). Thus, Shh both influences D-V patterning and regulates cell number within diverse tissues of the developing embryo.

The dorsal root ganglia (DRG) of the peripheral nervous system are derivatives of the neural crest that differentiate between the neural tube and somites. Previous work has indicated that numerous factors are required for the formation of neural crest cells and for their correct migration (Liem et al., 1997; Liem et al., 1995; Placzek and Furley, 1996; Sela-Donenfeld and Kalcheim, 1999; Wu et al., 2003). For example, exposure of premigratory crest to factors derived from the dorsal neural tube (most likely members of the Wnt-family) is required for neural crest cells to differentiate as peripheral neurons and glia (Dorsky et al., 1998; Honore et al., 2003; Yanfeng et al., 2003). Further, Shh promotes neural crest proliferation (Fu et al., 2004) and survival (Ahlgren and Bronner-Fraser, 1999; Ahlgren et al., 2002) in addition to regulating crest motility (Testaz et al., 2001). However, once crest cells have become committed to a sensory lineage and have arrested migration in the vicinity of the forming sensory ganglia, the factors controlling the subsequent patterning of sensory neurons within the DRG are poorly understood (Marmigere and Ernfors, 2007). Recent data suggests that the Runx transcription factors Runx1 and Runx3 play important roles in regulating the projections of nocioceptive, proprioceptive and mechano-receptive sensory neurons in the spinal cord (Chen et al., 2006; Inoue et al., 2007; Kramer et al., 2006; Levanon et al., 2002b). However, the Runx genes are not expressed until relatively late in sensory development, after neurons project into the periphery, and the factors controlling the initiation of Runx expression and the pattern of peripheral sensory projections are not known.

Our previous work has indicated that sensory neurons have unique behavior and patterns of gene expression very early in development, shortly after they are generated from neuronal progenitors and well before sensory growth cones extend into the periphery and interact with target tissues (Guan and Condic, 2003; Guan et al., 2003). These data indicate that the fates of sensory neurons do not depend on target-derived factors, but rather are determined either in the neural crest or during early DRG development. The early determination of sensory neuron subtypes is supported by embryological manipulations that demonstrate exposure to target-derived neurotrophins is not sufficient to cause sensory neurons to alter their central projection in the spinal cord (Oakley and Karpinski, 2002; Oakley et al., 2000), again indicating that the specification of sensory neurons has been largely accomplished prior to the establishment of peripheral projections. While these experiments establish the developmental window during which sensory specification occurs, they do not identify the factors responsible for the specification of sensory phenotypes.

By analogy to the D-V patterning of neural tube and somites, it is possible that sensory neurons in DRG are specified early in development at least in part as a consequence of diffusible cell signaling molecules, such as Shh. In humans (Josephson et al., 2001), rodents (Inoue et al., 2002; Levanon et al., 2002a) and avians (Guan et al., 2003; Rifkin et al., 2000), sensory neurons subtypes are distributed in a rough D-V pattern within the DRG (see Supplemental Figure). The early-born proprioceptive neurons are located in the ventral-lateral third of the DRG while later born cutaneous neurons are more broadly distributed along the D-V axis, including the extreme dorsal-medial (DM) region, and may arise from distinct populations of neural crest cells (Marmigere and Ernfors, 2007). Here we investigated whether Shh regulates this D-V patterning of sensory subtypes in DRG. We found that in vitro, Shh promoted both cell proliferation and PCD of early DRG cells. In vivo, exogenous Shh selectively promoted proliferation of ventrally located neural progenitors while inducing cell death in the dorsal region of the DRG. Moreover, increased Shh function expanded the expression of markers characteristic of ventral sensory neurons to more dorsal DRG positions. When loss of Shh function reduced the number of early arising ventral neurons, pathfinding of later arising afferents was aberrant. These results indicate that Shh can regulate DRG cell number, the distribution of sensory phenotypes and sensory pathfinding.

MATERIALS AND METHODS

Embryonic treatment and sectioning

All experiments were performed on white leghorn chickens. At stage 21 (Hamburger and Hamilton, 1951), a window was cut in the shell, the embryo was stained with 0.5% neutral red in phosphate-buffered saline (PBS). Shh (R&D Systems, Minneapolis, MN) at 0.75 μg/egg (Oppenheim et al., 1999) or Shh function-blocking antibody 5E1 [Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA] at 50 μg/egg was injected under the vitelline membrane. The embryo was moistened with Ringers (150 mM NaCl; 3 mM KCl; 17 mM NaHCO3; 1 mM MgCl2; 3 mM CaCl2; 12 mM dextrose; 10 mM HEPES; pH=7.4). Eggs were sealed with paraffin and a cover slip and returned to a 37°C incubator until desired stages. To examine cell proliferation, BrdU was injected at 30 μg/egg one hour prior to fixation. At the end of the incubation period, embryos were removed, staged (Hamburger and Hamilton, 1951), rinsed in PBS and fixed in 4% paraformaldehyde overnight at 4°C. After washing with PBS, the embryos were transferred through 5, 15, and 30% sucrose in PBS, embedded in OCT (Sakura Finetek, Torrance, CA), and cryosectioned to a thickness of 16 μm at the lumbar level.

Measurements of sectioned DRG

To measure the size of the first three lumbosacral DRG (LS1, LS2 and LS3) under different treatment conditions, the cross-sectional areas of the largest section was measured. Images of these sections were captured at 20× magnification and analyzed using the NIH Image software. To analyze the dorsal/ventral pattern of cell proliferation and death in DRG, we acquired a 20× image of every other section from LS1, LS2 and LS3 DRG. The midpoint of the longest axis of the DRG was determined, and a line was drawn from this midpoint to the bottom of the neural tube to divide the DRG into dorsal-medial and ventral-lateral regions, as illustrated in Fig. 2B and 3A. The total number of BrdU+ or TUNEL+ cells in each region of the DRG was determined for LS1, LS2 and LS3 DRG from at least three independent experiments.

Figure 2.

Figure 2

Open in a new tab

Exogenous Shh selectively stimulates programmed cell death of DM DRG cells in vivo. (A) At stage 23, no TUNEL+ cells were observed in DRG for any condition (not shown). However, numbers of TUNEL+ cells in the apical ectodermal ridge and distal mesenchyme of the limb were decreased by Shh and increased by 5E1 at this stage, indicating that these treatments affect cell death as expected (Chiang et al., 1996). (B) At stage 25, TUNEL staining was observed in DRG of control, Shh and 5E1 treated embryos. Cartoon illustrates the division of DRG into DM vs. VL regions, as described in MATERIALS AND METHODS. (C) TUNEL+ cells in DM and VL regions of DRG LS1, LS2 and LS3, with data from at least three independent experiments shown. *Significantly different from DM controls (t-test, p<0.01). #Significantly different from VL Shh treated (t-test, p<0.01).

Figure 3.

Figure 3

Open in a new tab

Exogenous Shh predominantly stimulates the proliferation of VL DRG cells. (A) Examples of BrdU labeling of DRG from control, Shh and 5E1 treated embryos at stage 25. Cartoon illustrates the division of DRG into DM vs. VL regions, as described in MATERIALS AND METHODS. (B) The number of BrdU+ cells in stage 23 LS2 ganglia is increased equally in both the DM and VL regions following Shh treatment at stage 21, while 5E1 treatment results in a reduction of BrdU+ cells throughout the ganglia. *Significantly different (p<0.01; t-test). Both treatments are also different from control ganglia (p<0.01; t-test). (C) At stage 25, Shh alters proliferation in a D/V pattern. BrdU+ cells in DM and VL regions of LS1, LS2 and LS3 DRG from control, Shh, 5E1 treated embryos, with data from at least three independent experiments shown. *Significantly different from 5E1 treated (t-test, p<0.02). #Significantly different from DM Shh treated (t-test, p<0.02).

Embryonic cell culture

Dissociated DRG cell cultures were prepared as previously described (Guan et al., 2003). Briefly, lumbar DRG at stage 23 were dissociated (0.2% trypsin in PBS for 20 min at 37°C), and the suspension was enriched for neurons by pre-plating on tissue culture plastic for 2 hr and harvesting the non-adherent neuronal cells. The DRG neurons were cultured on UV-sterilized cover slips (Fischer Scientific, Houston, TX) that had been baked previously at 350 °C for 12 hr and then coated with 50 μg/ml fibronectin (FN) (Invitrogen, Grand Island, NY) in PBS for 2 hr at room temperature. The dissociated DRG cells were cultured in neural basal medium (F-12 medium plus 10 ng/ml N2 additives, 500 μM L-glutamine, 25 μM glutamic acid, 10 U/ml penicillin, 10 U/ml streptomycin, all from Invitrogen) supplemented with 5 μg/ml Shh (Testaz et al., 2001). In control cultures, Shh was omitted. Cultures were incubated at 37°C in a humidified incubator with 5% CO2 for 48 hr.

TUNEL and BrdU labeling

Cultured cells

After culturing dissociated DRG cells for 48 hr, a 12 hr bromodeoxyuridine (BrdU) pulse at 10 μg/ml was given. Cover slips were fixed in 4% paraformaldehyde for 30 min, rinsed, incubated at room temperature in fresh 2N HCl for 10 min to denature DNA and so render the incorporated BrdU accessible to antibodies, then neutralized with 0.1 M Na2B4O7, pH=8.5 for another 10 min. Mouse anti-BrdU antibody G3G4 was obtained from DSHB and was used at 1:3 dilution in the blocking buffer (5% NGS, 0.1% Triton X-100 and 1% BSA) at 4°C overnight. The next day, the cells were rinsed with PBS, followed by goat anti-mouse Alexa-568 (Molecular Probes, Eugene, OR) secondary antibody at 1:200 in blocking buffer for 1 hr. Then the cultures were labeled with 4', 6'-diamidino-2-phenylindole (DAPI) for 1 hr at 10 μg/ml to reveal the nuclei. For TUNEL labeling, cover slips were washed with PBS, permeabilized in fresh 0.1% sodium citrate and 0.1% Triton X-100 solution for 2 min on ice, and incubated in TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, Roche, Mannheim, Germany) mixture at 37°C for 1hr. In some cases, cultures were double labeled with both TUNEL and BrdU. Finally, the cell culture cover slips were mounted in SlowFade medium (Molecular Probes). To determine the percentages of BrdU+ or TUNEL+ cells, at least 100 DAPI+ DRG cells were counted in each of 5 or 7 independent batches of cell cultures. Cryostat sectioned embryos were processed for TUNEL and BrdU staining as above. For quantifying cells double labeled with TUNEL and trkA (see below), every other section was scored at 40× by an observer blind to the experimental conditions. TUNEL+ in the dorsal most and ventral most thirds of the ganglia were scored as trkA+ or trkA−.

In situ hybridization on cryostat sections

Treated embryos were incubated to stage 25, dissected in DEPC-PBS (PBS with 0.1% Diethyl Pyrocarbonate, pH=7.1), fixed, embedded and sectioned. Sections were fixed in 4% paraformaldehyde for 20 min. After washing, the sections were permeabilized with proteinase K (1 μg/ml) for 10 min, acetylated with triethanolamine and acetic anhydride for 10 min, then hybridized overnight with Dig-labeled probes. The sections were incubated in AP conjugated anti-Dig antibody and reacted with NBT/BCIP. For all experiments, control sense probes were negative.

Immunohistochemistry on cryostat sections

The sections were incubated for 20 min at room temperature in TBS buffer (10 mM Tris, pH 7.4, 150 mM NaCl) containing 0.1% Triton X-100. Rabbit polyclonal antibodies to trkA and trkC were generously provided by Dr. Frances Lefcort (Montana State University, Bozeman, MT). TrkA or trkC staining was conducted using Tyramide Signal Amplification (TSA), according to the protocols of the manufacturer (NENTM Life Science Products, Inc., Boston, MA). Goat anti-rabbit conjugated to biotin was used at 1:600, FITC-avidin at 1:600 and goat anti-rabbit conjugated to Cy-3 at 1:2000 (all from Jackson) in NGS buffer (0.03M Tris, 0.15M NaCl, glycine at 10 mg/ml, 0.4% Triton X-100, 10% normal goat serum) and mounted as described above.

RESULTS

Sensory neurons in chick are generated in a specific temporal and spatial sequence. As early as stage 19−20 (embryonic day 3−3.5; E3−3.5) neurons in the ventral-lateral (VL) region of lumbar DRG have withdrawn from the cell cycle and are beginning to extend axons toward the plexus region of the hindlimb (Tosney and Landmesser, 1985; Wang and Scott, 2000), whereas neurons in the dorsal-medial (DM) region are generated as late as stage 32 (E7.5) (Carr and Simpson, 1978; Lawson and Biscoe, 1979; Lawson et al., 1974; Rifkin et al., 2000). Early born, VL neurons are a mixed population, containing the majority of the cells fated to be proprioceptive (Honig et al., 1986), as well as a minority (roughly 6%) of neurons that will project to skin (Oakley et al., 2000). Although neurotrophin receptor expression is not an exclusive marker for any single type of sensory neuron, the majority of neurons found in the VL domain express the neurotrophin receptor trkC (see Supplemental Figure). In contrast, the latest generated DM neurons are a more homogeneous population of cutaneous afferents that project largely to the dorsal body wall along the dorsal ramus (Davies, 1987; Davies and Lumsden, 1990). The majority of DM neurons express the trkA neurotrophin receptor. During DRG development, both neurons and glia are generated from the same multipotent progenitors (Marmigere and Ernfors, 2007), with neurons being generated first, and glia arising significantly later, from stage 27 on (Wakamatsu et al., 1999; Wakamatsu et al., 2000). While the time course of neurogenesis differs significantly between vertebrate species, the general temporal sequence (VL cells being born prior to DM) and spatial distribution of sensory cell fates along the D-V axis of the DRG is highly conserved in rodents (Levanon et al., 2002a; Mu et al., 1993; White et al., 1996; Wright and Snider, 1995) and in humans (Josephson et al., 2001).

Shh stimulates PCD of early DRG cells in vitro

To determine whether Shh directly affects early DRG cells, we treated cells derived from stage 23 DRG with Shh in vitro (Testaz et al., 2001). Dissociated DRG from this stage contain post-migratory neural crest, proliferating multi-potent progenitors (Marmigere and Ernfors, 2007) and small numbers of post-mitotic VL neurons that are largely fated to be proprioceptive (Carr and Simpson, 1978; Rifkin et al., 2000; Wakamatsu et al., 1999). Previous work indicates that Shh increases programmed cell death (PCD) in both spinal cord (Oppenheim et al., 1999) and limb bud (Sanz-Ezquerro and Tickle, 2000). We therefore examined PCD in Shh-treated cultures established from stage 23 DRG. Shh induced a significant increase (p<0.01; t-test) in TUNEL+ cells (Fig. 1A) compared to untreated cultures, demonstrating that Shh acts directly on early DRG cells to promote cell death.

Figure 1.

Figure 1

Open in a new tab

Programmed cell death and proliferation of early DRG cells are stimulated by Shh in vitro. (A) Top panel: Double labeling of cells with DAPI (blue) and TUNEL (green) in cultures derived from stage 23 dissociated DRG. Bottom panel: The percentage of TUNEL+ cells out of total DAPI+ DRG cells was determined from at least 100 DAPI+ cells in each of 7 independent experiments. (B) Top panel: Double labeling of cells with DAPI (blue) and BrdU (red) in cultures derived from stage 23 dissociated DRG. Bottom panel: The percentage of BrdU+ cells out of total DAPI+ DRG cells was determined from at least 100 DAPI+ cells in each of 5 experiments. *Shh significantly different from control (t-test, p<0.01). Arrowheads indicate doubly labeled cells. Arrows indicate cells unlabeled by TUNEL or BrdU.

Shh stimulates proliferation of early DRG cells in vitro

We also examined cell proliferation in Shh treated DRG cultures (Testaz et al., 2001) established from stage 23 embryos. Shh significantly (p<0.01; t-test) promoted proliferation of early DRG cells in vitro, roughly doubling the percentage of cells that incorporate BrdU in culture (Fig. 1B). These results support the conclusion that Shh acts directly on early DRG cells, and further indicate that Shh acts as a mitogen to stimulate proliferation in this population. Double labeling of control cultures with both BrdU and TUNEL indicated that there was very little overlap between proliferating cells and those undergoing PCD. On average, 28% of the cells were labeled with BrdU, but only 2% were double-labeled for both BrdU and TUNEL (data not shown). Shh treatment increased the number of doubly labeled cells very slightly, to between 4 and 10% of cells, suggesting that few of the cells induced to proliferate by Shh subsequently die. Thus in vitro, Shh appears to act on largely independent populations within the early DRG to promote either cell proliferation or cell death.

Early DRG cells in vivo respond to Shh

To determine whether Shh indeed acts on distinct populations of cells in developing DRG, we examined the effects of Shh on sensory development in vivo. We treated embryos with Shh (Oppenheim et al., 1999) or with Shh function-blocking antibody 5E1 (Ericson et al., 1996) at stage 21, and examined DRG at stage 25−27. These stages were chosen because in lumbar regions, the D-V patterning of both spinal cord (Briscoe et al., 2000) and somite (Brent and Tabin, 2002) are largely complete by stage 15, and the coalescence of neural crest into sensory ganglia is complete by stage 20 (Le douarin and Kalcheim, 1999). The earliest lumbar DRG neurons are born around stage 19 and begin to extend along the ventral sensory route starting at stage 20 (Le douarin and Kalcheim, 1999). Thus, manipulating Shh function at stage 21 allowed us to exclude any early effects of Shh on neural crest specification, migration or arrest, and to specifically examine the effects of Shh on DRG patterning and proliferation. During these stages, notocord and floorplate are the primary sources of Shh in the regions of the DRG (Marti et al., 1995; Oppenheim et al., 1999), but Shh is not found in the DRG itself (Oppenheim et al., 1999).

To determine the efficacy of Shh and 5E1 treatment under our experimental conditions, we examined the expression of chick patched-1 (ptc-1), a Shh receptor that is strongly induced by Shh (Marigo et al., 1996; Marigo and Tabin, 1996; Pearse et al., 2001). Consistent with previous work, we found that while very few cells express ptc-1 in DRG under control conditions, exogenous Shh greatly increased ptc-1 expression in the neural tube and DRG, while blocking Shh function with 5E1 decreased ptc-1 expression (Supplemental Figure). Together these results indicate that early DRG cells in vivo respond to Shh gain and loss of function manipulations, supporting the possibility that Shh influences early sensory development.

Shh alters DRG programmed cell death in a D-V pattern

To examine the effects of Shh on DRG cell death in vivo, embryos were treated with Shh at stage 21 and maintained in ovo until either stage 23 (approximately 18 hours post treatment) or stage 25 (approximately 36 hours post treatment). The embryos were removed from the eggs, staged to ensure all animals had developed to the same embryonic age, sectioned and processed for TUNEL staining.

Consistent with the normal period of cell death during the development of sensory ganglia (Caldero et al., 1998), we detected no TUNEL+ cells in DRG at stage 23 for any condition (not shown), although altering Shh function at this stage affected PCD in the apical ectodermal ridge and underlying mesoderm of the limb (Fig. 2A), as anticipated (Chiang et al., 1996; Kraus et al., 2001; Sanz-Ezquerro and Tickle, 2000; Todt and Fallon, 1987).

By stage 25, we observed TUNEL-labeling in the DRG of control and experimental animals. Images of TUNEL-labeled DRG were divided into DM and VL regions to analyze Shh-induced death along the D-V axis of the ganglion (Fig. 2B). Consistent with the ability of Shh to directly promote DRG cell death in vitro (Fig. 1), exogenous Shh greatly increased PCD in DM-DRG (where cells normally experience lower levels of Shh signaling due to their greater distance from the ventral structures that produce Shh in the embryo), while not altering cell death in VL-DRG (where Shh signaling is normally high; Fig. 2C). Increased cell death in DM regions could reflect the fact that dorsal DRG cells are born later than ventral cells (Carr and Simpson, 1978) and are therefore at an earlier developmental stage (see discussion of proliferation, below). However, the effects of Shh treatment were nearly identical in DRG along the rostrocaudal axis; in particular, Shh did not increase cell death in VL cells in more caudal (i.e. developmentally younger) ganglia (Fig. 2C). Thus, the differential effects of Shh on PCD in DM versus VL cells is unlikely to be due to the younger developmental age of DM cells. In addition, the fact that we did not observe an increase in cell death in stage 23 DRG by enhanced Shh function (see legend of Fig. 2) further supports the conclusion that the restriction of Shh-induced PCD to DM-DRG at stage 25 (Fig. 2C) represents a real difference in the response of DM and VL cells to Shh, rather than being merely due to developmental age of later born DM cells compared to developmentally “older” VL cells.

In contrast to increasing Shh function, blocking Shh function did not alter cell death in either the dorsal or ventral regions of the DRG. Failure to detect an effect of 5E1 treatment on cell death may reflect the very low numbers of cells undergoing apoptosis in the DRG at this stage (typically less than 10 TUNEL+ cells were observed in the DRG under control conditions). Alternatively, failure of 5E1 to block cell death may suggest that at stage 25, DRG cells are not dependent on Shh for survival. Nevertheless, the finding that exogenous Shh increases PCD in vivo indicates that, similar to our findings in vitro, Shh can regulate PCD in early DRG. Further, by stage 25 there are differences between sensory progenitors located in VL and DM DRG that are revealed by the differential response of cells along the D-V axis to Shh.

To further investigate the identity of cells that are induced to die in response to higher levels of Shh signaling, we double stained sections of embryos with TUNEL and antibodies directed against the neurotrophin receptor trkA, a marker for postmitotic neurons that predominantly project to skin (Henion et al., 1995; Rifkin et al., 2000). In control (saline treated) embryos, very few trkA+ cells were also TUNEL+ (a total of 4 cells observed in twenty-four ganglia from four animals). In contrast, Shh treatment greatly increased the numbers of trkA+ cells undergoing programmed cell death, with this effect being largest in the dorsal regions of the DRG (see Table 1). This suggests that postmitotic, trkA+ neurons are induced to die in response to high levels of Shh. The trkA−/TUNEL+ cells we observed could be either other classes of postmitotic neurons that are present in small numbers in dorsal DRG or progenitor cells.

Table 1.

Cells double labeled for TUNEL and TrkA.

Control Shh
 
 trkA-
 trkA+
 trkA−
 trkA+
Dorsal 4.3 0.5 7.2* 5.8*
Ventral 3.5 0.5 3.6 1.4
Open in a new tab

Embryos were treated with saline (control) or Shh at stage 21 and analyzed for TUNEL and trkA labeling at stage 25. Cells in either the dorsal-most or ventral-most third of the DRG were scored. Average number of TUNEL+ cells per animal that were either positive or negative for trkA labeling are given. At least twelve DRG (LS1-LS3) from at least four animals were counted for each condition

*

Dorsal Shh condition is significantly different from control (p<.005, Fisher's exact test).

Shh alters DRG proliferation in a D-V pattern

To examine the effects of Shh on DRG proliferation in vivo, embryos were treated with Shh and 5E1 as above. At the end of the incubation period, embryos were given a BrdU pulse, and BrdU incorporation in the DM and VL halves of the DRG was examined in cryosectioned specimens (Fig. 3A).

At stage 23, greater numbers of BrdU+ nuclei were observed in VL regions than in DM of control DRG [Table 2; (Carr and Simpson, 1978; Rifkin et al., 2000)]. Consistent with the ability of Shh to directly promote DRG proliferation in vitro (Fig. 1), Shh treatment increased proliferation in both VL and DM DRG, and proliferation was significantly reduced when Shh function was blocked (Fig. 3B), suggesting that at stage 23 Shh functions as a mitogen for early DRG progenitors regardless of their D-V location.

Table 2.

Numbers of BrdU+ cells in VL and DM regions of DRG.

Control Shh 5E1
 
 LS1
 LS2
 LS3
 LS1
 LS2
 LS3
 LS1
 LS2
 LS3
Stage 23
DM - 99±5 - - 138±6 - - 64±3 -
VL
 -
 141±10
 -
 -
 203±18
 -
 -
 108±10
 -
Stage 25
DM 138±14 127±17 125±23 198±20 183±31 194±42 147±16 150±16 135±7
VL 101±12 79±9 82±13 182±20 182±23 183±36 98±7 106±9 90±2
Open in a new tab

Embryos were treated as indicated at stage 21 and analyzed for BrdU incorporation following a pulse at stage 23 or 25. Average numbers of BrdU+ cells in DM and VL regions of DRG±SEM are given from at least three independent experiments. Shaded boxes are significantly different from corresponding control values (p<0.02; t-test).

By stage 25, most (but not all) progenitors in the VL region have exited the cell cycle in branchial DRG (Carr and Simpson, 1978; Rifkin et al., 2000), whereas the majority of progenitors in DM-DRG are still actively proliferating. Consistent with this finding, we observed greater numbers of BrdU+ nuclei in DM-DRG, compared to VL-DRG of control animals at this stage (Table 2). Treatment with Shh increased BrdU incorporation in both DM- and VL-DRG (Fig. 3C; Table 2). However, the percent increase observed in the VL region of the DRG was significantly greater than that observed in the DM region (Fig. 3C).

As noted above, VL cells are developmentally more mature than DM cells (Carr and Simpson, 1978), raising the possibility that differences in the extent to which Shh increased proliferation in dorsal versus ventral cells merely reflect differences in the developmental age of the progenitors. DRG also differ in age along the rostral-caudal axis, with numbers of BrdU+ cells being significantly greater in (developmentally older) LS1 ganglia than in LS3 at stage 25 (Table 2). However, Shh did not induce statistically greater DM proliferation in rostral (i.e. developmentally older) versus caudal ganglia (Fig. 3C), suggesting that developmental stage does not account for the differences in Shh-induced proliferation between DM and VL regions. These observations further support the conclusion that by stage 25, there are distinct dorsal and ventral populations of proliferating DRG progenitors that are differentially responsive to Shh.

Shh alters DRG size

DRG in embryos treated with Shh frequently appeared larger than control DRG from the same axial level (e.g. Fig. 2B, 3A). We measured DRG size on sections of control, Shh treated and 5E1 treated embryos at stage 25 (Supplemental Figure), and observed a significant increase in DRG area in embryos treated with Shh. DRG in animals treated with 5E1 were comparable in size to control DRG. Increased size of DRG was not due to increased cell size; average size of cells measured from hematoxylin-eosin stained paraffin sections was not statistically different (p>0.2, t-test) in Shh treated and control animals (average cell size ± SEM for at least 20 cells in LS1 was 75.6 μm2 ± 3.5 for Shh treated animals and 80.1 μm2 ± 4.4 for controls). Thus increased size of DRG is likely to reflect increased cell number in embryos treated with Shh.

Shh is required for proliferation of early trkC+ ventral cells

To determine whether Shh alters the development of different classes of sensory neurons, we examined trk expression at stage 23 and 25 in animals that had received Shh gain and loss of function treatments at stage 21. All DRG neurons initially express trkC, and beginning around stage 25, trkC starts to be restricted to proprioceptive neurons located in the ventral DRG (Guan et al., 2003; Rifkin et al., 2000). Previous work has demonstrated that a subset of migratory neural crest express trkC (Henion et al., 1995; Rifkin et al., 2000), suggesting that the earliest arising trkC+ neurons may be derived from a restricted population of trkC+ progenitors in the neural crest.

In animals with Shh gain of function, trkC expression was comparable to control DRG at stage 23, yet by stage 25, DRG were notably larger and trkC expression was expanded into a more DM domain (Fig. 4). In contrast, Shh loss of function resulted in decreased trkC expression and a loss of trkC+ axons as early as stage 23 in DRG (Fig. 4). These results indicate that alterations in Shh signaling can alter the normal pattern of trk expression.

Figure 4.

Figure 4

Open in a new tab

Expansion of trkC expressing cells in animals with altered Shh function. At stage 23, LS2 DRG stained for trkC show comparable numbers of labeled cells and axons in control and Shh treated animals, while animals treated with 5E1, a Shh function-blocking antibody, have reduced numbers of trkC+ cells. By stage 25, in Shh treated animals there is a notable increase in the number of trkC+ cells, and a corresponding decrease in 5E1-treated animals. All images are mid-ganglion sections of the LS2 DRG. White lines enclose trkC+ cells, to illustrate approximate distribution of these cells within the ganglion.

Together with our finding that loss of Shh function does not alter PCD at stage 23, and does not promote increased cell death in VL DRG at any stage (Fig. 2), the decrease of trkC+ DRG cells in 5E1-treated embryos at stage 23 (Fig. 4) suggests that Shh promotes proliferation of ventral, trkC+ DRG progenitors. These findings strongly suggest that generation of early arising VL proprioceptive neurons is significantly compromised in animals with reduced Shh function due to decreased proliferation of early progenitors that normally give rise to ventral neurons. This expansion of trkC expression suggests that Shh is driving cell proliferation, but does not necessarily require that Shh promotes generation of ventral cells at the expense of dorsal cells, since these populations may arise from distinct progenitor pools (Marmigere and Ernfors, 2007).

Shh induces expression of a ventral sensory marker

To investigate further whether Shh influences the D-V patterning of neuronal cell types in DRG, we examined expression of a marker characteristic of ventral sensory neurons. Our recent work has shown that cUnc-5H3, a Netrin-1 receptor that mediates repulsive signaling, is expressed by ventral DRG neurons (Guan et al., 2003). Similar to the expanded domain of trkC (Fig. 4), cUnc-5H3 was dramatically up-regulated following Shh treatment and was observed in more dorsal locations (Fig. 5). In contrast, blocking Shh function with 5E1 did not significantly alter the domain of cUnc-5H3 expression in DRG (not shown), suggesting that expression of this gene does not require Shh, that expression is determined prior to the onset of our treatment at stage 21, or that there is a “Shh threshold” for cUnc-5H3 expression in DRG. Although we decreased Shh signaling with 5E1 treatment, residual Shh signal may still be above the threshold required to induce cUnc-5H3 expression in DRG.

Figure 5.

Figure 5

Open in a new tab

Altered Shh function in vivo alters the normal D-V pattern of cUnc-5H3 expression. In situ hybridization on cryostat sections of stage 25 lumbar DRG from control and Shh treated embryos. Shh treatment increased the expression of cUnc-5H3 and expanded the domain of expression to more dorsal positions, an effect that appeared somewhat more prominent in more posterior ganglia (cf. LS2 to LS4). Lines outline the ganglion and show the DV midpoint, as in Figs. 2B and 3A. cUnc-5 expressing cells are concentrated near the outer margin of the ganglia and images reflect the maximum dorsal extent of cUnc-5 expressing cells for each condition. Because sections are not taken from the middle of the ganglia, the lines do not accurately reflect maximum DRG dimensions at the mid-point.

Pathfinding errors in animals with reduced Shh function

During normal development, trkA+ sensory afferents arise slightly later than the earliest trkC+ neurons and extend along preexisting axon tracks pioneered by the trkC+ axons (Guan et al., 2003). To determine whether the earliest arising DRG neurons that pioneer the ventral route are required for correct pathfinding by the later growing axons, we examined pathfinding in animals in which development of the earliest neurons was compromised by reducing Shh function. Animals treated with 5E1 at stage 21 (after Shh-dependent patterning of the dermomyotome and spinal cord are complete) were examined at stage 25 for pathfinding of later arising, trkA+ sensory neurons. In 83% of DRG examined, trkA+ afferents made significant pathfinding errors in their initial projection toward the limb (Fig. 6, Table 3). TrkA+ neurites extended in an aberrant dorsal-lateral direction towards the dermamyotome (Fig. 6B, 6C, arrowheads). Such projections are normally only seen at later stages in untreated control animals. Moreover, the ventral nerve of the DRG was not tightly fasciculated, as it is in control animals (Fig. 6A), but instead was frequently bifurcated and broad (Fig. 6C, 6D, brackets). Finally, neurites were often observed in an aberrant medial location, extending quite close to the ventral neural tube (Fig. 6C, 6D, arrows).

Figure 6.

Figure 6

Open in a new tab

Pathfinding of late arising trkA+ cells is disrupted in animals with reduced Shh function. (A) A control embryo treated with vehicle at stage 21 and stained for trkA at stage 25. Axons are tightly fasciculated (bracket) and extend along preexisting pioneers (Guan et al., 2003). (B-D) Animals treated with 5E1 to reduce Shh function. (B) TrkA+ fibers extend in an aberrant dorsal-lateral direction (arrowheads). (C) TrkA+ fibers misroute laterally (arrowhead), the ventral nerve is broad and bifurcated (bracket) with fibers extending in ectopic medial positions (arrow). Similar errors were seen in 29 out of 60 5E1-treated ganglia (see Table 3). (D) Ventral route is broad and bifurcated (bracket) with fibers extending in aberrant medial positions (arrow). Medially projecting axons were seen in 41 out of 60 5E1-treated ganglia (see Table 3). Scale bar in (A) is 50 μm.

Table 3.

TrkA+ axon pathfinding errors.

Control (n=49)
 Shh (n=40)
 5E1 (n=60)
DRG BN/BiN Med DRG BN/BiN Med DRG BN/BiN Med
w/ w/ w/
errors
  
  
 errors
  
  
 errors
  
  
18% 3 6 15% 5 2 83% 29 41
Open in a new tab

Embryos were treated as indicated at stage 21 and stained for trkA at stage 25. Percent of DRG with abnormal projections and total number of DRG examined from four independent experiments are given. BN/BiN = number of DRG with a broad or bifurcated nerve. Med = number of DRG with axons in abnormal medial positions. Single DRG could exhibit both defects. Because we cannot determine whether lateral axon projections in 5E1 treated animals were abnormal or merely precocious, we did not consider DRG with lateral projections abnormal unless they exhibited additional defects.

DISCUSSION

Shh plays a critical role in the patterning and growth cone guidance of central neurons (Salie et al., 2005). Here we show that Shh may also influence the distribution of neuronal subtypes in dorsal root ganglia, and contribute to sensory pathfinding. In vitro, Shh stimulates both PCD and proliferation in largely non-overlapping populations of early DRG cells, indicating that Shh acts directly on DRG cells and functions as a mitogen for a subset of sensory precursors. In vivo, progenitors in the dorsal and ventral regions of DRG respond differentially to Shh as early as stage 25 (summarized in Fig. 7). Exogenous Shh selectively stimulates proliferation of progenitors in ventral regions and expands the expression of markers for ventral sensory subtypes. In contrast, dorsal DRG cells undergo increased cell death in response to exogenous Shh. Reducing Shh function results in decreased BrdU incorporation and a reduction of trkC+ cells from early (stage 23) DRG. Together these data suggest that Shh, which is produced by the floor plate and notochord, contributes to the ventral patterning of DRG neurons. Finally, later arising trkA+ DRG neurons make significant pathfinding errors when Shh function is blocked, suggesting either that Shh directly affects sensory pathfinding or that trkA+ cutaneous afferents are unable to accurately pioneer the sensory nerve in the absence early arising, ventral proprioceptive trkC+ afferents, or that earlier arising motor or sensory axons project incorrectly, and thereby indirectly affect the pathfinding of later arising axons. Thus, our findings are consistent with Shh playing important roles in sensory development by affecting cell survival, proliferation, the D-V distribution of DRG neurons, and sensory axon pathfinding.

Figure 7.

Figure 7

Open in a new tab

Summary of findings. In control conditions, DM and VL DRG neurons express distinct markers (Guan and Condic, 2003; Guan et al., 2003) and predominantly extend to different targets (skin and muscle, respectively). For example, trkA+ neurons are present throughout the DRG at this stage, while trkC+ neurons are found predominantly in the VL regions, with trkB+ neurons in the middle third of the D-V extend of the ganglia (see Supplemental Figure). In Shh gain of function conditions, DRG size increases concomitant with an increase in proliferation throughout the DRG at stage 23 that is maintained in ventral regions until at least stage 25. Shh treatment induces an expansion of ventral markers, and causes a significant increase in dorsal cell death at stage 25. When Shh function is reduced, proliferation of early progenitors (at stage 23) is reduced and the expression of the ventral marker trkC is suppressed.

Shh regulates proliferation and survival in a D-V pattern

Our findings indicate that Shh promotes both proliferation and PCD of early DRG cells in vitro and in vivo. The role of Shh in regulating cell number in Shh-responsive tissues is complex. Several lines of evidence suggest that Shh acts to promote cell cycle progression in a variety of cell types (Kenney et al., 2003; Kenney et al., 2004; Lowrey et al., 2002; Oliver et al., 2003). In chick neural tube, previous work has shown that exogenous Shh induces increased proliferation and that subsequently excess cells are removed by apoptosis (Oppenheim et al., 1999), suggesting that Shh regulates both proliferation and death. Somewhat in contrast to these findings, several lines of evidence suggest that in cranial neural crest, blocking Shh signaling, either with antibodies (Ahlgren and Bronner-Fraser, 1999) or with cyclopamine (Ungos et al., 2003) increases cell death without affecting proliferation. In agreement with Shh having an anti-apoptotic function, recent work demonstrates that the hedgehog receptor patched-1 (ptc-1) plays an important role in preventing apoptosis; when the ptc-1 is unoccupied by ligand, death ensues (Thibert et al., 2003).

Our evidence suggests that Shh influences DRG cell number in several ways. Shh acts as mitogen for progenitors in both dorsal and ventral DRG, albeit more strongly for progenitors in ventral regions (Fig. 3). Conversely, exogenous Shh promotes cell death in dorsal, but not ventral DRG (Fig. 2; Table 1). Alterations in proliferation and death do not correlate with the rostrocaudal position of the DRG (with rostral DRG being developmentally older) or with the overall age of the embryo (Figs. 2 and 3), suggesting that the differential effects of exogenous Shh on dorsal and ventral DRG populations is not likely to reflect the differences in developmental stage between early born ventral cells and later born dorsal cells. Alternatively, Shh may be an endogenous mitogen in early DRG (eg. st. 24), but not a mitogen for the late wave of neurogenesis in the DRG (e.g. St. 25). Our results indicate that exogenous Shh induces trkA+ postmitotic neurons to die, as well as trkA− cells (either other classes of neurons or progenitors). The differential effect of altered Shh function on DM and VL cells supports the conclusion that DRG cells in these regions have phenotypic differences at early developmental stages prior to target innervation (Oakley and Karpinski, 2002; Oakley et al., 2000).

D-V patterning, cell lineage and progenitor specification in DRG

Shh signaling is known to influence the embryonic progenitors of DRG in several ways. Shh promotes neural crest survival (Ahlgren and Bronner-Fraser, 1999; Dunn et al., 1995), and may also induce arrest of migrating neural crest in the vicinity of the forming DRG (Testaz et al., 2001). Work in zebrafish suggests that formation of sensory progenitors from migratory crest cells depends on Shh function, perhaps through Shh-dependent induction of neurogenin-1 (Ungos et al., 2003) or regulation of members of the Runx family of transcription factors (Chen et al., 2006; Inoue et al., 2007; Kramer et al., 2006; Levanon et al., 2002b). Given that we see differences in the response of cells along the D-V axis of the DRG as early as stage 23, prior to projection of sensory neurons into the spinal cord (Figs. 3, 4), differences in Shh concentration experienced by DRG progenitors at different distances from the ventral floor plate and notochord may play a role in early DRG development and patterning. Our current study is the first to specifically examine the role of Shh in the patterning of sensory phenotypes within DRG.

Our finding that altering Shh function alters the expression of markers for ventral neurons in DRG (Figs. 4 and 5) does not conclusively address the origin of dorsal DRG cells expressing ventral neuronal markers that are formed in response to Shh treatment. Shh-induced expansion of ventral markers could occur through at least three distinct mechanisms: 1) increased proliferation of a specified ventral progenitor population in combination with selective death of (unspecified or differently specified) dorsal progenitors, or 2) respecification of dorsally located progenitors to a ventral fate or 3) by acting as a mitogen for early progenitors resulting in an expansion in the number of neurons derived from these progenitors.

Previous work has shown that both DM and VL cells in DRG share a common lineage. When pre-migratory chick neural crest are labeled with retrovirus and resulting DRG clones analyzed after sensory neurogenesis is complete, the majority of clones are mixed (i.e. contain both neurons and glia) and occupy both DM and VL positions, indicating that pre-migratory neural crest are not yet restricted (Frank and Sanes, 1991). This conclusion is supported by a study of fate determination within early DRG that suggests both neurons and glia are produced from a common Notch1+/Sox2+ progenitor that generates neurons at early developmental stages, and subsequently produces glia (Wakamatsu et al., 2000). Neither of these studies directly addresses whether or when DRG precursors become specified to produce particular subtypes of sensory neurons and what factors control this specification. Our results are consistent with the possibility that ventral and dorsal progenitors are distinct early in DRG development; Shh gain and loss of function manipulations have different effects on dorsal and ventral cells prior to stage 25.

Sensory pathfinding

Previous studies have demonstrated that numerous guidance molecules, including semaphorin3a (Kitsukawa et al., 1997; Masuda et al., 2000; Masuda et al., 2003; Taniguchi et al., 1997; White and Behar, 2000), axonin-1/Tag1/SC2 (Masuda et al., 2000) and CSPGs produced by the notochord (Masuda et al., 2004), contribute to the correct navigation of sensory afferents along their initial route between the spinal cord and the dermomyotome. We found that trkA+ neurons, which arise relatively late in embryogenesis, fail to pathfind accurately in Shh loss of function conditions.

It is unlikely that the pathfinding errors we observe arise from perturbation of signaling from surrounding tissues because D-V patterning of the spinal cord and somite is largely complete by stage 15 (Brent and Tabin, 2002; Briscoe et al., 2000). More likely, these observations suggest that Shh either directly or indirectly affects sensory pathfinding. Shh contributes to both ventral (Charron et al., 2003) and longitudinal guidance (Bourikas et al., 2005) of commissural axons in the spinal cord, suggesting Shh could directly affect the pathfinding of trkA+ sensory afferents.

Alternatively, Shh may indirectly affect the pathfinding of later arising trkA+ fibers, via its effects on earlier arising sensory axons that normally pioneer the ventral sensory nerve or on the pathfinding of early arising motor neurons. In normal animals, later arising, trkA+ afferents tightly fasciculate with preexisting axons [Fig. 6A; (Guan et al., 2003)] and therefore closely follow the route established by the early pioneer fibers. When Shh function is reduced, there is a dramatic loss of trkC+ axons at stage 23 (Fig. 4), strongly suggesting that generation of ventral proprioceptive neurons is compromised or delayed in the absence of Shh. Late arising afferents are cUnc-5H3-negative and express neogenin (Guan and Condic, 2003), suggesting that netrin-1 from the dermamyotome or ventral neural tube could misdirect these neogenin-expressing growth cones along inappropriate medial and lateral routes when they extend in an environment where the normal ventral pioneer fiber are compromised.

Acknowledgments

We thank Drs. M.L. Vetter and H. J. Yost for critical reading of this manuscript, Dr. K. Zhang for technical help with experiments and Dr. F. Lefcort for trk antibodies. The 5E1 antibody was provided by the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported by R01 NS 048382 to MLC and R01 NS 16067 to SAS.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Supplementary Material

01

Supplemental Figure: (A) Distribution of trk receptor expression in mature DRG. TrkA expressing cells are found throughout the DRG, but make up the majority of the neurons in the dorsal-medial (DM) region. TrkC expressing cells are primarily found in the ventral-lateral (VL) region, with trkB expressing cells in the middle region of the DRG (Guan et al., 2003; Rifkin et al., 2000). (B) In situ hybridization for Shh receptor ptc-1 message. At stage 21, embryos were treated with Shh, 5E1 or control Ringers solution and allowed to develop to stage 25. Cryostat sections of stage 25. Dorsal is up, medial is left. Shh treatment greatly increased ptc-1 expression in the neural tube and DRG. In contrast, blocking Shh function with 5E1 dramatically decreased ptc-1 expression in these structures. NT: neural tube; DRG: dorsal root ganglion; DM: dermomyotome. Arrowheads indicate ptc-1 expressing cells. (C) The area of the largest profile of DRG LS1, LS2 and LS3 from control, Shh and 5E1 treated embryos were measured with NIH software at stage 25. *Significantly larger than control DRG (t-test, p<0.01).

REFERENCES

  1. Ahlgren SC, Bronner-Fraser M. Inhibition of sonic hedgehog signaling in vivo results in craniofacial neural crest cell death. Curr Biol. 1999;9:1304–14. doi: 10.1016/s0960-9822(00)80052-4. [DOI] [PubMed] [Google Scholar]
  2. Ahlgren SC, Thakur V, Bronner-Fraser M. Sonic hedgehog rescues cranial neural crest from cell death induced by ethanol exposure. Proc Natl Acad Sci U S A. 2002;99:10476–81. doi: 10.1073/pnas.162356199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bourikas D, Pekarik V, Baeriswyl T, Grunditz A, Sadhu R, Nardo M, Stoeckli ET. Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat Neurosci. 2005;8:297–304. doi: 10.1038/nn1396. [DOI] [PubMed] [Google Scholar]
  4. Brent AE, Tabin CJ. Developmental regulation of somite derivatives: muscle, cartilage and tendon. Curr Opin Genet Dev. 2002;12:548–57. doi: 10.1016/s0959-437x(02)00339-8. [DOI] [PubMed] [Google Scholar]
  5. Briscoe J, Pierani A, Jessell TM, Ericson J. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell. 2000;101:435–45. doi: 10.1016/s0092-8674(00)80853-3. [DOI] [PubMed] [Google Scholar]
  6. Caldero J, Prevette D, Mei X, Oakley RA, Li L, Milligan C, Houenou L, Burek M, Oppenheim RW. Peripheral target regulation of the development and survival of spinal sensory and motor neurons in the chick embryo. J Neurosci. 1998;18:356–70. doi: 10.1523/JNEUROSCI.18-01-00356.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Capdevila J, Tabin C, Johnson RL. Control of dorsoventral somite patterning by Wnt-1 and beta-catenin. Dev Biol. 1998;193:182–94. doi: 10.1006/dbio.1997.8806. [DOI] [PubMed] [Google Scholar]
  8. Carr VM, Simpson SB., Jr. Proliferative and degenerative events in the early development of chick dorsal root ganglia. I. Normal development. J Comp Neurol. 1978;182:727–39. doi: 10.1002/cne.901820410. [DOI] [PubMed] [Google Scholar]
  9. Charron F, Stein E, Jeong J, McMahon AP, Tessier-Lavigne M. The Morphogen Sonic Hedgehog Is an Axonal Chemoattractant that Collaborates with Netrin-1 in Midline Axon Guidance. Cell. 2003;113:11–23. doi: 10.1016/s0092-8674(03)00199-5. [DOI] [PubMed] [Google Scholar]
  10. Chen AI, de Nooij JC, Jessell TM. Graded activity of transcription factor Runx3 specifies the laminar termination pattern of sensory axons in the developing spinal cord. Neuron. 2006;49:395–408. doi: 10.1016/j.neuron.2005.12.028. [DOI] [PubMed] [Google Scholar]
  11. Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature. 1996;383:407–13. doi: 10.1038/383407a0. [DOI] [PubMed] [Google Scholar]
  12. Davies AM. Molecular and cellular aspects of patterning sensory neurone connections in the vertebrate nervous system. Development. 1987;101:185–208. [PubMed] [Google Scholar]
  13. Davies AM, Lumsden A. Ontogeny of the somatosensory system: origins and early development of primary sensory neurons. Annu Rev Neurosci. 1990;13:61–73. doi: 10.1146/annurev.ne.13.030190.000425. [DOI] [PubMed] [Google Scholar]
  14. Dorsky RI, Moon RT, Raible DW. Control of neural crest cell fate by the Wnt signalling pathway. Nature. 1998;396:370–3. doi: 10.1038/24620. [DOI] [PubMed] [Google Scholar]
  15. Dunn MK, Mercola M, Moore DD. Cyclopamine, a steroidal alkaloid, disrupts development of cranial neural crest cells in Xenopus. Dev Dyn. 1995;202:255–70. doi: 10.1002/aja.1002020305. [DOI] [PubMed] [Google Scholar]
  16. Ericson J, Morton S, Kawakami A, Roelink H, Jessell TM. Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell. 1996;87:661–73. doi: 10.1016/s0092-8674(00)81386-0. [DOI] [PubMed] [Google Scholar]
  17. Ericson J, Muhr J, Jessell TM, Edlund T. Sonic hedgehog: a common signal for ventral patterning along the rostrocaudal axis of the neural tube. Int J Dev Biol. 1995;39:809–16. [PubMed] [Google Scholar]
  18. Fan CM, Tessier-Lavigne M. Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell. 1994;79:1175–86. doi: 10.1016/0092-8674(94)90009-4. [DOI] [PubMed] [Google Scholar]
  19. Frank E, Sanes JR. Lineage of neurons and glia in chick dorsal root ganglia: analysis in vivo with a recombinant retrovirus. Development. 1991;111:895–908. doi: 10.1242/dev.111.4.895. [DOI] [PubMed] [Google Scholar]
  20. Fu M, Lui VC, Sham MH, Pachnis V, Tam PK. Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut. J Cell Biol. 2004;166:673–84. doi: 10.1083/jcb.200401077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guan W, Condic ML. Characterization of Netrin-1, Neogenin and cUnc-5H3 expression during chick dorsal root ganglia development. Gene Expr Patterns. 2003;3:369–73. doi: 10.1016/s1567-133x(03)00007-3. [DOI] [PubMed] [Google Scholar]
  22. Guan W, Puthenveedu MA, Condic ML. Sensory neuron subtypes have unique substratum preference and receptor expression before target innervation. J Neurosci. 2003;23:1781–91. doi: 10.1523/JNEUROSCI.23-05-01781.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hamburger V, Hamilton H. A series of normal stages in the development of chick embryo. J. Morph. 1951;88:49–92. [PubMed] [Google Scholar]
  24. Henion PD, Garner AS, Large TH, Weston JA. trkC-mediated NT-3 signaling is required for the early development of a subpopulation of neurogenic neural crest cells. Dev Biol. 1995;172:602–13. doi: 10.1006/dbio.1995.8054. [DOI] [PubMed] [Google Scholar]
  25. Honig MG, Lance-Jones C, Landmesser L. The development of sensory projection patterns in embryonic chick hindlimb under experimental conditions. Dev Biol. 1986;118:532–48. doi: 10.1016/0012-1606(86)90024-2. [DOI] [PubMed] [Google Scholar]
  26. Honore SM, Aybar MJ, Mayor R. Sox10 is required for the early development of the prospective neural crest in Xenopus embryos. Dev Biol. 2003;260:79–96. doi: 10.1016/s0012-1606(03)00247-1. [DOI] [PubMed] [Google Scholar]
  27. Inoue K, Ito K, Osato M, Lee B, Bae SC, Ito Y. The transcription factor Runx3 represses the neurotrophin receptor TrkB during lineage commitment of dorsal root ganglion neurons. J Biol Chem. 2007;282:24175–84. doi: 10.1074/jbc.M703746200. [DOI] [PubMed] [Google Scholar]
  28. Inoue K, Ozaki S, Shiga T, Ito K, Masuda T, Okado N, Iseda T, Kawaguchi S, Ogawa M, Bae SC, Yamashita N, Itohara S, Kudo N, Ito Y. Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons. Nat Neurosci. 2002;5:946–54. doi: 10.1038/nn925. [DOI] [PubMed] [Google Scholar]
  29. Josephson A, Widenfalk J, Trifunovski A, Widmer HR, Olson L, Spenger C. GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia. J Comp Neurol. 2001;440:204–17. doi: 10.1002/cne.1380. [DOI] [PubMed] [Google Scholar]
  30. Kenney AM, Cole MD, Rowitch DH. Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors. Development. 2003;130:15–28. doi: 10.1242/dev.00182. [DOI] [PubMed] [Google Scholar]
  31. Kenney AM, Widlund HR, Rowitch DH. Hedgehog and PI-3 kinase signaling converge on Nmyc1 to promote cell cycle progression in cerebellar neuronal precursors. Development. 2004;131:217–28. doi: 10.1242/dev.00891. [DOI] [PubMed] [Google Scholar]
  32. Kitsukawa T, Shimizu M, Sanbo M, Hirata T, Taniguchi M, Bekku Y, Yagi T, Fujisawa H. Neuropilin-semaphorin III/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice. Neuron. 1997;19:995–1005. doi: 10.1016/s0896-6273(00)80392-x. [DOI] [PubMed] [Google Scholar]
  33. Kramer I, Sigrist M, de Nooij JC, Taniuchi I, Jessell TM, Arber S. A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification. Neuron. 2006;49:379–93. doi: 10.1016/j.neuron.2006.01.008. [DOI] [PubMed] [Google Scholar]
  34. Kraus P, Fraidenraich D, Loomis CA. Some distal limb structures develop in mice lacking Sonic hedgehog signaling. Mech Dev. 2001;100:45–58. doi: 10.1016/s0925-4773(00)00492-5. [DOI] [PubMed] [Google Scholar]
  35. Lawson SN, Biscoe TJ. Development of mouse dorsal root ganglia: an autoradiographic and quantitative study. J Neurocytol. 1979;8:265–74. doi: 10.1007/BF01236122. [DOI] [PubMed] [Google Scholar]
  36. Lawson SN, Caddy KW, Biscoe TJ. Development of rat dorsal root ganglion neurones. Studies of cell birthdays and changes in mean cell diameter. Cell Tissue Res. 1974;153:399–413. doi: 10.1007/BF00229167. [DOI] [PubMed] [Google Scholar]
  37. Le douarin NM, Kalcheim C. The neural crest. In: Bard JB, Barlow PW, Kirk DL, editors. “Developmental and cell biology”. Cambridge; Cambridge: 1999. pp. 153–197. [Google Scholar]
  38. Lee KJ, Jessell TM. The specification of dorsal cell fates in the vertebrate central nervous system. Annu Rev Neurosci. 1999;22:261–94. doi: 10.1146/annurev.neuro.22.1.261. [DOI] [PubMed] [Google Scholar]
  39. Levanon D, Bettoun D, Harris-Cerruti C, Woolf E, Negreanu V, Eilam R, Bernstein Y, Goldenberg D, Xiao C, Fliegauf M, Kremer E, Otto F, Brenner O, Lev-Tov A, Groner Y. The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons. Embo J. 2002a;21:3454–63. doi: 10.1093/emboj/cdf370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Levanon D, Bettoun D, Harris-Cerruti C, Woolf E, Negreanu V, Eilam R, Bernstein Y, Goldenberg D, Xiao C, Fliegauf M, Kremer E, Otto F, Brenner O, Lev-Tov A, Groner Y. The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons. Embo J. 2002b;21:3454–63. doi: 10.1093/emboj/cdf370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Liem KF, Jr., Tremml G, Jessell TM. A role for the roof plate and its resident TGFbeta-related proteins in neuronal patterning in the dorsal spinal cord. Cell. 1997;91:127–38. doi: 10.1016/s0092-8674(01)80015-5. [DOI] [PubMed] [Google Scholar]
  42. Liem KF, Jr., Tremml G, Roelink H, Jessell TM. Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell. 1995;82:969–79. doi: 10.1016/0092-8674(95)90276-7. [DOI] [PubMed] [Google Scholar]
  43. Lowrey JA, Stewart GA, Lindey S, Hoyne GF, Dallman MJ, Howie SE, Lamb JR. Sonic hedgehog promotes cell cycle progression in activated peripheral CD4(+) T lymphocytes. J Immunol. 2002;169:1869–75. doi: 10.4049/jimmunol.169.4.1869. [DOI] [PubMed] [Google Scholar]
  44. Marigo V, Scott MP, Johnson RL, Goodrich LV, Tabin CJ. Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development. 1996;122:1225–33. doi: 10.1242/dev.122.4.1225. [DOI] [PubMed] [Google Scholar]
  45. Marigo V, Tabin CJ. Regulation of patched by sonic hedgehog in the developing neural tube. Proc Natl Acad Sci U S A. 1996;93:9346–51. doi: 10.1073/pnas.93.18.9346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Marmigere F, Ernfors P. Specification and connectivity of neuronal subtypes in the sensory lineage. Nat Rev Neurosci. 2007;8:114–27. doi: 10.1038/nrn2057. [DOI] [PubMed] [Google Scholar]
  47. Marti E, Takada R, Bumcrot DA, Sasaki H, McMahon AP. Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo. Development. 1995;121:2537–47. doi: 10.1242/dev.121.8.2537. [DOI] [PubMed] [Google Scholar]
  48. Masuda T, Fukamauchi F, Takeda Y, Fujisawa H, Watanabe K, Okado N, Shiga T. Developmental regulation of notochord-derived repulsion for dorsal root ganglion axons. Mol Cell Neurosci. 2004;25:217–27. doi: 10.1016/j.mcn.2003.10.005. [DOI] [PubMed] [Google Scholar]
  49. Masuda T, Okado N, Shiga T. The involvement of axonin-1/SC2 in mediating notochord-derived chemorepulsive activities for dorsal root ganglion neurites. Dev Biol. 2000;224:112–21. doi: 10.1006/dbio.2000.9813. [DOI] [PubMed] [Google Scholar]
  50. Masuda T, Tsuji H, Taniguchi M, Yagi T, Tessier-Lavigne M, Fujisawa H, Okado N, Shiga T. Differential non-target-derived repulsive signals play a critical role in shaping initial axonal growth of dorsal root ganglion neurons. Dev Biol. 2003;254:289–302. doi: 10.1016/s0012-1606(02)00087-8. [DOI] [PubMed] [Google Scholar]
  51. Mu X, Silos-Santiago I, Carroll SL, Snider WD. Neurotrophin receptor genes are expressed in distinct patterns in developing dorsal root ganglia. J Neurosci. 1993;13:4029–41. doi: 10.1523/JNEUROSCI.13-09-04029.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Oakley RA, Karpinski BA. Target-independent specification of proprioceptive sensory neurons. Dev Biol. 2002;249:255–69. doi: 10.1006/dbio.2002.0775. [DOI] [PubMed] [Google Scholar]
  53. Oakley RA, Lefcort FB, Plouffe P, Ritter A, Frank E. Neurotrophin-3 promotes the survival of a limited subpopulation of cutaneous sensory neurons. Dev Biol. 2000;224:415–27. doi: 10.1006/dbio.2000.9804. [DOI] [PubMed] [Google Scholar]
  54. Oliver TG, Grasfeder LL, Carroll AL, Kaiser C, Gillingham CL, Lin SM, Wickramasinghe R, Scott MP, Wechsler-Reya RJ. Transcriptional profiling of the Sonic hedgehog response: a critical role for N-myc in proliferation of neuronal precursors. Proc Natl Acad Sci U S A. 2003;100:7331–6. doi: 10.1073/pnas.0832317100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Oppenheim RW, Homma S, Marti E, Prevette D, Wang S, Yaginuma H, McMahon AP. Modulation of early but not later stages of programmed cell death in embryonic avian spinal cord by sonic hedgehog. Mol Cell Neurosci. 1999;13:348–61. doi: 10.1006/mcne.1999.0755. [DOI] [PubMed] [Google Scholar]
  56. Pearse RV, 2nd, Vogan KJ, Tabin CJ. Ptc1 and Ptc2 transcripts provide distinct readouts of Hedgehog signaling activity during chick embryogenesis. Dev Biol. 2001;239:15–29. doi: 10.1006/dbio.2001.0430. [DOI] [PubMed] [Google Scholar]
  57. Placzek M, Furley A. Patterning cascades in the neural tube. Neural development. Curr Biol. 1996;6:526–9. doi: 10.1016/s0960-9822(02)00533-x. [DOI] [PubMed] [Google Scholar]
  58. Rifkin JT, Todd VJ, Anderson LW, Lefcort F. Dynamic expression of neurotrophin receptors during sensory neuron genesis and differentiation. Dev Biol. 2000;227:465–80. doi: 10.1006/dbio.2000.9841. [DOI] [PubMed] [Google Scholar]
  59. Roelink H, Porter JA, Chiang C, Tanabe Y, Chang DT, Beachy PA, Jessell TM. Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell. 1995;81:445–55. doi: 10.1016/0092-8674(95)90397-6. [DOI] [PubMed] [Google Scholar]
  60. Salie R, Niederkofler V, Arber S. Patterning molecules; multitasking in the nervous system. Neuron. 2005;45:189–92. doi: 10.1016/j.neuron.2005.01.002. [DOI] [PubMed] [Google Scholar]
  61. Sanz-Ezquerro JJ, Tickle C. Autoregulation of Shh expression and Shh induction of cell death suggest a mechanism for modulating polarising activity during chick limb development. Development. 2000;127:4811–23. doi: 10.1242/dev.127.22.4811. [DOI] [PubMed] [Google Scholar]
  62. Sela-Donenfeld D, Kalcheim C. Regulation of the onset of neural crest migration by coordinated activity of BMP4 and Noggin in the dorsal neural tube. Development. 1999;126:4749–62. doi: 10.1242/dev.126.21.4749. [DOI] [PubMed] [Google Scholar]
  63. Tanabe Y, Jessell TM. Diversity and pattern in the developing spinal cord. Science. 1996;274:1115–23. doi: 10.1126/science.274.5290.1115. [DOI] [PubMed] [Google Scholar]
  64. Taniguchi M, Yuasa S, Fujisawa H, Naruse I, Saga S, Mishina M, Yagi T. Disruption of semaphorin III/D gene causes severe abnormality in peripheral nerve projection. Neuron. 1997;19:519–30. doi: 10.1016/s0896-6273(00)80368-2. [DOI] [PubMed] [Google Scholar]
  65. Testaz S, Jarov A, Williams KP, Ling LE, Koteliansky VE, Fournier-Thibault C, Duband JL. Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched- Smoothened-Gli signaling pathway. Proc Natl Acad Sci U S A. 2001;98:12521–6. doi: 10.1073/pnas.221108698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Thibert C, Teillet MA, Lapointe F, Mazelin L, Le Douarin NM, Mehlen P. Inhibition of neuroepithelial patched-induced apoptosis by sonic hedgehog. Science. 2003;301:843–6. doi: 10.1126/science.1085405. [DOI] [PubMed] [Google Scholar]
  67. Todt WL, Fallon JF. Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation. Development. 1987;101:501–15. doi: 10.1242/dev.101.3.501. [DOI] [PubMed] [Google Scholar]
  68. Tosney KW, Landmesser LT. Growth cone morphology and trajectory in the lumbosacral region of the chick embryo. J Neurosci. 1985;5:2345–58. doi: 10.1523/JNEUROSCI.05-09-02345.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Ungos JM, Karlstrom RO, Raible DW. Hedgehog signaling is directly required for the development of zebrafish dorsal root ganglia neurons. Development. 2003;130:5351–62. doi: 10.1242/dev.00722. [DOI] [PubMed] [Google Scholar]
  70. Wakamatsu Y, Maynard TM, Jones SU, Weston JA. NUMB localizes in the basal cortex of mitotic avian neuroepithelial cells and modulates neuronal differentiation by binding to NOTCH-1. Neuron. 1999;23:71–81. doi: 10.1016/s0896-6273(00)80754-0. [DOI] [PubMed] [Google Scholar]
  71. Wakamatsu Y, Maynard TM, Weston JA. Fate determination of neural crest cells by NOTCH-mediated lateral inhibition and asymmetrical cell division during gangliogenesis. Development. 2000;127:2811–21. doi: 10.1242/dev.127.13.2811. [DOI] [PubMed] [Google Scholar]
  72. Wang G, Scott SA. The “waiting period” of sensory and motor axons in early chick hindlimb: its role in axon pathfinding and neuronal maturation. J Neurosci. 2000;20:5358–66. doi: 10.1523/JNEUROSCI.20-14-05358.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. White FA, Behar O. The development and subsequent elimination of aberrant peripheral axon projections in Semaphorin3A null mutant mice. Dev Biol. 2000;225:79–86. doi: 10.1006/dbio.2000.9822. [DOI] [PubMed] [Google Scholar]
  74. White FA, Silos-Santiago I, Molliver DC, Nishimura M, Phillips H, Barbacid M, Snider WD. Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia. J Neurosci. 1996;16:4662–72. doi: 10.1523/JNEUROSCI.16-15-04662.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Wright DE, Snider WD. Neurotrophin receptor mRNA expression defines distinct populations of neurons in rat dorsal root ganglia. J Comp Neurol. 1995;351:329–38. doi: 10.1002/cne.903510302. [DOI] [PubMed] [Google Scholar]
  76. Wu C, Shimo T, Liu M, Pacifici M, Koyama E. Sonic hedgehog functions as a mitogen during bell stage of odontogenesis. Connect Tissue Res. 2003;44(Suppl 1):92–6. [PubMed] [Google Scholar]
  77. Yanfeng W, Saint-Jeannet JP, Klein PS. Wnt-frizzled signaling in the induction and differentiation of the neural crest. Bioessays. 2003;25:317–25. doi: 10.1002/bies.10255. [DOI] [PubMed] [Google Scholar]
  78. Zeng L, Kempf H, Murtaugh LC, Sato ME, Lassar AB. Shh establishes an Nkx3.2/Sox9 autoregulatory loop that is maintained by BMP signals to induce somitic chondrogenesis. Genes Dev. 2002;16:1990–2005. doi: 10.1101/gad.1008002. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Supplemental Figure: (A) Distribution of trk receptor expression in mature DRG. TrkA expressing cells are found throughout the DRG, but make up the majority of the neurons in the dorsal-medial (DM) region. TrkC expressing cells are primarily found in the ventral-lateral (VL) region, with trkB expressing cells in the middle region of the DRG (Guan et al., 2003; Rifkin et al., 2000). (B) In situ hybridization for Shh receptor ptc-1 message. At stage 21, embryos were treated with Shh, 5E1 or control Ringers solution and allowed to develop to stage 25. Cryostat sections of stage 25. Dorsal is up, medial is left. Shh treatment greatly increased ptc-1 expression in the neural tube and DRG. In contrast, blocking Shh function with 5E1 dramatically decreased ptc-1 expression in these structures. NT: neural tube; DRG: dorsal root ganglion; DM: dermomyotome. Arrowheads indicate ptc-1 expressing cells. (C) The area of the largest profile of DRG LS1, LS2 and LS3 from control, Shh and 5E1 treated embryos were measured with NIH software at stage 25. *Significantly larger than control DRG (t-test, p<0.01).

NIHMS40749-supplement-01.tif (6.1MB, tif)

RESOURCES