Supporting Online Material for

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1 Supporting Online Material for A Frazzled/DCC-Dependent Transcriptional Switch Regulates Midline Axon Guidance Long Yang, David S. Garbe, Greg J. Bashaw* *To whom correspondence should be addressed. gbashaw@mail.med.upenn.edu This PDF file includes: Materials and Methods Figs. S1 to S10 Table S1 References Published 26 March 2009 on Science Express DOI: /science

2 Materials And Methods Genetics: The following stocks were used: 1) fra 3 /CyWgßgal, 2) fra 4 /CyWgßgal, 3) fra 3, UAS- TauMycGFP/CyTubGal80; eaglegal4, 4) fra 4, UAS-TauMycGFP/CyTubGal80; eaglegal4, 5) fra 6 /CyWgßgal; UAS-TauMycGFP, 6) fra 6 / CyTubGal80, eaglegal4, 7) comm e39, eaglegal4/tm3ßactin, 8) UAS-TauMycGFP/CyTubGal80; eaglegal4, 9) fra 4, UAS- TauMycGFP/CyOelavβgal; comm e39 /TM6Ubxβgal, 10) fra 4, UAS-TauMycGFP/CyOelavβgal; comm G485 /TM6Ubxβgal, 11) Df(2R)BSC3, UAS-TauMycGFP/CyWgßgal, 12) fra 3, UAS- FraMyc(#4.2)/CyWgßgal, 13) fra 3, UASFra C(#4)/CyWgßgal, 14) fra 3 /CyWgßgal; UASMyr- Fra-MyC(#1.1), 15) fra 3 /CyWgßgal; UASFra P1 P2 P3-Myc(#1.2), 16) apterousgal4, UAS- TauMycGFP/CyTubGal80, 17) Sp/CyO; UASFraMyc(#2), 18) neta,b/fm7ßactin, 19) neta,b/fm7, UASComm, 20) fra 3 /CyWgßgal, UASComm, 21) fra 3, robo GA285 /CyWgßgal, 22) fra 4, robo GA285, UAS-TauMycGFP/CyTubGal80; eaglegal4, 23) Sp/CyO; UASFra C(#2), 24) comm e39, elav/tm3ßactin. Antibody staining: Embryos were collected, fixed and stained as previously described (1). The following primary antibodies were used: 1) Ms-MAb BP102 [(Developmental Studies Hybridoma Bank (DSHB), 1:100], 2) Ms-anti-βgal (DSHB, 1:250), 3) Ms-anti-HA (Covance, 1:250), 4) Rb-anti-GFP (Molecular Probes, 1:500), 5) Rb-anti-HRP (MP Biomedicals, 1:1000). The following secondary antibodies were used: 1) AlexaFluor488 gt-anti-rb (Molecular Probes, 1:500), 2) Cy3 gt-anti-ms (Jackson Laboratories, 1:1000). Fluorescent images were taken using a Leica Confocal TCS SL microscope and processed by NIH Image J software. Fluorescent in situ hybridization and quantification: Fluorescent in situ hybridization was performed as previously described with digoxigenin/biotin-labeled probes(2). The comm and neta,b in situ probes were PCR amplified from full-length cdnas and transcribed from the PCR products. The 4.0 kb comm intron probes were PCR amplified from the genomic DNA using the following primers with T7 or SP6 tag: 5 -TAATACGACTCACTATAGGGAGAGAGGATGGCAGGACAGGCGAAGTTC-3, 5 - ATTTAGGTGACACTATAGAACGGACAGCCACCCAAATATAATCCCC-3 For fluorescence quantification, six random pairs of heterozygous and mutant siblings were isolated from the same embryo collection. Hybridized embryos were also labeled with Rb-anti- Myc (Sigma-Aldrich, 1:500) to display the eagle neurons. Fluorescence intensity is calculated as area multiplied by average fluorescence intensity using NIH Image J software. Myc staining was processed to generate a cell body mask for the eagle neurons. Relative fluorescent intensity of comm mrna is calculated as absolute comm mrna fluorescence intensity under the mask divided by absolute Myc fluorescence intensity under the mask. Quantitative real-time PCR: 10 nerve cords of stage 14 embryos of each genotype were live dissected. RNA was extracted by using TRIzol-LS reagent (Invitrogen). cdna was prepared using Verso TM cdna kit (Thermo scientific). Quantitative real-time PCR was carried out in triplic1tes from prepared cdna using TaqMan universal PCR Master Mix (Applied Biosystems). The following FAM dye-labeled probes were used: comm probe (#Dm _m1, Applied Biosystems), fra probe (#Dm _m1, Applied Biosystems). Expression of 18S rrna was used for cross-experiment normalization.

3 Fig. S1. Dominant genetic interaction between UASFraΔC and comm (A to E) Stage 16 embryos stained with MAb-BP102 [Magenta in (A to C)] to display all CNS axons and anti-ha [Green in (D) and (E)] to visualize the transgene expression. (F to H) Stage 16 embryos stained with anti-ha (F and G) or anti-gfp (H) visualize the eagle neurons. Expressing UASTau-MycGFP with the eaglegal4 driver allows visualization of a cluster of 3-4 neurons per hemi-segment (EW neurons) that project their axons across the midline in the posterior commissure and another cluster of neurons per hemi-segment (EG neurons) whose axons cross the midline in the anterior commissure (3). Removing one copy of comm enhances the axon guidance defect caused by over-expressing one copy of UASFraΔC in all neurons [compare arrowheads in (A) and (B)] or in the eagle neurons [compare arrows in (F) and (G)]. comm/+ animals have normal commissural formation (C) and normal EW trajectories (H). (I) Quantification of EW axon non-crossing defect. Error bars represent standard error of the mean. Asterisk denotes p < 1e-18 in a Student s t test. Scale bar in (A), 20 microns.

4 Fig. S2. Dose dependent genetic interaction between fra and comm Three different fra alleles and two different comm alleles were used. fra 3 and fra 4 are published null alleles, while fra 6 is a hypomorphic allele isolated from a collection of midline guidance mutants (4). comm e39 is a null allele and comm G485 is a strong hypomorphic allele (5). We reexamined the guidance defects in the previously identified fra null alleles, fra 3 and fra 4, by quantifying the EW axon non crossing defects. Surprisingly, fra 3 is likely to be hypomorphic allele rather than a null allele as shown by the significantly milder guidance defects in fra 3 mutants compared to that in Df(2R)BSC3/fra 3 mutants. Df(2R)BSC3 is a large deletion covering the entire fra gene locus. The nature of fra 4 remains unclear due to other unknown mutations on the same chromosome. (A to C) Stage 16 eglgal4::uastau-mycgfp embryos stained with MAb- BP102 (magenta) to display all CNS axons and anti-gfp (green) to visualize the eagle neurons. (D to F) Stage 16 embryos stained with anti-fra antibodies. (A) In fra 4 /+; comm e39 /+ embryos, EW and EG neurons project their axons across the midline in almost every segment. (B) Compared to fra 4 /fra 6, fra 4 /fra 3 embryos display frequent thin commissures (arrowheads) and stronger EW axon non-crossing defects (arrows). (C) Compared to fra 4 /fra 3, fra 4 /fra 3 ; comm e39 /+ embryos have more missing and thin commissures (arrowheads) and more EW axons also fail to cross the midline (arrows). fra 4 /fra 6 embryos have wild-type levels of Fra protein (E), while fra 4 /fra 3 have reduced, but detectable levels of Fra (F). (G) Quantification of EW defects. Error bars represent standard error of the mean. Asterisks denote p < 0.02 in a Student s t test. Scale bar in (A), 20 microns.

5 Fig. S3. comm RNA is strongly reduced in the nerve cords of fra mutants Quantitative real-time PCR analysis of the nerve cords of stage 14 embryos. Total comm mrna or fra mrna levels are normalized by total 18S rrna levels and are shown on the x axis. Genotypes are indicated on the Y axis. Error bars represent standard error of the mean. Asterisk denotes p < in a Student s t test.

6 Fig. S4. Quantification of comm mrna in fra mutants Expressing UASFra-Myc in the eagle neurons of fra mutants completely rescues comm mrna expression in the EWs, even in the EWs whose axon trajectories are not rescued [arrowheads in (A)]. (B) Quantification of relative fluorescence intensity of comm mrna in the eagle neurons. Error bars represent standard error of the mean. Asterisk denotes p < 0.01 and double asterisks denote p < 0.03 in a Student s t test. Scale bar in (A), 20 microns.

7 Fig. S5. Fra is required cell-autonomously in the EGs for comm mrna expression (A to I) Stage 14 eglgal4::uastau-mycgfp embryos double-labeled with RNA in situ probes for comm (green) and anti-myc (Magenta) to visualize the eagle neurons. Anterior is up. Confocal sections of the EGs are shown. White hash marks in each panel indicate the positions of the XZ and YZ sections displayed below and to the right of the main XY panels respectively. (A to C) comm mrna expression in the EGs of fra/+ embryos (arrowheads). (D to F) comm mrnas are dramatically reduced in the EGs of fra 4 /fra 3 mutants (arrowheads). (G to I) Over-expressing UASFra-Myc in the eagle neurons of fra mutants completely rescues comm mrna expression in the EGs (arrowheads). Scale bar in (A), 20 microns.

8 Fig. S6. comm pre-mrna is greatly reduced in the eagle neurons of fra mutants (A-F) Stage 14 eglgal4::uastau-mycgfp embryos double-labeled with RNA intron probes for comm (green) and anti-myc (Magenta) to visualize the eagle neurons. Anterior is up. Confocal sections of the EWs are shown. White hash marks in each panel indicate the positions of the XZ and YZ sections displayed below and to the right of the main XY panels respectively. (A-C) comm pre-mrna expression in the EWs of fra/+ embryos (arrowheads). (D-F) comm pre-mrna is significantly reduced in the EWs of fra 4 /fra 3 mutants (arrowheads). Scale bar in (A), 20 microns.

9 Fig. S7. Over-expression of Robo does not affect comm expression (A to F) Stage 14 eglgal4::uastau-mycgfp embryos labeled with RNA in situ probes for comm (green) and anti-myc (magenta) to visualize the eagle neurons. Anterior is up. Confocal sections of the EWs are shown. White hash marks in each panel indicate the positions of the XZ and YZ sections displayed below and to the right of the main XY panels respectively. (A to C) comm mrna expression in the EWs of eaglegal4/+ embryos (arrowheads). (D to F) Over-expressing UASRobo in the eagle neurons prevents the EW axons from crossing the midline (arrows) yet maintains normal comm expression (arrowheads). Scale bar in (A), 20 microns.

10 Fig. S8. Netrins are not the ligands for Fra to induce comm mrna expression (A-C) Stage 14 eglgal4::uastau-mycgfp embryos triple-labeled with RNA in situ probes for comm (green) and netrinab (blue), and anti-myc (magenta) to visualize the eagle neurons. Anterior is up. Confocal sections of the EWs are shown. White hash marks in each panel indicate the positions of the XZ and YZ sections displayed below and to the right of the main XY panels respectively. (A and A ) Expressing UASMyr- Fra-Myc in the eagle neurons of fra mutants completely rescues comm mrna expression (arrowhead indicates an EW that has rescued comm mrna expression). (B and B ) Expressing UASFraΔP1ΔP2ΔP3-Myc in the eagle neurons of fra mutants completely rescues comm mrna expression (arrowhead indicates an EW that has rescued comm mrna expression). (C and C ) Expressing UASFraΔC-HA in the eagle neurons of fra mutants does not rescue comm mrna expression (arrowhead indicates an EW that has reduced comm mrna expression). (D) Quantification of relative fluorescence intensity of comm mrna in the EWs. Error bars represent standard error of the mean. Asterisk denotes p < in a Student s t test. Scale bar in (A), 20 microns.

11 Fig. S9. Overexpression of UASFraΔC in the eagle neurons of wild type embryos mildly decreases comm mrna expression in the EWs (A-F) Stage 14 eglgal4::uastau-mycgfp embryos double-labeled with RNA in situ probes for comm (green) and anti-myc (Magenta) to visualize the eagle neurons. Anterior is up. Confocal sections of the EWs are shown. White hash marks in each panel indicate the positions of the XZ and YZ sections displayed below and to the right of the main XY panels respectively. (A-C) comm mrna expression in the EWs of wild type embryos (arrowheads). (D-F) comm mrnas are mildly reduced in the EWs of embryos with UASFraΔC over-expressed in the eagle neurons. Scale bar in (A), 20 microns.

12 Fig. S10. A model of dual roles for Fra in midline guidance. Fra mediates chemoattraction in response to Netrin and also acts independently of Netrin to regulate comm transcription. Comm, in turn, negatively regulates Robo surface levels in pre-crossing commissural axons ensuring midline crossing.

13 Supplementary Table 1. Quantification of EW axon guidance defects Genotype 1 Total embryos scored Total segments scored 2 % of EW axon non-crossing defects 3 Corresponding figures fra 4 / fig 1A, 1D comm e39 / Suppl fig 2G fra 4 /+; comm e39 / Suppl fig 2A, 2G fra Suppl fig 2G fra 4 /fra fig 1B, 1D fra 4 /fra 6 ; comm G485 /+ fra 4 /fra 6 ; comm e39 / fig 1D fig 1C, 1D fra 4 /fra Suppl fig 2B, 2G fra 4 /fra 3 ; comm G485 /+ fra 4 /fra 3 ; comm e39 /+ fra 3 Df(2R)BSC3/fra 3 netab/+; UASComm/ Suppl fig 2G Suppl fig 2C, 2G Suppl fig 2G Suppl fig 2G fig 5E, 5I fra 4 /fra 3 ; UASComm/ fig 5C, 5I fra 4 /fra 3 ; UASMyr-Fra(#1.1)/+ fra 4 /fra 3 ;UASFraΔP1ΔP2ΔP3(#1.2)/+ fra 4 /fra 3,UASFraΔC(#4) fra 4,robo GA285 /fra 3,robo GA *(p=7e-4) 32.6*(p=2e-3) Suppl fig 7A Suppl fig 7B Suppl fig 7C fig 5D, 5I netab fig 5F, 5I netab; UASComm/ ** (p=0.096) fig 5G, 5I UASFraΔC(#2)/ suppl fig 1F, 1I UASFraΔC(#2)/comm e suppl fig 1G, 1I Late stage 15- early stage 16 embryos were scored. 1 Indicated genotypes also contain one copy of UASTau-MycGFP and eaglegal4. 2 Eight abdominal segments were scored in each animal.

14 3 The EW neurons labeled with Tau-MycGFP were scored for defects in normal midline crossing. Defects are defined as a complete loss of the commissural EW bundle. Percentage of EW non-crossing defects is calculated as total number of defective segments divided by total segments scored. * Statistically different from fra 4 /fra 3 in a two-sample Student s t test. The p value is indicated. ** Not statistically different from netab in a two-sample Student s t test. The p value is indicated. References 1. T. Kidd et al., Cell 92, 205 (1998a). 2. J. P. Labrador et al., Curr Biol 15, 1413 (Aug 9, 2005). 3. S. Higashijima, E. Shishido, M. Matsuzaki, K. Saigo, Development 122, 527 (Feb, 1996). 4. M. Seeger, G. Tear, D. Ferres-Marco, C. S. Goodman, Neuron 10, 409 (1993). 5. G. Tear et al., Neuron 16, 501 (1996).