Development Of Vertebral Column Research Articles

Development of the axial skeleton in the bay snook Petenia splendida Günther, 1862 (Perciformes: Cichlidae)

We report the first description of the ontogenic development of the axial skeleton and unpaired fins of hatchery reared bay snook (Petenia splendida, Perciformes: Cichlidae) from hatching (5.3–5.5 mm in standard length, SL) to the juvenile stage (16.1–17.5 mm SL) by means of alcian blue-alizarin red staining. The study of axial skeleton development was complemented by digital analysis of its level of ossification (number of red pixels) in order to identify major changes in vertebral column and caudal fin complex development. The main events of the skeletogenesis of the vertebral column and unpaired fins in P. splendida larvae were comprised between the onset of notochord segmentation and completion of the caudal fin complex at 6 (171 degree days post hatch, ddph; 6.4–6.5 mm SL) and 24 days post hatching (dph) (684 ddph, 8.9–9.4 mm SL), respectively; whereas the anal and dorsal fins completed morphogenesis at older ages (36 dph, 1026 ddph, 11.0–11.8 mm SL). In this sense, the development of a subcarangiform swimming mode in early juveniles was achieved with complete ossification of vertebral bodies and caudal lepidotrichia, as well as partial ossification of the caudal-fin complex elements (hypurals, epurals, uroneural and parahypural). This information can be considered as a reference for other studies evaluating the quality of larvae and the influence of rearing biotic and abiotic factors in the skeletogenesis of this cichlid species and the occurrence of skeletal deformities.

Pecularities of bony skeleton development in Asian clawed salamanders (Onychodactylus, Hynobiidae) related to embryonization

We studied skull, vertebral column, and limb skeleton development in Japanese clawed sala� mander Onychodactylus japonicus (Hynobiidae). The study is based on the ontogenetic series of embryos and larvae obtained from wildcaptured adults by artificial induction of breeding using hormonal stimulation. The first stages of the skeleton formation in O. japonicus are shifted to the late embryonic period and hatching lar� vae already possess a wellossified vertebral column, large number of skull ossifications and show signs of ossi� fication in the forelimb skeleton. Compared to the primitive pattern of the skeleton development typical for other hynobiid salamanders, O. japonicus shows a number of heterochronies related to embryonization. In particular, this species is characterized by an earlier ossification of the vertebral column compared to that of the skull and by the delayed development and early reduction of the coronoid. Our results, along with the pre� viously reported data on the skeleton development in the Fischer's clawed salamander O. fischeri (Smirnov and Vassilieva, 2002), indicate that the genus Onychodactylus is characterized by the loss or reduction of sev� eral skeletal features typically found at early larval stages in other Hynobiidae species. In particular, provi� sional bones (especially the coronoid) and their dentition are underdeveloped. In addition, it is corroborated that the first tooth generation is absent in Onychodactylus, whereas such monocuspid nonpedicellate tooth generation normally develops at the early larval stages of other caudate amphibians. Since similar patterns of skeleton ontogeny are observed in other caudate groups with different extent of embryonization, it is pro� posed that, in different lineages of Urodela, the evolution of ontogeny followed similar pathways and was accompanied by the same changes in skeletogenesis.

An Essential Role for B1 Integrin Mediated Cell-Matrix Interactions in the Development and Maintenance of the Nucleus Pulposus

IntroductionIn humans, the almost total disappearance of notochordal cells in the nucleus pulposus (NP) correlates with early degenerative changes in the intervertebral disk which include changes in the extracellular matrix (ECM). Recent cell lineage studies in mice suggest cells within the NP are derived from the early embryonic notochord. Since notochordal cells are surrounded by a rich ECM, the functions of the notochord and the NP are likely mediated via cell-matrix interactions. Integrin heterodimers are transmembrane proteins and are the major adhesion receptors that mediate cell-matrix interactions. They connect the ECM to the intracellular cytoskeleton and transduce bidirectional signals between the extracellular matrix and intracellular compartments. At least 24 integrin heterodimers have been identified, and among them receptors containing b 1 integrin comprise the largest integrin-subfamily. Materials and MethodsHere, we used Cre recombinase to specifically inactivate b 1 integrin gene in the notochord in mice, to understand the role of b 1 integrin-mediated cell-matrix interactions in the development and maintenance of the notochord and NP. The development and fate of the notochordal cells were analyzed at embryonic stages E8.5-E10.5 using key expression markers such as Shh, Brachyury, and Noto and cell apoptotic assay. NP formation was studied by histological analysis. The function of b 1 integrin in cell-matrix interactions in notochord cells isolated by FACS were analyzed using a functional blocking antibody specific to b 1 integrin. Fibronectin matrix and Cadherin-2 expression pattern were investigated by immunofluorescence and confocal microscopy. ResultsMice lacking b 1 integrin subunit in notochordal cells display abnormal vertebral column development with reduced size or total absence of the notochord descendents and NP in the intervertebral disks (IVD). This abnormality is accompanied with disorganized kinked patterning of the tail vertebrae and hemivertebrae. These defects are related to defects in the development and maintenance of the notochord at early embryonic stages from E8.25. Abnormalities in localization of the notochord were found, such as displacement from D-V or L-R midline axes, and failure to separate from the foregut endoderm. Assembly of ECM components was also impaired. Impaired cell matrix assembly and cell-cell contacts found in the mutants may possibly contribute to the notochord mallocalization. Increased cell apoptosis together with impaired cell-cell adhesion may potentially contribute to the defective notochord tissue integrity. b 1 integrin antibody blocking assays on notochordal cells display impaired cell adhesion, delayed maturation of focal adhesion complexes and decreased migration ability on matrix substrates. ConclusionMaintenance of b 1 integrin mediated cell-matrix interactions is essential for the proper development and maintenance of the notochord and NP, and later the proper patterning of the axial skeleton. Impaired cell-matrix interactions which affect cell survival, cell polarity, and migration may contribute to the progressive loss of NP cells during disk degeneration.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declared

An Essential Role for B1 Integrin Mediated Cell-Matrix Interactions in the Development and Maintenance of the Nucleus Pulposus

Introduction In humans, the almost total disappearance of notochordal cells in the nucleus pulposus (NP) correlates with early degenerative changes in the intervertebral disk which include changes in the extracellular matrix (ECM). Recent cell lineage studies in mice suggest cells within the NP are derived from the early embryonic notochord. Since notochordal cells are surrounded by a rich ECM, the functions of the notochord and the NP are likely mediated via cell-matrix interactions. Integrin heterodimers are transmembrane proteins and are the major adhesion receptors that mediate cell-matrix interactions. They connect the ECM to the intracellular cytoskeleton and transduce bidirectional signals between the extracellular matrix and intracellular compartments. At least 24 integrin heterodimers have been identified, and among them receptors containing b 1 integrin comprise the largest integrin-subfamily. Materials and Methods Here, we used Cre recombinase to specifically inactivate b 1 integrin gene in the notochord in mice, to understand the role of b 1 integrin-mediated cell-matrix interactions in the development and maintenance of the notochord and NP. The development and fate of the notochordal cells were analyzed at embryonic stages E8.5-E10.5 using key expression markers such as Shh, Brachyury, and Noto and cell apoptotic assay. NP formation was studied by histological analysis. The function of b 1 integrin in cell-matrix interactions in notochord cells isolated by FACS were analyzed using a functional blocking antibody specific to b 1 integrin. Fibronectin matrix and Cadherin-2 expression pattern were investigated by immunofluorescence and confocal microscopy. Results Mice lacking b 1 integrin subunit in notochordal cells display abnormal vertebral column development with reduced size or total absence of the notochord descendents and NP in the intervertebral disks (IVD). This abnormality is accompanied with disorganized kinked patterning of the tail vertebrae and hemivertebrae. These defects are related to defects in the development and maintenance of the notochord at early embryonic stages from E8.25. Abnormalities in localization of the notochord were found, such as displacement from D-V or L-R midline axes, and failure to separate from the foregut endoderm. Assembly of ECM components was also impaired. Impaired cell matrix assembly and cell-cell contacts found in the mutants may possibly contribute to the notochord mallocalization. Increased cell apoptosis together with impaired cell-cell adhesion may potentially contribute to the defective notochord tissue integrity. b 1 integrin antibody blocking assays on notochordal cells display impaired cell adhesion, delayed maturation of focal adhesion complexes and decreased migration ability on matrix substrates. Conclusion Maintenance of b 1 integrin mediated cell-matrix interactions is essential for the proper development and maintenance of the notochord and NP, and later the proper patterning of the axial skeleton. Impaired cell-matrix interactions which affect cell survival, cell polarity, and migration may contribute to the progressive loss of NP cells during disk degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared

Vaccinated farmed Atlantic salmon are susceptible to spinal and skull deformities

St Petersburg State University, St Petersburg,RussiaSummaryThe aim of this study was to investigate the effects ofvaccination, and starvation after vaccination, on the morphol-ogy of skull bones and vertebral bodies, bone mineralizationand the development of intra-muscular melanin deposits infarmed Atlantic salmon. For this purpose, triplicate groups ofvaccinated (V) or unvaccinated (UV) underyearling smolts(38 g) were fed normal rations (F), or starved for 3 weeks aftervaccination (S) (September 2004), leaving four experimentalgroups (V-F, V-S, UV-F, UV-S). After termination of thestarvation period, all groups were fed a normal ration until thefish reached a normal harvest size (January 2006). There wereno long term effects of starvation on body weight, bonemineralization or deformity incidence. However, at termina-tion, V fish (pooled mean V-F and V-S, 4.4 kg) were overallsignificantlysmallerthanUVfish(pooledmeanUV-FandUV-S, 5.1 kg), and they also developed overall significantly moredeformities in both the skull (pooled mean UV-F and UV-S27%,pooledmeanV-FandV-S81%)andthevertebralcolumn(pooledmeanUV-FandUV-S21%,pooledmeanV-FandV-S41%). These deformities were site-specific in both compart-ments; skull deformities were clearly observed in the ceratohy-ale anterior, while those in the vertebral column were mainlylocated in the tail region (V31–49). There were no significantcorrelations among individual relationships between the skulland vertebral column deformities. UV fish had overall signif-icantly higher vertebral bone total ash content than V fish attermination, butthere werenoeffectsonvaccinationontheashcontentor morphology ofthe vertebrae2 monthsafter transferto seawater. The incidences of intra-muscular melanin depositswere not significantly different between UV and V fish. Theseresults show that vaccination can induce deformities in thevertebral column and ⁄ or skull in farmed Atlantic salmon. Thesite-specificlocationofthedeformitiesinrelationtomechanicalloading is discussed. Furthermore, the results show thatvaccination is not likely the main cause of the current melaninspot problem in Atlantic salmon aquaculture.IntroductionAll farmed Norwegian Atlantic salmon are vaccinated with oil-based adjuvant vaccines before they complete smoltification.This cause a short-term decrease in appetite (Sorum andDamsga˚rd, 2004), induces abdominal lesions that adhere andembed the visceral organs (Poppe and Breck, 1997; Midtlyng,1998) and may induce vertebral deformities (Berg et al., 2006a;Aunsmo et al., 2008), but also gives the fish protectiveimmunity against several diseases (Midtlyng et al., 1996a;Midtlyng, 1998; Lillehaug et al., 2003). Vaccination has alsobeen associated with muscle inflammation and melanin accu-mulation (Koppang et al., 2005), granulomatous uveitis(Koppang et al., 2004), and systemic autoimmunity (Koppanget al., 2008; Haugarvoll et al., 2010). Intra-muscular melanindeposits are a major problem in Atlantic salmon farming, butthe link with vaccination has never been studied. Themechanism behind vaccine induced vertebral deformities hasnot yet been identified. It is not clear whether the vaccine-induced deformities start to develop immediately after vacci-nation or later in the production cycle during on-growth inseawater. Small fish size at vaccination is identified as a riskfactor for vertebral deformities (Va˚gsholm and Djupvik, 1998;Berg et al., 2006a). Haugarvoll et al. (2010) suggest thatvaccine induced deformities can be coupled with systemicautoimmunity. Furthermore, low appetite combined withrelatively good growth in body length has been suggested toimpair bone mineralization and increase the risk for vertebraldeformities in Atlantic salmon (Fjelldal et al., 2007a; Griniet al., 2011). Hence, the short-term decrease in appetite aftervaccination (Sorum and Damsga˚rd, 2004), may make vacci-nated salmon prone to developing vertebral deformities.However, effects of a restricted ration or starvation onpathogenesis in the vertebral column in Atlantic salmon hasnever been tested. In addition, the presence of vaccine-inducedmalformations in other skeletal structures than the vertebralcolumn would indicate a link to a systemic autoimmune origin.The question of whether vaccination affects skeletal structuresother than the vertebral column has never been studied.This study examined the effects of vaccination and post-vaccination starvation on the development of deformities inthe skull and vertebral column. Triplicate groups of vaccinatedand un-vaccinated Atlantic salmon underyearling smolts werefed normal rations or starved for 3 weeks post-vaccination. Allgroups were thereafter fed a normal ration until harvest sizeand their growth performance, severity of abdominal lesions(lesion score), intra-muscular melanin deposits, malformationsin the vertebral column and skull, and mineralization of thevertebral bodies were measured.Materials and methods

Development of Vertebral Column and Caudal Skeleton of the Red Sea Bream, Pagrus major

Development of Vertebral Column and Caudal Skeleton of the Red Sea Bream, Pagrus major

Mutation of the fucose-specific β1,3 N-acetylglucosaminyltransferase LFNG results in abnormal formation of the spine

Notch signaling is an evolutionarily conserved mechanism that determines cell fate in a variety of contexts during development. This is achieved through different modes of action that are context dependent. One mode involves boundary formation between two groups of cells. With this mode of action, Notch signaling is central to vertebrate evolution as it drives the segmentation of paraxial mesoderm in the formation of somites, which are the precursors of the vertebra. In this case, boundary formation facilitates a mesenchymal to epithelial transition, leading to the creation of a somite. In addition, the boundary establishes a signaling center that patterns the somite, a feature that directly impacts on vertebral column formation. Studies in Xenopus, zebrafish, chicken and mouse have established the importance of Notch signaling in somitogenesis, and indeed in mouse how perturbations in somitogenesis affect vertebral column formation. Spondylocostal dysostosis is a congenital disorder characterized by formation of abnormal vertebrae. Here, mutation in Notch pathway genes demonstrates that Notch signaling is also required for normal somite formation and vertebral column development in humans; of particular interest here is mutation of the LUNATIC FRINGE (LFNG) gene, which causes SCD type 3. LUNATIC FRINGE encodes for a fucose-specific β1,3-N-acetylglucosaminyltransferase, which modifies Notch receptors and alters Notch signaling activity. This review will focus on Notch glycolsylation, and the role of LUNATIC FRINGE in somite formation and vertebral column development in mice and humans.

Classification and subclassification of funnel and pigeon chest

The Willital's clinical classification of funnel- and pigeon-chest deformities can be subclassified by special measurements of the chest. This easy technique enables to differentiate between: Sternocranial, sternocaudal, infrasternal, symmetric and asymmetric marginal and parasternal deformities. This further classification includes deformities of the vertebral column and deviatory development as well. The exact analysis of the abnormal chest in three different planes can be performed by external chest wall measurements that have been developed in our hospital.

Sox5andSox6are required for notochord extracellular matrix sheath formation, notochord cell survival and development of the nucleus pulposus of intervertebral discs

The notochord has major roles in vertebral column formation: indirectly by inducing sclerotome cell differentiation; and directly by forming the nucleus pulposus of intervertebral discs. Sox5 and Sox6 encode Sry-related HMG box transcription factors that act redundantly to promote chondroblast differentiation in all cartilages of the mouse embryo. We show that Sox5 and Sox6 are expressed in the notochord cell lineage and required for notochord late development. In Sox5(-/-)/Sox6(-/-) embryos, the notochord formed a typical rod-like structure. It fulfilled its inductive functions, as indicated by expression of sonic hedgehog and sclerotome specification. However, the notochord failed to become surrounded with an extracellular matrix sheath. This phenotype was associated with a downregulation of extracellular matrix genes, including the genes for collagen 2, aggrecan and perlecan in both notochord cells and surrounding chondrocytic cells of presumptive inner annuli and vertebral bodies. The mutant notochord then underwent an aberrant, fatal dismantling after sclerotome cell migration. Its cells became removed first from intervertebral spaces and then from vertebral bodies, and it progressively underwent apoptosis. Meanwhile, the development of inner annuli and vertebral bodies was dramatically impaired. Consequently, the vertebral column of Sox5(-/-)/Sox6(-/-) fetuses consisted of a very deficient cartilage and was devoid of nuclei pulposi. In Sox5(-/-)/Sox6(+/-) and more severely in Sox5(+/-)/Sox6(-/-) embryos, the notochord sheath was thinner, but cells survived. By birth, nuclei pulposi were rudimentary, and its cells poorly swelled and still expressing sonic hedgehog. Hence, Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and formation of nuclei pulposi. Through these roles and essential roles in cartilage formation, they are central transcriptional regulators of vertebral column development.

Segmental relationship between somites and vertebral column in zebrafish.

The segmental heritage of all vertebrates is evident in the character of the vertebral column. And yet, the extent to which direct translation of pattern from the somitic mesoderm and de novo cell and tissue interactions pattern the vertebral column remains a fundamental, unresolved issue. The elements of vertebral column pattern under debate include both segmental pattern and anteroposterior regional specificity. Understanding how vertebral segmentation and anteroposterior positional identity are patterned requires understanding vertebral column cellular and developmental biology. In this study, we characterized alignment of somites and vertebrae, distribution of individual sclerotome progeny along the anteroposterior axis and development of the axial skeleton in zebrafish. Our clonal analysis of zebrafish sclerotome shows that anterior and posterior somite domains are not lineage-restricted compartments with respect to distribution along the anteroposterior axis but support a 'leaky' resegmentation in development from somite to vertebral column. Alignment of somites with vertebrae suggests that the first two somites do not contribute to the vertebral column. Characterization of vertebral column development allowed examination of the relationship between vertebral formula and expression patterns of zebrafish Hox genes. Our results support co-localization of the anterior expression boundaries of zebrafish hoxc6 homologs with a cervical/thoracic transition and also suggest Hox-independent patterning of regionally specific posterior vertebrae.

Osteological development of the vertebral column and of the fins in Diplodus sargus (Teleostei: Perciformes: Sparidae)

The osteological development of vertebral column and fins in white sea bream, Diplodus sargus (Linnaeus, 1758) was studied. Vertebral ontogeny started at 5.7 mm total length (TL, post-fixation), with the formation of the first cartilaginous neural and haemal arches, and was completed by the full attainment of dorsal ribs (20.8 mm TL). The formation of vertebral centra occurred between 7.3 and 9.4 mm TL, but was fully completed only after the development of post- and pre-zygapophyses (26.0 mm TL). Pectoral supports were the first fin elements that started to develop (3.8 mm TL), followed by those of the caudal fin (5.2 mm TL), dorsal and anal fins (6.7 mm TL), and then by those of the pelvic fin (9.5 mm TL). The caudal fin of D. sargus was the first to develop fin rays and attain the full count of lepidotrichia (5.6–7.8 mm TL), but the last to attain the full count of dermatotrichia (7.9–16.0 mm TL). The next fins starting to present rays were the pectoral (6.5 mm TL) and the dorsal and anal fins (7.5 mm TL), while the pelvic fins were the last (11.5 mm TL). Following the caudal lepidotrichia (7.8 mm TL), the anal (10.5 mm TL), dorsal (12.0 mm TL), pectoral (11.4 mm TL) and pelvic fins (12.5 mm TL) were the next with fully completed ray counts. Finally, fin meristics were fully developed with the caudal dermatotrichia. The results are discussed in comparison with the fin and vertebral ontogeny of other teleosts.

The development of the avian vertebral column.

Segmentation of the paraxial mesoderm leads to somite formation. The underlying molecular mechanisms involve the oscillation of "clock-genes" like c-hairy-1 and lunatic fringe indicative of an implication of the Notch signaling pathway. The cranio-caudal polarity of each segment is already established in the cranial part of the segmental plate and accompanied by the expression of genes like Delta1, Mesp1, Mesp2, Ulicx-1, and EphA4 which are restricted to one half of the prospective somite. Dorsoventral compartmentalization of somites leads to the development of the dermomyotome and the sclerotome, the latter forming as a consequence of an epithelio-to-mesenchymal transition of the ventral part of the somite. The sclerotome cells express Pax-1 and Pax-9, which are induced by notochordal signals mediated by sonic hedgehog (Shh) and noggin. The craniocaudal somite compartmentalization that becomes visible in the sclerotomes is the prerequisite for the segmental pattern of the peripheral nervous system and the formation of the vertebrae and ribs, whose boundaries are shifted half a segment compared to the sclerotome boundaries. Sclerotome development is characterized by the formation of three subcompartments giving rise to different parts of the axial skeleton and ribs. The lateral sclerotome gives rise to the laminae and pedicles of the neural arches and to the ribs. Its development depends on signals from the notochord and the myotome. The ventral sclerotome giving rise to the vertebral bodies and intervertebral discs is made up of Pax-1 expressing cells that have invaded the perinotochordal space. The dorsal sclerotome is formed by cells that migrate from the dorso-medial angle of the sclerotome into the space between the roof plate of the neural tube and the dermis. These cells express the genes Msx1 and Msx2, which are induced by BMP-4 secreted from the roof plate, and they later form the dorsal part of the neural arch and the spinous process. The formation of the ventral and dorsal sclerotome requires directed migration of sclerotome cells. The regionalization of the paraxial mesoderm occurs by a combination of functionally Hox genes, the Hox code, and determines the segment identity. The development of the vertebral column is a consequence of a segment-specific balance between proliferation, apoptosis and differentiation of cells.

Pax1 and Pax9 synergistically regulate vertebral column development.

The paralogous genes Pax1 and Pax9 constitute one group within the vertebrate Pax gene family. They encode closely related transcription factors and are expressed in similar patterns during mouse embryogenesis, suggesting that Pax1 and Pax9 act in similar developmental pathways. We have recently shown that mice homozygous for a defined Pax1 null allele exhibit morphological abnormalities of the axial skeleton, which is not affected in homozygous Pax9 mutants. To investigate a potential interaction of the two genes, we analysed Pax1/Pax9 double mutant mice. These mutants completely lack the medial derivatives of the sclerotomes, the vertebral bodies, intervertebral discs and the proximal parts of the ribs. This phenotype is much more severe than that of Pax1 single homozygous mutants. In contrast, the neural arches, which are derived from the lateral regions of the sclerotomes, are formed. The analysis of Pax9 expression in compound mutants indicates that both spatial expansion and upregulation of Pax9 expression account for its compensatory function during sclerotome development in the absence of Pax1. In Pax1/Pax9 double homozygous mutants, formation and anteroposterior polarity of sclerotomes, as well as induction of a chondrocyte-specific cell lineage, appear normal. However, instead of a segmental arrangement of vertebrae and intervertebral disc anlagen, a loose mesenchyme surrounding the notochord is formed. The gradual loss of Sox9 and Collagen II expression in this mesenchyme indicates that the sclerotomes are prevented from undergoing chondrogenesis. The first detectable defect is a low rate of cell proliferation in the ventromedial regions of the sclerotomes after sclerotome formation but before mesenchymal condensation normally occurs. At later stages, an increased number of cells undergoing apoptosis further reduces the area normally forming vertebrae and intervertebral discs. Our results reveal functional redundancy between Pax1 and Pax9 during vertebral column development and identify an early role of Pax1 and Pax9 in the control of cell proliferation during early sclerotome development. In addition, our data indicate that the development of medial and lateral elements of vertebrae is regulated by distinct genetic pathways.

Notochord-Dependent Expression of MFH1 and PAX1 Cooperates to Maintain the Proliferation of Sclerotome Cells during the Vertebral Column Development

During axial skeleton development, the notochord is essential for the induction of the sclerotome and for the subsequent differentiation of cartilage forming the vertebral bodies and intervertebral discs. These functions are mainly mediated by the diffusible signaling molecule Sonic hedgehog. The products of the paired-box-containingPax1and the mesenchyme forkhead-1 (Mfh1) genes are expressed in the developing sclerotome and are essential for the normal development of the vertebral column. Here, we demonstrate thatMfh1likePax1expression is dependent on Sonic hedgehog signals from the notochord, andMfh1andPax1act synergistically to generate the vertebral column. InMfh1/Pax1double mutants, dorsomedial structures of the vertebrae are missing, resulting in extreme spina bifida accompanied by subcutaneous myelomeningocoele, and the vertebral bodies and intervertebral discs are missing. The morphological defects inMfh1/Pax1double mutants strongly correlate with the reduction of the mitotic rate of sclerotome cells. Thus, both theMfh1and thePax1gene products cooperate to mediate Sonic hedgehog-dependent proliferation of sclerotome cells.

Genetic interactions suggest that Danforth's short tail (Sd) is a gain‐of‐function mutation

Danforth's short tail (Sd) is a semidominant mutation on mouse chromosome 2 that acts cell autonomously in the notochord and leads to its disintegration, and thus causes severe defects in somite patterning and vertebral column development. The molecular nature of the Sd gene and mutation is unknown, and it is unclear whether Sd is a loss-of-function mutation and the semidominant inheritance of the Sd phenotype is due to haploinsufficiency, or whether Sd represents a gain-of-function mutation in a gene essential for notochord development and maintenance. Here, we report on the genetic interaction between Sd and an insertional mutation called Etl4lacZ, which provides genetic evidence that Sd is a gain-of-function mutation. Etl4lacZ is an enhancer trap insertion, which gives rise to lacZ expression in distinct cell types, including the notochord. In homozygosity, the lacZ insertion leads to abnormal vertebrae in the caudal part of the vertebral column. Etl4lacZ maps approximately 0.75 cM distal to Sd, and in double heterozygotes modifies the Sdphenotype contrarily, depending on the chromosomal configuration of the Sd and Etl4lacZ mutations: when Etl4lacZ is present on the chromosome wild type for Sd (Sd +/+ Etl4lacZ; trans configuration), the Sd phenotype is enhanced, i.e., vertebral malformations extend to more anterior positions and the vertebral body of the axis is further reduced. Conversely, when Etl4lacZ is present on the same chromosome as Sd (Sd Etl4lacZ /+ +; cis configuration), the Sd phenotype is attenuated, i.e., vertebral malformations are confined to more posterior levels, and the dens axis, which is severely reduced or absent in Sd heterozygotes, is restored. The different effect of the Etl4lacZ insertion on Sd, depending on its presence in trans or cis, suggests a direct interaction of the transgene insertion with the Sd gene. Additionally, the attenuation of the Sd phenotype by Etl4lacZ in cis suggests that Sd is a gain-of-function mutation and lends support to the idea that Etl4lacZ is a new allele of Sd. Dev. Genet. 23:86–96, 1998. © 1998 Wiley-Liss, Inc.

Genetic interactions suggest that Danforth's short tail (Sd) is a gain-of-function mutation.

Danforth's short tail (Sd) is a semidominant mutation on mouse chromosome 2 that acts cell autonomously in the notochord and leads to its distintegration, and thus causes severe defects in somite patterning and vertebral column development. The molecular nature of the Sd gene and mutation is unknown, and it is unclear whether Sd is a loss-of-function mutation and the semidominant inheritance of the Sd phenotype is due to haploinsufficiency, or whether Sd represents a gain-of-function mutation in a gene essential for notochord development and maintenance. Here, we report on the genetic interaction between Sd and an insertional mutation called Etl4lacZ, which provides genetic evidence that Sd is a gain-of-function mutation. Etl4lacZ is an enhancer trap insertion, which gives rise to lacZ expression in distinct cell types, including the notochord. In homozygosity, the lacZ insertion leads to abnormal vertebrae in the caudal part of the vertebral column. Etl4lacZ maps approximately 0.75 cM distal to Sd, and in double heterozy gotes modifies the Sd phenotype contrarily, depending on the chromosomal configuration of the Sd and Etl4lacZ mutations: when Etl4lacZ is present on the chromosome wild type for Sd (Sd+/+ Etl4lacZ; trans configuration), the Sd phenotype is enhanced, i.e., vertebral malformations extend to more anterior positions and the vertebral body of the axis is further reduced. Conversely, when Etl4lacZ is present on the same chromosome as Sd (Sd Etl4lacZ/+ +; cis configuration), the Sd phenotype is attenuated, i.e., vertebral malformations are confined to more posterior levels, and the dens axis, which is severely reduced or absent in Sd heterozygotes, is restored. The different effect of the Etl4lacZ insertion on Sd, depending on its presence in trans or cis, suggests a direct interaction of the transgene insertion with the Sd gene. Additionally, the attenuation of the Sd phenotype by Etl4lacZ in cis suggests that Sd is a gain-of-function mutation and lends support to the idea that Etl4lacZ is a new allele of Sd. Dev. Genet. 23:86-96, 1998.

Cloning and developmental expression analysis of the murine homolog of the spinocerebellar ataxia type 1 gene (Sca1).

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat which encodes glutamine in the novel protein ataxin-1. In order to characterize the developmental expression pattern of SCA1 and to identify putative functional domains in ataxin-1, the murine homolog (Sca1) was isolated. Cloning and characterization of the murine Sca1 gene revealed that the gene organization is similar to that of the human gene. The murine and human ataxin-1 are highly homologous but the CAG repeat is virtually absent in the mouse sequence suggesting that the polyglutamine stretch is not essential for the normal function of ataxin-1 in mice. Cellular and developmental expression of the murine homolog was examined using RNA in situ hybridization. During cerebellar development, there is a transient burst of Sca1 expression at postnatal day 14 when the murine cerebellar cortex becomes physiologically functional. There is also marked expression of Sca1 in mesenchymal cells of the intervertebral discs during development of the spinal column. These results suggest that the normal Sca1 gene, has a role at specific stages of both cerebellar and vertebral column development.

Expression pattern of type II collagen mRNA during early vertebral development in the human embryo.

The role of extracellular matrix molecules in ontogenetic differentiation processes is a matter of increasing interest. In cartilage development, type II collagen is suspected of promoting chondrogenic differentiation, since its expression has been demonstrated in a range of precartilaginous tissues of vertebrate species. Up to now, no studies supplying a coherent description of type II collagen expression in the skeletogenesis of human embryos including early embryonic stages have been published. In this work, we examine the temporal and spatial distribution of type II collagen mRNA during vertebral column development in human embryos from 4 to 12 weeks of gestation using non-radioactive in situ hybridization. The onset of gene expression was demonstrable in the 5th week in precartilaginous mesenchymal cells and in notochordal cells. Additionally, we found a weaker hybridization signal in the mesenchymal precursors of the intervertebral discs. In the anlagen of the axial skeleton, type II collagen expression decreased during osteogenic reconstruction in the 11th/12th week, whereas expression continued in the notochordal remnants of the future nuclei pulposi. The results suggest a relatively late occurrence of type II collagen in human vertebral development compared with other vertebrate species. The distribution of gene expression is concordant with a possible role of this molecule in promoting differentiation of mesenchymal cells into chondrocytes. The mechanism of this influence remains to be established.

Pax1 is expressed during development of the thymus epithelium and is required for normal T-cell maturation

Pax1 is a transcriptional regulatory protein expressed during mouse embryogenesis and has been shown to have an important function in vertebral column development. Expression of Pax1 mRNA in the embryonic thymus has been reported previously. Here we show that Pax1 protein expression in thymic epithelial cells can be detected throughout thymic development and in the adult. Expression starts in the early endodermal epithelium lining the foregut region and includes the epithelium of the third pharyngeal pouch, a structure giving rise to part of the thymus epithelium. In early stages of thymus development a large proportion of thymus cells expresses Pax1. With increasing age, the proportion of Pax1-expressing cells is reduced and in the adult mouse only a small fraction of cortical thymic stromal cells retains strong Pax1 expression. Expression of Pax1 in thymus epithelium is necessary for establishing the thymus microenvironment required for normal T cell maturation. Mutations in the Pax-1 gene in undulated mice affect not only the total size of the thymus but also the maturation of thymocytes. The number of thymocytes is reduced about 2- to 5-fold, affecting mainly the CD4+8+ immature and CD4+ mature thymocyte subsets. The expression levels of major thymocyte surface markers remains unchanged with the exception of Thy-1 which was found to be expressed at 3- to 4-fold higher levels.

The inheritance of patterns of metameric variation in the mouse (Mus musculus) vertebral column

In this paper, we examine the patterns of metameric variation shown by different dimensions of vertebrae from the mouse vertebral column. We examine differences in metameric variation between variables within an inbred strain and compare patterns of metameric variation between inbred strains and between inbred strains and their F1 S. We make both visual and numerical comparisons, the latter being based upon a novel application of Fourier analysis. Comparisons between dimensions within a strain reveal two distinct patterns of metameric variation within the column. The first, shown only by the antero‐posterior (dorso‐ventral) diameter of the neural canal, is relatively simple: dimensions become progressively smaller caudally. The second is more complex, with two peaks, and appears more closely related to somatic structures. The second pattern may be modified according to the temporal pattern of development of different vertebral elements. Comparisons between mouse strains suggest that those variables which complete growth early, and which preserve the basic patterns of metameric variation in a relatively unmodified form, are best suited to discriminating between inbred strains. The Fl S between inbred strains show patterns of metameric variation which in some cases are more like those of one parental strain and in others more like those of the other parental strain. We consider the significance of these findings in relation to further studies of mammalian vertebral column genetics, development and evolution.