Raghavan R , Coppola U , Wu Y , Ihewulezi C , Negrón-Piñeiro LJ , Maguire JE , Hong J , Cunningham M , Kim HJ , Albert TJ , Ali AM , Saint-Jeannet JP , Ristoratore F , Dahia CL , Di Gregorio A .

R03 HD098395 NICHD NIH HHS , P30 DE020754 NIDCR NIH HHS, T32 HD007520 NICHD NIH HHS , F32 GM123700 NIGMS NIH HHS , R01 AR077145 NIAMS NIH HHS , R01 AG070079 NIA NIH HHS , S10 OD026763 NIH HHS

	Fig. 1 Identification of genes expressed during notochord development in Ciona robusta. Ciona robusta embryos ranging from early gastrula to late tailbud, hybridized in situ with antisense RNA probes specific for the genes reported on top of panels A, F, K, P, R, W (Table S1). Gene models are indicated in the bottom left corners. Insets in (D, H, M, T) show embryos at different developmental stages. Insets in (I, J) show a higher magnification view of the regions boxed in red in the main panels, to display staining in notochord cells, which is clearer in the trunk because it is not obscured by the staining in muscle cells. Stained territories are denoted by arrowheads, color-coded as follows: red, notochord; light pink, fading notochord staining; white, no detectable notochord staining; blue, CNS; purple, mesenchyme; orange, muscle. Red lines underneath the panels indicate the approximate time span of notochord expression throughout development. In (A, F, K, R, W) dashed curved lines delineate the location of notochord precursors. Scale bar: 50 µm. (AB) Dot plot summary of the published scRNA-Seq data [50] available for the genes in (A-AA), compared to the scRNA-Seq data available for Ciona Brachyury, which was used as a reference for notochord expression [51]. In the dot plot, each dot represents two values: the mean expression of each gene (visualized by color) and the fraction of cells expressing each gene (visualized by the size of the dot). The embryonic stages used by Cao et al. [50] to generate the scRNA-Seq dataset reflect only approximately the stages that we used for WMISH. Abbreviations: init., initial; md., middle; ea., early; G, gastrula; N, neurula; Tb, tailbud
	Fig. 2 Phylogenetic reconstruction of the evolutionary relationships within the Rgm gene family. ML phylogenetic tree showing the relationships among members of different Rgm classes. Proteins encoded by genes present in invertebrates in single copy and equally related to the RgmA-D classes have been indicated as RgmA/B/C/D. RgmA, RgmB, and RgmC were found in all the vertebrates analyzed in this study, while RgmD genes have only been reported, thus far, in teleosts and cartilaginous fish. Distinct colors highlight the family members analyzed in this study. Values reported at the branching points indicate replicates obtained using the aLRT method
	Fig. 3 Phylogenetic reconstruction of the evolutionary relationships within the Tmod gene family. ML phylogenetic tree displaying the relationships among members of different Tmod classes. Proteins encoded by genes present in invertebrates in single copy and equally related to the 1–4 classes have been tentatively indicated as Tmod1/2/3/4. Tmod1, Tmod2, Tmod3 and Tmod4 have been found in all the vertebrates analyzed here. Distinct colors highlight the family members analyzed in this study. Values reported at the branching points indicate replicates obtained using the aLRT method
	Fig. 4 Phylogenetic reconstruction of the evolutionary relationships between proteins of the Rgs6/7 and Rgs9/11 subfamilies. A global phylogenetic study of the Rgs proteins found in Ciona robusta is available [38]. This ML phylogenetic tree is centered on the Rgs6/7 subfamily, since the expression of some of its members has been studied here, and on the Rgs9/11 subfamily, because of its close relationship to the Rgs6/7 subfamily. Distinct colors highlight the family members whose expression was analyzed in this study. Values at the branches indicate replicates obtained using the aLRT method
	Fig. 5 Phylogenetic reconstruction of the evolutionary relationships within the Coronin gene family. ML phylogenetic tree of the Coronin (Coro) family. The Coro7 subfamily is highlighted by a light blue box. Distinct colors indicate the family members whose expression was analyzed in this study. Values reported at the branching points indicate replicates obtained using the aLRT method
	Fig. 6 Expression of phip, rgmD, tmod2, rgs7a and coro7a in Danio rerio. A-E Whole-mount zebrafish embryos at the stages indicated at the bottom of each panel, hybridized in situ with probes specific for the genes indicated on the bottom right of panels A-E. (A’-E”) Close-ups of the tails of stained embryos, either magnified from (A-E) or acquired from representative embryos from the same batch as those in (A-E). Arrowheads are color-coded as follows: blue, nervous system; red, notochord. All panels show lateral views, anterior to the left. Scale bar: 150 µm
	Fig. 7 Expression of phip, rgmb, tmod2, rgs6 and coro7 in Xenopus laevis. A-E Whole-mount in situ hybridization of NF stage 28 embryos. Lateral views, anterior to the right, dorsal on top. (A’-E’) Transverse sections of NF stage 32 embryos. Dorsal is on top. Arrowheads color code: red, notochord; blue, nervous system; orange, somites; green, branchial arches; violet, pronephros. Scale bars: 500 µm (A); 200 µm (A’)
	Fig. 8 Gene expression analysis in mouse nuclei pulposi. A Normalized read counts in E12.5 notochord and P0 NP cells from the GSE100934 dataset [82]. B-E Multiplex qPCR analysis showing the expression of Phip (B), Rgmb (C), Tmod1 (D), and Coro7 (E) relative to Gapdh in one-week (n = 3), one-year (n = 6) and two-year (n = 6) old mouse NP cells from lumbar IVDs of wild-type mice. Results are presented as scatter dot plots with mean and SD for each cohort. The qPCR results plotted in (B-E) were analyzed by ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. * = p < 0.05
	Fig.9 Expression of the genes of interest in human nuclei pulposi. A Normalized chip signals plotted as gene expression measures from microarray data (E-MTAB-6868; [88]) obtained from notochordal cells from human embryonic (7.5–8.5 weeks post-conception, n = 3) and fetal (12–14 weeks post-conception, n = 2) stages. B Log2 normalized read counts plotted as gene expression measures from the RNA-seq data of NP samples from lumbar disc herniation (DH, n = 5), and lumbar disc spondylolisthesis (DS, n = 5) obtained from the GEO database (GSE146904; [89]). Expression of PHIP (C), RGMB (D), TMOD1 (E), RGS6 (F), and CORO7 (G) relative to GAPDH, measured by qPCR analysis in the NP tissue collected from less degenerated (Grade 1–3) or moderately to severely degenerated (Grade 4–5) lumbar disc from human male and female of various age groups (see Methods). Results are presented as scatter dot plots with mean and standard deviation for each cohort. The qPCR results plotted in (C-G) were analyzed by unpaired t -test. * = p < 0.05. p, post-conception
	tmod2 (tropomodulin 2) expression in Xenopus laevis embryo, NF stage 28 (top) assayed via in situ hybridization, lateral view, anterior right, dorsal up, and transverse sections of mid trunk region of NF stage 32
	phip (pleckstrin homology domain interacting protein) expression in Xenopus laevis embryo, NF stage 28 (top) assayed via in situ hybridization, lateral view, anterior right, dorsal up, and transverse sections of mid trunk region of NF stage 32 (below), dorsal up.
	rgmb (repulsive guidance molecule BMP co-receptor b) expression in Xenopus laevis embryo, NF stage 28 (top) assayed via in situ hybridization, lateral view, anterior right, dorsal up, and transverse sections of mid trunk region of NF stage 32
	rgs6 (regulator of G-protein signaling 6) expression in Xenopus laevis embryo, NF stage 28 (top) assayed via in situ hybridization, lateral view, anterior right, dorsal up, and transverse sections of mid trunk region of NF stage 32
	coro7 (coronin 7) expression in Xenopus laevis embryo, NF stage 28 (top) assayed via in situ hybridization, lateral view, anterior right, dorsal up, and transverse sections of mid trunk region of NF stage 32

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