Phox2a Defines a Developmental Origin of the Anterolateral System in Mice and Humans

Abstract

Anterolateral system neurons relay pain, itch, and temperature information from the spinal cord to pain-related brain regions, but the differentiation of these neurons and their specific contribution to pain perception remain poorly defined. Here, we show that most mouse spinal neurons that embryonically express the autonomic-system-associated Paired-like homeobox 2A (Phox2a) transcription factor innervate nociceptive brain targets, including the parabrachial nucleus and the thalamus. We define the Phox2a anterolateral system neuron birth order, migration, and differentiation and uncover an essential role for Phox2a in the development of relay of nociceptive signals from the spinal cord to the brain. Finally, we also demonstrate that the molecular identity of Phox2a neurons is conserved in the human fetal spinal cord, arguing that the developmental expression of Phox2a is a prominent feature of anterolateral system neurons.

Keywords: LSN; Phox2a; anterolateral tract; autonomic; dI5; dorsal horn; lamina I; lamina V; pain; spinoparabrachial.

Figure 1.. Spinal Phox2a^Cre Neurons Reside in Lamina I, Lamina V, and LSN All images are of Phox2a^Cre; R26^LSL-tdT/+ mice.

(A) BAC recombination strategy: Cre-PolyA insertion 3′ to the Phox2a ATG codon in the BAC RP23–333J21. (B) tdTomato (tdT)+ neurons in lamina I, lamina V, and LSN of the cervical spinal cord of adult mice. (B’) Magnified box in (B) showing lamina I Neurotrace, tdT, and NeuN co-labeling. (C) Expression of tdT and Phox2a in E11.5, E16.5, and adult mouse spinal cord. (D) Percentage of tdT+ neurons that express Phox2a, as well as percentage of Phox2a+ neurons that express tdT at E11.5 and E16.5 and in adult mice. (E) Percentage of tdT+ neurons that express Phox2a, as well as percentage of Phox2a+ neurons that express tdT in the superficial and deep dorsal horn of E16.5 mouse spinal cords. n = 4 E11.5 mice, n = 4 E16.5 mice, and n = 3 adult Phox2a^Cre; R26^LSL-tdT/+ mice. Data are represented as mean ± SEM. Scale bars: 500 μm in (B), 100 μm in (B’), 100 μm in (C), and 25 μm in insets. SG, sympathetic ganglia.

Figure 2.. Spinal Phox2a^Cre Neurons Innervate AS Targets

(A) Intersectional genetic strategy to visualize spinofugal axons with tdT. Phox2a^Cre; R26^LSL-tdT/+ mice have tdT cellular expression in the brain and spinal cord, while Phox2a^Cre; Cdx2^FlpO; R26^{FSF-LSL-tdT/+} mice have cellular tdT expression only in spinal Phox2a neurons. (B–F’) In (B)–(F): expression of cellular tdT in the brain and spinal cord of Phox2a^Cre; R26^LSL-tdT/+ mice in comparison to (B’–F’) the lack of tdT expression in the brain and spinal cord of Phox2a^Cre; Cdx2^FlpO; R26^{FSF-LSL-tdT/+} mice, except caudal to the cervical level. In (E’), arrow indicates presumptive anterolateral tract (ALT) axons in white matter. Insets in (E), (F), (E’), and (F’) correspond to stippled boxes and show tdT+ cell bodies (white arrows) (G–P) Prominent targets of tdT+ spinofugal axons. Higher magnifications are shown in (M’) and (N’). (Q–Q”) vGluT2 and tdT immunohistochemistry in the thalamus demonstrates putative excitatory synaptic termini arising from spinofugal axons. The image in (Q) is duplicated from (H) and is used as a reference for (Q’) and (Q”). (R) Diagram summarizing the termination sites of tdT+ spinofugal axons. n = 3 Phox2a^Cre; R26^LSL-tdT/+ adult mice, and n = 3 Phox2a^Cre; Cdx2^FlpO; R26^{FSF-LSL-tdT/+} adult mice. Scale bars: 100 μm, except 25 μm for (Q’) and (Q”). AP, area postrema; CU, cuneate nucleus; DR, dorsal raphe; DRt, dorsal reticular nucleus; GRN, gigantocellular reticular nucleus; ic, internal capsule; IRN, intermediate reticular nucleus; LH, lateral habenula; LP, lateral posterior thalamus; LRN, lateral reticular nucleus; MEV, midbrain trigeminal nucleus; MH, medial habenula; mo, molecular layer of the cerebellum; MV, medial vestibular nucleus; mVII, facial motor nucleus; mX, vagal motor nucleus; mXII, hypoglossal motor nucleus; NLL, nucleus of the lateral lemniscus; nVII, facial motor nerve; PAS, parasolitary nucleus; pBdm, dorsal-medial parabrachial nucleus; PCG, pontine central gray; PRN, pontine reticular nucleus; PRP, nucleus prepositus; PVT, paraventricular thalamus; RT, reticular thalamic nucleus; SCi, superior colliculus, intermediate laminae; scp, superior cerebellar peduncle; SCs, superior colliculus, superficial laminae; SPV, spinal trigeminal nucleus; ZI, zona incerta.

Figure 3.. Spinal Phox2a^Cre Neurons Are Predominantly AS Neurons

(A and B) Adult Phox2a^Cre; R26^LSL-tdT/+ mice were injected with FG in the VPL thalamus (A) and CTb-488 in the parabrachial nucleus (B). (C and D) Percentage of cervical spinal cord dorsal horn projection neurons expressing tdT, classified as those labeled with either tracer (C; All PNs) or selectively with FG or CTb (D). (E) Diagram of the locations of tdT+ only, retrograde-labeled only (FG or CTb), or tdT+ and tracer-labeled (violet) neurons, in 5 non-sequential 25-μm sections of the cervical spinal cord of one representative animal. (F–G’) In (F) and (G): representative images of the cervical spinal cord demonstrating tdT+ neuron labeling by retrograde tracers. See Figures S3A and S3B for more examples. (F’ and G’) High magnification of boxed areas in (F) and (G), depicting retrograde-labeled lamina I (F’) or lamina V/LSN (G’) tdT+ neurons (indicated by yellow arrows). (H–N) Laminar analysis of neuron location in the cervical spinal cord ipsilateral or contralateral to tracer injection. (H–K) Percentage of tracer-labeled neurons in lamina I (H and I) or lamina V/LSN (J and K) also expressing tdT, separated by tracer type. (L–N) Percentage of tdT-labeled neurons labeled with one or both tracers in all laminae (L), in lamina V/LSN (M), or in lamina I (N). (O) Diagram depicting overlap between tdT and retrograde tracer in lamina I of a representative mouse. (P) Diagrams illustrating the estimated percentages of cervical and lumbar lamina I Phox2a^Cre neurons projecting to mouse pB, VPL, or both. Stippled line represents spinal midline. n = 7 Phox2a^Cre; R26^LSL-tdT/+ adult mice (4 male, 3 female). Mann-Whitney test in (I) and (K); **p < 0.01; ns, non-significant. Data are represented as mean ± SEM. Scale bars: 250 μm in (A) and (B), 100 μm in (F) and (G), and 50 μm in (F’) and (G’).

Figure 4.. Heterogeneity of Spinal Phox2a Neuron Migration, Sensory Afferent Interaction, and Birth Time

(A) Migration of Phox2a+ and tdT+ neurons in embryonic spinal cords of Phox2a^Cre; R26^LSL-tdT/+ mice between E10.5 and E15.5. Boxed regions in upper panels are magnified below. (B–B”) Location of tdT+ neurons in the dorsal horn of Phox2a^Cre; R26^LSL-tdT/+ spinal cords at E13.0, highlighting contacts (B’ and B”) between lamina I neurons and TrkA+ sensory afferents (white arrowheads). (C–E) Spinal Phox2a neuron (white arrows) location in E14.5 TrkA^+/+, TrkA^+/‒, and TrkA^−/− mouse embryos. Boxed regions in (C) are magnified in (C’). (D and E) Counts of Phox2a neurons in lamina I (D) and lamina V/LSN (E). (F–M) Birthdating of spinal Phox2a (F–I) or tdT (J–M) neurons in E16.5 Phox2a^Cre; R26^LSL-tdT/+ mouse embryos, exposed to BrdU at E9.5 (F and J), E10.5 (G and K) or E11.5 (H and L). (I and M) Phox2a+/BrdU+ (I) or tdT+/BrdU+ (M) neurons as a percentage of all Phox2a+ or tdT+ neurons in either lamina I, lamina V/LSN, or laminae II/III (“Antenna”-like neurons) and compared between groups. (N) Diagram of migration of spinal Phox2a neuron subpopulations. Phox2a^Cre; R26^LSL-tdT/+ embryos: (A) n = 3 E10.5, n = 3 E11.5, n = 3 E12.5, n = 3 E13.5, n = 3 E14.5, and n = 3 E15.5; (B–B”) n = 3 E13.0 embryos; (F–M) n = 4–5 E16.5 embryos per condition. (C–E) n = 3 TrkA^+/+, n = 5 TrkA^+/−, and n = 3 TrkA^−/− E14.5 embryos. In (D) and (E), one-way ANOVA, with Tukey’s multiple comparisons test; in (I) and (M), individual one-way ANOVAs for each neuron type with Tukey’s multiple comparisons test. **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM. Scale bars: 100 μm in (A) and (C); 50 μm in (B), (C’), (F)–(H), and (J)–(L); 10 μm in (B’) and insets in (F)–(H) and (J)–(L); and 1 μm in (B”). NF-L: Neurofilament-L.

Figure 5.. The Molecular Identity and Specification of Spinal Phox2a Neurons

(A) Co-expression of Phox2a with embryonic spinal neuron markers in the E10.5 Phox2a^Cre; R26^LSL-tdT/+ spinal cord using immunohistochemistry. (B) Quantification of each marker as a percentage of Phox2a+/Lmx1b+ neurons. (C–F) Phox2a expression in control (C), Ascl1 null (D), and Ptf1a null (E) E11.5 spinal cords, with the average number of Phox2a+ cells per section quantified in (F). (C’), (D’), and (E’) show high magnification of boxed regions in (C), (D), and (E), respectively. (G–J) Representative images from transverse sections of E4 chick neural tubes co-electroporated with the ePhox2a-GFP reporter and expression plasmids: control (Myc-tag only) (G), ^MycAscl1 (H), ^MycAscl1 and Prdm13 (I), or ^MycPtf1a (J). Insets show control Myc-tag, Prdm13, or Ptf1a expression. (K) Diagram of Phox2a expression regulation showing direct transcriptional upregulation by Ascl1, upregulation by Lmx1b (Figure S5), and repression by Ptf1a. (L–P) Single-cell RNA-seq data analysis of dI5 (Lmx1b+) neurons (Delile et al., 2019). (L and M) UMAP analysis (L) of Lmx1b+ neurons from E9.5–E13.5 compared between each other, with volcano plot of cluster-6-enriched mRNAs (M) compared to all other neurons. (N–P) UMAP analysis (N) of Lmx1b+ neurons from E9.5–E11.5 compared between each other, with volcano plots of cluster-2-enriched mRNAs (O) and cluster-3-enriched mRNAs (P) compared to all other neurons. (Q–V) In situ hybridization of select mRNAs enriched in dI5 (Q; E11.5) and lamina I neurons (T; E16.5) based on UMAP analyses. The percentage of Phox2a+ neurons co-expressing selected mRNAs are quantified at both embryonic time points in (R) and (U). Tac1 is present in dI5 Lmx1b+ neurons and lamina I neurons that do not express Phox2a, as depicted in diagrams in (S) and (V). Phox2a^Cre; R26^LSL-tdT/+ embryos: (A and B) n = 3 E10.5; (Q and R) n = 3 E11.5; (T and U) n = 3–4 E16.5 embryos. (C–F) n = 6 control, n = 3 Ascl1^GFP/GFP, and n = 4 Ptf1a^Cre/Cre embryos. (G–J) n = 6 E4 chicken embryos for each condition. Student’s t test in (F); **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM. Data in (L)–(P) are derived from Delile et al. (2019); data processing and statistics are described in STAR Methods. Scale bars: 50 μm for all, except 10 μm for insets in (A). DRG, dorsal root ganglion; FC, fold change; MN, motor neuron.

Figure 6.. Phox2a Is Required for the Normal Development and Function of a Subset of AS Neurons

(A) The parabrachial nucleus (pB) of control (Hoxb8^Cre; Phox2a^+/+; R26^LSL-tdT/+; Ctrl, top row) and Phox2a^cKO (Hoxb8^Cre; Phox2a^f/f; R26^LSL-tdT/+; cKO, bottom row) adult mice, depicting spinoparabrachial axons labeled via Hoxb8^Cre-driven axonal tdT expression. Right panels show magnified views of the left panels, with insets depicting tdT axons (red arrows). (B) Diagram depicting the loss of spinal afferents (magenta) to the pB of Phox2a^cKO mice. (C–E) Spinal neurons labeled by CTb injections in the pB, in lamina I (left column of C, quantified in D) and lamina V/LSN (right column of C, quantified in E), in control (top row) and Phox2a^cKO (bottom row) adult mice. (F and G) Co-expression of Phox2a and Lmx1b with dI5-enriched mRNAs in control (top row) and Phox2a^cKO (bottom row) E11.5 spinal cords (F), quantified as a percentage of Phox2a+ neurons (G). See Figure S6 for additional images. (H and I) Distribution of Phox2a+ neurons in E16.5 control and Phox2a^cKO mouse embryos. (H) Insets show individual Phox2a+ cells magnified. (I) Individual Phox2a neuron locations (left) and density plots of Phox2a+ neurons (right) derived from 3–5 sections (10 μm) of 4 control (gray) and 4 Phox2a^cKO (black) E16.5 spinal cords. Coordinates and mediolateral distribution are normalized to the width and height of an idealized spinal cord. Individual lines on density plots represent single animals, dotted lines represent mean distribution of 4 animals, gray lines represent control embryos, and black lines represent Phox2a^cKO Phox2a+ cells. Black arrows point to the bimodal distribution of Phox2a^cKO Phox2a+ cells in deep laminae. Average mediolateral positions of lamina I and lamina V/LSN neurons are statistically compared. (J and K) Co-expression of Phox2a with dI5-enriched mRNAs in lamina I of control (top row) and Phox2a^cKO (bottom row) E16.5 spinal cords (J), quantified as a percentage of Phox2a+ neurons (K). (L and M) Co-expression of Phox2a and candidate lamina V/LSN neuron marker mRNAs in lamina V/LSN neurons of control (top row) and Phox2a^cKO (bottom row) E16.5 spinal cords (L), quantified as a percentage of Phox2a+ neurons (M). See Figure S6 for additional images. (N–X) Behavioral tests in control and Phox2a^cKO mice. (N) Radiant heat paw-withdrawal assay; n = 11 control, and n = 12 Phox2a^cKO. (O) Hot-water tail flick assay; n = 11 control, and n = 12 Phox2a^cKO. (P) von Frey test; n = 10 control, and n = 10 Phox2a^cKO. (Q) Adhesive removal latency; n = 11 control, and n = 10 Phox2a^cKO. (R and S) Time spent licking (R) and latency to any response (S) during the hot-plate test; n = 13 control, and n = 14 Phox2a^cKO. (T) Acetone test; n = 11 control, and n = 11 Phox2a^cKO. (U) Foot clip test; n = 15 control, and n = 16 Phox2a^cKO. (V) Capsaicin test; n = 11 control, and n = 12 Phox2a^cKO. (W and X) Formalin test (acute phase in W; late phase in X); n = 11 control, and n = 12 Phox2a^cKO. In (A), n = 4 control, and n = 4 Phox2a^cKO adult mice. (C–E) n = 4 control, and n = 6 Phox2a^cKO adult mice. (F and G) n = 3 control, and n = 5 Phox2a^cKO E11.5 mice. (H and I) n = 4 control, and n = 4 Phox2a^cKO E16.5 mice. (J and K) n = 3–4 control, and n = 3 Phox2a^cKO E16.5 mice. (L and M) n = 3–8 control, and n = 3–8 Phox2a^cKO E16.5 mice. (N–X) The ns are given above. In (D) and (E), two-way ANOVA with Tukey’s multiple comparisons test; in (I), unpaired t test; in (G), (K), and (M), multiple t tests using the Holm-Sidak method; in (Q), mixed-effects analysis with Sidak’s multiple comparisons test; and Mann-Whitney test in (N)–(P) and (R)–(X). ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM. Scale bars: 250 μm in (A); 50 μm in (C); (H) 100 μm in (H); 20 μm in insets for (H); 50 μm in (F), (J), and (L); and 10 μm in insets for (F), (J), and (L). Images in Figure 5Q have been re-used in Figure 6F, and images in Figure 5T have been re-used in Figure 6J. Data from Figures 5R and 5U representing the above images are re-used in Figures 6G and 6K, respectively. These data were collected and analyzed as a single experiment.

Figure 7.. Phox2a Neuron Molecular Identity Is Conserved in the Developing Human Spinal Cord

(A) Sections of GW7.3–GW8.4 human spinal cords showing Phox2a, Lmx1b, TrkA, and Pax2 expression. Location of higher magnification panels is shown in boxed regions. (B–B”’) In (B): Phox2a, Lmx1b, Pax2, Pou4F2, Tlx3, Lbx1, and TrkA expression in the GW7.3 spinal cord, demonstrating co-labeling of Phox2a neurons with dorsal horn markers Lmx1b and Lbx1, but not with Pax2, Pou4F2, or Tlx3, in a Phox2a^LamI/Phox2a^DeepEarly-like cluster (B’), the deep dorsal horn (B”), and a Phox2a^DeepLate-like cluster near the roof plate (B”’). Top right panel shows apposition of Phox2a cells with TrkA+ sensory afferents, similar to that in mouse (Figure 4). Insets show higher magnification of boxed regions (TrkA and Phox2a channels split and inverted). In (A), three human embryonic spinal cords (GW7.3, GW7.4, and GW8.4) are represented. In (B), one GW7.3 human embryonic spinal cord from (A) is represented. Scale bars: 200 μm in (A), 100 μm in (B), and 10 μm in insets in (B’)–(B”’)