Expression of Brn3a in the spinal cord at embryonic stages.

(A-D) Immunostaining of Brn3a (green) was performed on the transverse sections of the thoracic spinal cord from C57BL/6J mice at E12.5 (A), E14.5 (B), E16.5 (C), and E18.5 (D). The sections were counterstained by Hoechst 33342 (blue). Brn3a-void region in the dorsal horn is marked by square brackets. Spinal cords on the right side are shown. Arrows in D indicate Brn3a-positive neurons in the marginal region. Schematic diagrams of the spinal cord are shown in the insets. Red dotted squares indicate the area shown in the image. (E-J) Immunostaining of Brn3a (green), together with Brn3b (magenta), was performed on the transverse sections of the thoracic spinal cord from C57BL/6J mice at E12.5 (E-G) and E18.5 (H-J). Scale, 200 μm. (K) The number of Brn3a (red)- and Brn3b (blue)-positive cells in the hemi-spinal cord at E12.5 and E18.5 is shown. (L) The percentage of Brn3a- and Brn3b-double positive cells among Brn3a-positive cells at E12.5 and E18.5 is shown. Data are presented as the mean ± SEM.</p

AP assay of spinal cord sections of Lbx1-Cre;Brn3a-cKOAP mice.

AP assay was performed on the transverse sections the spinal cord of Lbx1Cre/+;Brn3acKOAP/+ (A) and Lbx1Cre/+;Brn3acKOAP/cKOAP(B) at E18.5. Nonspecific recombination occurred in a population of DRG neurons (red arrows). Scale, 500 μm. (PDF)</p

Labeling of Brn3a-KO neurons by using Brn3a-cKOAP mice.

(A) pCAG-Cre together with pCAG-nlsEGFP were introduced into spinal dorsal horn neurons of Brn3a-cKOAP mice at E11.8 by in utero electroporation, and the spinal cord was dissected out at E18.5. AP assay was performed on the transverse section of the spinal cord to visualize the distribution of Brn3a-KO neurons. (B-D) AP signal (magenta; B, D) and nlsEGFP fluorescence (green; C, D) on the right spinal dorsal horn are shown. Arrows indicate double-positive neurons. Scale, 200 μm. (PDF)</p

Effect of Brn3a overexpression on the axonal extension of Brn3a-lineage neurons.

pCAG-LSL-EGFP with or without pCAG-Brn3a was introduced into the spinal dorsal horn neurons of Brn3aCre/+ mice, as shown in Fig 6. The number of EGFP-positive axons normalized by that of EGFP-positive cell bodies in each section was defined as the index. The index of control (n = 5) and Brn3a-overexpressing (n = 5) mice in the DF on the ipsilateral side (A) as well as in the VF and VLF on the ipsilateral (B) and contralateral (C) sides is shown. Significant differences were assessed using the Mann–Whitney U-test. (A) p = 0.6905. (B) p = 0.0079. (C) p = 0.0317. “ns” = non-significant. *p p < 0.01. Data are the mean ± SEM.</p

Effect of Brn3a overexpression on the localization of Brn3a-lineage neurons.

(A, B) pCAG-LSL-EGFP without (A, control) or with pCAG-Brn3a (B, Brn3a overexpression) was unilaterally introduced into the spinal dorsal horn neurons of Brn3aCre/+ mice at E12.5 via in utero electroporation, and the transverse sections of the spinal cord at E18.5 were prepared to analyze the localization of EGFP-positive neurons. Arrows indicate EGFP-positive fibers derived from Brn3a-overexpressing neurons in the VF and VLF. Scale, 200 μm. (C) Five zones were defined in the spinal dorsal horn as shown in Fig 3G. (D) Percentage of EGFP-positive neurons localized in each zone of the spinal cord in control (n = 5) and Brn3a-overexpressing (n = 5) mice is shown. Significant differences were assessed using the Mann–Whitney U-test. Zone 1, p = 0.0397; Zone 2, p = 0.0079; Zone 3, p = 0.3889; Zone 4, p = 0.0079; Zone 5, p = 0.0079. *p p < 0.01. Data are the mean ± SEM.</p

Spinal dorsal horn-specific gene transfer by in utero electroporation.

pCAG-mCherry was unilaterally introduced into spinal dorsal horn neurons of Brn3aCre/+ mice at E12.5 by in utero electroporation, and the spinal cord of the mice was dissected at E18.5. Fluorescence of mCherry (magenta; A, B) and Hoechst 33342 (blue; B) on the transverse section of the sample is shown. White dotted lines indicate the outline of the spinal cord and DRG. Note that mCherry-positive cells are only found in the spinal cord but not DRG. Scale, 200 μm. (PDF)</p

The percentage of control and Brn3a-overexpressed neurons localized in Zone 1–5.

The percentage of control and Brn3a-overexpressed neurons localized in Zone 1–5.</p

S10 Fig -

Rostrocaudal axonal extension of Brn3a-overexpressed neurons (A) pCAG-LSL-EGFP together with pCAG-Brn3a were focally introduced into spinal dorsal horn neurons of Brn3aCre/+ mice at E12.5 by in utero electroporation (0.5 mm round electrode). The spinal cord of the mice was dissected out at E18.5 to analyze the rostrocaudal axonal extension of Brn3a-overexpressed neurons. (B) The number of EGFP-positive cell bodies in the spinal dorsal horn as well as EGFP-positive axons in the ipsilateral VLF and VF was analyzed on the serial transverse sections of the spinal cord of 2 mice (mouse #4 and #7). The number of EGFP-positive cell bodies (blue) and EGFP-positive axons (orange) on each section is shown. The left and right sides of each graph indicate the sections located rostral and caudal to the electroporation sites, respectively. (PDF)</p

Analysis of axonal tracts and terminals of Brn3a-persistent spinal dorsal horn neurons.

(A) AAV carrying FLEX-switched tdTomato and SypEGFP genes was injected into the lumbar spinal cord (~L5) of Brn3aCre/+ mice. This experiment enables labeling of Brn3a-persistent neurons and their presynaptic terminals by tdTomato and SypEGFP, respectively. (B-M) Three weeks after AAV injection, the spinal cord and whole brain of the mice were dissected out to analyze the distribution of axonal tracts and terminals of Brn3a-persistent neurons. tdTomato-positive cells were distributed in the lumbar spinal dorsal horn on the right. (B) Dorsal view of the spinal cord of AAV-injected mice. Solid and broken lines indicate the outline and the center of the spinal cord, respectively. (C) A transverse section of the lumbar spinal cord. A solid line indicates the outline of the spinal cord whereas a broken line indicates the boundary between the gray and white matters. Scale, 500 μm. (D) Distribution of tdTomato (left) and SypEGFP (right) fluorescence throughout the spinal cord of AAV-injected mouse. The area where tdTomato-positive cells were distributed was about 1.5 mm along the rostrocaudal axis. Fluorescence image of the representative transverse section in this area is shown in “injection site”. Fluorescence images rostrally (+2.0 cm, +1.5 cm, +1.0 cm, and +0.5 cm) and caudally (-0.5 cm and -1.0 cm) away from the injection site are also shown. The axonal tract labeled with tdTomato is distributed in the ipsilateral dorsal funiculus (DF), ipsilateral dorsolateral funiculus (DLF), ipsilateral ventrolateral funiculus (VLF), contralateral ventral funiculus (VF), contralateral VLF, or ipsilateral deep dorsal horn (circled by dotted magenta line). The fluorescence of tdTomato and SypEGFP is pseudocolored in black. The axonal tract on the contralateral side was mainly distributed in the VF near the injection site (+0.5 cm), but was found in the VLF on the section distant from the injection site (+2.0 cm, +1.5 cm, and +1.0 cm). Throughout the spinal cord, accumulation of presynaptic terminal of Brn3a-persistent neurons labeled with SypEGFP was found in the ipsilateral deep dorsal horn 0.5 cm and 1.0 cm rostral to the injection site (dotted green line). This region roughly corresponds to the Clarke nucleus in the thoracic spinal cord. Solid red lines indicate the outline of the spinal cord, whereas dotted red lines indicate the boundary between gray and white matters. Data are representative of images from 2 mice. (E-M) The distribution of presynaptic terminals of Brn3a-persistent spinal dorsal horn neurons in the medulla (E-I) and pons (J-M). (E) Schematic of the medulla with the location of nuclei (dotted red line) are shown. GN, gracile nucleus; CuN, cuneate nucleus; NTS, nucleus of the solitary tract; LRN, lateral reticular nucleus; IO, inferior olivary nucleus. Fluorescence images of SypEGFP (marked in E) around the dorsal (F, G) and ventral (H, I) medulla are shown. Strong EGFP signal was found in the ipsilateral GN, whereas weaker signal was also seen in the contralateral GN, and NTS, LRN, and IO on both sides. Solid and dotted red lines indicate the outline of the brain and the boundary of nuclei, respectively. (J) Schematic of the pons with the location of nuclei (dotted red line) is shown. Fluorescence images of SypEGFP (marked in J) around the dorsal (K, L) and ventral (M) pons are shown. EGFP signal was seen in the lateral parabrachial nucleus on both sides (arrows) and ventral area of the pons on the ipsilateral side. Red dotted lines indicate the outline of neuronal tracts and nuclei. scp, superior cerebellar peduncle; vsc, ventral spinocerebellar tract; un, uncinate fasciculus of the cerebellum; 7n, facial nerve; rs, rubrospinal tract; DPO, dorsal periolivary nucleus; LSO, lateral superior olive. Scale in F-I and K-M, 200 μm. Data are representative of images from 2 mice. (PDF)</p

Axonal extension index of control and Brn3a-overexpressed neurons.

Axonal extension index of control and Brn3a-overexpressed neurons.</p