Developmental Cell, Volume 51

Supplemental Information

Sema3a-Nrp1 Signaling Mediates

Fast-Twitch Myofiber Specificity of Tw2+ Cells

Stephen Li, Dileep Karri, Efrain Sanchez-Ortiz, Priscilla Jaichander, Rhonda Bassel-Duby, Ning Liu, and Eric N. Olson

Figure S1. Related to Figure 1 (A) tSNE and (B) violin plots of droplet-based single cell RNA

sequencing of limb muscle from Tabula Muris depicting Nrp1, Twist2, and Sema3a expression.

(C) Immunostaining of Nrp1 (red), wheat germ agglutinin (WGA) (white), and Hoechst (blue) on

transverse sections of gastrocnemius muscle from 3-month-old C57Bl6 wild-type mice. Scale:

50 µm.

Figure S2. Related to Figure 2. Transverse sections of tongue muscle from 3-month-old C57Bl6

WT mice were co-immunostained with My32 (green), Sema3a (red), and Hoechst (blue). Scale:

100 µm.

Figure S3. Related to Figure 3. (A) 10T1/2 fibroblasts, C2C12 myoblasts, mouse embryonic

fibroblasts (MEFs), and Cos7 cells were fixed and stained with Hoechst (blue) 1 day after seeding

onto Sema3a stripes (green). Scale: 100 µm. (B) Quantification of Sema3a avoidance for 10T1/2

fibroblasts, C2C12 myoblasts, mouse embryonic fibroblasts, and Cos7 cells. The dashed line

represents the baseline for cells unresponsive to Sema3a. Three fields per sample per experiment

were quantified. Source data for S3B is provided in Supplementary Table 1.

Figure S4. Related to Figure 4. Quantitative real-time PCR confirming over-expression of Nrp1

in Tw2+ cells infected with Nrp1 expressing retrovirus. *: p < 0.05. Source data for S4 is provided

in Supplementary Table 1.

Figure S5. Related to Figure 5. (A) Quantitative real-time PCR confirming knockdown of Nrp1

using two separate shRNAs. 3 separate experiments were performed. *p < 0.05. (B) Schematic

showing the Nrp1 genomic locus and the locations of the 2 sgRNAs (sgNrp1-2 and sgNrp1-5) that

target exon 2. Locations of primers for T7E1 assay are depicted in red. (C) sgNrp1-2 and sgNrp1-

5 were cloned into cas9-containing lentiviral vectors to knockout Nrp1 in Tw2+ cells. T7E1 assay

was used to validate cutting efficiency. (D) TOPO TA cloning and sequencing of sgNrp1-2 and

sgNrp1-5 infected Tw2+ cells. Representative clones containing indels are shown. Cas9-sgRNA

targeting sites are depicted on the template strand. Source data for S5A is provided in

Supplementary Table 1.

Figure S6. Related to Figure 6. (A) Quantitative real-time PCR confirms overexpression of

Sema3a in primary myoblasts infected with Sema3a. Sema3a expression levels were normalized

to the Tw2-Puro condition. *: p < 0.05. (B) Tw2+ cells (red) infected with retroviruses expressing

control empty vector, shNrp1-2, or Nrp1 were differentiated for 7 days before fixing and staining

fast myosin (white) and Hoechst (blue). Scale bar: 100 µm. (C) Primary myoblasts infected with

retroviruses expressing EGFP control (green) or Sema3a-EGFP (green) were differentiated for 7

days before fixing and staining for fast myosin (white) and Hoechst (blue). Scale bar: 100 µm. (D)

Primary myoblasts over-expressing Sema3a (green) were mixed with Tw2+ cells (red) infected

with Nrp1 (top), control empty vector (middle), or shNrp1-2 (bottom) and differentiated for 7 days.

Cells were fixed and stained with Hoechst (blue). Scale bar: 100 µm. Source data for S6A is

provided in Supplementary Table 1.

Figure S7. Related to Figure 7. Quantitative real-time PCR of tibialis anterior (TA) and

quadriceps muscle depicting over-expression of Sema3a in MCK-Sema3a transgenic mice

compared to WT mice. n = 4 for each genotype; *: p < 0.05. Source data for S7 is provided in

Supplementary Table 1.