Supporting Information - PNAS · Supporting Information Huang et al. 10.1073/pnas.0911838107 SI...
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Supporting Information Huang et al. 10.1073/pnas.0911838107 SI Materials and Methods Mice. Shh cre/+ (1), and Ptf1a cre/+ (2) lines have been described previously. Ptch lacZ/+ (3), R26R (4), Nestin-cre (5), Shh f/f (6), Wnt1-cre (7), SmoM2 (8), Smo f/f (8), and mT/mG (9) mice were obtained from the Jackson Laboratory. Retrieval of Embryonic CSF and Shh ELISA. WT embryos from CD1 females, at embryonic day (E)12.5–E15.5, were dissected in sterile ice-cold PBS and then placed on Kimwipe with hindbrain ventricle facing upward. CSF was aspirated using a mouth- operated micropipette placed inside the hindbrain ventricle. Because CSF is generated from the choroid plexi of lateral, third, and fourth ventricles, and all brain ventricles are con- nected, it is not possible to isolate CSF selectively in the fourth ventricle. Therefore, we determined the presence of Sonic hedgehog (Shh) protein in the context of the entire CSF. We typically retrieved 2–4 μL of CSF per embryo, depending on the age. CSF was kept on ice and pooled prior to centrifugation for 10 min at 16,000 × g and then transferred to a precooled tube before storage at -80 °C. Approximately 150 μL CSF was pooled for each stage, followed by ELISA. Shh concentration in CSF was measured according to the manufacturer’s instructions (DuoSet ELISA Development System; R&D Systems) using 5E1 monoclonal antibody (DSHB). ELISA was performed on 2-fold dilution of CSF at a starting concentration of 1:1.83 (reagent diluent: CSF) for E12.5 and E15.5 and at 1:1.14 and 1:1.27 for E13.5 and E14.5, respectively. The colorimetric optical density was measured at 450 nm (FLUOstar Omega; BMG Labtech). We also included purified Shh protein (ShhNp, starting at 0.063 nM) as positive control and NIH 3T3 cell-conditioned media as negative control in the analysis. NIH 3T3 cells do not express Shh endogenously. As shown in Fig. 5 (main text), Shh protein is present in the CSF at a concentration range of 100–300 pg/mL. The local concentration surrounding the hindbrain choroid plexus (hChP) and cerebellar ventricular zone (VZ) could be much higher. Immunohistochemistry. All immunohistochemistry analyses were performed on tissue sections collected from OCT (optimum cutting temperature)- or paraffin-embedded embryos. The pri- mary antibodies were mouse anti-Lhx1/5 (DSHB, 1:10), mouse anti-CyclinD1 (BD Pharmingen; 1:100), rabbit anti-Sox9 (gift of Michael Wegner; 1:2,000), rabbit anti-Sox2 (Chemicon; 1:1,000), rabbit anti-Pax2 (Zymed; 1:500), rabbit anti-Ptf1a (gift of Chris Wright; 1:1,000), mouse anti-Mash1 (gift of Jane Johnson; 1:40), rabbit anti–brain lipid binding protein (BLBP) (Chemicon; 1:500), mouse anti-β-galactosidase (Promega; 1:500), mouse anti- acetylated Tubulin (Sigma; 1:500), and rabbit anti-Arl13b (gift of Tamara Caspary; 1:1,000). Cell Counting and Statistics. Defined areas selected for cerebellar cell counting are shown in Fig. S8. Boxed region indicates unit area defined by ImageJ. For cell countings from stainings that de- termine the proliferative capacity of cerebellar regions, we se- lected an 800 × 80 region (approximately 10–12 cells from the ventricular surface, which parallels the Ki67+ layer) along the medial VZ in E13.5 and E14.5 cerebella, because almost all proliferative activities occur in the VZ at this stage. For E16.5 and E18.5 embryos, we arbitrarily selected a broader 1,000 × 100 re- gion for vermis VZ (vVZ) and lateral VZ (lVZ) region to assess the proliferative effect of Shh signaling, as indicated by BrdU and CyclinD1 stainings. For stainings that determine GABAergic progenitors at E16.5, we selected the 1,000 × 800 region for both vermal and lateral cerebellum, because reduced GABAergic cell types are considered to be a cumulative result of defective VZ radial glial proliferation. The numbers of labeled cells vary greatly depending on markers; we therefore further normalized all mutant cell counts to those of the wild type, presenting a percentage comparison for clarity. Three independent stainings were performed for each marker, and at least 10 sections in each region were counted to generate a statistical comparison. To assess differences among groups, statistical analyses were performed using a one-way ANOVA with Microsoft Excel and significance accepted at P < 0.05. Results are presented as mean ± SD. X-Gal Staining and Transcript Detection. X-gal staining for β-gal- actosidase was performed according to standard protocol. The following cDNAs were used as templates for synthesizing digox- ygenin-labeled riboprobes: Shh, Patched1, Gli1, and Ngn2. Real-Time RT-PCR. Whole cerebella were dissected from E12.5– E16.5 WT embryos. Total RNA was extracted using RNAeasy Mini Kit (Qiagen) and treated with DNase I to remove genomic DNA. Two micrograms of RNA was reverse transcribed to cDNA using Promega AMV-RT reverse transcriptase. All DNA and RNA concentrations were measured by Gene Spec I. Real- time detection and quantification of cerebellar cDNA were performed with the iCycler instrument (Bio-Rad). Quantitative PCR was performed in a 50-μL reaction mixture containing 1× SYBR 4 Green DNA polymerase mixture (Stratagene), 0.4 μM of each pair of primers (see below), and 1 μL of cDNA template. Forty cycles of amplification were performed according to the manufacturer’s instructions. Fluorescence data were collected at annealing stages and real-time analysis performed with iCycler iQ Optical System Software V3.0a. Serial dilutions of cDNAs were used for construction of the standard curve. Ct values were determined with automatically set baseline and manually ad- justed fluorescence threshold. Gene expressions of Shh, Gli1, and Ihh were normalized with that of GAPDH. All experiments were repeated three times, and statistical values were calculated using Microsoft Excel. Primers used were as follows: GAPDH (5′TTCACCACCATGGAGAAGGC3′F; 5′GGCATGGACTG- TGGTCATGA3′R), Shh (5′TCTGTGATGAACCAGTGGC- C3′F; 5′GCCACGGAGTTCTCTGCTTT3′R); Gli1 (5′CTGG- AGAACCTTAGGCTGGA3′F; 5′CGGCTGACTGTGTAAG- CAGA3′R); and Ihh (5′CCCCAACTACAATCCCGACATC3′ F; 5′ CGCCAGCAGTCCATACTTATTTCG3′R) (Fig. S6). Shh-LIGHT2 Cells and hChP Coculture with Modulators of Shh Signaling. We used Shh-LIGHT2 cells with stably incorporated Gli-luc reporter and TK-Renilla vectors. Shh-LIGHT2 cells were grown in 96-well plates and cultured in DMEM with 10% FBS. Freshly dissected hChPs retrieved from E16.5 embryos of one CD1 female were cocultured with Shh-LIGHT2 cells as a mon- olayer at >95% confluency. The hChPs and Shh-LIGHT2 cells were further cultured in DMEM with 10% FBS for another 2 h before switching to DMEM with 0.5% calf serum. Shh pathway agonist SAG, Shh pathway antagonist cyclopamine, and Shh blocking antibody 5E1 were added at the indicated concen- trations (Fig. S6). Luciferase Assay. Shh-LIGHT2 cells and hChPs were cocultured for 48 h. hChPs were then carefully removed before generating cell lysates. Luciferase assays were performed according to the Huang et al. www.pnas.org/cgi/content/short/0911838107 1 of 8
Transcript of Supporting Information - PNAS · Supporting Information Huang et al. 10.1073/pnas.0911838107 SI...
Supporting InformationHuang et al. 10.1073/pnas.0911838107SI Materials and MethodsMice. Shhcre/+ (1), and Ptf1acre/+ (2) lines have been describedpreviously. PtchlacZ/+ (3), R26R (4), Nestin-cre (5), Shhf/f (6),Wnt1-cre (7), SmoM2 (8), Smof/f (8), and mT/mG (9) mice wereobtained from the Jackson Laboratory.
Retrieval of Embryonic CSF and Shh ELISA. WT embryos from CD1females, at embryonic day (E)12.5–E15.5, were dissected insterile ice-cold PBS and then placed on Kimwipe with hindbrainventricle facing upward. CSF was aspirated using a mouth-operated micropipette placed inside the hindbrain ventricle.Because CSF is generated from the choroid plexi of lateral,third, and fourth ventricles, and all brain ventricles are con-nected, it is not possible to isolate CSF selectively in the fourthventricle. Therefore, we determined the presence of Sonichedgehog (Shh) protein in the context of the entire CSF. Wetypically retrieved 2–4 μL of CSF per embryo, depending on theage. CSF was kept on ice and pooled prior to centrifugation for10 min at 16,000 × g and then transferred to a precooled tubebefore storage at −80 °C. Approximately 150 μL CSF was pooledfor each stage, followed by ELISA. Shh concentration in CSFwas measured according to the manufacturer’s instructions(DuoSet ELISA Development System; R&D Systems) using 5E1monoclonal antibody (DSHB). ELISA was performed on 2-folddilution of CSF at a starting concentration of 1:1.83 (reagentdiluent: CSF) for E12.5 and E15.5 and at 1:1.14 and 1:1.27 forE13.5 and E14.5, respectively. The colorimetric optical densitywas measured at 450 nm (FLUOstar Omega; BMG Labtech).We also included purified Shh protein (ShhNp, starting at 0.063nM) as positive control and NIH 3T3 cell-conditioned media asnegative control in the analysis. NIH 3T3 cells do not expressShh endogenously. As shown in Fig. 5 (main text), Shh protein ispresent in the CSF at a concentration range of 100–300 pg/mL.The local concentration surrounding the hindbrain choroidplexus (hChP) and cerebellar ventricular zone (VZ) could bemuch higher.
Immunohistochemistry. All immunohistochemistry analyses wereperformed on tissue sections collected from OCT (optimumcutting temperature)- or paraffin-embedded embryos. The pri-mary antibodies were mouse anti-Lhx1/5 (DSHB, 1:10), mouseanti-CyclinD1 (BD Pharmingen; 1:100), rabbit anti-Sox9 (gift ofMichael Wegner; 1:2,000), rabbit anti-Sox2 (Chemicon; 1:1,000),rabbit anti-Pax2 (Zymed; 1:500), rabbit anti-Ptf1a (gift of ChrisWright; 1:1,000), mouse anti-Mash1 (gift of Jane Johnson; 1:40),rabbit anti–brain lipid binding protein (BLBP) (Chemicon;1:500), mouse anti-β-galactosidase (Promega; 1:500), mouse anti-acetylated Tubulin (Sigma; 1:500), and rabbit anti-Arl13b (gift ofTamara Caspary; 1:1,000).
Cell Counting and Statistics. Defined areas selected for cerebellarcell counting are shown in Fig. S8. Boxed region indicates unit areadefined by ImageJ. For cell countings from stainings that de-termine the proliferative capacity of cerebellar regions, we se-lected an 800 × 80 region (approximately 10–12 cells from theventricular surface, which parallels the Ki67+ layer) along themedial VZ in E13.5 and E14.5 cerebella, because almost allproliferative activities occur in the VZ at this stage. For E16.5 andE18.5 embryos, we arbitrarily selected a broader 1,000 × 100 re-gion for vermis VZ (vVZ) and lateral VZ (lVZ) region to assessthe proliferative effect of Shh signaling, as indicated by BrdU andCyclinD1 stainings. For stainings that determine GABAergic
progenitors at E16.5, we selected the 1,000 × 800 region for bothvermal and lateral cerebellum, because reduced GABAergic celltypes are considered to be a cumulative result of defective VZradial glial proliferation.The numbers of labeled cells vary greatly depending on
markers; we therefore further normalized all mutant cell counts tothose of the wild type, presenting a percentage comparison forclarity. Three independent stainings were performed for eachmarker, and at least 10 sections in each region were counted togenerate a statistical comparison. To assess differences amonggroups, statistical analyses were performed using a one-wayANOVA with Microsoft Excel and significance accepted at P <0.05. Results are presented as mean ± SD.
X-Gal Staining and Transcript Detection. X-gal staining for β-gal-actosidase was performed according to standard protocol. Thefollowing cDNAs were used as templates for synthesizing digox-ygenin-labeled riboprobes: Shh, Patched1, Gli1, and Ngn2.
Real-Time RT-PCR. Whole cerebella were dissected from E12.5–E16.5 WT embryos. Total RNA was extracted using RNAeasyMini Kit (Qiagen) and treated with DNase I to remove genomicDNA. Two micrograms of RNA was reverse transcribed tocDNA using Promega AMV-RT reverse transcriptase. All DNAand RNA concentrations were measured by Gene Spec I. Real-time detection and quantification of cerebellar cDNA wereperformed with the iCycler instrument (Bio-Rad). QuantitativePCR was performed in a 50-μL reaction mixture containing 1×SYBR4 Green DNA polymerase mixture (Stratagene), 0.4 μM ofeach pair of primers (see below), and 1 μL of cDNA template.Forty cycles of amplification were performed according to themanufacturer’s instructions. Fluorescence data were collected atannealing stages and real-time analysis performed with iCycleriQ Optical System Software V3.0a. Serial dilutions of cDNAswere used for construction of the standard curve. Ct values weredetermined with automatically set baseline and manually ad-justed fluorescence threshold. Gene expressions of Shh, Gli1,and Ihh were normalized with that of GAPDH. All experimentswere repeated three times, and statistical values were calculatedusing Microsoft Excel. Primers used were as follows: GAPDH(5′TTCACCACCATGGAGAAGGC3′F; 5′GGCATGGACTG-TGGTCATGA3′R), Shh (5′TCTGTGATGAACCAGTGGC-C3′F; 5′GCCACGGAGTTCTCTGCTTT3′R); Gli1 (5′CTGG-AGAACCTTAGGCTGGA3′F; 5′CGGCTGACTGTGTAAG-CAGA3′R); and Ihh (5′CCCCAACTACAATCCCGACATC3′F; 5′ CGCCAGCAGTCCATACTTATTTCG3′R) (Fig. S6).
Shh-LIGHT2 Cells and hChP Coculture with Modulators of ShhSignaling. We used Shh-LIGHT2 cells with stably incorporatedGli-luc reporter and TK-Renilla vectors. Shh-LIGHT2 cells weregrown in 96-well plates and cultured in DMEM with 10% FBS.Freshly dissected hChPs retrieved from E16.5 embryos of oneCD1 female were cocultured with Shh-LIGHT2 cells as a mon-olayer at >95% confluency. The hChPs and Shh-LIGHT2 cellswere further cultured in DMEM with 10% FBS for another 2 hbefore switching to DMEM with 0.5% calf serum. Shh pathwayagonist SAG, Shh pathway antagonist cyclopamine, and Shhblocking antibody 5E1 were added at the indicated concen-trations (Fig. S6).
Luciferase Assay. Shh-LIGHT2 cells and hChPs were coculturedfor 48 h. hChPs were then carefully removed before generatingcell lysates. Luciferase assays were performed according to the
Huang et al. www.pnas.org/cgi/content/short/0911838107 1 of 8
manufacturer’s protocol. All reporter assays were normalized us-ing Renilla luciferase internal control (luminometric detection,
Promega dual luciferase assay kit). Five independent experimentswere performed, and results are presented as means ± SD.
1. Li Y, Zhang H, Litingtung Y, Chiang C (2006) Cholesterol modification restricts thespread of Shh gradient in the limb bud. Proc Natl Acad Sci USA 103:6548–6553.
2. Kawaguchi Y, et al. (2002) The role of the transcriptional regulator Ptf1a in convertingintestinal to pancreatic progenitors. Nat Genet 32:128–134.
3. Goodrich LV, Milenković L, Higgins KM, Scott MP (1997) Altered neural cell fates andmedulloblastoma in mouse patched mutants. Science 277:1109–1113.
4. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. NatGenet 21:70–71.
5. Graus-Porta D, et al. (2001) Beta1-class integrins regulate the development of laminaeand folia in the cerebral and cerebellar cortex. Neuron 31:367–379.
6. Lewis PM, et al. (2001) Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell 105:599–612.
7. Jiang X, Rowitch DH, Soriano P, McMahon AP, Sucov HM (2000) Fate of the mammaliancardiac neural crest. Development 127:1607–1616.
8. Jeong J, Mao J, Tenzen T, Kottmann AH, McMahon AP (2004) Hedgehog signaling inthe neural crest cells regulates the patterning and growth of facial primordia. GenesDev 18:937–951.
9. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent Crereporter mouse. Genesis 45:593–605.
CB
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Fig. S1. Characterization of Lhx1/5+ cells in embryonic cerebellum and Shh-lineage cells in the midbrain. (A) As shown by Lhx1/5 and Pax2+ double labeling inE16.5 WT cerebellum, Lhx1/5+ cells include both Pax2- cells that presumably are developing Purkinje neurons, and Pax2+ developing GABAergic interneurons.(B) GFP signal in Shhcre/+;mT/mG mice marks both the cell body and cellular processes of Shh-lineage cells. Note that even the cellular processes of the Shh-lineage cells in the midbrain (Mid) localize quite far away from the rostral cerebellum. CB, cerebellum.
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Fig. S2. Ptf1acre/+;Smof/- mutants did not display apparent VZ proliferation phenotype. E16.5 Ptf1acre/+;Smof/- mutant cerebellum developed numbers ofCyclinD1+ VZ progenitors and Pax2+ GABAergic interneuron progenitors comparable to those of the WT embryos.
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Fig. S3. Wnt1-cre;Shhf/- mutant cerebella display severe VZ phenotypes at E16.5. (A) Shh signaling in E14.5 Wnt1-cre;Shhf/- mutant cerebellum is essentiallyablated. Red boxes represent magnified vermal and lateral VZ regions in adjacent panels. (B) E16.5 Wnt1-cre;Shhf/- mutant cerebellar VZ shows severelyimpaired radial glial population, proliferative activity, and GABAergic progenitor expansion compared with WT. BLBP+ and Sox9+ cells represent radial glialpopulation. BrdU (vermis region: 100% ± 2.8% vs. 10.2% ± 0.5%, P < 0.001; lateral VZ region: 100% ± 4.6% vs. 40.2% ± 4.1%, P < 0.001) and CyclinD1 stainingsindicate proliferative activity. Ptf1a (vermis region: 100% ± 6.4% vs. 46.7% ± 3.5%, P < 0.001; lateral VZ region: 100% ± 8.1% vs. 24.7% ± 6.6%, P < 0.001),Pax2 (vermis region: 100% ± 6.4% vs. 55.3% ± 6.7%, P < 0.001; lateral VZ region: 100% ± 15.5% vs. 67.2% ± 7.7%, P < 0.01) and Lhx1/5 mark GABAergicprogenitor cells. (C) Statistical comparisons between WT and Wnt1-cre;Shhf/- mutant cerebella for BrdU+, Pax2+, and Ptf1a+ cells.
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Fig. S4. Wnt1-cre;Shhf/- mutants display impaired radial glial cell proliferation. (A) To determine whether Shh signaling promotes the self-renewal capability ofcerebellar VZ progenitors, WT andWnt1-cre; Shhf/- mutant were pulsed with 24-h BrdU and analyzed for the proportion of progenitor cells that remain in the cellcycle. The percentage of progenitors that remain in the cell cycle was calculated by the ratio of BrdU+;Ki67+ cells over total BrdU+ cells. Wnt1-cre;Shhf/- mutantcerebellar VZ progenitors show premature cell cycle exit as indicated by more than 2-fold reduction in BrdU+/Ki67+ cells. (B) Quantification of percentage of VZprogenitor cells that remained in the cell cycle from E13.5 to E14.5 inWnt1-cre;Shhf/- mutant and WT (6.1% ± 2.3% vs. 15.3% ± 3%, P < 0.01, n = 3). (C)Wnt1-cre;Shhf/- or Nestin-cre;SmoM2 mutants exhibit impaired or enhanced BrdU incorporation in BLBP+ or Sox9+ radial glial cells, respectively. (D) Quantification ofproliferating radial glial cell number inWnt1-cre;Shhf/- mutant andWT (45.5% ± 7.9% vs. 100% ± 4.5% for BLBP+ cells, P < 0.001; 54.4% ± 4.4% vs. 100% ± 5.2%
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Fig. S5. Wnt1-cre;Shhf/- mutant cerebella developed largely reduced Ptf1a+, Ngn2+ and Mash1+ VZ progenitors. Similarly, the number of Lhx1/5 that marksdifferentiating GABAergic neurons is also reduced. Note that Lhx1/5 ectopically occupies the Wnt1-cre;Shhf/- mutant cerebellar VZ at E13.5, which is neverobserved in WT. Statistical comparison between Wnt1-cre;Shhf/- and WT cerebella for Ptf1a+ cells (64% ± 4.6% vs. 100% ± 9.2%, P < 0.001, n = 5) and Mash1+cells (60.8% ± 3.4%. vs. 100% ± 4.3% in vermal VZ and 65.7% ± 7.9% in lateral VZ, P < 0.001, n = 5).
for Sox9+ cells, P < 0.001, n = 4). In contrast, VZ radial glial cells in Nestin-cre;SmoM2 displayed approximately 2-fold higher proliferative activity thanWT (224.1%± 5.8% vs. 100% ± 10.1% for BLBP+ cells, P < 0.001; 198% ± 10.4% vs. 100% ± 7.2% for Sox9+ cells, P < 0.001, n = 4). CB, cerebellum.
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Fig. S7. Proposed model of transventricular delivery of Shh on cerebellar VZ development. A schematic illustration of a coronal section of the midbrain–cerebellum–medulla region at E14.5 is shown. Red regions represent the VZ neuroepithelium. We propose that the hChP epithelium, depicted in yellow,secretes functional Shh protein into the fourth ventricle, which is then transventricularly delivered to the cerebellar VZ to promote the proliferation of itsresident progenitors. At this stage there is no endogenous cerebellar tissue expressing Shh, and the nascent external granule neuron layer (EGL) is shown inblue. Note that VZ-derived progenitors migrate radially, whereas granule neural precursors migrate tangentially (arrows).
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Fig. S6. hChP secretes Shh to elicit pathway activity in vitro. hChP-secreted Shh elicits signaling activity when cocultured with Shh-LIGHT2 cells; induction canbe ablated by adding Shh blocking antibody 5E1 or cyclopamine.
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For cell countings on E13.5/E14.5 WT and mutant embryo cerebellar VZ
For cell countings from BrdU and CyclinD1 stainings on E16.5 WT and mutant cerebella
Vermal VZ region Lateral VZ region
For cell countings from Pax2, Lhx1/5 stainings on E16.5 WT and mutant cerebella
Vermal CB region Lateral CB region
Fig. S8. Representative regions selected for cell countings and statistical analyses. Boxed region indicates unit area defined by ImageJ. For cell countings fromstainings that determine the proliferative capacity of cerebellar regions, we selected an 800 × 80 region (approximately 10–12 cells from the ventricularsurface, which parallels the Ki67+ layer) along the medial VZ in E13.5 and E14.5 cerebella, because almost all proliferative activities occur in the VZ at this stage.For E16.5 and E18.5 embryos, we arbitrarily selected a broader 1,000 × 100 region for vermis VZ (vVZ) and lateral VZ (lVZ) region to assess the proliferativeeffect of Shh signaling, as indicated by BrdU and CyclinD1 stainings. For stainings that determine GABAergic progenitors at E16.5, we selected the 1,000 × 800region for both vermal and lateral cerebellum, because reduced GABAergic cell types are considered to be a cumulative result of defective VZ radial glialproliferation.
Huang et al. www.pnas.org/cgi/content/short/0911838107 8 of 8
