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• W2788094033 abstract "•The notochord sheath segments into alternating cartilaginous and mineralizing areas•Segmented activation of Notch patterns the mineralized sheath domains•Osteoblasts are selectively recruited to mineralized sheath segments•Notochord sheath segmentation provides a template for spine patterning The spine is a segmented axial structure made of alternating vertebral bodies (centra) and intervertebral discs (IVDs) assembled around the notochord. Here, we show that, prior to centra formation, the outer epithelial cell layer of the zebrafish notochord, the sheath, segments into alternating domains corresponding to the prospective centra and IVD areas. This process occurs sequentially in an anteroposterior direction via the activation of Notch signaling in alternating segments of the sheath, which transition from cartilaginous to mineralizing domains. Subsequently, osteoblasts are recruited to the mineralized domains of the notochord sheath to form mature centra. Tissue-specific manipulation of Notch signaling in sheath cells produces notochord segmentation defects that are mirrored in the spine. Together, our findings demonstrate that notochord sheath segmentation provides a template for vertebral patterning in the zebrafish spine. The spine is a segmented axial structure made of alternating vertebral bodies (centra) and intervertebral discs (IVDs) assembled around the notochord. Here, we show that, prior to centra formation, the outer epithelial cell layer of the zebrafish notochord, the sheath, segments into alternating domains corresponding to the prospective centra and IVD areas. This process occurs sequentially in an anteroposterior direction via the activation of Notch signaling in alternating segments of the sheath, which transition from cartilaginous to mineralizing domains. Subsequently, osteoblasts are recruited to the mineralized domains of the notochord sheath to form mature centra. Tissue-specific manipulation of Notch signaling in sheath cells produces notochord segmentation defects that are mirrored in the spine. Together, our findings demonstrate that notochord sheath segmentation provides a template for vertebral patterning in the zebrafish spine. Segmentation of the vertebrate trunk starts during embryogenesis with the formation of somites within the presomitic mesoderm (PSM) (Hubaud and Pourquié, 2014Hubaud A. Pourquié O. Signalling dynamics in vertebrate segmentation.Nat. Rev. Mol. Cell Biol. 2014; 15: 709-721Crossref PubMed Scopus (220) Google Scholar, Pourquié, 2011Pourquié O. Vertebrate segmentation: from cyclic gene networks to scoliosis.Cell. 2011; 145: 650-663Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). This process is controlled in part by the segmentation clock, which acts on the PSM to produce somites in a sequential and highly coordinated fashion (Holley et al., 2002Holley S.A. Jülich D. Rauch G.J. Geisler R. Nüsslein-Volhard C. her1 and the notch pathway function within the oscillator mechanism that regulates zebrafish somitogenesis.Development. 2002; 129: 1175-1183Crossref PubMed Google Scholar, Oates et al., 2012Oates A.C. Morelli L.G. Ares S. Patterning embryos with oscillations: structure, function and dynamics of the vertebrate segmentation clock.Development. 2012; 139: 625-639Crossref PubMed Scopus (258) Google Scholar). Synchronization of the segmentation clock between cells that assemble into discrete somites depends on the Notch pathway, which coordinates gene expression oscillations across cell boundaries (Jiang et al., 2000Jiang Y.J. Aerne B.L. Smithers L. Haddon C. Ish-Horowicz D. Lewis J. Notch signalling and the synchronization of the somite segmentation clock.Nature. 2000; 408: 475-479Crossref PubMed Scopus (422) Google Scholar). The Notch pathway also regulates the period of the segmentation clock and thus also controls somite size and number (Liao et al., 2016Liao B.K. Jörg D.J. Oates A.C. Faster embryonic segmentation through elevated Delta-Notch signalling.Nat. Commun. 2016; 7: 11861Crossref PubMed Scopus (36) Google Scholar). Following somitogenesis, re-segmentation of the paraxial mesoderm (PM) and condensation of the ventral-medial portion, the sclerotome, around the notochord is thought to underlie the generation of discrete vertebral bodies (centra) (Fleming et al., 2015Fleming A. Kishida M.G. Kimmel C.B. Keynes R.J. Building the backbone: the development and evolution of vertebral patterning.Development. 2015; 142: 1733-1744Crossref PubMed Scopus (91) Google Scholar, Renn et al., 2013Renn J. Büttner A. To T.T. Chan S.J. Winkler C. A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization.Dev. Biol. 2013; 381: 134-143Crossref PubMed Scopus (47) Google Scholar). The concept of re-segmentation was introduced by Remak in 1855 (Remak, 1855Remak R. Untersuchungen uber die Entwickling der Wirbelthiere. Verlag von G. Reimer, 1855: 40-44Google Scholar) to explain the spatial mismatch between the segments of the PM and the vertebral bodies of the spine. Since then, this paradigm has been re-visited, validated, and modified in different vertebrate animal models (Bagnall et al., 1988Bagnall K.M. Higgins S.J. Sanders E.J. The contribution made by a single somite to the vertebral column: experimental evidence in support of resegmentation using the chick-quail chimaera model.Development. 1988; 103: 69-85PubMed Google Scholar, Ewan and Everett, 1992Ewan K.B. Everett A.W. Evidence for resegmentation in the formation of the vertebral column using the novel approach of retroviral-mediated gene transfer.Exp. Cell Res. 1992; 198: 315-320Crossref PubMed Scopus (37) Google Scholar, Morin-Kensicki et al., 2002Morin-Kensicki E.M. Melancon E. Eisen J.S. Segmental relationship between somites and vertebral column in zebrafish.Development. 2002; 129: 3851-3860Crossref PubMed Google Scholar, Ward et al., 2017Ward L. Evans S.E. Stern C.D. A resegmentation-shift model for vertebral patterning.J. Anat. 2017; 230: 290-296Crossref PubMed Scopus (17) Google Scholar). However, the cellular and molecular processes that underlie the transition from the metameric pattern of the PM to that of the spine remain unknown. Particularly, how sclerotome-derived cells condense around the notochord to produce precise vertebral segments with sharp boundaries separated by regular gaps remains unresolved. Moreover, although mutations in the Notch pathway lead to somite segmentation and to vertebral patterning defects in mice and humans (Sparrow et al., 2012Sparrow D.B. Chapman G. Smith A.J. Mattar M.Z. Major J.A. O’Reilly V.C. Saga Y. Zackai E.H. Dormans J.P. Alman B.A. et al.A mechanism for gene-environment interaction in the etiology of congenital scoliosis.Cell. 2012; 149: 295-306Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, Turnpenny et al., 2007Turnpenny P.D. Alman B. Cornier A.S. Giampietro P.F. Offiah A. Tassy O. Pourquié O. Kusumi K. Dunwoodie S. Abnormal vertebral segmentation and the notch signaling pathway in man.Dev. Dyn. 2007; 236: 1456-1474Crossref PubMed Scopus (112) Google Scholar), it is unclear whether the segmentation machinery acts primarily on the PM, the notochord, or both. Furthermore, it is also unclear if those Notch pathway mutations also affect other cues that may instruct vertebral patterning later in development. Embryological experiments in chick and zebrafish (Fleming et al., 2004Fleming A. Keynes R. Tannahill D. A central role for the notochord in vertebral patterning.Development. 2004; 131: 873-880Crossref PubMed Scopus (157) Google Scholar, Haga et al., 2009Haga Y. Dominique 3rd, V.J. Du S.J. Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model.Transgenic Res. 2009; 18: 669-683Crossref PubMed Scopus (44) Google Scholar, Ward et al., 2017Ward L. Evans S.E. Stern C.D. A resegmentation-shift model for vertebral patterning.J. Anat. 2017; 230: 290-296Crossref PubMed Scopus (17) Google Scholar, Watterson et al., 1954Watterson R.L. Fowler I. Fowler B.J. The role of the neural tube and notochord in development of the axial skeleton of the chick.Am. J. Anat. 1954; 95: 337-399Crossref PubMed Scopus (100) Google Scholar) and anatomical studies in salmon (Grotmol et al., 2003Grotmol S. Kryvi H. Nordvik K. Totland G.K. Notochord segmentation may lay down the pathway for the development of the vertebral bodies in the Atlantic salmon.Anat. Embryol. (Berl.). 2003; 207: 263-272Crossref PubMed Scopus (106) Google Scholar, Wang et al., 2014Wang S. Furmanek T. Kryvi H. Krossøy C. Totland G.K. Grotmol S. Wargelius A. Transcriptome sequencing of Atlantic salmon (Salmo salar L.) notochord prior to development of the vertebrae provides clues to regulation of positional fate, chordoblast lineage and mineralisation.BMC Genomics. 2014; 15: 141Crossref PubMed Scopus (36) Google Scholar) have suggested that the notochord is somehow linked to spine patterning. Furthermore, mouse genetic manipulations have implicated the notochord in spine development (Choi and Harfe, 2011Choi K.S. Harfe B.D. Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs.Proc. Natl. Acad. Sci. U S A. 2011; 108: 9484-9489Crossref PubMed Scopus (101) Google Scholar). However, it is not known whether or how the notochord influences the segmentation pattern of the spine. Determining whether the notochord instructs spine patterning will shed light on spine morphogenesis and the origin of vertebral defects. Here, we use live imaging of transgenic zebrafish, transcriptomic analyses, and tissue-specific genetic manipulations to investigate the role of the notochord in spine patterning. We show that the outer epithelial cell layer of the zebrafish notochord, the sheath, segments into alternating cartilage-like and mineralizing domains prior to the formation of centra. Similar to somitogenesis, notochord sheath segmentation occurs sequentially in an anteroposterior direction. Subsequently, osteoblasts are recruited to the surface of the mineralized sheath domains to form mature vertebral bodies. Gene expression analyses and pharmacological and notochord-specific genetic manipulations indicate that the Notch pathway controls the segmentation of the notochord sheath that guides the patterning of the spine. Our work reveals a central role for the notochord in morphogenesis of the segmented spine in zebrafish. The zebrafish notochord consists of a core of large vacuolated cells surrounded by an epithelial sheath that secretes a thick extracellular matrix (Ellis et al., 2013bEllis K. Hoffman B.D. Bagnat M. The vacuole within: how cellular organization dictates notochord function.Bioarchitecture. 2013; 3: 64-68Crossref PubMed Google Scholar, Yamamoto et al., 2010Yamamoto M. Morita R. Mizoguchi T. Matsuo H. Isoda M. Ishitani T. Chitnis A.B. Matsumoto K. Crump J.G. Hozumi K. et al.Mib-Jag1-Notch signalling regulates patterning and structural roles of the notochord by controlling cell-fate decisions.Development. 2010; 137: 2527-2537Crossref PubMed Scopus (67) Google Scholar). These notochord sheath cells have also been referred to as notochord epithelial cells or chordoblasts in the past. Vacuole inflation and integrity contribute to embryonic axis elongation and straight spine axis formation by providing a hydrostatic scaffold (Ellis et al., 2013aEllis K. Bagwell J. Bagnat M. Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis.J. Cell Biol. 2013; 200: 667-679Crossref PubMed Scopus (113) Google Scholar, Navis and Bagnat, 2015Navis A. Bagnat M. Developing pressures: fluid forces driving morphogenesis.Curr. Opin. Genet. Dev. 2015; 32: 24-30Crossref PubMed Scopus (45) Google Scholar). The notochord sheath is key for scaffold formation and is essential for repairing the notochord after mechanical damage (Garcia et al., 2017Garcia J. Bagwell J. Njaine B. Norman J. Levic D.S. Wopat S. Miller S.E. Liu X. Locasale J.W. Stainier D.Y.R. et al.Sheath cell invasion and trans-differentiation repair mechanical damage caused by loss of caveolae in the zebrafish notochord.Curr. Biol. 2017; 27: 1982-1989.e3Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, Gray et al., 2014Gray R.S. Wilm T.P. Smith J. Bagnat M. Dale R.M. Topczewski J. Johnson S.L. Solnica-Krezel L. Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations.Dev. Biol. 2014; 386: 72-85Crossref PubMed Scopus (70) Google Scholar). Previously, we generated col9a2 zebrafish transgenic reporters that specifically label notochord sheath cells (Garcia et al., 2017Garcia J. Bagwell J. Njaine B. Norman J. Levic D.S. Wopat S. Miller S.E. Liu X. Locasale J.W. Stainier D.Y.R. et al.Sheath cell invasion and trans-differentiation repair mechanical damage caused by loss of caveolae in the zebrafish notochord.Curr. Biol. 2017; 27: 1982-1989.e3Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). At early stages in development, col9a2:GFPCaaX was uniformly expressed in the plasma membrane of all sheath cells (Figure 1A). However, in larvae as early as 4.0 mm standard length (SL) (Parichy et al., 2009Parichy D.M. Elizondo M.R. Mills M.G. Gordon T.N. Engeszer R.E. Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.Dev. Dyn. 2009; 238: 2975-3015Crossref PubMed Scopus (508) Google Scholar), we observed a segmented pattern of col9a2-driven GFPCaaX expression that generated an alternating pattern of col9a2+ and col9a2-negative domains. Temporally, this pattern emerged first in anterior regions and moved in a wave toward the posterior (Figure 1A). At late stages (6.5 mm SL), col9a2:GFPCaax+ domains exclusively labeled the fully developed intervertebral discs (IVDs) (Figure 1A). To understand the role of col9a2 segmentation in spine patterning, we crossed the col9a2 reporter to a transgenic line that labels osteoblasts, Tg(osx:mcherry-NTR) (Singh et al., 2012Singh S.P. Holdway J.E. Poss K.D. Regeneration of amputated zebrafish fin rays from de novo osteoblasts.Dev. Cell. 2012; 22: 879-886Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Interestingly, we observed that osteoblasts were specifically recruited from the PM to patches of col9a2-negative regions in an anteroposterior fashion, prior to the formation of mature centra (Figures 1A and 1B). Previous work has shown that before the appearance of ossified centra, mineralized rings, called chordacentra, are formed around the zebrafish notochord sheath (Fleming et al., 2015Fleming A. Kishida M.G. Kimmel C.B. Keynes R.J. Building the backbone: the development and evolution of vertebral patterning.Development. 2015; 142: 1733-1744Crossref PubMed Scopus (91) Google Scholar). To visualize col9a2 segmentation in relation to the chordacentra, we stained 5.5 mm and 6.0 mm SL col9a2:mcherry fish with calcein, which labels calcified structures. This clearly showed that col9a2 expression was specific for non-mineralized segments of the notochord (Figure 1C). Because notochord sheath mineralization has been shown to require ectonucleoside triphosphate/diphosphohydrolase, Entpd5a (Huitema et al., 2012Huitema L.F. Apschner A. Logister I. Spoorendonk K.M. Bussmann J. Hammond C.L. Schulte-Merker S. Entpd5 is essential for skeletal mineralization and regulates phosphate homeostasis in zebrafish.Proc. Natl. Acad. Sci. U S A. 2012; 109: 21372-21377Crossref PubMed Scopus (76) Google Scholar), and chordacentra are likely made by the notochord sheath (Fleming et al., 2004Fleming A. Keynes R. Tannahill D. A central role for the notochord in vertebral patterning.Development. 2004; 131: 873-880Crossref PubMed Scopus (157) Google Scholar, Grotmol et al., 2006Grotmol S. Kryvi H. Keynes R. Krossøy C. Nordvik K. Totland G.K. Stepwise enforcement of the notochord and its intersection with the myoseptum: an evolutionary path leading to development of the vertebra?.J. Anat. 2006; 209: 339-357Crossref PubMed Scopus (50) Google Scholar), we used TgBAC(entpd5a:pkRED) animals to visualize the col9a2-negative notochord sheath segments. Similar to the downregulation of the col9a2 reporter, segmented expression of entpd5a proceeded in an anteroposterior direction starting at about 4 days post-fertilization (dpf), or approximately 3.5 mm SL (Figures 2A and S1). Newly formed entpd5a+ segments typically manifested as one-cell-wide vertical stripes of cells overlapping the col9a2+ domain. Over the course of domain segregation, entpd5a+ segments expanded at the expense of the col9a2 domain, while cells at the border of the two domains retained expression of both transgenes (Figures 2B–2D). Because of perdurance of GFPCaaX, segmented expression of entpd5a:pkRED appeared to precede col9a2 downregulation. These observations indicate that alternating notochord segments arise from a seemingly homogeneous cell population, initially labeled by uniform expression of col9a2:GFPCaaX. Then, cells fated to form chordacentra transition to expressing both transgenes before exclusively expressing entpd5a:pkRED. Tracking the appearance of new entpd5a+ segments over time revealed that these segments were formed sequentially, approximately every 8 hr during the initial linear phase (r2 = 0.95), in a manner reminiscent of somite segmentation (Figure 2E). We then imaged notochord segmentation together with the osteoblast reporter Tg(osx:mTagBFP-2A-CreER) (Singh et al., 2012Singh S.P. Holdway J.E. Poss K.D. Regeneration of amputated zebrafish fin rays from de novo osteoblasts.Dev. Cell. 2012; 22: 879-886Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). We observed that the alternating domains of the notochord sheath were established prior to centra formation and that osteoblasts were specifically recruited to the entpd5a:pkRED+ domains (Figure 2F). At later stages (7.25 mm SL), we observed that osteoblasts associated with the centra completely overlaid the entpd5a+ domains, while col9a2:GFPCaaX labeled the mature IVDs that were devoid of osteoblasts (Figure 2G). Taken together, these data indicate that notochord sheath cells undergo dynamic changes to generate alternating segments, one of which possesses the capacity to mineralize and recruit osteoblasts to form centra. To identify transcriptional programs underlying notochord segmentation and spine patterning, we used fluorescence-activated cell sorting (FACS) to isolate from 13 dpf larvae sheath cell populations unique to the col9a2+ domain, the entpd5a+ domain, or the transitional (col9a2+/entpd5a+ double-positive) cells. These double-positive cells include cells from both new segments and overlapping domain boundaries from older segments (Figure 3A). Isolated cell populations were then used to generate transcriptomes for each domain (Figure 3A). Principal-component and differential expression analyses confirmed that each population had unique enrichment signatures (Figures 3B and S2A). Profiling using Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that genes in the col9a2+ domain were associated with cartilage development (e.g., sox9b, col2a1, and matn4) (Figures 3C and 3D; Tables S1 and S2). The transitional (col9a2+/entpd5a+ double-positive) domain was enriched for Notch target genes known to function in somite segmentation, such as mespbb, notch1a, and ripply1 (Figures 3C, 3D, and S2B), and also expressed low levels of her1. By contrast, the entpd5a+ domain was enriched in genes implicated in tissue mineralization and osteoblast differentiation, including scpp1, sp7, and runx2b (Figures 3C and 3D; Tables S1 and S2). To validate our findings and interrogate the dynamics of sheath segmentation, we generated a transgenic line to label the transitional population by isolating a promoter sequence from id2a, a known regulator of proliferation and differentiation (Uribe et al., 2012Uribe R.A. Kwon T. Marcotte E.M. Gross J.M. Id2a functions to limit Notch pathway activity and thereby influence the transition from proliferation to differentiation of retinoblasts during zebrafish retinogenesis.Dev. Biol. 2012; 371: 280-292Crossref PubMed Scopus (13) Google Scholar). Early in development (4.0 mm SL), id2a:GFPCaax exhibited a segmented pattern of expression labeling cells that later gave rise to new entpd5a+ segments (Figure 3E). Then, as the new entpd5a+ segments expanded (4.25 mm SL), id2a:GFPCaaX expression was restricted to the cells at the boundary between col9a2+ and entpd5a+ sheath domains. These observations are consistent with the dynamics of entpd5a+ and col9a2+ domains and the distinct transcriptional profile we found for the double-positive cells. Additionally, we found that sox9b:eGFP (Plavicki et al., 2014Plavicki J.S. Baker T.R. Burns F.R. Xiong K.M. Gooding A.J. Hofsteen P. Peterson R.E. Heideman W. Construction and characterization of a sox9b transgenic reporter line.Int. J. Dev. Biol. 2014; 58: 693-699Crossref PubMed Scopus (16) Google Scholar), a known transcriptional regulator of cartilage development (Yan et al., 2005Yan Y.L. Willoughby J. Liu D. Crump J.G. Wilson C. Miller C.T. Singer A. Kimmel C. Westerfield M. Postlethwait J.H. A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in craniofacial and pectoral fin development.Development. 2005; 132: 1069-1083Crossref PubMed Scopus (246) Google Scholar), was enriched in the col9a2+ domain, further supporting our transcriptomic analyses (Figure 3E). These data reveal the existence of well-defined alternating domains of gene expression in the notochord sheath corresponding to cartilage-like and mineralizing domains. One of the most highly upregulated genes in the transitional population with respect to the entpd5a+ and col9a2+ populations was mespbb, a known target of Notch signaling (Cutty et al., 2012Cutty S.J. Fior R. Henriques P.M. Saúde L. Wardle F.C. Identification and expression analysis of two novel members of the Mesp family in zebrafish.Int. J. Dev. Biol. 2012; 56: 285-294Crossref PubMed Scopus (12) Google Scholar, Sawada et al., 2000Sawada A. Fritz A. Jiang Y.J. Yamamoto A. Yamasu K. Kuroiwa A. Saga Y. Takeda H. Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites.Development. 2000; 127: 1691-1702Crossref PubMed Google Scholar). During zebrafish embryogenesis, mespbb is expressed at the determination front (somite S-I), and as segmentation proceeds its expression keeps shifting posteriorly to remain always at the anterior end of the PSM (Cutty et al., 2012Cutty S.J. Fior R. Henriques P.M. Saúde L. Wardle F.C. Identification and expression analysis of two novel members of the Mesp family in zebrafish.Int. J. Dev. Biol. 2012; 56: 285-294Crossref PubMed Scopus (12) Google Scholar, Sawada et al., 2000Sawada A. Fritz A. Jiang Y.J. Yamamoto A. Yamasu K. Kuroiwa A. Saga Y. Takeda H. Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites.Development. 2000; 127: 1691-1702Crossref PubMed Google Scholar). In contrast, during notochord segmentation, mespbb expression was clearly detected in newly formed and mature segments at similar levels (Figure S3), suggesting that its expression does not oscillate in the notochord, as it does during PSM segmentation. We also attempted to image the expression of her1:her1-venus during notochord segmentation. Expression of this reporter has been shown to oscillate during somitogenesis (Delaune et al., 2012Delaune E.A. François P. Shih N.P. Amacher S.L. Single-cell-resolution imaging of the impact of Notch signaling and mitosis on segmentation clock dynamics.Dev. Cell. 2012; 23: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, expression levels were too low for live imaging, and we could not determine whether this gene oscillates in the notochord or not. The transition from cartilage-like to mineralizing domains is associated with the expression of Notch target genes and mutations in Notch pathway genes have been linked to vertebral patterning defects in mouse and humans (Sparrow et al., 2012Sparrow D.B. Chapman G. Smith A.J. Mattar M.Z. Major J.A. O’Reilly V.C. Saga Y. Zackai E.H. Dormans J.P. Alman B.A. et al.A mechanism for gene-environment interaction in the etiology of congenital scoliosis.Cell. 2012; 149: 295-306Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, Turnpenny et al., 2007Turnpenny P.D. Alman B. Cornier A.S. Giampietro P.F. Offiah A. Tassy O. Pourquié O. Kusumi K. Dunwoodie S. Abnormal vertebral segmentation and the notch signaling pathway in man.Dev. Dyn. 2007; 236: 1456-1474Crossref PubMed Scopus (112) Google Scholar). Therefore, we tested whether activation of Notch signaling occurs during notochord segmentation. To this end, we examined the expression pattern of a previously validated reporter of Notch activity that consists of a short-half-life form of the fluorescent protein Venus (VenusPEST) under the control of a Notch-responsive element (TP1) (Ninov et al., 2012Ninov N. Borius M. Stainier D.Y. Different levels of Notch signaling regulate quiescence, renewal and differentiation in pancreatic endocrine progenitors.Development. 2012; 139: 1557-1567Crossref PubMed Scopus (143) Google Scholar). Interestingly, robust Notch activation could be detected in the sheath, completely overlapping with entpd5a+ expression domains, including newly forming and mature segments (Figures 4A and 4B ). Next, we tested the effect of Notch inhibition on sheath segmentation. We found that incubation of 7 dpf larvae with the γ-secretase inhibitor DAPT for 24 hr blocked entpd5a induction in the sheath, whereas in larvae that were treated with DMSO, two or three new segments were formed during the same period of time (Figure 4C). Remarkably, once DAPT was washed out, entpd5a expression completely recovered in the segments that had been stalled (Figure 4C). To better understand the dynamics of Notch activation in the notochord, we photobleached single TP1:VenusPEST segments and assayed for recovery of reporter expression. We observed partial recovery of the Notch reporter expression after just 4 hr (Figure 4D) and full recovery by 24 hr in the presence of DMSO, but not when DAPT was added following photobleaching (Figure 4E). Thus, the strong signal of the Notch reporter within entpd5a+ notochord sheath segments was due to continuous activation of Notch and not to perdurance of Venus-PEST. Together, these data reveal that the transition from cartilage-like to mineralizing domains in the notochord sheath depends on the activation of Notch signaling at regular intervals. These data also indicate that Notch activation and sheath segmentation are not strictly linear. Our gene expression analyses, Notch signaling reporter, and DAPT inhibition data revealed that a Notch-dependent mechanism controls segmentation of the notochord sheath. However, DAPT inhibits Notch signaling globally and cannot be sustained for long enough to allow analysis of its effect on spine patterning. Therefore, to test whether notochord and spine segmentation could be altered by manipulating Notch signaling specifically in notochord sheath cells, we used the QF2/QUAS system (Subedi et al., 2014Subedi A. Macurak M. Gee S.T. Monge E. Goll M.G. Potter C.J. Parsons M.J. Halpern M.E. Adoption of the Q transcriptional regulatory system for zebrafish transgenesis.Methods. 2014; 66: 433-440Crossref PubMed Scopus (36) Google Scholar, Potter et al., 2010Potter C.J. Tasic B. Russler E.V. Liang L. Luo L. The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.Cell. 2010; 141: 536-548Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) to drive effector genes in this tissue. This experimental approach also allowed us to avoid somitogenesis defects (Figures S4A and S4B). Expression of GFP, or the system components QF2 or QUAS alone, did not lead to defects in either notochord or vertebral segmentation (Figures 5A and S4C). In contrast, constitutive activation of Notch signaling in the col9a2 domain by expression of notch1a intracellular domain (nlsVenus-V2a-notch1a) (Eom et al., 2015Eom D.S. Bain E.J. Patterson L.B. Grout M.E. Parichy D.M. Long-distance communication by specialized cellular projections during pigment pattern development and evolution.eLife. 2015; 4: 4Crossref Scopus (76) Google Scholar) produced ectopic and indistinct patches of entpd5a:pkRED at 14 dpf, as opposed to well-defined segmented domains (Figure 5B). At later stages, these early defects resolved into wedge, short, and pebble vertebrae, as visualized by staining with alizarin red (Figures 5B and S5A). These more severe phenotypes are strikingly similar to the pebble vertebrae observed in humans with mutations in Notch pathway genes (Turnpenny et al., 2007Turnpenny P.D. Alman B. Cornier A.S. Giampietro P.F. Offiah A. Tassy O. Pourquié O. Kusumi K. Dunwoodie S. Abnormal vertebral segmentation and the notch signaling pathway in man.Dev. Dyn. 2007; 236: 1456-1474Crossref PubMed Scopus (112) Google Scholar). When Notch signaling was inhibited in the col9a2 domain by targeted expression of a dominant-negative suppressor of hairless (nVenus-V2a-SuHDN) cassette (Eom et al., 2015Eom D.S. Bain E.J. Patterson L.B. Grout M.E. Parichy D.M. Long-distance communication by specialized cellular projections during pigment pattern development and evolution.eLife. 2015; 4: 4Crossref Scopus (76) Google Scholar), we observed that entpd5a+ sheath segments were missing or" @default.
• W2788094033 created "2018-03-06" @default.
• W2788094033 creator A5007067001 @default.
• W2788094033 creator A5008787691 @default.
• W2788094033 creator A5025373775 @default.
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• W2788094033 date "2018-02-01" @default.
• W2788094033 modified "2023-10-12" @default.
• W2788094033 title "Spine Patterning Is Guided by Segmentation of the Notochord Sheath" @default.
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