Fig 2: Foxp1 misexpression induces LMC motor pool markers.(a) Analysis of cervical LMC motor pools in E12.5 Foxp1+/+, Foxp1−/− and Hb9::Foxp1; Foxp1−/− embryos. All LMC motor pools were lost in Foxp1−/− embryos. The Pou3f1+ MNs in Foxp1−/− embryos represent the expanded HMC MN population (identified by their medial position). Insets depict Islet1 co-staining of the same section. The Etv4 motor pool was restored in Hb9::Foxp1; Foxp1−/− embryos (white arrows), with some Foxp1+/Etv4+ MNs located more dorsally (arrowhead). The Nkx6.1 and Pou3f1 motor pools were not restored in Hb9::Foxp1; Foxp1−/− embryos, although a small number of Foxp1+/Pou3f1+ MNs were identified (yellow arrows). Scale bar, 50 μm. (b) Control Hb9::GFP ESC-derived MNs did not express any LMC motor pool markers. Hb9::Foxp1 ESC-derived MNs expressed Etv4 and Pou3f1 at high levels, but did not express Nkx6.1. Boxed regions are depicted as single-colour images below each image. Scale bars, 50 μm. (c) Quantification of motor pool markers in ESC-derived MNs (mean±s.e.m.; n=2 independent experiments, 938 Hb9::GFP MNs and 723 Hb9::Foxp1 MNs total; Student's t-test). Etv4: **P=0.009. Pou3f1: **P=0.008. (d) Foxp1 misexpression did not alter the percentage of Pou3f1+/Foxp1− ESC-derived HMC-like MNs (mean±s.e.m.; n=3 independent experiments, 390 Hb9::GFP MNs and 255 Hb9::Foxp1 MNs total).

Fig 3: Tissue opacity is effectively cleared using the SCALE method on chips. (A) Schematic of the SCALE process modified for organ-chip use. (B) Comparison of ICC vs. SCALE phase imaging d28 (scale bar = 200 μm). (C) Phase imaging of ICC vs. SCALE after undergoing their respective staining protocols, and quantification of opacity changes from phase images of chips processed using either ICC or SCALE (scale bar = 200 μm) (n = 10 chips for fixation, n = 5 chips for SCALE and ICC, one-way ANOVA adjusted P values are p < 0.0001 fixation vs. SCALE, p = 0.0131 fixation vs. ICC, and p < 0.0001 SCALE vs. PBS, error bars = SEM). (D) Confocal images of ICC and SCALE chips from Fig 2B, top panels indicate DAPI. White and yellow arrows highlight cells in the top channel and bottom channel, respectively. The middle panels show non-phosphorylated neurofilament heavy chain (SMI32) and islet1 (ISL1)-positive spinal motor neurons, as well as Ki67-postive proliferating cells in the top neuronal channel. Yellow box indicates area quantified for E. Bottom panel is a top-down view of the neuronal channel. All images shown in B–D are taken from the same chips. (E) Quantification of area of visible stain in the top channel (n = 12 sites per condition, 4 individual chips per condition, scale bar z-axis = 50 μm, scale bar top channel = 30 μm, p < 0.0001, unpaired t-test, error bars = SEM).

Fig 4: Nociceptor deletion of Tsc2 preferentially upregulates cJun and Atf3 expression in IB4-positive neurons. A, Immunohistochemistry of L4 DRG contralateral and ipsilateral to a sciatic nerve transection (SN injury) at 3 d post-injury stained for cJun, Isl1 (all neurons), and IB4. Arrows point to cJun-positive, IB4-negative neurons, and arrowheads point to cJun, IB4 double-positive neurons in uninjured Tsc2 cKO DRG. Scale bars: 50 µm. B, Quantification of percentage of cJun-positive neurons from A. C, Immunohistochemistry of L4 DRG for Atf3, Isl1 (all neurons) and IB4. Arrows point to Atf3-positive, IB4-negative neurons and arrowheads point to Atf3, IB4 double-positive neurons in uninjured Tsc2 cKO DRG. Scale bars: 50 µm. D, Quantification of percentage of Atf3-positive neurons from C. E, Western blotting of uninjured control and Tsc2 cKO L4/L5 DRG from mice receiving daily vehicle or rapamycin treatment for 3 d. F, Quantification of protein expression from E. Log2 fold change relative to uninjured control from the same biological replicate. N.S., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Extended Data Figure 5-1 shows data values of mean and SEM, number of replicates, statistical tests, and values for all comparisons.

Fig 5: EMSA with Isl1 truncations and Pou4f2.Left: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on SBRN3(W), but not with Isl1D1 and Isl1D2. Right: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on CBRN3(W), but not with Isl1D1 and Isl1D2. F indicates free probes.