Guo T , Mandai K , Condie BG , Wickramasinghe SR , Capecchi MR , Ginty DD .

NS34814 NINDS NIH HHS , Howard Hughes Medical Institute , R01 GM021168-38 NIGMS NIH HHS , R01 GM021168 NIGMS NIH HHS , R01 NS034814 NINDS NIH HHS , R37 NS034814 NINDS NIH HHS

	Figure 2. Hoxd1 instructs development of mammal-specific features of nociceptor axonal connections in the skin(a) Diagram showing the distinct patterns of nociceptor endings in the skin of mammals and birds. (b) Anti-peripherin staining of mouse hairy skin from proximal limb. Upper panels: transverse sections; lower panels: cross sections. (Hair is autofluorescent.) Scale bar = 40 μm and 50 μm in upper and lower panels, respectively. (c) A dramatic reduction in the number of nociceptor free nerve endings that penetrate the epidermis of hindlimb footpad glabrous skin of Hoxd1−/− mice was detected by anti-CGRP staining, which labels peptidergic nociceptors. Arrows indicate epidermal nociceptor endings. Scale bar = 40 μm. (d) Double fluorescent labeling of Mrgprb4 mRNA and CGRP protein in L3 DRGs of WT and Hoxd1−/− mice. (e) Quantification of the number (±SEM) of transverse lanceolate endings per mm2 skin area in serial sections of hindlimb back thigh hairy skin. * p < 0.001 by Student’s t-test. (f) Percentage (±SEM) of Mrgprb4+ neurons in WT and Hoxd1−/− mice that co-express CGRP or TrkA. * p < 0.001 by Student’s t-test. (g) Quantification of the percentage (±SEM) of lumbar DRG neurons that express Mrgprb4. * p < 0.001 by Student’s t-test. (h) Quantification of the average (±SEM) number of CGRP+ free nerve endings crossing the dermal-epidermal boundary per unit length (300 μm) of hindlimb footpad glabrous skin. * p < 0.005 by Student’s t-test.
	Figure 3. Hoxd1 controls a species-specific pattern of nociceptor axonal connectivity within the spinal cord(a, b) Anti-CGRP staining of the dorsal spinal cord of P0 mice and stage 39 (e13) chick. (a) Deep nociceptor central projections are abnormally increased in the spinal cord of Hoxd1−/− compared to WT mice revealed by anti-CGRP, a peptidergic nociceptor marker. More numerous deep projections were found at all axial levels, including cervical (Cer) and thoracic (Th) segments. V, lamina V; X, lamina X. Arrows point to aberrant deep projections extending into a region near the central canal (lamina X). Some CGRP+ axons normally reach lamina V similarly in WT and Hoxd1−/− mice mostly from ventral projections emanating from the dorsal funiculus. (b) Anti-CGRP staining of nociceptor central projections in stage 39 (e13) chick spinal cord at lower cervical and thoracic levels. Arrows indicate prominent horizontally-projecting deep nociceptor axon bundles in the spinal cord of the chick that resemble the abnormally increased fibers in Hoxd1−/− mice. Arrowhead indicates axon bundles that first project ventrally and then turn medially. Scale bar = 50 μm in (a) and (b). (c) Quantification of (a) comparing the fraction of 20μm sections of the spinal cord at different axial levels that contains bundles of deep nociceptor axons in WT (n = 5 animals) and Hoxd1−/− (n = 5 animals) mice. Numbers denote the numbers of sections examined. (d) Quantification of (a) comparing the average (±SEM) numbers of individual deep CGRP+ fibers detected per section at different axial levels * p < 0.05 by Student’s t-test. (n = 5 animals in each group.)
	Figure 4. NGF signaling controls a subset of central axonal projections of mammalian nociceptors in the spinal cord(a) Aberrant nociceptor axons, labeled with anti-Peripherin, project horizontally into deep regions of the spinal cord in Ngf−/−; Bax−/− mice. Lower panels represent high-magnification of the images shown in upper panels. Dashed boxes indicate excessive deep nociceptor axons. cc, the central canal. (Inset) Peripherin is expressed at high levels in nociceptors19,38. Note that in Ngf−/−; Bax−/−mice, in addition to excessive horizontal projections, there is also an abnormally large number of aberrant deep nociceptor axons that project ventrally from the medial edge of the dorsal horn (arrowhead), some of which extend beyond the central canal. Scale bar = 60 μm and 30 μm in low- and high-magnification panels, respectively. (b) Diagram representing nociceptor central projection defects of Ngf−/−; Bax−/− mice in comparison to Bax−/− controls. (c, d) Quantification of (a). (c) Fluorescent intensities are measured from areas represented by dashed rectangular boxes in (a). Average (±SEM) fluorescent intensities of Peripherin+ axons in deep ventral and horizontal projections are shown. * p ≤ 0.001 by Student’s t-test (n ≥ 5 animals in each group). (d) Fraction (%) of level-matched lumbar spinal cord sections that show anti-Peripherin fluorescent intensity in horizontal projections.
	Figure 5. Ectopic expression of Hoxd1 in chick nociceptors impairs their axonal ingrowth into the lateral spinal cord(a) DNA constructs used for in ovo electroporation. (b) Sox10 enhancer-driven in ovo electroporation directs ectopic gene expression in developing chicken DRGs but not in the spinal cord. Left panel: A whole-mount lateral view of GFP fluorescence in stage 26 chick embryos 3 days after electroporation. Right panel: Transverse sections of embryos processed as in the left panel, stained with anti-GFP. sp, spinal cord. drg, dorsal root ganglion. (c) The pattern of central projections of chick nociceptors is altered when Hoxd1 is expressed in the chick DRG. Upper panel: A spinal cord section labeled with anti-TrkA at stage 35 (e9); Lower panels: high-magnification views of the boxed areas of the same spinal cord section. The left side is control, and DRGs of the right side of the embryo are electroporated with Hoxd1. Scale bar = 50 μm and 25 μm in low- and high-magnification panels, respectively. (d) Quantification of (c) shows the ratio between spinal cord areas occupied by TrkA+ fibers in the electroporated side versus the control side. * p = 0.0001 by Student’s t-test (n > 4 embryos for each group; sections are 25 μm thick and are sampled at least 200 μm apart). cHoxd1, chicken Hoxd1 gene. cΔ, a chicken Hoxd1 gene construct lacking the homeobox motif. mHoxd1, mouse Hoxd1 gene. mΔ, a mouse Hoxd1 construct lacking homeobox. (e) Diagram of the different patterns of spinal cord innervation by mammalian and avian nociceptors.
	Figure 6. Behavioral responses of Hoxd1−/− mice to somatosensory stimuli(a) Hoxd1−/− mice show a defect in their avoidance response to extreme cold. The paw licking or flinching response latency following exposure of mice to a 0°C cold plate is significantly increased in Hoxd1−/− mice compared to their WT littermate controls (n = 14 for WT, 13 for KO). *** p < 0.001 by Student’s t-test. (b–d) Hoxd1−/− mice respond normally to acute noxious thermal stimuli. Response latencies in (b) the Hargreaves (n = 12 for WT, 11 for KO), (c) hot-plate (50°C; n = 9 per genotype) and (d) tail-immersion (50°C; n = 9 per genotype) tests do not differ between WT and Hoxd1−/− mice. (e) The paw withdrawal threshold of Hoxd1−/− mice to punctate mechanical stimuli (von Frey test) is comparable to that of WT mice (n = 12 per genotype). (f) Hoxd1−/− mice show prolonged thermal hyperalgesia 24 hours after intraplantar injection of 1% carrageenan as compared with WT mice (10 μl; n = 9 per genotype). Pre, preinjection. Data are presented as Mean (±SEM). * p < 0.01 Two-way ANOVA.

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