Supporting Information - PNAS · Supporting Information Inta et al. 10.1073/pnas.0807059105 SI...

Supporting InformationInta et al. 10.1073/pnas.0807059105SI MethodsGeneration of 5-HT3-EGFP Transgenic Mice. A mouse 129SV strainBAC library (Research Genetics) was screened for the 5-HT3Agene by hybridization with an exon 9-derived 285-bp probegenerated by PCR with primers 5-HT3 probe1 (5�-TGCAGC-CATGTTGGAC-3�) and probe2 (5�-TCAAGAATAATGC-CAAATGGAC-3�). Hybridizing BACs were further analyzedfor size following NotI digestion and pulse-field gel electrophore-sis (PFGE) (CHEF-DRIII; Bio-Rad) and for the extent of5�-f lanking and 3�-f lanking region of the 5-HT3A gene bySouthern blots of PGFE-resolved PacI-digested and of ApaI-digested BAC DNA with suitable PCR-generated probes. Of theisolated BAC clones, clone 310N1 with an approximate size of195 kb and containing �50 kb upstream and �30 kb downstreamof the 5-HT3A gene was chosen for transgene construction. Atargeting vector was constructed to introduce a cassette of theEGFP-coding sequence fused to the bovine growth hormonepolyadenylation signal into the 5-HT3A gene on BAC 310N1 suchthat the initiator methionine codon became the initiator codonfor EGFP. The cassette was flanked by recombinogenic arms(RAs) spanning 1,134 bp 5� and 1,063 bp 3� of the initiator codon(Fig. 1), and generated by PCR with primers 5-HT3-5�RA1(5�-TTGACCTCGGAGGGCTTGGTGC-3�) and 5-HT3-5�RA2 (5�-GAGTTCCATGGCAAGCTTCCC-3�) and primers5-HT3-3�RA1 (5�-CATCCCGCAGGTGCTGTTG-3�) and5-HT3- 3� RA2 (5�-GCCCAGGAGGATAAATAGCA-3�).EGFP coding cassette and RAs were assembled by standardcloning techniques, and the correct sequence assembly wascloned into the SalI site of the pSV1recA shuttle vector. Forhomologous recombination of the targeting cassette into BACclone 310N1, cointegrates were generated by transforming310N1-containing DH10B cells with pSV1.RecA-5�3�RA-EGFPDNA. Cointegration and resolution steps were as described (18).The modified BAC DNA separated from bacterial genomicDNA by cesium chloride gradient ultracentrifugation was re-leased from the vector by NotI-digestion and isolated by Sepha-rose column chromatography with column fractions analyzed byPFGE.

Purified, linearized BAC DNA (0.7 �g/ml) was microinjectedinto B6D2F2 mouse zygotes. Tail DNA of founders served to testintegrity of the integrated BAC by PCR and Southern blot.Presence of the EGFP gene was assessed with the primersEGFP-1 (5�-ATGGTGAGCAAGGGCGAGCT-3�) andEGFP-2 (5�-GCCGAGAGTGATCCCGGCGGCGGT-3�).Breeding transgenic with C57BL/6 mice produced wild-type andtransgenic mice at Mendelian ratios. The EGFP expressionpattern was analyzed in transgenic mice, starting with the F2generation. Three independent transgenic lines analyzed showedidentical EGFP expression patterns. The insertion of the EGFPcDNA followed by the polyadenylation sequence prevents tran-scription of the 5-HT3 sequence located downstream on theBAC. Thus, only EGFP, but not 5-HT3, expression is controlledby the 5-HT3 promoter located on the BAC. The latter isregulated by the native 5-HT3 receptor promoter. The excellentcellular correspondence between the two signals that are con-

trolled by two 5-HT3 promoters (EGFP by the promoter on theBAC and 5-HT3 receptors by the native promoter) denotes thecorrect expression of the transgene.

In situ Hybridization. Radioactive in situ hybridization experi-ments were performed as described by using [�-35S] dATP-end-labeled oligonucleotides that corresponded to nucleotides 181–225 and 334–378 of the mouse 5-HT3A and EGFP codingsequences, respectively.

Slice Preparation and Electrophysiology. Parasagittal brain sections(250–350 �m-thick for recordings in hippocampus and cortex) orcoronal brain sections (for recordings in amygdala) were madefrom transgenic mice (P8–P42) by using a vibratome (LeicaVT1000S). Whole-cell voltage and current clamp recordingswere made from visually identified, EGFP-positive neurons byusing an EPC9 amplifier (HEKA) and an upright microscope(Zeiss Axioskop FS2) equipped with IR-DIC and standardepifluorescence. Patch pipettes had a resistance of 2–6 M� whenfilled with (in mM): 105 K-gluconate, 30 KCl, 0.5 CaCl2, 5EGTA, 2 Mg-ATP, and 10 Hepes. Slices were continuouslysuperfused with ACSF containing (in mM): 120 NaCl, 3.5 KCl,2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, 25 NaHCO3, and 25glucose. The presence of functional 5-HT3 receptors was as-sessed by application of 100 �M 5-HT (dissolved in ACSF) viapressure ejection from a second pipette connected to a pico-spritzer. Cells were voltage clamped at �60 mV. Signals werefiltered at 1–5 kHz, sampled at 2–10 kHz, and off-line analysiswas performed by using Igor Pro (Wavemetrics).

Immunohistochemistry. Immunostaining was carried out on 50 �mor 100 �m free-floating sections. The following primary anti-bodies were used: Polyclonal rabbit anti-EGFP antibody,1:10,000 (Molecular Probes); the polyclonal chicken anti-EGFPantibody (Abcam), 1: 2,000, or the monoclonal anti-EGFPantibody, 1:300 (Chemicon); mouse anti-GAD67, 1:1,000(Chemicon); mouse anti-cholecystokinin antibody, 1:5,000(UCLA RIA Core); mouse anti-parvalbumin antibody, 1:3,000(Sigma); rat anti-somatostatin antibody, 1:1,000 (Chemicon);mouse or rabbit anti-calretinin antibody, each at 1:5,000 (Swant);mouse anti-calbindin antibody, 1:5,000 (Swant); mouse anti-glialfibrillary acidic protein (GFAP), 1:100 (Chemicon); mouseanti-NeuN, 1:1,000 (Chemicon); mouse anti-2�, 3�-cyclic nucle-otide, 3�-phosphodiesterase (CNPase), 1:5,000 (Sternberger);and rat anti-BrdU antibody, 1:400 (Accurate). For visualizationof primary antibodies, slices were incubated with FITC 488 orAlexa 488 (Molecular Probes) conjugated anti-rabbit, anti-chicken, anti-mouse, or anti-goat IgG; anti-mouse, anti-goat, oranti-rat Cy3 coupled secondary antibody; and anti-mouse oranti-rabbit Cy5 coupled secondary antibody (Jackson ImmunoResearch Laboratories). Sections were analyzed by using anupright fluorescent microscope (Zeiss Axioplan 2), a LEICATCS-NT confocal microscope (Leica Microsystems), or a ZeissAxiovert 200M confocal microscope. The antibody #9303 raisedagainst Gastrin/CCK was provided by Antibody/RIA Core,CURE/Digestive Diseases Center, Los Angeles.

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Fig. S1. Generation of transgenic mice and specificity of EGFP expression. (A) Representation of the native and modified 5-HT3A gene structure, containingthe EGFP coding sequence followed by a polyadenylation signal located on a BAC. (B) Application of 100 �M 5-HT to whole-cell voltage clamped EGFP-positiveneurons evoke a 5-HT3 receptor mediated-inward current that can be blocked by the specific antagonist MDL72222 (n � 5). (C) All 39 randomly chosenEGFP-positive neurons in different brain areas had functional 5-HT3 receptor-mediated inward currents. Bars represent the average peak amplitude. The numberof recorded cells is displayed in the bars. (D and E) Radioactive in situ hybridization experiments show similar mRNA expression pattern of EGFP and 5-HT3A

receptors in brain coronal (D) and sagittal (E) sections from adult mice. Note the signal of scattered cells in the cortex, the high labeling in the hippocampus andbaso-lateral amygdala (BLA), as well as the lack of mRNA expression in the thalamus (D) and the high signal in the RMS (E). The exposure time for the sectionsshown in D was longer than for those in E accounting for the difference in the intensity of the signal in the cortex. (F–I) Specific EGFP (green) staining in thehippocampus (F), cortex (G), BLA (H), and OB (I) of adult transgenic mice. (J and K) EGFP staining in embryonic coronal sections (E14.5) of transgenic mice. (Insets)Drawings indicate the plane of sectioning. Cx, cortex; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare; LGE,lateral ganglionic eminence; MGE, medial ganglionic eminence; and CGE, caudal ganglionic eminence. (Scale bars: 200 �m.)



Fig. S2. Colocalization of EGFP with specific GABAergic interneuron markers in the adult cortex. (A–A�) Colocalization of EGFP (green) and NeuN (red) and nocolocalization of EGFP (green) with the astrocytic GFAP (red) (B–B�) or the marker for oligodendrocytes CNP (red) (C–C�) in the cortex of transgenic animals. (D–G)Double-labeling showing colocalization in the cortex of EGFP (green) and GAD67 (red) (D–D�) and CR (E), but not with parvalbumin (F) and somatostatin oftransgenic mice (G). CC, Corpus callosum; Cx, Cortex. (Scale bars: 50 �m.)



Fig. S3. Location of migrating cells visible in Movies S1 and S5. (A) RMS and VMP on sagittal slices from P16-old animals. (Inset) Location of the 20� image onsagittal slices is indicated. This represents one frame of Movie S1 in which neuroblast migration in the streams and in the striatum are demonstrated. (B) Locationof the boxed cortical area with respect to the RMS in sagittal slices from a P10-old animal. Time-lapse imaging of the boxed area in this 20� magnification isshown as a 40� magnification in Movie S5. This area was chosen to demonstrate migration not only in areas in immediate vicinity to the streams but also withinthe cortical layers.


http://www.pnas.org/content/vol0/issue2008/images/data/0807059105/DCSupplemental/SM1.mpg




Fig. S4. Chain-like migration of neuroblasts exiting the streams. 5-HT3/EGFP-positive neuroblasts labeled with antibodies against EGFP (green in A, C, D, F, andH), DCX (red in B, C, E, F, and H), and DAPI staining (blue in G and H) showing chain-like migration from the RMS into the corpus callosum toward the cortexin fixed sagittal sections of a P12 transgenic mouse. (D–H) Enlargements of the boxed area in C. CC, Corpus callosum. (Scale bars: 20 �m.)



Fig. S5. Adult neurogenesis of cortical 5-HT3/EGFP-positive cells. (A) Numerous EGFP-positive cells (green) with the specific morphology of neuroblasts locatedoutside of the RMS in the corpus callosum and directed toward the cortex in sagittal sections from P90 transgenic mice. (B) DCX-positive cells (red) in the RMSand vicinity in P90 wild-type mice. Insets (A, Lower Left and B, Upper Right) show enlargements of the boxed areas. (C–G�) Adult transgenic mice were injectedwith BrdU and 14 days (C–D�) or 28 days (E–G�) later, EGFP (D, F, and G), BrdU (D�, F�, and G�) and Calretinin-positive cells (F� and G�) were found in lower corticallayers. D, F, and G show magnifications of the boxed areas in C and E. Cx, cortex; CPu, caudate putamen. [Scale bars: (A and B) 200 �m; (C and D) 50 �m; (D�, F�,and G�) 20 �m.]



Fig. S6. Embryonically and postnatally generated 5-HT3/EGFP-positive cells represent distinct subgroups of GABAergic interneurons. Schematic representationindicating the localization of embryonically (A) and postnatally (D) generated 5-HT3/EGFP-positive cells (green dots) in sagittal and coronal sections of aone-month-old animal. Bipolar 5-HT3/EGFP-positive cells in upper layers are labeled with BrdU when injected at embryonic development (B–B�) and thisinterneuronal type is positive for CR (C). Monopolar 5-HT3/EGFP-positive cells in lower layers are labeled with postnatal BrdU injections (E–E�), and this cell typeis also positive for CR (F). A minor proportion of P4 BrdU/5-HT3/EGFP-labeled cells are found in layers II–IV; these multipolar cells are positive for NeuN (G–G�).Green dots in A and D indicate the approximate position of the cells shown in the pictures. (Scale bars: 20 �m.)



Fig. S7. Neuroblast migratory patterns in wild-type mice are similar to that in transgenic animals. (A–A�) Expression pattern of DCX in neuroblasts along theexternal capsule in horizontal sections (A), in cells emerging from the RMS toward the cortex (A�), and along the DMP (A�) in sagittal sections, all from P10wild-type animals. (A) Enlargement (Bottom Right) of the boxed area (Middle). (A� and A�) Examples of individual or groups of migratory cells in regions adjacentto the streams are indicated by arrows. (A and A�) Plane of the sections is indicated in the White Insets. (B and C) Retrovirus-RFP was delivered into the SVZ ofP3–4 wild-type mice, and the brains were fixed and immunostained either 6 days (B–B�) or 2 weeks (C–C�) after injection. B shows an example of a viral-infected(red) neuroblast stained with DCX (green) migrating out of the RMS; B� and B� are enlargements of the boxed area in B (Upper Left). The scheme in B illustratesthe injection site and brain area shown in the White Inset. (C) Reconstruction of a sagittal section from a P19 wild-type mouse after 2 weeks of retrovirus-RFPinfection, showing RFP labeling. (C� and C�) Higher magnifications of the boxed areas in C, showing examples of mature NeuN neurons (stained in green)expressing RFP (red) in the cortex. [Scale bars: (A) 200 �m, (A� and A�) 100 �m, (B–B�) 50 �m, (C) 200 �m, (C�) 20 �m.]



Table S1. Colocalization of EGFP with interneuron-specific GABAergic interneuron markers in the indicated regions in the adult brain

Region CB* CR* CCK* CB† CR† CCK†

CA1, CA3 77.5% 76.9% 60.4% 4.2% 15.5% 66%(n � 89) (n � 134) (n � 106) (n � 167) (n � 187) (n � 94)

Dentate gyrus 17.3% 8.6% 68.6% 4.8% 12.33% 73.7%(n � 52) (n � 35) (n � 35) (n � 63) (n � 146) (n � 38)

Entorhinal cortex 9.5% 76.8% 52.1% 4.1% 33.9% 48.5%(n � 74) (n � 99) (n � 71) (n � 49) (n � 189) (n � 66)

Temporal cortex 3.1% 65.8% 55.6% 1% 31.3% 32%(n � 65) (n � 38) (n � 54) (n � 96) (n � 112) (n � 97)

Auditory cortex 7% 58.5% 43.6% 7.7% 23.7% 35.6%(n � 81) (n � 65) (n � 62) (n � 39) (n � 93) (n � 59)

Somatosensory cortex 1.6% 40.6% 37% 0% 22.5% 27.4%(n � 64) (n � 96) (n � 46) (n � 21) (n � 89) (n � 62)

*Data shows the percentage of 5-HT3/EGFP-positive cells that are CB-, CR-, and CCK-positive and indicates that the majority of 5-HT3/EGFP-positive interneuronsare CR- or CCK-positive. n indicates cell number.

†Data shows the percentage of CB-, CR-, and CCK-positive cells that are also 5-HT3/EGFP-positive and indicates that only a subpopulation of CR- and CCK-positiveinterneurons also express the 5-HT3 receptor. n indicates cell number.



Movie S1 (MPG)

Movie S1. Neuroblast migration in the RMS and VMP at P16. Time-lapse imaging at 20� magnification in sagittal slices. Location of the imaged area withinsagittal slices in indicated in Fig. S3A. Total length of movie is 3 h.



http://www.pnas.org/cgi/data/0807059105/DCSupplemental/Supplemental_PDF#nameddest=SF3


Movie S2 (MPG)

Movie S2. Directed migration from the RMS into cortex at P7. Time-lapse imaging at 20� magnification in sagittal slices demonstrates massive migration atthis age. Chain-like migration of groups of cells is more pronounced at this age compared to later developmental stages. Total length of movie is 20 h.




Movie S3 (MPG)

Movie S3. Path lengths and trajectory of migratory neuroblasts into the cortex. Clearly identifiable and moving cells from Movie S2 were selected and theirmigratory trajectory is visualized by using manual tracking plug-in of ImageJ software.





Movie S4 (MPG)

Movie S4. Directed migration from the DMP into cortex at P10. Time-lapse imaging at 20� magnification in sagittal slices shows migration within the DMPand migration of neuroblasts that exit the stream and migrate toward the cortex. Migration within lower cortical layers is also visible. Total length of movie is5 h.




Movie S5 (MPG)

Movie S5. Migration of neuroblasts from layer V toward layer IV at P10. Time-lapse imaging at 40� of a group of neuroblasts within the cortex. The locationof the imaged neuroblasts with respect to the RMS in the sagittal slice is indicated as boxed area in the 20� image shown in Fig. S3B. This movie demonstratesa frequently noticed pattern of movement, for example, one neuroblast migration ‘‘on top’’ of another one. These neuroblasts were imaged for �7 h: 2 h at20� followed by 4 h at 40� and then again for another 2 h at 20�. During the 4 h shown here, several neuroblasts exhibit migration. The neuroblast with thehorizontally oriented leading process directed to the left exhibits movement of the leading process, but not of the soma. The outgrowth of a secondary branchis visible. During the final phase of this time-lapse imaging study (last 20� movie, data not shown), the somal translocation of this neuroblast also occurs.



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Movie S6 (MPG)

Movie S6. Movement patterns of neuroblasts at P19. Time-lapse imaging at 40� of several neuroblasts that have exited the RMS. Advancement and retractionof the leading process with growth cone-like structure at its tip, before the translocation of the soma occurs. Migration of isolated neuroblasts and chain-likemigration of a small group of cells can also be seen. Total length of the video is 3 h.




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