The backbone, or spine, is an integral part of the human body, providing support to our torsos so that we can sit, stand, bend and twist. If this structure does not form correctly, it can lead to pain, neurologic problems, and mobility issues. The spine normally has curves, but these can become deformed for many reasons, including genetic and muscular factors. There are also cases in which the cause of a spine distortion is unknown, such as in scoliosis (where the spine twists to the side), lordosis (where the lower part of the spine curves excessively), and kyphosis (where the upper part of the spine shows extreme curvature).

The structure of the spine is laid out during embryonic development and maintained throughout life. Experiments in zebrafish have shown that a crucial element in preserving the shape of the spine is the flow of cerebrospinal fluid or CSF. Propelled by the movement of little ‘hairs’ at the surface of specialized cells, this liquid runs through our central nervous system along a cavity lined with neurons. These nerve cells produce Urp1 and Urp2, two short molecules (or peptides) built from the same components as proteins. In zebrafish embryos, lowering the levels of these peptides had previously been shown to cause early body deformities. But what role, if any, do Urp1 and Urp2 play in maintaining the shape of the spine in adult zebrafish?

Bearce et al. set out to answer this question. First, they generated mutant zebrafish which did not carry either Urp1, Urp2 or both peptides. Contrary to previous findings, all three of these mutants developed normally as embryos. Once they were adults, zebrafish lacking Urp1 exhibited normal spines, while those lacking Urp2 had slightly deformed curves. However, zebrafish lacking both peptides had prominent curves in the tail-region of their spines, somewhat akin to lordosis in humans. This indicates that both peptides are necessary for adult spine structure, but work in a semi-redundant manner. Interestingly, the defects observed first appeared in adolescent fish and gradually worsened as they grew; many forms of human spinal abnormalities follow a similar trajectory.

Bearce et al. also tested the role of the protein Uts2r3, a receptor for peptides which belong to the urotensin family (such as Urp1 and Urp2). Fish lacking this protein showed normal spine structure as embryos, but distorted spinal curves as adults, suggesting that Urp1 and Urp2 might control spine morphology by signaling via the Uts2r3 receptor.

Together, Bearce et al.’s observations show that disturbing urotensin signaling leads to a lordosis-like condition in adult zebrafish, with evident deformities in the tail-region of the spine. Considering the broad similarities in structures between the zebrafish and the human spine, these results point to a possible involvement of urotensin signaling in spine distortion in humans. More studies using zebrafish will likely provide further insights into the principles that control the shape of the spine and what goes wrong when it breaks down.

Understanding how the shape of organisms is acquired is a central goal of developmental biology. The chordate body axis forms during embryonic development, when it is based around the rod-like notochord (Stemple, 2005). Later, the vertebrate axis comprises a column of repeating vertebrae which grows during juvenile and adolescent phases and is then maintained during adult life for up to several decades in some species (Bagnat and Gray, 2020). While a great deal has been learned about how the body axis emerges during embryogenesis, less is known about how spine morphology is maintained during growth and adulthood.

A breakdown of spine morphology occurs in scoliosis, lordosis, and kyphosis. Scoliosis is medically defined as lateral curvatures of the spine greater than 10° (Cheng et al., 2015; Mesiti, 2021; Wise et al., 2008) and can be caused by congenital defects of vertebral patterning or as a secondary consequence of neuromuscular disease (Pourquié, 2011; Wishart and Kivlehan, 2021). However, most cases of scoliosis are idiopathic in nature, with no known etiology: approximately 3% of children are afflicted by idiopathic scoliosis, which most often onsets during adolescence (Cheng et al., 2015; Labrom et al., 2021). By contrast, kyphosis and lordosis occur when there is excessive curvature of the thoracic and lumbar regions of the vertebral column, respectively, resulting in a hunched upper back (kyphosis) or a concave lower back (lordosis) (Ogura et al., 2021) without vertebral structural defects. Since these categories of curves can co-occur, there are likely to be overlapping as well as distinct causes.

A challenge to understanding the origin of spinal curvature has been the dearth of suitable animal models recapitulating disease states. Recently, teleost fishes, in particular zebrafish (Danio rerio), have emerged as prominent animal models of spinal deformity (Bagnat and Gray, 2020; Bearce and Grimes, 2021; Boswell and Ciruna, 2017; Gorman and Breden, 2009; Roy, 2021). Using zebrafish, it was found that motile cilia-generated cerebrospinal fluid (CSF) flow is essential for maintaining body and spine morphology (Grimes et al., 2016). Mutants with defective motile cilia failed to undergo axial straightening during embryogenesis and so developed a misshapen early embryonic body axis called ‘curly tail down’ (CTD; Brand et al., 1996). If rescued during this early stage, mutants went on to develop three-dimensional spinal curves that recapitulated some features of idiopathic scoliosis, including adolescent-stage onset in the absence of vertebral patterning defects (Grimes et al., 2016; Marie-Hardy et al., 2021; Wang et al., 2022). Precisely how motile cilia and CSF flow maintain spine morphology during growth is not understood, but it is known that during early larval stages cilia motility is essential for the assembly of the Reissner fiber (RF), an extracellular thread-like structure composed predominantly of the large glycoprotein SCOspondin (encoded by sspo) which sits in the CSF in brain ventricles and the central canal (Cantaut-Belarif et al., 2018; Rodríguez et al., 1998). Zebrafish sspo mutants exhibited CTD as embryos while hypomorphic mutants which can survive beyond embryonic stages also manifested spinal curves (Cantaut-Belarif et al., 2018; Lu et al., 2020; Rose et al., 2020; Troutwine et al., 2020).

The URPs, Urp1 and Urp2, may also function downstream of motile cilia in the central canal. Urp1 and Urp2 are 10-amino acid cyclic peptides previously linked to heart disease and mental illness (Sugo et al., 2003; Konno et al., 2013; Nobata et al., 2011; Parmentier et al., 2011; Quan et al., 2021; Tostivint et al., 2006; Vaudry et al., 2010). In zebrafish, Urp1 and Urp2 are expressed in CSF-contacting neurons (CSF-cNs), flow sensory neurons in the central canal, and their expression is increased by motile cilia function and the RF (Cantaut-Belarif et al., 2020; Lu et al., 2020; Quan et al., 2015; Zhang et al., 2018). Morpholino knockdown of Urp1/Urp2 results in embryonic CTD phenotypes while addition of Urp1/Urp2 peptides can rescue the CTD of cilia motility- and RF-deficient mutants (Lu et al., 2020; Zhang et al., 2018). This suggested that Urp1 and Urp2 act downstream of cilia motility to promote early axial straightening (Grimes, 2019; Lu et al., 2020; Zhang et al., 2018).

Here, we set out to address whether Urp1 and Urp2 function beyond embryogenesis in maintaining body and spine morphology during growth and adulthood. By generating zebrafish mutants lacking Urp1 and Urp2 peptides, we found that they are essential, in a semi-redundant fashion, for adult spine morphology. Loss of Urp1 and Urp2 together led to the onset of spinal curves during adolescent stages and, by adulthood, resulted in planar curves in the caudal region of the spine that occurred without vertebral patterning defects or significant structural malformations. A similar phenotype was present upon mutation of the urotensin receptor (UT) gene, uts2r3, suggesting that Urp1 and Urp2 signal via Uts2r3 to maintain spine morphology. Urotensin-associated curves were quantitatively distinct from the curves displayed by cfap298 mutants, which lack cilia motility, and pkd2l1 mutants in which a CSF-cN-localized ion channel is mutated, suggestive of overlapping but distinct roles of these components. Moreover, RF breakdown preceded curve formation in cfap298 mutants while RF structure was maintained before and after curves appeared in uts2r3 mutants. Overall, this demonstrates that Urp1 and Urp2 peptides control the morphology of the zebrafish spine. We suggest that urotensin-deficient zebrafish model human spinal deformities and will be important tools for deciphering how the spine is maintained and how this process goes wrong in disease.

Urotensin II (UII) is a cyclic peptide that was first identified from the teleost urophysis (Pearson et al., 1980) and subsequently found to exist in amphibians (Conlon et al., 1992) and mammals (Coulouarn et al., 1998). A highly similar peptide, called URP was then isolated from the brains of rodents (Sugo et al., 2003). UII and URP both signal via the UT, a G-protein-coupled receptor (Ames et al., 1999; Liu et al., 1999; Mori et al., 1999; Nothacker et al., 1999). UIIs, URPs, and UTs have been linked to cardiovascular function and inflammation, but their roles in the development of morphology are little understood.

In this study, we discovered a role for two of the URPs, Urp1 and Urp2, in zebrafish spine morphology. To do so, we generated urp1^∆P and urp2^∆P mutants that lacked the genetic region coding for the Urp1 and Urp2 peptides, respectively, then phenotyped skeletal morphology by µCT. This revealed that Urp1 and Urp2 function semi-redundantly to control spine morphology, with double mutants, as well as Uts2r3 receptor mutants, developing spinal curves in the caudal region during adolescent growth. By contrast, single urp1^∆P and urp2^∆P mutants developed more subtle curves that nevertheless worsened with age. The lack of major vertebral defects, the location and direction of the curves coupled with phenotypic differences compared with mutants that model AIS and kyphosis, suggested that urotensin-deficient mutants model a lordosis-like condition.

The RF, a long proteinaceous thread-like structure which sits in the central canal and is mostly made from SCOspondin (encoded by sspo), has been implicated in controlling body axis and spine morphology (Cantaut-Belarif et al., 2018; Lu et al., 2020; Rose et al., 2020; Troutwine et al., 2020). We find that RF breaks down prior to curve formation in the cilia motility cfap298^tm304 mutant. Given other studies linking presence of the RF with a linear body axis, this strongly suggests that RF breakdown is a major factor driving spinal curves in cfap298^tm304 mutants. By contrast, Uts2r3-deficient mutants exhibited an intact RF, demonstrating that curves are not formed by RF breakdown in urotensin pathway mutants and nor do the presence of curves significantly disrupt RF structure. This coheres with a model in which urotensin signals act downstream of RF function in controlling spine morphology. Similarly, urp1 and urp2 expression are known to be controlled by RF function during embryonic phases (Figure 1F; Cantaut-Belarif et al., 2020; Lu et al., 2020; Rose et al., 2020; Zhang et al., 2018).

Intriguingly, motile cilia mutants and sspo mutants exhibit three-dimensional spinal curves, with dorso-ventral and medio-lateral curvature (Grimes et al., 2016; Lu et al., 2020; Rose et al., 2020; Troutwine et al., 2020). By contrast, we find that urotensin-deficient mutants exhibit largely planar curves, only in the dorso-ventral direction, thereby potentially uncoupling two systems controlling posture (Picton et al., 2021). Urp1 and Urp2 are expressed in CSF-cNs (Figure 1D; Quan et al., 2015), a cell type which consists of both dorsal and ventral subpopulations. While Urp1 and Urp2 are co-expressed in ventral CSF-cNs, only Urp2 is expressed in dorsal CSF-cNs (Figure 1D; Quan et al., 2015). It is therefore tempting to speculate that dorso-ventral spine shape is mediated specifically by ventral CSF-cNs which express higher amounts of Urp1/Urp2 peptides, resulting in dorso-ventral curves in urotensin-deficient mutants. This also suggests, as above, that while Urp1/Urp2 expression is in part controlled by upstream cilia motility and RF function, decreased urotensin signaling cannot account fully for the spinal curve phenotypes that occur upon loss of cilia motility or the RF. Indeed, the RF is required for both dorsal and ventral CSF-cN function (Orts-Del’Immagine et al., 2020), which may explain why dorso-ventral and medio-lateral curves occur when RF is disrupted either by cilia motility mutations or mutations to SCOspondin. This scenario is further complicated by the finding that increased urp1 and urp2 expression occurs upon mutation of rpgrip1l, a gene encoding a component of the ciliary transition zone (Vesque et al., 2019 — preprint). As such, various cilia-dependent signals likely control the precise levels of Urp1/Urp2 peptides, and controlling those levels appears critical for maintaining the shape of the spine.

In addition to exhibiting phenotypic differences in terms of curve direction compared with cilia motility and RF mutants, urotensin-deficient mutants also showed gradual worsening of spinal phenotypes upon aging. urp1^∆P mutants exhibited no obvious phenotypes at 3 mpf but by 12 mpf showed abnormal curves, while urp2^∆P showed mild curves at 3 mpf and more severe deformity at 12 mpf. This implies that Urp1/Urp2 function throughout adulthood and aging to maintain spine morphology. Since double mutants were more severe at 3 mpf than either single mutant, partial redundancy between Urp1 and Urp2 appears to occur while overall dose likely sets how early phenotypes manifest. In contrast to this long-term role for urotensin peptides, temperature-shift experiments in which motile cilia were inactivated after 34 dpf showed no role for motile cilia beyond this stage in maintaining the spine (Grimes et al., 2016). It is worth noting, however, that this result does not preclude a role for CSF flow or the RF during long-term spine morphostasis because it has not been determined if motile cilia are required for CSF flow or RF formation in adult fish.

While the study of zebrafish spinal curve mutants holds great promise for understanding the basic science of spine morphology, it will also be important for the field to grapple with the question of how closely zebrafish spinal deformity mutants truly recapitulate human spinal curve diseases. The spines of humans and zebrafish are broadly similar, and it has been suggested that spinal loads are comparable (Gorman and Breden, 2009). Moreover, zebrafish spines, like human spines, seem predisposed to curvature, with high levels of scoliosis-like curves naturally developing with age (Bearce and Grimes, 2021; Gorman and Breden, 2009). The overall shape of the zebrafish spine is also similar to human, with a natural kyphotic curve in the pre-caudal (rib-bearing) vertebrae and a compensatory, albeit very minor, lordotic curve in the most anterior caudal vertebrae. However, these curves are not as pronounced as in humans. Moreover, zebrafish also exhibit some fish-specific structures such as the Weberian apparatus (Dietrich et al., 2021). Nevertheless, zebrafish cilia motility mutants appear to model several features of AIS including the three-dimensional nature of curves, lack of vertebral patterning defects or significant vertebral structural malformations, adolescent-onset and, in some cases, sex bias (Grimes et al., 2016; Marie-Hardy et al., 2021; Wang et al., 2022). The curves of urp1^∆P;urp2^∆P mutants display some of these features as well but, importantly, are not three-dimensional. Instead, urotensin pathway-deficient mutants display primarily planar curves, with little or no lateral deviation. This is more similar to what occurs in hyper-kyphosis and hyper-lordosis in humans, when natural curves are accentuated. Given this planarity, and since curves are mostly present in the caudal vertebrae, we suggest that urp1^∆P;urp2^∆P mutants model some aspects of lordosis and so refer to this phenotype as lordosis-like. However, we note that human lumbar vertebrae and zebrafish caudal vertebrae are structurally distinct (Boswell and Ciruna, 2017), and humans have a significant natural lordotic curve that allows for an efficient upright walking gait, whereas zebrafish do not. Thus, urotensin-deficient mutants recapitulate some aspects of lordosis but clearly cannot mimic human-specific aspects of hyper-lordotic curves.

A surprising finding from our work was that Urp1 and Urp2 peptides are genetically dispensable for embryonic axial straightening. This interpretation is challenged by morpholino knockdown of Urp1/Urp2, which does result in failure of axial straightening in some individuals, resulting in a CTD phenotype (Zhang et al., 2018). One possibility is that the CTD of morphants results from morpholino off-target effects. However, this seems unlikely for three reasons: (1) adding exogenous Urp1/Urp2 peptides to the central canal can rescue the CTD phenotype of cilia motility- and RF-deficient mutants (Lu et al., 2020; Zhang et al., 2018), suggesting the involvement of Urp1/Urp2 in axial straightening, at least in gain-of-function experiments; (2) since motile cilia and the RF are required for both axial straightening during embryogenesis and for the maintenance of spine morphology during adolescence, it seems parsimonious that Urp1/Urp2 peptides would also function across these two life stages; and (3) urp1 and urp2 transcript levels are reduced in motile cilia and RF mutants (Figure 1F; Cantaut-Belarif et al., 2020; Lu et al., 2020; Zhang et al., 2018), suggestive of a link between Urp1 and Urp2 upregulation and axial straightening.

Nevertheless, the lack of CTD in our mutants, in which the Urp1 and Urp2 peptide coding sequences were entirely removed, is clear: single, double, and maternal-zygotic urp1^∆P and urp2^∆P mutants all underwent normal axial straightening. This strongly argues that Urp1 and Urp2 are dispensable for straightening. Since the deletions were induced toward the end of the protein, it does leave open the possibility that the pro-domain sequences are required for straightening. However, this seems unlikely for three reasons: (1) urp1^∆P and urp2^∆P mutants showed urp1 and urp2 transcript downregulation, respectively, in addition to deletion of the peptide coding regions; (2) urp1 and urp2 single and double crispants, in which gRNAs targeted several regions of the gene, also showed normal straightening; and (3) exogenous addition of Urp1 and Urp2 peptides, without pro-domains, rescued CTD of a cilia motility and RF mutant (Lu et al., 2020; Zhang et al., 2018), suggesting that it is the peptide itself and not some other region which is functional.

One possibility is that genetic compensation explains the mutant/morphant phenotypic differences (Rossi et al., 2015; Sztal and Stainier, 2020). In this putative scenario, a feedback response in the cell buffers otherwise harmful mutations, preventing their effects from manifesting phenotypically. A recently discovered compensation mechanism is transcriptional adaptation in which mutant mRNA is decayed and the products of that decay are, via a sequence-dependent mechanism, recruited to genes with similar sequences where they promote transcriptional upregulation (El-Brolosy et al., 2019; Ma et al., 2019). The upregulation of adapting genes then masks phenotypes in mutants but not morphants. We did find slight upregulation of urp2 in urp1^∆P mutants (Figure 1—figure supplement 2E), which may indicate transcriptional adaptation, although this alone cannot explain the lack of CTD phenotypes in urp1^∆P mutants because urp1^∆P;urp2^∆P double mutants also lacked CTD. It will be informative in the future to determine whether other urotensin II-encoding peptides (Figure 1—figure supplement 1A–B) are able to compensate, during embryonic phases, for loss of urp1 and urp2 or if other factors explain the mutant/morphant discrepancies.

While we were in the final stages of preparing this manuscript, a study was released which made several complementary findings, also concluding that Urp1 and Urp2 function redundantly to maintain spine shape (Gaillard et al., 2022 — preprint). Importantly, and in agreement with our work, Gaillard and colleagues observed no embryonic axial defects upon genetic loss of Urp1 and Urp2. Moreover, they found that other urotensin II-encoding genes were not upregulated in urp1 and urp2 mutants, arguing against phenotypic masking by genetic compensation. Our findings and those of Gaillard and colleagues together therefore strongly argue that Urp1 and Urp2 are not essential for axial straightening during embryogenesis but are instead required for the maintenance of the body axis during growth and adulthood. As such, other mechanisms, currently unknown, likely operate downstream of cilia motility and RF function to mediate embryonic axial straightening.

Future efforts will be required to discern which tissues respond to Urp1/Urp2 signals during the control of spine morphology. During embryonic stages, Uts2r3 is expressed in dorsal muscle (Zhang et al., 2018), but it remains unclear how Urp1/Urp2 peptides released by CSF-cNs could signal to effect muscle during adolescent stages. Moreover, based on single-cell RNA-sequencing gene expression atlases (Farnsworth et al., 2020), uts2r3 is expressed in several other cell types in addition to muscle. Tissue-specific ablations and rescue experiments should be used to untangle precisely where Uts2r3-dependent Urp1/Urp2 signaling occurs to control spine morphology. It will also be critical to determine the timing of action of urotensin signaling. While we observe phenotypes first appearing between 9 and 11 dpf in urp1^∆P;urp2^∆P mutants, it is possible that the underlying defect in mutants is caused by an earlier event which only phenotypically manifests later. One candidate is subtle disruptions to the notochord, which may later result in spinal curves (Bagwell et al., 2020). On the other hand, our experiments in which we aged urp1^∆P and urp2^∆P single mutants argue that Urp1 and Urp2 peptides function throughout life, and not only during early growth, to maintain spine morphology.

Another major question is whether the function of urotensin signaling in spine morphology is conserved in other species, and whether our findings are directly relevant to humans. These questions will require future work to answer, but two recent studies shed some light on these matters. First, in the frog Xenopus laevis, disruption of Utr4, a counterpart of Uts2r3, causes abnormal curvature of the body axis (Alejevski et al., 2021). Second, a human genetics study reported that rare mutations in UTS2R are significantly associated with spinal curvature, being discovered within AIS patient cohorts (Dai et al., 2021). Thus, a deeper understanding of the role of urotensin signaling in maintaining spine shape will not only provide insight into principles of morphogenesis but potentially also human disease.

All data generated or analysed during this study are included in the manuscript and supporting file.

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Author details

1. Elizabeth A Bearce

   Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, United States

   Contribution

   Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

2. Zoe H Irons

   Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, United States

   Contribution

   Funding acquisition, Validation, Methodology

   Competing interests

   No competing interests declared

3. Johnathan R O'Hara-Smith

   Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, United States

   Contribution

   Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Colin J Kuhns

   Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, United States

   Contribution

   Validation, Investigation

   Competing interests

   No competing interests declared

5. Sophie I Fisher

   Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. William E Crow

   Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2991-3076

7. Daniel T Grimes

   Institute of Molecular Biology, Department of Biology, University of Oregon, Eugene, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   dtgrimes@uoregon.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0173-1887

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