www.sciencemag.org/cgi/content/full/1171320/DC1

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. E-mail: gbashaw@mail.med.upenn.edu

Published 26 March 2009 on Science Express DOI: 10.1126/science.1171320

This PDF file includes:

Materials and Methods Figs. S1 to S10 Table S1 References

Materials And Methods Genetics: The following stocks were used: 1) fra3/CyWgßgal, 2) fra4/CyWgßgal, 3) fra3, UAS-TauMycGFP/CyTubGal80; eagleGal4, 4) fra4, UAS-TauMycGFP/CyTubGal80; eagleGal4, 5) fra6/CyWgßgal; UAS-TauMycGFP, 6) fra6/ CyTubGal80, eagleGal4, 7) comme39, eagleGal4/TM3ßactin, 8) UAS-TauMycGFP/CyTubGal80; eagleGal4, 9) fra4, UAS-TauMycGFP/CyOelavβgal; comme39/TM6Ubxβgal, 10) fra4, UAS-TauMycGFP/CyOelavβgal; commG485/TM6Ubxβgal, 11) Df(2R)BSC3, UAS-TauMycGFP/CyWgßgal, 12) fra3, UAS-FraMyc(#4.2)/CyWgßgal, 13) fra3, UASFra∆C(#4)/CyWgßgal, 14) fra3/CyWgßgal; UASMyr-Fra-MyC(#1.1), 15) fra3/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) fra3/CyWgßgal, UASComm, 21) fra3, roboGA285/CyWgßgal, 22) fra4, roboGA285, UAS-TauMycGFP/CyTubGal80; eagleGal4, 23) Sp/CyO; UASFra∆C(#2), 24) comme39, 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 VersoTM 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 (#Dm01822861_m1, Applied Biosystems), fra probe (#Dm01818218_m1, Applied Biosystems). Expression of 18S rRNA was used for cross-experiment normalization.

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 10-12 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.

Fig. S2. Dose dependent genetic interaction between fra and comm Three different fra alleles and two different comm alleles were used. fra3 and fra4 are published null alleles, while fra6 is a hypomorphic allele isolated from a collection of midline guidance mutants (4). comme39 is a null allele and commG485 is a strong hypomorphic allele (5). We reexamined the guidance defects in the previously identified fra null alleles, fra3 and fra4, by quantifying the EW axon non crossing defects. Surprisingly, fra3 is likely to be hypomorphic allele rather than a null allele as shown by the significantly milder guidance defects in fra3 mutants compared to that in Df(2R)BSC3/fra3 mutants. Df(2R)BSC3 is a large deletion covering the entire fra gene locus. The nature of fra4 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 fra4/+; comme39/+ embryos, EW and EG neurons project their axons across the midline in almost every segment. (B) Compared to fra4/fra6, fra4/fra3 embryos display frequent thin commissures (arrowheads) and stronger EW axon non-crossing defects (arrows). (C) Compared to fra4/fra3, fra4/fra3; comme39/+ embryos have more missing and thin commissures (arrowheads) and more EW axons also fail to cross the midline (arrows). fra4/fra6 embryos have wild-type levels of Fra protein (E), while fra4/fra3 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.

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 < 0.003 in a Student’s t test.

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.

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 fra4/fra3 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.

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 fra4/fra3 mutants (arrowheads). Scale bar in (A), 20 microns.

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.

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 < 0.003 in a Student’s t test. Scale bar in (A), 20 microns.

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.

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.

Supplementary Table 1. Quantification of EW axon guidance defects

Genotype1 Total

embryos scored

Total segments scored2

% of EW axon non-crossing

defects3

Corresponding figures

fra4/+ 10 78 0 fig 1A, 1D

comme39/+ 11 88 0 Suppl fig 2G

fra4/+; comme39/+ 25 185 2.8 Suppl fig 2A, 2G

fra6 25 195 0 Suppl fig 2G

fra4/fra6 25 196 24.6 fig 1B, 1D

fra4/fra6; commG485/+

fra4/fra6; comme39/+

20

24

154

192

46.9

57.3

fig 1D

fig 1C, 1D

fra4/fra3 27 200 54.0 Suppl fig 2B, 2G

fra4/fra3; commG485/+

fra4/fra3; comme39/+

fra3

Df(2R)BSC3/fra3

netAB/+; UASComm/+

20

25

23

23

12

157

197

183

183

95

80.8

72.1

43.9

64.6

0

Suppl fig 2G

Suppl fig 2C, 2G

Suppl fig 2G

Suppl fig 2G

fig 5E, 5I

fra4/fra3; UASComm/+ 26 201 31.8 fig 5C, 5I

fra4/fra3; UASMyr-Fra(#1.1)/+

fra4/fra3;UASFraΔP1ΔP2ΔP3(#1.2)/+

fra4/fra3,UASFraΔC(#4)

fra4,roboGA285/fra3,roboGA285

25

21

33

26

196

165

252

192

28.1*(p=7e-4)

32.6*(p=2e-3)

100

33.6

Suppl fig 7A

Suppl fig 7B

Suppl fig 7C

fig 5D, 5I

netAB 27 216 34.3 fig 5F, 5I

netAB; UASComm/+ 27 212 26.8** (p=0.096) fig 5G, 5I

UASFraΔC(#2)/+ 26 190 19.5 suppl fig 1F, 1I

UASFraΔC(#2)/comme39 26 197 96.4 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.

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 fra4/fra3 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).