Figures and data
Expression of Sp1 and its coactivators in chick and mouse LMC motor neurons.
(A-O) mRNA detection in consecutive sections of spinal cords: all chick sections represent the HH stage 25/26 lumbar spinal cord, while all mouse sections represent the e11.5 lumbar spinal cord. (A, B) Detection of Isl1 (A) and Lim1 (B) mRNA in the chick spinal cord, marking the medial and lateral LMC neurons, respectively. (C-E) mRNA detection of Sp1 (C) and its coactivators p300 (D) and CBP (E) in both medial and lateral LMC neurons of the chick. (F, G, K, L) mRNA detection of Isl1 (F, K) and Lim1 (G, L) in mouse LMC neurons. (H-J, M-O) mRNA detection of Sp1 (H, M), p300 (I, N), and CBP (J, O) in both medial and lateral LMC neurons in the mouse.
M, medial; L, lateral. Scale bar, μm (A-O) 30 μm
Sp1 function is required for the selection of limb axon trajectory.
All images are from chick HH st. 28/29 lumbar levels.
(A-C) Detection of Foxp1, Isl1, GFP, and Sp1 in LMC neurons of chick embryos electroporated with either GFP (A), scrambled [Sp1]siRNA and GFP (B), or [Sp1]siRNA and GFP (C).
(D-F) Detection of Foxp1, Sp1, and GFP in cultured LMC neurons from chick embryos electroporated with GFP (A), scrambled [Sp1]siRNA and GFP (B), or [Sp1]siRNA and GFP (C).
(G) Quantification of the effects of GFP, scrambled [Sp1]siRNA and GFP, or [Sp1]siRNA + GFP electroporation on Sp1 mRNA levels. The ratio of immunoreactivity signal between the LMC regions of electroporated and un-electroporated contralateral sides (e/u ratio) was measured in at least 15 sections from 5 embryos.
(H-J) Detection of Foxp1, Isl1, GFP protein, and mouse Sp1 mRNA in LMC neurons of chick embryos at HH stage 28/29 electroporated with GFP (H), Sp1 and GFP (I), or Sp1T278A, T739A and GFP (J) expression plasmids.
(K) Quantification of axon outgrowth effects following electroporation of GFP, scrambled [Sp1]siRNA and GFP, [Sp1]siRNA and GFP, Sp1 and GFP, or Sp1T278A, T739A and GFP. GFP+ LMC axon lengths were compared to the GFP control group, with measurements taken from at least 15 sections of 5 embryos.
(L) The number of LMC motor neurons was expressed as the average count of Foxp1+ LMC neurons per section (# Foxp1+ MNs/section), with 5 embryos per group.
(M, N) The number of medial (Foxp1+ Isl1+) and lateral (Foxp1+ Isl1-) LMC motor neurons in the lumbar spinal cord was expressed as a percentage of total motor neurons [Foxp1+ MNs (%)]
(M) or electroporated motor neurons [GFP+ MNs (%)] (N), with 5 embryos per group.
(O-V) Detection of neurofilament and GFP in the limb nerve branches of the crural plexus in chick embryos electroporated with different plasmids and siRNAs, including GFP (O), scrambled [Sp1]siRNA and GFP (P), [Sp1]siRNA and GFP (Q), Sp1 and GFP (R), [Sp1]siRNA, mouse Sp1 and GFP (S), Sp1T278A, T739A and GFP (T), medial LMC axonal marker, e[Isl1]::GFP (U), or [Sp1]siRNA and e[Isl1]::GFP (V). GFP fluorescence signals were quantified in dorsal and ventral limb nerves, with the percentages (GFP Fluo [%]) presented for each electroporation condition. The number of embryos was n = 5 for all groups.
d, dorsal; v, ventral; n.s. = not significant; *** = p<0.001; ** = p<0.01; * = p<0.05; statistical significance computed using Fisher’s exact test (A-N) or Mann-Whitney U test (O-U); solid lines in each scatterplot show the median difference. Scale bar: (A-C, H-J) 45 μm; (D-F) 35μm; (O-U) 150 μm
Sp1 is required for the fidelity of LMC motor axon trajectory selection.
Retrograde labeling of LMC neurons via HRP injections into dorsal or ventral limb muscles of mouse embryos at e12.5.
(A-H) Detection of Lim1 (green), Foxp1 (red), and Sp1 in the LMC region at the brachial level in mouse Sp1F/F (A-D) or Syn1-Cre:Sp1F/F(E-H) embryos.
(I) Quantification of LMC motor neurons as the average number of total (Foxp1+) LMC neurons per section (# FoxP1+/section). The number of embryos used: n = 5 for all groups.
(J) Quantification of total medial (FoxP1+ Lim1-) and lateral (FoxP1+ Lim1+) LMC motor neurons in the brachial spinal cord, expressed as a percentage of total motor neurons [FoxP1+ MNs (%)]. The number of embryos used: n = 5 for all groups.
(K-N) Detection of HRP (red), Isl1 (blue), and Foxp1 (green) in the LMC regions of Sp1F/F (K, L) and Syn1-Cre:Sp1F/F(M, N) embryos following HRP injection into the dorsal forelimb muscles.
(O) Quantification of retrogradely labeled medial LMC axon projections. The graph displays the percentage of HRP+ motor neurons expressing the medial LMC marker Isl1 after dorsal limb injection. The number of embryos used: n = 5 for all groups.
(P) Schematic summary showing medial LMC projections in Syn1-Cre:Sp1F/F mice, highlighting the significant misrouting of medial LMC axons into the dorsal limb.
(Q-T) Detection of HRP (red), Lim1 (blue), and Foxp1 (green) in the LMC regions of Sp1F/F (Q, R) and Syn1-Cre:Sp1F/F(S, T) embryos following HRP injection into the ventral forelimb muscles.
(U) Quantification of retrogradely labeled lateral LMC axon projections. The graph displays the percentage of HRP+ motor neurons expressing the lateral LMC marker Lim1 after ventral limb injection. The number of embryos used: n = 5 for all groups.
(V) Schematic summary showing lateral LMC projections in Syn1-Cre:Sp1F/F mice, depicting significant misrouting of lateral LMC axons into the ventral limb.
HRP, horseradish peroxidase; Syn1, Synapsin1; n.s. = not significant; ** = p<0.01; *** = p<0.001; statistical significance computed using Fisher’s exact test; solid lines in each scatterplot show the median difference. Scale bar: (A-H, K-N, Q-T) 20 μm
Sp1-mediated gene expression and pathways analysis in neuronal development
(A) Volcano plot showing the differential gene expression between Syn1Tg/0/Sp1f/fand Syn1Tg/0 conditions. The x-axis represents the Log2 fold-change in gene expression, and the y-axis shows the −Log (p-value). Significantly upregulated genes (in red) and downregulated genes (in green) are displayed, with 333 and 353 genes identified as upregulated and downregulated, respectively.
(B) Pathway enrichment analysis was performed on the differentially expressed genes (DEGs) using Ingenuity Pathway Analysis, focusing on pathways related to organismal growth and development. The top 10 enriched pathways and signaling events are presented.
(C) Network representation of key pathways, including Ephrin-Receptor Signaling and Axonal Guidance Signaling. DEGs are represented, with red nodes indicating upregulated genes and green nodes showing downregulated genes. The network highlights the interactions between these genes, with different node shapes corresponding to the functional classes of the gene products (e.g., transcription factors, receptors, enzymes).
(D) RNA sequencing analysis of gene expression in primary mouse neurons revealed significant changes in genes related to axon guidance. Specifically, ephrin-B2 (Efnb2) and ephrin-A5 (Efna5) expression decreased, while Sema7A increased following Sp1 siRNA knockdown (left panel). A ChIP assay using Sp1 antibody confirmed Sp1 binding at the ephrin-B2 promoter region (right panel).
(E-G) Diagrams illustrate the predicted Sp1-binding sites on the ephrin-B2 promoter (E). Both promoter activation and protein levels were assessed in NSC-34 cells, indicating Sp1’s regulatory role (F, G).
Sp1 function is specifically required for ephrin-mediated guidance of cultured LMC neurites.
Growth preference of medial or lateral LMC axons on protein stripes. Each experiment consists of three panels (left, middle, and right) and one quantification.
(A-H) Left panels: Detection of medial (GFP+) LMC neurites from explants grown on stripes of eA5/Fc (A), EA4/Fc (C), N/Fc (E), or S3F/Fc (G), and explants co-electroporated with [Sp1]siRNA on stripes of eA5/Fc (B), EA5/Fc (D), N/Fc (F), or S3F/Fc (H) stripes. Middle panels: Inverted images showing GFP signals as dark pixels overlaid on the substrate stripes. Right panels: Superimposed images of five representative explants from each experimental group, illustrating the distribution of medial LMC neurites. Quantification shows medial (GFP+) LMC neurites on the first (pink) and second (pale) stripes as a percentage of total GFP signals. A minimum of 88 neurites was analyzed. Number of embryos: n = 5 for all groups. A minimum of 3 explants per embryo was used.
(I-P) Left panels: Detection of lateral (GFP+ EphA4+) LMC neurites from explants grown on stripes of eB2/Fc (I), EB1/Fc (K), N/Fc (M), or GN/Fc (O), and explants co-electroporated with [Sp1]siRNA on stripes of eB2/Fc (J), EB1/Fc (L), N/Fc (N), or GN/Fc (P). Middle panels: Inverted images showing EphA4 signals as dark pixels overlaid on the substrate stripes. Right panels: Superimposed images of five representative explants from each experimental group, demonstrating the distribution of lateral LMC neurites. Quantification shows lateral (EphA4+) LMC neurites on the first (pink) and second (pale) stripes as a percentage of total EphA4 signals. A minimum of 85 neurites was analyzed. Number of embryos: n = 5 for all groups. A minimum of 3 explants per embryo was used.
eA5, ephrin-A5-Fc; EA4, EphA4-Fc; N, Netrin-1; S3F. Semaphorin-3F; eB2, ephrin-B2-Fc; EB1, EphB1-Fc; GN, GDNF; *** = p<0.001; ** = p<0.01; n.s. = not significant; statistical significance computed using Mann-Whitney U test; solid lines in each scatterplot show the median difference. Scale bar: (A-P) 150 μm
Sp1 coactivators, p300 and CBP, are required for Sp1-regulated guidance of cultured LMC neurites.
(A-L) All images are from chick embryos at HH stage 28/29, lumbar levels.
(A-C) Detection of Foxp1, Isl1, GFP, and p300 in LMC neurons from chick embryos electroporated with GFP (A), scrambled [p300]siRNA and GFP (B), or [p300]siRNA and GFP (C).
(D-F) Detection of Foxp1, Isl1, GFP, and CBP in LMC neurons from chick embryos electroporated with GFP (A), scrambled [CBP]siRNA and GFP (B), or [CBP]siRNA and GFP (C).
(G, H) Quantification of the effects of GFP, scrambled [p300]siRNA and GFP, or [p300]siRNA + GFP electroporation on p300 mRNA levels (G) and the effects of GFP, scrambled [CBP]siRNA and GFP, or [CBP]siRNA + GFP electroporation on CBP mRNA levels (H). The ratio of immunoreactivity between electroporated and un-electroporated contralateral sides (e/u ratio) was measured from at least 15 sections of 5 embryos.
(I) Quantification of the effects of GFP, scrambled [p300]siRNA and GFP, [p300]siRNA and GFP, scrambled [CBP]siRNA and GFP, or [CBP]siRNA and GFP electroporation on axon outgrowth. GFP+ LMC axon lengths were compared to the GFP control group, with measurements taken from at least 15 sections of 5 embryos.
(J) The number of LMC motor neurons is expressed as the average number of total (Foxp1+) LMC neurons per section (# Foxp1+ MNs/section). Numbers of embryos: n = 5 for all groups.
(K, L) The number of total or electroporated medial (Foxp1+ Isl1+) and lateral (Foxp1+ Isl1-) LMC motor neurons in the lumbar spinal cord is expressed as a percentage of total motor neurons [Foxp1+ MNs (%)] (K) or electroporated motor neurons [GFP+ MNs (%)] (L). Numbers of embryos: n = 5 for all groups.
(M-T) Growth preferences of medial or lateral LMC axons on protein stripes. Each experiment includes three panels (left, middle, and right) and one quantification.
(M-P) Left panels: Detection of medial (GFP+) LMC neurites from explants electroporated with GFP plasmid electroporated explants (M), Sp1 expression plasmid co-electroporated explants (N), Sp1 + scrambled [CBP]siRNA + scrambled [p300]siRNA (O), or Sp1 + [CBP]siRNA + [p300]siRNA (P) on eB2/Fc stripes. Middle panels: Inverted images showing GFP signals as dark pixels overlaid on substrate stripes. Right panels: Superimposed images of five representative explants from each group, highlighting the distribution of medial LMC neurites. Quantification of medial (GFP+) LMC neurites on the first (pink) and second (pale) stripes, expressed as a percentage of total GFP signals. A minimum of 88 neurites were analyzed. Numbers of embryos: n = 5 for all groups, with a minimum of 3 explants per embryo. (Q-T) Left panels: Detection of lateral (GFP+ EphA4+) LMC neurites from explants electroporated with GFP plasmid (Q), Sp1 expression plasmid (R), Sp1 + scrambled [CBP]siRNA + scrambled [p300]siRNA (S), or Sp1 + [CBP]siRNA + [p300]siRNA stripes. Middle panels: Inverted images showing EphA4 signals as dark pixels overlaid on substrate stripes. Right panels: Superimposed images of five representative explants from each group, highlighting the distribution of lateral LMC neurites. Quantification of lateral (EphA4+) LMC neurites on the first (pink) and second (pale) stripes, expressed as a percentage of total EphA4 signals. A minimum of 85 neurites were analyzed. Numbers of embryos: n = 5 for all groups, with a minimum of 3 explants per embryo.
eB2, ephrin-B2-Fc; eA5, ephrin-A5-Fc; *** = p<0.001; ** = p<0.01; * = p<0.05; statistical significance computed using Fisher’s exact test (A-L) or Mann-Whitney U test (M-T); solid lines in each scatterplot show the median difference. Scale bar: (A-F) 45 μm; (M-T) 150 μm
Sp1 mutant mice exhibit muscle strength and locomotor defects due to its regulation of ephrin activity in spinal motor neurons.
(A, B) Open field test: The total distance traveled (A) and the time spent in the center (B) were recorded over a 60-minute period.
(C) Y-maze test: The percentage of alternation between the arms was measured to assess cognitive performance.
(D) Rota-rod test: Mice were tested on an accelerating rota-rod (4-40 rpm) for a maximum duration of 300 seconds. The time until the mice fell from the apparatus was recorded as a measure of motor coordination.
(E) Grip strength test: Grip strength was measured using a grip strength meter, and the value was normalized to the body weight to represent the relative grip strength of the mice.
(F) Sp1, in combination with its co-activators p300 and CBP, regulates the accurate trajectory of LMC axons into the limb by controlling ephrin gene expression. This regulation mediates ephrin/Eph cis-attenuation and ephrin:Eph trans-signaling, ensuring proper axon guidance. Numbers of embryos: n = 8 for both groups in open field, Y-maze, and grip tests; n = 7 for Sp1F/F, and 11 for Syn1-Cre:Sp1F/Fgroups in Rota-rod test.
