Combinatorial expression of Brn3 transcription factors in somatosensory neurons: genetic and morphologic analysis

Abstract

The three members of the Brn3 family of POU-domain transcription factors (Brn3a/Pou4f1, Brn3b/Pou4f2, and Brn3c/Pou4f3) are expressed in overlapping subsets of visual, auditory/vestibular, and somatosensory neurons. Using unmarked Brn3-null alleles and Brn3 conditional alleles in which gene loss is coupled to expression of an alkaline phosphatase reporter, together with sparse Cre-mediated recombination, we describe the following: (1) the overlapping patterns of Brn3 gene expression in somatosensory neurons; (2) the manner in which these patterns correlate with molecular markers, peripheral afferent arbor morphologies, and dorsal horn projections; and (3) the consequences for these neurons of deleting individual Brn3 genes in the mouse. We observe broad expression of Brn3a among DRG neurons, but subtype-restricted expression of Brn3b and Brn3c. We also observe a nearly complete loss of hair follicle-associated sensory endings among Brn3a(-/-) neurons. Together with earlier analyses of Brn3 gene expression patterns in the retina and inner ear, these experiments suggest a deep functional similarity among primary somatosensory neurons, spiral and vestibular ganglion neurons, and retinal ganglion cells. This work also demonstrates the utility of sparse genetically directed labeling for visualizing individual somatosensory afferent arbors and for defining cell-autonomous mutant phenotypes.

Figure 1.

Conditional Brn3c allele with an AP reporter reveals central targets of Brn3c-expressing RGCs. A, Gene targeting strategy. I, WT Brn3c gene; II, targeted Brn3c gene with a PGK-Neo cassette flanked by frt sites; III, Brn3c^CKOAP allele with the Neo cassette excised by germline Flp recombination; and IV, Brn3c^AP allele following Cre-mediated deletion of the Brn3c coding region. Filled black rectangles, 5′ UTR and coding region; open rectangle, 3′ UTR; red arrow labeled “pA,” additional polyadenylation sites added to the 3′ UTR; ATG, initiator methionine codon; AP, AP coding region and 3′ UTR. The line above the gene indicates the structure of the spliced Brn3c transcript. B, BamHI. The bar labeled “3′ probe” indicates the location of the Southern blot hybridization probe. Arrows flanking the 5′ loxP site show the locations of PCR primers. Right, Genotyping by Southern blot (top) and PCR (bottom). B, Cre-mediated deletion of the Brn3c coding region in Brn3c^CKOAP/+ or Brn3c^CKOAP/− retinas by Pax6αCre activates AP expression in a subset of RGCs and simultaneously eliminates Brn3c protein from RGCs in Brn3c^CKOAP/− retinas as determined by immunostaining of adult retinas with anti-AP (green; plasma membrane) and anti-Brn3c (red; nuclei) antibodies. White arrowheads point to AP+ RGC somas; green fluorescent anti-mouse secondary antibodies also decorate intraretinal capillaries. INL, Inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. C, Central projections of Brn3a- versus Brn3c-expressing RGCs in coronal sections of adult brain histochemically stained for AP (purple). Retina-specific Cre-mediated recombination is conferred by Pax6αCre. Matched pairs of serial sections (e.g., a and f, b and g) proceed from anterior at the mid-LGN (a, f; approximately bregma −2.5) to posterior at the anterior colliculus (e, j; approximately bregma −3.5). Brn3a^AP/+ RGC axons (a–e). Red arrows, medial terminal tract and nucleus; green arrow, lateral aspect of the accessory optic tract. Brn3c^AP/+ RGC axons (f–l). k, The optic chiasm and optic tracts (bottom) are populated by Brn3c-expressing RGC axons, but the SCN (purple arrow) is not targeted by axons of Brn3c-expressing RGCs. l, The boxed LGN region in g is enlarged; the intergeniculate leaflet is seen as a central unstained region. Scale bars: B, 40 μm; Ca–j, 500 μm; Ck, 500 μm; Cl, 200 μm.

Figure 2.

Expression of Brn3a, Brn3b, and Brn3c in Brn3a^AP/+, Brn3a^AP/−, Brn3b^AP/+, Brn3b^AP/−, Brn3c^AP/+, and Brn3c^AP/− RGCs and DRG neurons determined by anti-AP and anti-Brn3 double immunostaining. A, Adult retina sections stained for cell surface AP (green) and nuclear Brn3a, Brn3b, or Brn3c (red). Note that green fluorescent anti-mouse secondary antibodies also decorate intraretinal capillaries. INL, Inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 40 μm. B, Quantification of the patterns of Brn3 immunostaining (labels at left) in adult WT and KO RGCs of the indicated genotypes (labels at top). Pie charts quantify the fraction of AP+ cells that were positive (blue) or negative (red) for the indicated transcription factor (TF). The number of RGCs that were counted in each category is indicated. Data for Brn3a and Brn3b expression in Brn3a^AP/+ and Brn3a^AP/− RGCs were described by Badea et al. (2009a) and are included here for completeness. Definitions of red and blue pie chart colors apply to B, D, and F. C, P5 DRGs following mosaic Cre-mediated recombination in Brn3a^CKOAP/+;R26CreER, Brn3b^CKOAP/+;R26CreER or Brn3c^CKOAP/+;R26CreER mice stained for cell surface AP (red) and nuclear Brn3a, Brn3b, or Brn3c (green). Scale bar, 40 μm. D, Quantification of data from C; color code as in B. E, P0–P1 DRGs following mosaic Cre-mediated recombination in Brn3a^CKOAP/+;R26rtTACreER, Brn3a^CKOAP/−;R26rtTACreER, Brn3b^CKOAP/+;R26rtTACreER, Brn3b^CKOAP/−;R26rtTACreER, Brn3c^CKOAP/+;R26rtTACreER, and Brn3c^CKOAP/−;R26rtTACreER mice stained for cell surface AP (green) and nuclear Brn3a, Brn3b, or Brn3c (red). Scale bar, 40 μm. F, Quantification of data from E; color code as in B.

Figure 3.

Correlation among Brn3a, Brn3b, and Brn3c expression and DRG neuron subtype as defined by molecular markers. A, B, Double immunostaining for AP (red) and the indicated molecular markers (green) in DRGs at P5 following mosaic Cre-mediated recombination in Brn3a^CKOAP/+;R26CreER, Brn3b^CKOAP/+;R26CreER, and Brn3c^CKOAP/+;R26CreER mice. Scale bar, 40 μm. C, Quantification of data from A and B. D, Quantification of adult WT DRGs using anti-Brn3a, anti-Brn3b, and anti-Brn3c immunostaining with the same series of markers as in A–C. Color code refers to C and D.

Figure 4.

Axons of Brn3a-, Brn3b-, and Brn3c-expressing DRG neurons target distinct territories in the dorsal horn of the spinal cord. A–H, Double immunostaining for AP (red) and the indicated molecular markers (green) in the adult dorsal horn following Cre-mediated recombination in Brn3a^CKOAP/+;Sox2Cre, Brn3b^CKOAP/+;Sox2Cre and Brn3c^CKOAP/+;Sox2Cre mice. AP staining at the dorsal edge of the spinal cord is derived from DRG fibers that have not yet entered the dorsal horn or are passing between spinal segments. A, B, Brn3a^AP fibers project broadly within the dorsal horn. C–E, Brn3b^AP fibers project to laminae ventral to the narrow zones of CGRP localization and IB4 staining, and overlapping the dorsal ∼50% of the broad zone of PKCγ localization. F–H, Brn3c^AP fibers are largely overlapping with the distribution of CGRP in the dorsal-most laminae. Scale bar, 100 μm.

Figure 5.

Individual Brn3a^AP/+, Brn3b^AP/+, and Brn3c^AP/+ somatosensory arbors visualized histochemically following sparse Cre-mediated recombination. A–N, Brn3a^CKOAP/+ afferents. A, 200 μm transverse section of P1 spinal cord and DRGs. In addition to DRG cell bodies and their processes, several large multipolar interneurons are labeled within the spinal cord. B–E, Innervation of muscle and tendons: hemisected P1 foot with the palmar surface at the bottom (B); adult diaphragm (C) and adult esophagus (D); and isolated muscle spindle in subdermal muscle at P1 (E). F–N, P1 abdominal skin flatmounts showing individual sensory arbors (F–K, M, N); tangential section of P1 skin showing a guard hair with a single sensory ending (green arrow) (L). F–N, Blue arrows indicate individual afferent fibers. F, A single elaborate follicle associated ending. M, A large arbor with a mixture of follicle-associated and nonassociated endings. Scale bars: A, C, 500 μm; B, D, E, G, N, 200 μm. F–L are at the same scale, and M and N are at the same scale. O–V, Brn3b^CKOAP/+ afferents. O–T, P1 abdominal skin flatmounts. Blue arrows indicate individual afferent fibers. U, Innervation of glabrous skin in the P1 foot in cross section. V, Brn3b-expressing fibers in a P1 Brn3b^AP/+ cochlea (following germline recombination) innervate sensory hair cells in the organ of Corti. Scale bars: O, 500 μm; P–U, 200 μm. W–Y, Brn3c^CKOAP/+ afferents. I–K, Brn3c^AP/+ afferents in P1–P3 abdominal skin flatmounts from Brn3c^CKOAP/+;NFL-CreER mice. Red arrows show low-level read-through AP expression from the un-recombined Brn3c^CKOAP allele in Merkel cells adjacent to each guard hair. Blue arrows indicate individual afferent fibers. Scale bar, 200 μm.

Figure 6.

Three-dimensional reconstructions of afferent arbors from Brn3a-, Brn3b-, and Brn3c- expressing DRG neurons in P1–P3 skin. A–L, Each dendritic arbor reconstruction is assigned a letter (A–L), and two projections—one in the plane of the skin (top) and one perpendicular to that plane (bottom)—are shown. For the territories encompassing arbors A and C and arbors E and I, the two neighboring arbors are shown in yellow and green. Outlines of guard hair follicles are shown in gray, and the point of contact between each follicle and the epidermis is marked by a pink horizontal line in the transverse projection. Stratification levels of individual subdermal muscle fibers are represented by dark red bars in the transverse projections. The most proximal point of each afferent fiber reconstruction is marked by a black arrow. Small C-shaped hair follicle-associated structures are present in arbors D, F, and K, and are highlighted in blue; more elaborate hair follicle associated structures are present in E, G, and H. Distinct morphological types representative of Brn3a^AP/+, Brn3b^AP/+, and Brn3c^AP/+ arbors are outlined in green, red, and blue, respectively. All morphologies were observed among Brn3a^AP/+ arbors. Types A and B were also observed among Brn3c^AP/+ arbors, types C–H were also observed among Brn3b^AP/+ arbors, and types I–L were unique to Brn3a^AP/+ arbors. The specific examples shown are derived from Brn3a^CKOAP/+;R26^rtTACreER/+ (A–F, H–L) and Brn3b^CKOAP/+;R26^rtTACreER/+ (G) P1 skins. Scale bar, 100 μm.

Figure 7.

Loss of Brn3a but not Brn3b or Brn3c causes a loss of dorsal horn projections and hair follicle-associated sensory endings in the skin. A, AP histochemistry of vibratome sectioned spinal cords from adult Brn3a^CKOAP/+;R26^rtTACreER, Brn3a^CKOAP/−;R26^rtTACreER, Brn3b^CKOAP/+;R26^rtTACreER, Brn3b^CKOAP/−;R26^rtTACreER, Brn3c^CKOAP/+;R26^rtTACreER, and Brn3c^CKOAP/−;R26^rtTACreER/+ mice following Cre-mediated recombination at E9.5. Red arrow points to the loss of Brn3a^CKOAP/− fibers in the dorsal horn. B, AP histochemistry of flatmounted abdominal skins from Brn3a^CKOAP/+;R26^rtTACreER and Brn3a^CKOAP/−;R26^rtTACreER P1 mice. Red arrowheads point to AP+ hair follicle-associated C-shaped sensory endings; these endings are common among Brn3a^AP/+ arbors (left) but are extremely rare among Brn3a^AP/− arbors (right). The right panel shows a region of Brn3a^CKOAP/−;R26^rtTACreER skin with a somewhat higher density of AP+ sensory arbors relative to the Brn3a^CKOAP/+;R26^rtTACreER skin in the left panel to emphasize the point that that the low density of C-shaped endings does not arise from a lower density of sensory arbors. C, Flatmount images of an isolated Brn3a^AP/− sensory arbor (two focal planes at left) and its reconstruction (Fig. 6, right; as), with relatively simple branching and small area, similar to the example shown in Figure 6C. D, Box plots quantifying the density of AP+ follicle-associated C-shaped sensory endings in the skin of Brn3a^CKOAP/+;R26^rtTACreER and Brn3a^CKOAP/−;R26^rtTACreER P1 mice. Regions with similar overall densities of labeled arbors were chosen for comparison. The central red mark is the median, the blue edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points. For each genotype, eight fields of 2.6 × 2.6 mm were scored. p = 1.56 × 10⁻⁴. Scale bars: A, 300 μm; B, C, 100 μm.

Figure 8.

Combinatorial expression of Brn3a, Brn3b, and Brn3c in projection neurons in the visual, auditory/vestibular, and somatosensory systems. A, Venn diagrams summarizing the relative numbers of expressing neurons and the patterns of overlapping expression among Brn3 family members in the retina, inner ear, and DRG. B, Central targets of RGCs that express different combinations of Brn3a, Brn3b, and Brn3c. For illustrative purposes only a subset of the many accessory optic areas is shown. For example, the central targets that control pupil constriction are not shown; these receive input from Brn3b-expressing RGCs and are functionally impaired in Brn3b^−/− mice (Badea et al., 2009a). C, In the auditory and vestibular systems, sensory hair cells express Brn3c (Xiang et al., 1997), and most or all spiral and vestibular ganglion cells express both Brn3a and Brn3b (Huang et al., 2001). D, In the somatosensory system, Brn3a is expressed by all or nearly all classes of sensory neurons, Brn3b is expressed by sensory neurons that have follicle-associated C-shaped endings, and Brn3c is expressed by sensory neurons that have endings not associated with hair follicles.