Transcriptional codes define distinct temporal and spatial progenitor domains in the early embryonic ventral midbrain
In the ventral spinal cord, distinct progenitor domains have been identified based on different expression patterns of homeodomain transcription factors, such as Nkx2.2, Nkx6.1, and Olig2. These progenitor domains generate distinct classes of neurons in response to Shh signaling [24
]. To determine if the developing vMB also contains different progenitor domains, we analyzed the temporal and spatial expression patterns of several transcriptional factors, including Lmx1a, Foxa2, Nkx6.1, and Nkx2.2, which have been implicated in neurogenesis in this region [17
]. As described below, we found that a combinatorial set of transcription factor expression did indeed define distinct progenitor domains in vMB. These progenitor domains, termed ventral midbrain domain (D)1 to D4, followed a medial to lateral expansion as the embryos became more mature. Upon closure of neural tube at E8 to E8.5, the vMB contained two distinct domains, with the medial domain D1 expressing Foxa2 and Nkx6.1, whereas the immediately adjacent lateral domain D2 expressed Nkx6.1 only. At this stage, there was no detectable expression of Lmx1a or Nkx2.2 (Figure A,B,K). At E9.5, expression of Lmx1a emerged, and its coexpression with Foxa2 defined the newly formed medial domain D1, whereas the Foxa2 and Nkx6.1 co-expressing cells shifted laterally to become the D2 domain (Figure C,D,K). Immediately adjacent to the domain D2 was a small D3 domain that expressed Nkx2.2, although a small number of the Nkx2.2+ cells could also be detected within the D2 domain (Figure D and inset). After E9.5, the vMB showed tremendous expansion, and now contained four distinct domains defined by these transcription factors. From E10.5 to E12.5, the medial-most domain D1 expressed Lmx1a+Foxa2+ cells, and the adjacent D2 domain expressed Foxa2+Nkx6.1+ cells. The more lateral D3 domain expressed Nkx2.2, whereas a small population of cells positive for Nkx6.1 only defined the D4 domain (Figure E-K). Interestingly, unlike in spinal cord, expression of Olig2 and Pax6 could not be detected in midbrain at E10.5, suggesting that progenitor domains in vMB contained distinctly different transcriptional profiles from those in spinal cord.
Figure 1 Distinct progenitor domains in the developing ventral midbrain (vMB) defined by a combinatorial code of transcription factors. (A-D) Double immunostaining for (A,C) Foxa2/Nkx6.1, (B,D) Lmx1a/Nkx2.2 and (inset) Foxa2/Nkx2.2 in vMB at embryonic day (E)8.5 (more ...)
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
]. 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.
Consistent with previous studies [13
], we found that Shh expression, detected by immunohistochemistry and in situ
hybridization, showed robust and dynamic expression in vMB from E8 to E12.5. At E8 to E8.5, Shh proteins and Shh
mRNA were detected mainly in the most medial region in vMB, and this expression domain expanded laterally from E9.5 to E10.5. Interestingly, from E11.5 to E12.5, Shh
mRNA expression diminished in the most medial vMB D1 domain, and became more restricted to the ventricular zone (VZ) of the lateral D2 domain in vMB (Figure A-D, and insets in Figure B-D). Similar to our previous results [29
], Shh proteins were detected in radial glial processes extending from the ventricular zone to the marginal zone at E11.5 (Figure D).
Figure 2 Dynamic expression patterns of Shh signaling components in the developing ventral midbrain (vMB). (A) Immunohistochemical staining and in situ hybridization (insets) revealed Shh expression in the midline of vMB at embryonic day (E)8.5 (10 somites). ( (more ...)
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 E-H). In addition to examining Smoothened
, we also examined the expression patterns of several Shh signaling effectors, including Patched
, 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 I-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 Q), and shifting laterally and dorsally from E9.5 onwards (Figure R-T). Finally, Gli3, the major repressor of Shh signaling, showed low and diffuse expression in the vMB at E8 to E8.5 (Figure U), but its expression became restricted to the dorsal part of midbrain after E9.5 (Figure V-X).
Because the expression patterns of Shh, Patched and Gli1 showed a medial to lateral expansion from E8.5 to E12.5 (Figure ; Figure ), we investigated if they might overlap with the vMB progenitor domains. Consistent with this idea, we found that Shh proteins were close to or partially overlapping with the Nkx6.1+Nkx2.2+ D2 domain at E9.5 and E10.5 (Figure A,B,D,E). By E11.5, the Shh proteins showed extensive overlapping with the Nkx6.1+ D2, domain and were immediately adjacent to the Nkx2.2+ D3 Domain (Figure C,F). Using combined in situ hybridization and immunohistochemistry, we found that the expression of Patched mRNA covered the D2 and D3 progenitor domains at E9.5 and E10.5 (Figure G,H,J,K), but became more restricted to the ventricular zone after E11.5 (Figure I,L). Similar to Patched expression, Gli1 mRNA also showed extensive overlap with the D2 and D3 domains at E9.5 and E10.5 (Figure M,N), but at E11.5, the Gli1 mRNA continued its expression in the D3 and D4 domains (Figure O).
Figure 3 Spatial distribution of Sonic hedgehog (Shh) signaling effectors in ventral midbrain progenitor domains. (A-C) Double immunofluorescence staining of Shh and Nkx6.1 reveal that Shh proteins were expressed mainly in (A) the D1 region at embryonic day (E)9.5,( (more ...)
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
], 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
To examine the roles of Shh signaling in the development of ventral midbrain, we generated conditional knockout mice in which the Shh receptor, Smoothened, was removed in vMB using Shh-Cre
). Consistent with the previously reported activity of Shh-Cre
in vMB [21
], we found that the Shh-Cre
recombination pattern completely covered the Lmx1a+ D1 domain and the majority of the Foxa2+ D2 domain from E9.5 to E11.5 (Figure A-F). Using in situ
hybridization, we confirmed complete removal of Smoothened
mRNA in vMB from E9.5 to E11.5 (Figure G-I’). The reduction in levels of Smoothened
mRNA in Shh-Cre;Smofl/fl
mutants began as early as E8.5 (eight-somite stage) (Figure G-G’, insets); this reduction was partial.
Figure 4 Region-specific removal of Smoothened in the ventral midbrain of Shh-Cre;Smofl/flmutants. (A-F) Using the R26R reporter line, we found that Shh-Cre drives recombination in D1 domain and the majority of D2 domain from embryonic day (E)9.5 to E11.5. (G-I (more ...)
To analyze the effects of ablating Smoothened, we quantified the numbers of progenitors in each vMB domain (D1 to D4). Surprisingly, despite the robust expression of Shh signaling effectors in vMB, we found no detectable reduction in the number of progenitor cells at E8.5 to E9.5 (Figure A-B’, and data not shown). Beginning at E10.5, there was a consistent decrease in the number of Lmx1a+, Foxa2+, Nurr1+, Nkx6.1+ and Nkx2.2+ progenitors in D1 to D4 domains in Shh-Cre;Smofl/fl embryos (Figure C-D’,I-N). Despite this reduction, the spatial arrangement of D1 to D4 domains was not altered (Figure C-D’). Furthermore, we did not detect any changes in the Sox2+ progenitors (Figure E-E’). The changes were not due to changes in cell death or proliferation, because there were no detectable changes in caspase 3 staining or 2-hour BrdU incorporation (data not shown). The effects of Smoothened loss of function on most progenitors appeared to be transient, thus by E12.5, there were no detectable differences in the total number of progenitors, including those for Lmx1a, Foxa,2 and Nkx6.1 (Figure G-L). Only the Nkx2.2+ progenitors in the D3 domain continued to show a significant reduction at E12.5 (Figure G-G’,H,K). Together, these data support the idea that the loss of Smoothened had a transient effect on the expansion of most progenitors in vMB at E10.5, except for the Nkx2.2+ progenitors, which showed persistent reduction at E12.5.
Figure 5 Transient reduction of dopaminergic (DA) progenitors in Shh-Cre;Smofl/flmutants. (A-B’) At embryonic day (E)9.5, Shh-Cre;Smofl/fl mutants showed no detectable reduction in progenitors expressing (A’) Lmx1a/Nkx2.2, or (B’) Foxa2/Nkx6.1. (more ...)
Loss of Smoothened in ventral midbrain affects the generation of neurons in red nucleus, oculomotor nucleus, and raphe nuclei, but not dopaminergic neurons
Given the progressively restricted expression of Shh effectors to the lateral domains in vMB, we investigated if the effects of Smoothened loss of function on the progenitors at E10.5 and E12.5 might affect distinct classes of neurons arising from different vMB domains (Figure , Figure , Figure ). The embryonic vMB gives rise to four major subtypes of neurons: 1) DA neurons, 2) neurons in the red nucleus, 3) oculomotor neurons, and 4) serotonergic neurons. It is known that the DA neurons are generated from the Foxa2+Lmx1a+ D1 domain from E10.5 to E12.5, the oculomotor neurons from the Nkx6.1+Foxa2+ D2 domain, and the red nucleus neurons from the Nkx6.1+Foxa2+ D2 domain, while the serotonergic neurons partially arise from the Nkx2.2+ progenitors in the caudal vMB [17
]. To confirm these results, we used genetic-fate mapping to investigate whether these four groups of neurons derived from Shh-expressing cells. Using anti-β-Gal antibody or colorimetric LacZ expression in Shh-Cre;R26R/+
mice, we could detect β-GAL coexpressed with all the TH+, Brn3a+, Islet1+ and 5-HT+ neurons at E12.5 in the midbrain. By post-natal day (P)0, LacZ expression could still be detected in most TH+ and Brn3a+ neurons, and in a significant number of Isl1+ and 5-HT+ neurons (Figure A-L). These results indicated that DA neurons (TH+), red nucleus neurons (Brn3a+), oculomotor neurons (Isl1+) and serotonergic neurons (5-HT+) were completely or partially derived from Shh-expressing progenitors, and Shh-Cre
could be an effective tool to target these groups of neurons.
Figure 6 Fate mapping of dopaminergic neurons, red nucleus neurons, oculomotor neurons, and serotonergic neurons from Sonic hedgehog (Shh)-expressing cells. Using the Shh-Cre;R26R reporter line, we found that (A) tyrosine hydroxylase (TH)+ DA neurons, (D) Brn3a+ (more ...)
Using cell type-specific markers, we found that the number of TH+ DA neurons and Nurr1+ DA intermediate progenitors, which were derived from Lmx1a+Foxa2+ progenitors in the D1 domain, were transiently reduced in the Shh-Cre;Smofl/fl mutants at E10.5 (Figure F-F’). Interestingly, by E12.5, there was no detectable reduction in the population of either cell type (Figure A-B’, I-J). By contrast, the numbers of Brn3a+ red nucleus neurons and Isl1+ oculomotor neurons, which both arose from the Nkx6.1+/Foxa2+ progenitors in the D2 domain, were significantly decreased at E12.5, and this decrease persisted at P0. Furthermore, we also detected a persistent reduction of serotonergic (5-HT+) neurons at E12.5 and P0 (Figure C-H’,K-M). Taken together, these data showed that the loss of Smoothened in the vMB had a transient and modest effect on the generation of DA neurons and DA progenitors at E10.5, but a more persistent effect on the generation of Brn3a+, Isl1+, and 5-HT+ neurons from the more lateral D2 and D3 domains.
Figure 7 Persistent loss of neurons in the red nucleus, oculomotor nucleus and the serotonergic neurons, but not dopaminergic neurons, in Shh-Cre;Smofl/flmutants at embryonic day (E)12.5 to post-natal day (P)0. (A-B’) Nurr1 and tyrosine hydroxylase (TH) (more ...)
Constitutive activation of smoothened transiently expand progenitors in ventral midbrain
To further examine the role of Shh signaling in controlling vMB development, we generated mice that expressed constitutively active Smoothened receptor under the control of Shh-Cre
]. Owing to the constitutive activation of Smoothened in the Shh expression domains, essentially all the Shh-Cre;SmoM2
showed limb malformations, with abnormal growth of cartilage in the patterning center of the developing limbs (data not shown). As expected, the expression of an extra copy of Smoothened transcript led to more intense Smoothened
mRNA signals detected by in situ
hybridization (Figure A-A’).
Figure 8 Constitutive activation of Sonic hedgehog (Shh) signaling in Shh-Cre;SmoM2 mutants leads to transient expansion of progenitors in ventral midbrain. (A-A’) In situ hybridization of Smoothened indicated overexpression of Smoothened in Shh-Cre;SmoM2 (more ...)
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 C-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 E). 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 B-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 , Figure ), 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
Given the fact that there was no detectable difference in DA lineage cells between either Smofl/fl
at E12.5, the results (Figure , Figure , Figure , Figure ) suggested that other signaling pathway(s) might have stage-dependent effects in regulating DA neuron development after E12.5. It has been shown that in addition to Shh and Wnt signaling, FGF8 is required for the patterning of MHB, induction of midbrain DA neurons, and regulation of DA progenitor domains [12
]. By examining FGF8
mRNA on whole-mount animal and sections from E9.5 to E12.5, we found that no difference in FGF8
expression could be detected in MHB, either in Shh-Cre
mutants (Figure A-D’ and data not shown). These results were distinctly different from those reported in En1-Cre;Smofl/fl
mutants, in which a marked reduction in the expression of FGF8 in midbrain-hindbrain region caused a profound patterning defect [8
Figure 9 Loss and gain of function in Smoothened affects Wnt1, but not fibroblast growth factor (FGF)8, expression, in ventral midbrain (vMB). (A-D’) In situ hybridization showed no detectable change in FGF8 expression in MHB in either (A’,C’) (more ...)
Previously, we reported that stabilizing the canonical Wnt signaling antagonized Shh expression in vMB to control the temporal development of DA neurons [29
]. 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
mutants. Consistent with our prediction, at both E10.5 and E12.5, Wnt1
mRNA levels were increased in the vMB of Shh-Cre
compared with control (Figure E-F’). By contrast, in the Shh-Cre;SmoM2
mRNA level was modestly downregulated at E10.5, but returned to the control levels at E12.5 (Figure G-H’).
To further investigate the effects of Smoothened on Wnt signaling, we generated SmoM2;BAT-GAL
mice, in which the Wnt signaling reporter, BAT-GAL
, could be used as a surrogate for canonical Wnt activity [20
]. 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 I-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.