The pelvic ganglion does not contain parasympathetic neurons and is made of sympathetic-like neurons.

(A) UMAP of cells isolated from 3 sympathetic ganglia (lumbar, stellate and celiac), a parasympathetic ganglion (sphenopalatine) and the pelvic ganglion dissected from postnatal day 5 mice. The pelvic ganglion is sharply divided into 4 clusters (P1-4), none of which co-segregates with sympathetic or parasympathetic neurons. (B) Heatmap of the highest scoring 100 genes in an all-versus-all comparison of their dichotomized expression pattern among the 4 ganglia and 4 pelvic clusters (see Material and Methods), excluding genes specific to the pelvic ganglion (shown in Fig. S2), and keeping only the top-scoring comparison for genes that appear twice. For overall legibility of the figure, the three largest cell groups (lumbar, stellate and sphenopalatine) are subsampled and genes are ordered by expression pattern (designated on the left), rather than score. “cholinergic” and “noradrenergic” genes are those that are coregulated with ChAT or Th, regardless of known function. “Other“ refers to various groupings that split sympathetic ganglia and are thus not informative as to a sympathetic or parasympathetic identity. Transcription factors are indicated in bold face. White arrowhead: pelvic P3 cluster; S, sympathetic; ParaS, parasympathetic. (C) Pie chart of the top 100 genes, counted by expression pattern in the all-versus-all comparison. Genes specific for the P4 cluster dominate (see heatmap in Fig. S1), followed by those which are “parasympathetic-not-pelvic” and “sympathetic-and-pelvic” (seen in 1B). The three genes marked in white are the only ones that are compatible with the current dogma of a mixed sympathetic/parasympathetic pelvic ganglion, by being expressed in the sphenopalatine ganglion and a subset of pelvic clusters (other than the full complement of cholinergic ones).

Pelvic ganglion cells express sympathetic but not parasympathetic markers.

Combined immunohistochemistry for Phox2b and in situ hybridization for 7 sympathetic markers including 6 transcription factors (left panels) or 7 parasympathetic markers including 5 transcription factors (right panels), in two parasympathetic ganglia (sphenopalatine and otic), the lumbar sympathetic chain, and the pelvic ganglion, at low and high magnifications (inset on the left) in E16.5 embryos. Ebf3 is expressed in both, the parasympathetic ganglia and the mesenchyme surrounding all ganglia. Sst is expressed in a salt and pepper fashion. Zbtb16, a zinc-finger transcriptional repressor, appeared after the 100 highest scorer gene of our screen, but was spotted as expressed in the sphenopalatine in Genepaint. Some transcription factors detected by the RNA-Seq screen at P5 (Satb2, Dlx6) were expressed below the detection limit by in situ hybridization at E16.5.

The lumbar outflow targets both cholinergic and noradrenergic pelvic ganglionic cells.

(A-E) Section (A, low magnification, B-D high magnifications of selected regions) through the pelvic ganglion of an adult male mouse stereotactically injected with Dextran at the L1 level of the lumbar spinal cord (inset) and showing dextran filled boutons decorating both ChAT+ (B-C) and Th+ cells (D). Whether they are filled by Dextran or not, cholinergic boutons (green), presumably from spinal preganglionics (lumbar or sacral), are present on most cells. In the inset, levels of the vertebral column are indicated on the right, levels of the spinal cord on the left. (E) Quantification of Th and ChAT cells among total or bouton-decorated ganglionic cells. ChAT+ cells represent 51% of total cells and 50% of decorated cells (for a total of 3186 counted cells, among which 529 decorated cells, on 48 sections in 4 mice).

Deployment of the divisions of the autonomic nervous system on the rostro-caudal axis.

Cg, celiac ganglion; mg, mesenteric ganglion; pelvic gg, pelvic ganglion. Only the target organs of the pelvo-sympathetic pathway are represented. The pelvic ganglion is shown with its lumbar input (through the hypogastric nerve), and sacral input (through the pelvic nerve).

Uniform Manifold Approximation and Projection (UMAP) of all ganglionic neurons.

(A) UMAP of neurons where the sample origin of cells is color-coded to show that both samples of the pelvic ganglion contribute to each of the P1-4 pelvic clusters. (B) UMAP of neurons where the clusters as defined by Seurat are color-coded. Clusters 1, 7, 15 and 19 correspond to ganglion clusters P1, P2, P3, P4 in the text.

Genes specific for pelvic ganglionic cells among the top 100 genes of an all-versus-all comparison.

These 42 genes correspond to pattern VII of main text. Transcription factors are indicated in bold face.

Expression of all Hox genes captured by the single-cell RNA-Seq data set.

Apart from Hoxb2, all Hox genes are excluded from the sphenopalatine and are expressed either in all sympathetic and pelvic cells (8 genes), caudal sympathetic and pelvic cells (3 genes), or only in pelvic cells (12 genes). Hoxb2 appears in 177th position as {P124/Sphenopalatine} versus {P3/Lumbar/Coeliac/Stellate}, thus as a cholinergic gene in the dichotomized comparison).

Five top genes for each of the 4 individual pelvic clusters in an all-versus-all comparison.

P1 appears as the cluster the least sharply defined by specific genes. The only two transcription factors (indicated in bold face) among these top genes are Gata5, expressed in P2 and MECOM expressed in P3. Noradrenergic and cholinergic cells are indicated by Th and ChAT expression in the lower panel.