Temporal and spatial requirements of Smoothened in ventral midbrain neuronal development
© Tang et al.; licensee BioMed Central Ltd. 2013
Received: 18 January 2013
Accepted: 5 April 2013
Published: 26 April 2013
Several studies have indicated that Sonic hedgehog (Shh) regulates the expansion of dopaminergic (DA) progenitors and the subsequent generation of mature DA neurons. This prevailing view has been based primarily on in vitro culture results, and the exact in vivo function of Shh signaling in the patterning and neurogenesis of the ventral midbrain (vMB) remains unclear.
We characterized the transcriptional codes for the vMB progenitor domains, and correlated them with the expression patterns of Shh signaling effectors, including Shh, Smoothened, Patched, Gli1, Gli2 and Gli3.
While Shh and its downstream effectors showed robust expression in the neurogenic niche for DA progenitors at embryonic day (E)8 to E8.5, their expression shifted to the lateral domains from E9.5 to E12.5. Consistent with this dynamic change, conditional mutants with region-specific removal of the Shh receptor Smoothened in the vMB progenitors (Shh-Cre;Smo fl/fl ) showed a transient reduction in DA progenitors and DA neurons at E10.5, but had more profound defects in neurons derived from the more lateral domains, including those in the red nucleus, oculomotor nucleus, and raphe nuclei. Conversely, constitutive activation of Smoothened signaling in vMB (Shh-Cre;SmoM2) showed transient expansion of the same progenitor population. To further characterize the nature of Shh-Smoothened signaling in vMB, we examined the BAT-GAL reporter and the expression of Wnt1 in vMB, and found that the antagonistic effects of Shh and Wnt signaling critically regulate the development of DA progenitors and DA neurons.
These results highlight previously unrecognized effects of Shh-Smoothened signaling in the region-specific neurogenesis within the vMB.
The mechanisms that govern the patterning of the neural tube and the subsequent generation of diverse neuronal subtypes have attracted intense attention. Because of its highly conserved structure, the developing spinal cord has provided an elegant model system to identify cell intrinsic and extrinsic cues that control the expansion of progenitors and differentiation of neurons . Sonic hedgehog (Shh) is a potent morphogen that controls the development of spinal cord [1, 2]. It is well established that temporal adaption to the graded Shh signals determines the progenitor and neuron identity in the ventral spinal cord [3–5]. Furthermore, the transcriptional network acting downstream of Shh provides important clues to the molecular logics that govern the diversity of ventral neural tube development .
In addition to the spinal cord, Shh has also been shown to regulate cell fate, expansion, and self-renewal of progenitors in the ventral forebrain, midbrain, and midbrain/hindbrain boundary (MHB) [7–10]. For instance, exogenous Shh, together with fibroblast growth factor (FGF) 8, can induce midbrain dopaminergic (DA) neurons in culture [11, 12]. Furthermore, fate-mapping studies show that Shh-expressing progenitors give rise to different neurons in the ventral midbrain (vMB) [13–16]. Indeed, several conditional mutants have been developed to remove Shh or the Shh receptor Smoothened using the Engrail1-Cre (En1-Cre;Smo fl/fl ) mutation, which specifically targets the mid/hindbrain region [8, 17]. These mutants show severe defects in the DA neurons, but it remains unclear if these defects are directly due to the effects of Shh in promoting DA neuron development or are caused by the loss of FGF8 and by profound MHB patterning defects in En1-Cre;Smo fl/fl mutants [18–20]. Thus, the exact role of Shh signaling in the development of DA and other progenitors in vMB remains unclear.
In this study, we used a set of transcription factors to define four distinct progenitor domains in vMB. Shh and its downstream effectors also showed robust expression in the neurogenic niche for DA progenitors at embryonic day (E)8 to E8.5, but their expression became progressively restricted to the lateral domains in vMB from E9.5 to E12.5. Interestingly, conditional mutants with vMB-specific removal of the Shh receptor Smoothened (Shh-Cre;Smo fl/fl ) showed a transient reduction in DA progenitors and DA neurons at E10.5, but had more profound defects in neurons derived from the more lateral progenitor domains. Conversely, constitutive activation of Smoothened signaling in vMB (Shh-Cre;SmoM2) showed a transient expansion of the same progenitor population. The transient effects of Shh-Smoothened signaling in vMB were due to the antagonistic effects of Shh and Wnt signaling that critically regulate the development of DA progenitors and DA neurons. Together, our results provide comprehensive views of the effects of Shh signaling on neurogenesis in vMB.
All procedures were approved by the University of California, San Francisco Institutional Animal Care and Use Committee. Shh-Cre, Smoothened fl/fl (Smo fl/fl ), SmoothenedM2 (SmoM2), Rosa26 (R26R) and BAT-GAL mice (stock numbers 005622, 004526, 005130, 003474 and 005317, respectively; the Jackson Laboratory, Bar Harbor, ME, USA). To generate conditional mutant mice that lacked Smoothened in the ventral neurogenic niche for DA neurons, Smo fl/fl mice were first crossed with Shh-Cre to generate Shh-Cre;Smo fl/+ mice, then Shh-Cre;Smo fl/+ mice were crossed with Smo fl/fl to generate the Shh-Cre;Smo fl/fl mutant. We also used the same Cre line to generate conditional mutants in which the constitutive active Smoothened receptor was expressed in the Shh-Cre domain (Shh-Cre;SmoM2).
Histology and immunohistochemistry
Histology and immunohistochemistry (IHC) were performed as described previously with minor modifications [21, 22]. Specifically, mouse embryos were collected E8.5, E9.5, E10.5, E11.5, and E12.5, then fixed in 4% paraformaldehyde (PFA) for 0.5 to 2 hours, followed by cryoprotection in 15 to 30% sucrose solutions, and sectioned on a cryostat (Leica, Heerbrugg, Switzerland). Primary antibodies in this study were: anti-Brn3a antibody (1:1,000; ), anti-β-galactosidase (β-Gal; 1:20; #40-1a; Developmental Hybridoma Study Bank (DHSB), Iowa City, IA, USA), anti-Foxa2 (1:500; #3143; Cell Signaling Technology, Danvers, MA, USA), anti-5-hydroxytryptamine (anti-5-HT; 1:500; #20080; ImmunoStar Inc., Hudson, WI, USA), anti-Lmx1a (1:1,000; gift of Dr Mike German, UCSF), anti-Islet1 (1:50; 39.4D5; DHSB), anti-Nkx2.2 (1:50; 74.5A5; DHSB), anti-Nkx6.1 (1:50; F55A10; DHSB), anti-Nurr1 (1:500; sc-990; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Olig2 (1:1,000; Gift of Dr David Rowitch, UCSF), anti-Pax6 (1:50; Pax6; DHSB), anti-Shh (1:200; #2207; Cell Signaling Technology), anti-Sox2 (1:200; AB5603; Millipore Corp., Billerica, MA, USA), and anti-tyrosine hydroxylase (anti-TH; 1:500; ab113; Abcam, Cambridge, MA, USA). For immunofluorescence staining, sections were incubated with primary antibody overnight, followed by secondary antibodies conjugated with Alexa fluorophores 488 and 568 (Invitrogen Corp., Carlsbad, CA, USA) for 1 hour to detect signals. For chromogen staining, sections were incubated with primary antibody overnight, followed by incubation for 1 hour with biotinylated IgG and avidin–biotin complex (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine (DAB) solution was used to visualize the results. Images were captured using a confocal microscope (LSM 510l Carl Zeiss 510 Microimaging, Jena, Germany), or a microscope (BX41 Olympus, Tokyo, Japan) equipped with a charge-coupled device (CCD) camera (DP70; Olympus).
In situ hybridization
RNA probes for in situ hybridization were prepared using plasmids that contained cDNA for Smoothened, Patched, Gli1, Gli2, Gli3 (gifts from Dr. Arturo Alvarez-Buylla, UCSF), FGF8, or Wnt1. The plasmids were linearized with appropriate restriction enzymes, and transcribed with SP6, T7, or T3 polymerase using digoxigenin (DIG)-labeling reagents and a DIG RNA labeling kit (Roche Diagnostics, Basel, Switzerland). For in situ hybridization, embryos were fixed overnight at room temperature in 4% PFA in diethylpyrocarbonate (DEPC)-treated PBS, cryoprotected in 15% and 30% sucrose, and embedded in optimal cutting temperature (OCT) compound, then sections were cut at 10 μm on Leica CM1950 crystat (Leica Microsystems, Buffalo Grove, IL, USA). During hybridization, sections were first post-fixed with 4% PFA, then washed with acetylation solution and 1% Triton X-100. Sections were incubated with hybridization buffer (Amresco LLC, Solon, OH, USA) for 2 to 4 hrs before applying hybridization buffer containing DIG-labeled riboprobes (200 to 400 ng/ml) at 65°C overnight. On the second day, slides were washed twice for 30 minutes each with 0.2 × SSC (0.1% Tween 20, pH 4.5) at 65°C, then twice for 10 minutes each with a solution of 100 mmol/l Maleic acid, 150 mmol/l NaCl, 2 mmol/l levamisole and 0.1% Tween (pH 7.5). Sections were blocked for 1 hour and incubated with anti-DIG antibody overnight at 4°C. For visualizing the in situ hybridization results, we used BM purple (Boehringer Mannheim, Mannheim, Germany). Finally, the slides were dried at room temperature and mounted (Clear Mount; Electron Microscopy Sciences, Hatfield, PA, USA).
Data were analyzed by two-tailed Student’s t test. Values were expressed as mean ± s.e.m. Changes were considered as significant at P<0.05.
Transcriptional codes define distinct temporal and spatial progenitor domains in the early embryonic ventral midbrain
Taken together, these results highlighted the dynamic expansion of the progenitor domains from E8.5 to E12.5 in the developing vMB. Furthermore, they provided an important framework to investigate potential effects of exogenous and intrinsic mechanisms that might affect the generation of DA neurons and other neuron subtypes at these critical developmental stages.
Dynamic expression of Shh and Shh downstream effectors in the developing ventral midbrain
It has been well established that temporal adaption to gradient Shh signaling specifies the formation of different progenitor domains in the ventral spinal cord, and thereby controls the generation of different classes of neurons [1, 5, 25, 28]. To understand the roles of Shh signaling in controlling the formation of progenitor domains and generation of different classes of neurons in the developing vMB, we characterized the spatial and temporal expression patterns of Shh and Shh downstream signaling effectors, including Smoothened, Patched, Gli1, Gli2, and Gli3.
Unlike the dynamic changes of Shh expression in vMB, we found that Smoothened, one of the receptors for Shh, showed a rather diffuse expression pattern that covered both ventral and dorsal parts of the developing midbrain from E8.5 to E10.5. From E10.5 onward, Smoothened expression became more restricted to the ventricular zone within the vMB (Figure 2E-H). In addition to examining Smoothened, we also examined the expression patterns of several Shh signaling effectors, including Patched, Gli1, Gli2, and Gli3. We found that Patched and Gli1 were both transiently expressed in the ventral medial region at E8 to E8.5. From E9.5 onward, the expression of Patched and Gli1 shifted laterally, and became more prominent in the ventricular zone of vMB D3 and D4 domains (Figure 2I-P). The expression pattern of Gli2 resembled those of Patched and Gli1, with a very robust level in the D1 and D2 domains at E8 to E8.5 (Figure 2Q), and shifting laterally and dorsally from E9.5 onwards (Figure 2R-T). Finally, Gli3, the major repressor of Shh signaling, showed low and diffuse expression in the vMB at E8 to E8.5 (Figure 2U), but its expression became restricted to the dorsal part of midbrain after E9.5 (Figure 2V-X).
Based on the dynamic, yet significant overlapping, expression of Shh signaling effectors in the vMB progenitor domains, these results suggest that Shh signaling might affect the temporal and spatial development of medial progenitors before E10.5. After E11.5, the lateral domains were the major regions receiving Shh signals. These results are reminiscent of the medial to lateral shift of progenitor domains in ventral spinal cord [4, 5], and suggest that the effects of Shh on the progenitors and neurons arising from the medial domains could be transient, whereas the effects on progenitors and neurons arising from lateral domains could last longer.
Removal of Smoothened in ventral midbrain leads to a transient reduction in ventral progenitors
Loss of Smoothened in ventral midbrain affects the generation of neurons in red nucleus, oculomotor nucleus, and raphe nuclei, but not dopaminergic neurons
Constitutive activation of smoothened transiently expand progenitors in ventral midbrain
Because removal of Smoothened led to a transient reduction in vMB progenitors, we investigated if constitutive activation of Smoothened might have the opposite effect. As anticipated, we detected an expansion of vMB in Shh-Cre;SmoM2 mice. Interestingly, several lines of evidence indicated that the expansion of vMB in Shh-Cre;SmoM2 mice occurred along the anterior-posterior (A-P) axis. First, by sectioning E10.5 vMB in the coronal plane at 60 μm intervals, we detected more sections that contained the progenitor domains in vMB of Shh-Cre;SmoM2 mice compared with SmoM2 controls (Figure 8C-D,c1-3,d1-4). As a consequence, the total number of progenitors in vMB labeled by Lmx1a, Foxa2, Nkx2.1, and Nkx6.1 showed significant increases at E10.5 (Figure 8E). Second, when examined on sagittal planes, the Lmx1a expression domain was seen to extend anteriorly in Shh-Cre;SmoM2 mice, whereas the posterior boundary of he Lmx1a+ domain ended in the same MHB in both control and Shh-Cre;SmoM2 mutants (Figure 8B-B’). Such expansion of the vMB was not detected at E9.5 or E12.5 (data not shown). Together, these results were consistent with the Smoothened loss-of-function data (Figure 5, Figure 7), and further confirmed that the effect of Smoothened signaling had transient effects on the development of progenitors in vMB.
Smoothened antagonizes Wnt signaling in dopaminergic neuron development in ventral midbrain
Previously, we reported that stabilizing the canonical Wnt signaling antagonized Shh expression in vMB to control the temporal development of DA neurons . To examine the effect of Shh signaling on Wnt1 expression, we performed in situ hybridization to examine the expression of Wnt1 mRNA in both Shh-Cre;Smo fl/fl and Shh-Cre;SmoM2 mutants. Consistent with our prediction, at both E10.5 and E12.5, Wnt1 mRNA levels were increased in the vMB of Shh-Cre;Smo fl/fl compared with control (Figure 9E-F’). By contrast, in the Shh-Cre;SmoM2 mutant, Wnt1 mRNA level was modestly downregulated at E10.5, but returned to the control levels at E12.5 (Figure 9G-H’).
To further investigate the effects of Smoothened on Wnt signaling, we generated SmoM2;BAT-GAL and Shh-Cre;SmoM2;BAT-GAL mice, in which the Wnt signaling reporter, BAT-GAL, could be used as a surrogate for canonical Wnt activity [20, 34]. Consistent with the Wnt1 mRNA changes in Shh-Cre;SmoM2 mutants, quantification of the number of β-Gal+ cells showed a significant reduction at E10.5 in Shh-Cre;SmoM2;BAT-GAL, but returned to the control level at E12.5 (Figure 9I-J). Collectively, these data suggest that there is a mutual antagonism effect between Shh and Wnt signaling in vMB. Perturbations in the Shh signaling mechanism triggered a transient, compensatory activation of Wnt signaling on vMB at E10.5.
Dynamic progression of progenitor domains in early ventral midbrain development
Several lines of evidence indicate that the ventral region of the developing neural tube contain progenitors that can be divided into distinct domains based on the expression of cell type-specific transcriptional factors, which are required for the development of different groups of neurons in the ventral neural tube [1, 4, 5]. Although previous studies attempted to define the vMB progenitor domains based on the expression of transcriptional factors , their results do not provide the temporal resolution of these progenitor domains in the developing vMB at the stages when patterning, expansion, and differentiation of these progenitors are active. By contrast, our results show that a combinatorial code of cell type-specific transcription factors defines discrete progenitor domains in vMB that are distinctly different from the ventral progenitors in spinal cord. First, the progenitor domains in vMB, marked by Lmx1a, Foxa2, Nkx2.1, and Nkx6.1, are identified along the midline at E8 to E8.5 (D1 and D2 domains), and subsequently expand to the lateral domains from E9.5 to E12.5 (D3 and D4 domains) (Figure 1, Figure 3). Although such medial to lateral expansion in vMB is similar to the ventral to dorsal expansion in the spinal cord, the Foxa2+ progenitor domain undergoes a tremendous expansion in vMB as neurogenesis progresses, compared with its progressively more restricted pattern in the most ventral region of the spinal cord. Second, unlike the spinal cord, the Foxa2+ progenitors in vMB show extensive coexpression with Nkx6.1 and transient coexpression with Nkx2.2. Finally, our results showed no detectable Olig2 expression in vMB at E10.5, whereas Olig2 was expressed in the motor neuron progenitor (pMN) domain in spinal cord (Figure 1). Furthermore, the Pax6+ progenitors, which could be detected from pMN to p0 domains in the spinal cord, were distinctly absent in the vMB at E10.5. Together, our data clearly delineate the dynamic expansion of the vMB progenitor domains, which show important differences from those in the spinal cord.
Sonic hedgehog-Smoothened signaling and neuronal development in ventral midbrain
Several studies have identified the Shh-expressing domain in vMB as an enriched source that gives rise to many neurons in the adult midbrain, including the DA neurons and neurons in the red nucleus [13–15]. Indeed, our results confirm and extend these findings by showing that the Shh signaling effectors are expressed in the medial D1 and D2 domains at E8 to E8.5. Interestingly, the Shh receptor Smoothened continued to show broad expression in vMB from E9.5 to 10.5, but became more restricted to the ventricular zone, especially in the neurogenic niche for DA progenitors, at E12.5 (Figure 2). By contrast, expression of Patched and Gli1 shifted to the lateral D3 and D4 domains in vMB from E9.5 to 11.5, whereas expression of Gli2 and Gli3 was present primarily in the dorsal midbrain (Figure 2, Figure 3). Furthermore, our fate-mapping data show that the majority of the neurons in the red nucleus, oculomotor nucleus, and raphe nuclei are derived from progenitors that respond to Shh signaling (Figure 6). These results represent the first comprehensive view of the dynamic changes in the expression of Shh signaling effectors, and provide an important framework to understand how perturbation of Shh signaling might affect the development of neurons from the progenitors in vMB.
Intriguingly, despite the broad expression of Shh signaling effectors in vMB at E8 to E8.5, removal of Smoothened using Shh-Cre resulted in only a transient reduction in DA progenitors at E10.5. The delay in the onset of detectable loss of DA progenitors in the vMB of Shh-Cre;Smo fl/fl mutants may be related to the slow turnover of Smoothened proteins after Cre recombination. Alternatively, the onset of Shh-Cre-mediated removal of Smoothened may not have completely removed Smoothened from the DA progenitors. Regardless of the exact mechanism, the DA progenitors in Shh-Cre;Smo fl/fl mutants returned to the control level by E12.5. This modest and transient loss of the DA progenitors and DA neurons in Shh-Cre;Smo fl/fl mutants is different from the severe DA neuron deficits seen in the En1-Cre;Smo fl/fl or En1-Cre;Shh fl/fl mutants [8, 17], most likely due to the general patterning defects in dorsal and ventral midbrain caused by the En1-Cre. Consistent with this notion, FGF8 expression, which is present in the MHB, is severely perturbed in both En1-Cre;Smo fl/fl and En1-Cre;Shh fl/fl mutants. By contrast, we did not observe any changes in FGF8 expression either in Shh-Cre;Smo fl/fl or Shh-Cre;SmoM2 mutants (Figure 9A-D). FGF8 has been shown to be required for the patterning of MHB, expansion of DA progenitors, and the induction of DA neurons [12, 20, 33]. Hence, perturbation to FGF8 expression in En1-Cre;Smo fl/fl and En1-Cre;Shh fl/fl mutants is likely to have a lasting effect on DA neurons owing to non-cell autonomous effects.
In contrast to the modest, transient phenotype in DA neurons, a pronounced and persistent deficit was noted in neurons derived from the more lateral D2 and D3 domains, including red nucleus neurons, oculomotor neurons, and serotonergic neurons (Figure 5, Figure 6, Figure 7). These results are consistent with the temporal and spatial requirements of Shh signaling in digit formation and ventral spinal cord development that have been shown previously by fate-mapping and genetic-ablation studies [5, 37]. In addition, our results support the evolutionarily conserved function of Shh signaling on midbrain neuron development in chicks and mammals [38, 39]. Perturbations to Shh-Smoothened signaling are likely to contribute to congenital defects involving midbrain neurons that are critical for extraocular movement, autonomic functions, and control of locomotion and respiratory rhythms [40–42].
Antagonistic effects between Shh and Wnt signaling in dopaminergic neuron development
Both loss-of-function and gain-of-function analyses of β-catenin in vMB have shown that canonical Wnt signaling antagonizes Shh expression during the neurogenesis of DA neurons [29, 35]. Such effects of Wnt and Shh have also been confirmed for the generation of DA neurons from stem cells . Using in situ hybridization for Wnt1 expression, we found increased Wnt1 expression in the neurogenic niche for DA neurons in Shh-Cre;Smo fl/fl mutants (Figure 9E-F’). Conversely, the Smoothened gain-of-function mutants Shh-Cre;SmoM2 mutants exhibited reduced BAT-GAL reporter activity, indicating that the canonical Wnt activity is reduced in these mutants (Figure 9). Despite the increase in Wnt1 expression, however, Shh-Cre;Smo fl/fl mutants showed a decrease in the DA progenitors at E10.5, suggesting that Shh-Smoothened activity, but not canonical Wnt signaling, has a more dominant effect in regulating the DA progenitor development in vMB at this stage (Figure 5). These results are consistent with our previous observations that stabilization of Wnt-β-catenin signaling using Shh-Cre expands DA progenitors only after E12.5, despite the fact that Shh-Cre recombination occurs as early as E9.5 .
In conclusion, our study shows that region-specific removal of Smoothened in vMB has a surprisingly modest and transient effect in the development of DA progenitors and DA neurons. By contrast, loss of Smoothened has more severe and persistent effects on the neurons derived from lateral domains of the vMB. These results provide important insights to the previously unrecognized roles of Shh-Smoothened in the development of neurons that are critical to the control of extra-ocular movement, locomotion, and respiratory rhythms.
This work was supported by grants from the National Institute of Health OD010927, the Department of Veterans Affairs Merit Review Award BX001108, and the University of California Multicampus Research Programs and Initiatives. We thank Dr David Rowitch for the Olig2 antibody, Dr Arturo Alvarez-Buylla for in situ probes, Dr Mike German for the Lmx1a antibody, and members of the Huang laboratory for helpful discussions.
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