Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton.

The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.

Unveiling the tale of the tail: an illustration of spinal dysraphisms.

Spinal dysraphism is an umbrella term describing herniation of meninges or neural elements through defective neural arch. They can be broadly categorized into open and closed types. MRI is the investigation of choice to study neural abnormalities and to assess the severity of hydrocephalus and Chiari malformation. Knowledge of the embryology of these disorders is valuable in correctly identifying the type of dysraphism. The aim of surgery is untethering and dural reconstruction. Accurate depiction of the abnormal anatomy in cases of spinal dysraphism is of utmost importance for surgical management of these patients. MRI makes this possible due to its excellent soft tissue contrast resolution and multiplanar capability, allowing the radiologist to evaluate the intricate details in small pediatric spinal structures. Imaging enlightens the surgeons about the status of spinal cord and other associated abnormalities and helps detect re-tethering in operated cases. Besides, antenatal surgery to repair myelomeningoceles has made detection of open dysraphisms on fetal MRI and antenatal ultrasound critical. The purpose of this review is to describe the development of spine, illustrate the myriad imaging features of open and closed spinal dysraphisms, and enlist the reporting points the operating surgeon seeks from the radiologist.

Non-neural surface ectodermal rosette formation and F-actin dynamics drive mammalian neural tube closure.

The mechanisms underlying mammalian neural tube closure remain poorly understood. We report a unique cellular process involving multicellular rosette formation, convergent cellular protrusions, and F-actin cable network of the non-neural surface ectodermal cells encircling the closure site of the posterior neuropore, which are demonstrated by scanning electron microscopy and genetic fate mapping analyses during mouse spinal neurulation. These unique cellular structures are severely disrupted in the surface ectodermal transcription factor Grhl3 mutants that exhibit fully penetrant spina bifida. We propose a novel model of mammalian neural tube closure driven by surface ectodermal dynamics, which is computationally visualized.

Deletion of the Fanconi Anemia C Gene in Mice Leads to Skeletal Anomalies and Defective Bone Mineralization and Microarchitecture.

Fanconi anemia (FA) is a rare genetic disorder associated with a progressive decline in hematopoietic stem cells leading to bone marrow failure. FA is also characterized by a variety of developmental defects including short stature and skeletal malformations. More than half of children affected with FA have radial-ray abnormalities, and many patients have early onset osteopenia/osteoporosis. Although many Fanconi anemia genes have been identified and a molecular pathway defined, the underlying mechanism leading to bone defects remains elusive. To understand the role of FA genes in skeletal development and bone microarchitecture, we evaluated bone physiology during embryogenesis and in adult FancA- and FancC-deficient mice. We found that both FancA-/- and FancC-/- embryos have abnormal skeletal development shown by skeletal malformations, growth delay, and reduced bone mineralization. FancC-/- adult mice present altered bone morphology and microarchitecture with a significant decrease in cortical bone mineral density in a sex-specific manner. Mechanical testing revealed that male but not female FancC-/- mice show reduced bone strength compared with their wild-type littermates. Ex vivo cultures showed that FancA-/- and FancC-/- bone marrow-derived mesenchymal stem cells (BM MSC) have impaired differentiation capabilities together with altered gene expression profiles. Our results suggest that defective bone physiology in FA occurs in utero and possibly results from altered BM MSC function. These results provide valuable insights into the mechanism involved in FA skeletal defects. © 2018 American Society for Bone and Mineral Research.

Dichorionic twins discordant for body-stalk anomaly: a management challenge.

Body-stalk anomaly is a sporadic and rare maldevelopment disorder characterised by large abdominal wall defect, spinal deformity and rudimentary umbilical cord. It is considered a lethal condition as there are only few reports of survival but there was at least one case of long-term survival after neonatal surgery.Differential diagnosis includes isolated omphalocele or gastroschisis, short umbilical cord, amniotic band, limb body-wall complex and other polymalformative syndromes.There are few reports about the expectant prenatal management of the body stalk anomaly as the majority of prenatal diagnosed cases undergo early elective termination. Twin pregnancies discordant for the anomaly represent a challenge to prenatal management as a healthy fetus should also be considered.We describe a case of dichorionic-diamniotic twins discordant for body stalk anomaly which underwent selective feticide of the affected fetus late in pregnancy, in accordance with parents' decision focused on the neonatal well-being of the unaffected twin.

The sp7 gene is required for maturation of osteoblast-lineage cells in medaka (Oryzias latipes) vertebral column development.

Sp7 is a zinc finger transcription factor that is essential for osteoblast differentiation in mammals. To verify the characteristic features of osteoblast-lineage cells in teleosts, we established medaka sp7 mutants using a transcription activator-like effector nuclease (TALEN) genome editing system. These mutants showed severe defects in the formation of skeletal structures. In particular, the neural and the hemal arches were not formed, although the chordal centra were formed. Analysis of the transgenic medaka revealed that sp7 mutant had normal distribution of type X collagen a1 a (col10a1a)-positive osteoblast-like cells around the centrum and at the proximal region of the vertebral arch. The sp7 mutant phenotype could be rescued by exogenous sp7 expression in col10a1a-positive cells, as well as in sp7-positive osteoblast cells. Furthermore, runx2-positive osteoblast progenitors were observed on the vertebral arches, but not on the centrum, during vertebral column development. In addition, these osteoblast progenitors differentiated into the col10a1a-positive cells. In sp7 mutant, the runx2-positive cells were normally distributed at the region of unformed vertebral arch but failed to differentiate into col10a1a-positive cells. These results indicate that osteoblast-lineage cells undergo two distinct differentiation processes during development of the vertebral arch and the centrum. Nevertheless, our results verified that sp7 gene expression in osteoblast-lineage cells is required for differentiation into mature osteoblasts to form the vertebral column and other skeletal structures.

Morphometric development of the vertebral segments of the vertebral column in the goat (Capra hircus) during the fetal period / Desarrollo morfométrico de los segmentos vertebrales de la columna vertebral en cabra (Capra hircus) durante el periodo fetal

Understanding normal fetal growth rates of the vertebral column, and between species, provide a basis to establish reference values for evaluation of body development and estimation of fetal age by ultrasonography. Goats are also widely used in biomedical perinatal research and are still considered a key to understanding the skeletal development. This study was carried out to clarify growth of length of vertebral column at segmental, regional, and total level of vertebral column in the fetal goats during different gestational age. The length of each vertebral segment of 25 goat fetuses, aged between 6 and 20 weeks were measured for each vertebra using a digital caliper. Our study demonstrated differences among various fetal ages in terms of regional, segmental, and total growth rate of the length of vertebral segments. With increase of fetal age, the relative length of vertebral segments of cervical, thoracic and lumbar regions diminished, whereas sacral and caudal regions increased in relative length. The thoracic vertebrae were the longest followed by cervical, lumbar, caudal, and sacral regions except at the oldest fetuses where caudal region became longer than lumbar region. Although the longest and shortest vertebral segments in cervical and lumbar regions were consistent among age groups, the trend of segmental growth of the vertebral regions was variable. Based on these detailed findings, the relative regional lengths of vertebral column were essentially different among fetal goats, humans, and neonatal rats. There is also a general trend of increasing segmental and regional initial growth and there is a relatively significant increase in growth rate caudally along the column during fetal period. This research yield important results that may be also useful for future orthopedic studies that contemplate the use of goat as a new model for the human spine.

Entender el crecimiento fetal normal de la columna vertebral entre las especies, proporciona una base para establecer valores de referencia en la evaluación del desarrollo corporal y la estimación de la edad fetal por ultrasonografía. Las cabras se utilizan frecuentemente en investigaciones biomédicas perinatales y son consideradas clave en el estudio del desarrollo esquelético. Este estudio se realizó con el objetivo de determiner el crecimiento de longitud de la columna vertebral a nivel segmentario, regional y total de la columna vertebral en cabras fetales durante diferentes etapas gestacionales. La longitud de cada segmento vertebral de 25 fetos de cabra, con edades comprendidas entre las 6 y 20 semanas se midió utilizando un calibre digital. Nuestro estudio demostró diferencias entre varias edades fetales en términos de tasa de crecimiento regional, segmentario y total de longitud de los segmentos vertebrales Con el aumento de la edad fetal, la longitud relativa de los segmentos vertebrales de las regiones cervical, torácica y lumbar disminuyó, mientras que las regiones sacras y caudales aumentaron en longitud relativa. Las vértebras torácicas fueron las más largas seguidas por las regiones cervical, lumbar, caudal y sacral excepto en los fetos más antiguos donde la región caudal se hizo más larga que la región lumbar. Aunque los segmentos vertebrales más largos y más cortos en las regiones cervical y lumbar fueron consistentes entre los grupos de edad, la tendencia de crecimiento segmentario de las regiones vertebrales fue variable. En base a estos resultados, las longitudes relativas de columna vertebral fueron esencialmente diferentes entre cabras fetales, humanos y ratas neonatas. También existe una tendencia general de aumento del crecimiento inicial segmentario y regional, como tambien un aumento relativamente significativo en la tasa de crecimiento a lo largo de la columna durante el período fetal. Esta investigación arroja importantes resultados que también pueden ser útiles para futuros estudios ortopédicos que contemplan el uso de la cabra como un nuevo modelo para la columna vertebral humana.

Absence of collagen IX accelerates hypertrophic differentiation in the embryonic mouse spine through a disturbance of the Ihh-PTHrP feedback loop.

Collagen IX (Col IX) is a component of the cartilage extracellular matrix and contributes to its structural integrity. Polymorphisms in the genes encoding the Col IX ɑ2- and ɑ3-chains are associated with early onset of disc degeneration. Col IX-deficient mice already display changes in the spine at the newborn stage and premature disc degeneration starting at 6 months of age. To determine the role of Col IX in early spine development and to identify molecular mechanisms underlying disc degeneration, the embryonic development of the spine was analyzed in Col IX -/- mice. Histological staining was used to show tissue morphology at different time points. Localization of extracellular matrix proteins as well as components of signaling pathways were analyzed by immunohistochemistry. Developing vertebral bodies of Col IX -/- mice were smaller and already appeared more compact at E12.5. At E15.5, vertebral bodies of Col IX -/- mice revealed an increased number of hypertrophic chondrocytes as well as enhanced staining for the terminal differentiation markers alkaline phosphatase and collagen X. This correlates with an imbalance in the Ihh-PTHrP signaling pathway at this time point, reflected by an increase of Ihh and a concomitant decrease of PTHrP expression. An accelerated hypertrophic differentiation caused by a disturbed Ihh-PTHrP signaling pathway may lead to a higher bone mineral density in the vertebral bodies of newborn Col IX -/- mice and, as a result, to the early onset of disc degeneration.

Notochord isolation using laser capture microdissection.

BACKGROUND: Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. METHODS: Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. RESULTS: This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. CONCLUSION: LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis.

Tracheal Atresia with Segmental Esophageal Duplication: An Unusual Anatomic Arrangement.

An unusual anatomic configuration of segmental tracheal agenesis/atresia with esophageal duplication on autopsy in a fetus that demised in utero at 29 weeks is reported. The mother was scanned initially for a cardiac anomaly at 20 weeks and on follow-up scan at 27 weeks had polyhydramnios and underwent amnioreduction. The final autopsy diagnosis was vertebral, ano-rectal, cardiac, tracheoesophageal, renal, and limb malformations (VACTERL). We discuss the autopsy findings along with the embryological mechanisms and compare the configuration with Floyd's classification for tracheal agenesis. The difficulties in prenatal diagnosis are discussed.

Desarrollo osteológico de la columna vertebral y del complejo caudal de larvas de Lutjanus guttatus (Perciformes: Lutjanidae) en condiciones de cultivo / Osteological development of the vertebral column and caudal complex of Lutjanus guttatus (Perciformes: Lutjanidae) larvae under rearing conditions

The spotted rose snapper (Lutjanus guttatus) is an important commercial species in Mexico with good culture potential. The osteological study at early stages in this species is an important tool to confirm normal bone structure and for the detection of malformations that may occur during early development. This study was carried out in order to evaluate and describe the normal osteological development of the vertebral column and caudal complex of this species grown under controlled conditions. For this, a total of 540 larvae of L. guttatus, between 2.1 and 17.5mm of total length (TL), were cultured during 36 days; culture conditions were 28ºC, 5.74mg/L oxygen and 32.2ups salinity with standard feeding rates. To detect growth changes, a sample of 15 organisms was daily taken from day one until day 36 of post-hatch (DPH). Samples were processed following standard techniques of clearing, and cartilage (alcian blue) and bone staining (alizarin red). Results showed that the vertebral column is composed of ten vertebrae in the abdominal region, and 14 vertebrae including the urostyle in the caudal region. The development of the axial skeleton starts with the neural arches and haemal arches at 3.8mm TL. Caudal elements such as the hypurals and parahypural began to develop at 4.1mm TL. Pre-flexion and flexion of the notochord and the formation of all hypurals were observed between 5.3 and 5.8mm TL. Ossification of the vertebrae in the abdominal region and in some neural arches initiated at 9.5mm TL. In the caudal region, all the neural and haemal arches ossified at 10.2mm TL. All the abdominal vertebrae and their respective neural arches and parapophyses ossified at 11.2mm TL, while the elements of the caudal complex that ossified were the hypurals, parahypurals and modified haemal spines. All caudal fin rays, 12 neural spines and 3 haemal arches were ossified by 15.5mm. The complete ossification process of this specie under laboratory culture conditions was observed when larvae reached 17.3mm TL on 36 DPH. Detailed analysis of the osteological structures will allow a reference description to evaluate and detect malformations that may occur during the larval culture of the spotted rose snapper.

El pargo flamenco (Lutjanus guttatus) es una especie de importancia comercial en México con un gran potencial para su cultivo. El estudio osteológico en estadios tempranos de esta especie bajo condiciones controladas, es una herramienta importante para el conocimiento de su estructura ósea normal y poder detectar las malformaciones que se puedan presentar. El objetivo del presente trabajo se realizó para conocer y describir el desarrollo osteológico normal de la columna vertebral y el complejo caudal de 540 larvas de 2.1 a 17.5mm de longitud total (LT) bajo condiciones de cultivo a 28°C, 5.74mg/L de oxígeno y 32.2UPS de salinidad. Diariamente se tomó una muestra de 15 organismos desde el día uno hasta el 36 después de la eclosión (DDE) y se procesaron con las técnicas de clareado y tinción de cartílago (azul aciano) y hueso (rojo alizarina) para llevar a cabo la descripción de las estructuras. La columna vertebral se divide en región abdominal con diez vértebras y región caudal compuesta por 14 vértebras incluido el urostilo. El desarrollo del esqueleto axial inicia con la formación de los arcos neurales y hemales a los 3.8mm de LT. A los 4.1mm de LT empieza la formación de los hipurales y parahipural que son elementos caudales. Entre los 5.3 y 5.8mm de LT se observó en pre-flexión y flexión del notocordio y la formación de todos los hipurales. La osificación de las vértebras en la región abdominal y en algunos arcos neurales inició a los 9.5mm de LT. A los 10.2mm de LT se osificó la región caudal y todos los arcos neurales y hemales. A los 11.2mm LT se osificaron todas las vértebras abdominales con sus respectivos arcos neurales y los parapófisis, mientras que los elementos del esqueleto caudal que se osificaron fueron los hipurales, parahipurales y las espinas hemales modificadas. A los 15.5mm de LT se osificaron los radios de la aleta caudal y 12 espinas neurales y 3 hemales. El proceso de osificación de las larvas de esta especie en condiciones de cultivo se completó a los 17.3mm LT o 36 DDE. El análisis detallado de las estructuras osteológicas, permitirá una descripción de referencia para evaluar y detectar las malformaciones que se puedan presentar durante el cultivo larvario.

MBTPS1/SKI-1/S1P proprotein convertase is required for ECM signaling and axial elongation during somitogenesis and vertebral development†.

Caudal regression syndrome (sacral agenesis), which impairs development of the caudal region of the body, occurs with a frequency of about 2 live births per 100 000 newborns although this incidence rises to 1 in 350 infants born to mothers with gestational diabetes. The lower back and limbs can be affected as well as the genitourinary and gastrointestinal tracts. The axial skeleton is formed during embryogenesis through the process of somitogenesis in which the paraxial mesoderm periodically segments into bilateral tissue blocks, called somites. Somites are the precursors of vertebrae and associated muscle, tendons and dorsal dermis. Vertebral anomalies in caudal regression syndrome may arise through perturbation of somitogenesis or, alternatively, could result from defective bone formation and patterning. We discovered that MBTPS1/SKI-1/S1P, which proteolytically activates a class of transmembrane transcription factors, plays a critical role in somitogenesis and the pathogenesis of lumbar/sacral vertebral anomalies. Conditional deletion of Mbtps1 yields a viable mouse with misshapen, fused and reduced number of lumbar and sacral vertebrae, under-developed hind limb bones and a kinky, shortened tail. We show that Mbtps1 is required to (i) maintain the Fgf8 'wavefront' in the presomitic mesoderm that underpins axial elongation, (ii) sustain the Lfng oscillatory 'clock' activity that governs the periodicity of somite formation and (iii) preserve the composition and character of the somitic extracellular matrix containing fibronectin, fibrillin2 and laminin. Based on this spinal phenotype and known functions of MBTPS1, we reason that loss-of-function mutations in Mbtps1 may cause the etiology of caudal regression syndrome.

Fetal hemivertebra: associations and perinatal outcome.

OBJECTIVES: To assess the accuracy of antenatal diagnosis of hemivertebra, to quantify the association with coexisting anomalies and to determine the perinatal outcome. METHOD: This was a retrospective observational study of all cases of suspected fetal or neonatal hemivertebra identified via the UK Southwest Congenital Anomaly Register (SWCAR) between 2002 and 2012. RESULTS: From a total of 88 cases of hemivertebra identified during the study period, data were obtained for 67 of them: 45 (10 isolated and 35 with coexisting anomalies) cases were suspected antenatally and 22 (10 isolated and 12 with coexisting anomalies) were diagnosed postnatally. Of the cases detected postnatally, five (four with coexisting anomalies) were unsuspected and diagnosed at postmortem examination. The most commonly associated anomalies included additional skeletal abnormalities (n = 16), genitourinary abnormalities (n = 10), VATER/VACTERL association (n = 5), cardiac abnormalities (n = 4) and central nervous system abnormalities (n = 4). In cases with coexisting anomalies there was a 48% fetal/neonatal loss, compared to 19% in cases with isolated hemivertebra. CONCLUSIONS: Although antenatal diagnosis of hemivertebra was accurate, a third of the cases were diagnosed only postnatally. These data suggest a difficulty in antenatal diagnosis of the condition. The majority of cases of hemivertebra had coexisting anomalies, and in these cases the rate of perinatal loss was high. These data should be useful in providing additional information for counseling when a diagnosis of hemivertebra is made.

Foetal "black bone" MRI: utility in assessment of the foetal spine.

OBJECTIVE: Foetal CT has recently been added to the foetal imaging armamentarium, but this carries with it the risks of ionizing radiation, both to the mother and the foetus. Foetal "black bone" MRI is a new technique that allows assessment of the foetal skeleton without the risk of exposure to ionizing radiation and is a potential new sequence in foetal MRI examination. METHODS: Retrospective review of all foetal MRI studies over the past 4- to 5-year period identified 36 cases where susceptibility weighted imaging was used. Cases were selected from this group to demonstrate the potential utility of this sequence. RESULTS: This sequence is most frequently useful not only in the assessment of spinal abnormalities, most commonly the bony abnormalities in myelomeningocele, but also in cases of scoliosis, segmentation anomalies and sacrococcygeal teratoma. CONCLUSION: Although the utility of this sequence is still being evaluated, it provides excellent contrast between the mineralized skeleton and surrounding soft tissues compared with standard half Fourier acquisition single-shot turbo-spin echo sequences. Further assessment is required to determine whether black bone MRI can more accurately evaluate the level of bony defect in spina bifida aperta, an important prognostic factor. Potential further uses include the assessment of skeletal dysplasias, evaluation of the skull base and craniofacial skeleton in certain congenital anomalies and the post-mortem evaluation of the foetal skeleton potentially obviating the need for necropsy. ADVANCES IN KNOWLEDGE: Foetal black bone MRI can be performed using susceptibility weighted imaging and allows better demonstration of the mineralized skeleton compared with standard sequences.

Spine ultrasound imaging in the newborn.

Ultrasound of the spine in the neonate is widely used as the initial modality to evaluate spinal canal anatomy, anatomical variants, and congenital malformations. The spinal canal and its contents are best visualized in the newborn and young infant owing to incomplete ossification of the posterior vertebral elements.

Transient infection of the zebrafish notochord with E. coli induces chronic inflammation.

Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation.

Runx2 regulates endochondral ossification through control of chondrocyte proliferation and differentiation.

Synthesis of cartilage by chondrocytes is an obligatory step for endochondral ossification. Global deletion of the Runx2 gene results in complete failure of the ossification process, but the underlying cellular and molecular mechanisms are not fully known. Here, we elucidated Runx2 regulatory control distinctive to chondrocyte and cartilage tissue by generating Runx2 exon 8 floxed mice. Deletion of Runx2 gene in chondrocytes caused failure of endochondral ossification and lethality at birth. The limbs of Runx2(ΔE8/ΔE8) mice were devoid of mature chondrocytes, vasculature, and marrow. We demonstrate that the C-terminus of Runx2 drives its biological activity. Importantly, nuclear import and DNA binding functions of Runx2 are insufficient for chondrogenesis. Molecular studies revealed that despite normal levels of Sox9 and PTHrP, chondrocyte differentiation and cartilage growth are disrupted in Runx2(ΔE8/ΔE8) mice. Loss of Runx2 in chondrocytes also impaired osteoprotegerin-receptor activator of NF-κB ligand (OPG-RANKL) signaling and chondroclast development. Dwarfism observed in Runx2 mutants was associated with the near absence of proliferative zone in the growth plates. Finally, we show Runx2 directly regulates a unique set of cell cycle genes, Gpr132, Sfn, c-Myb, and Cyclin A1, to control proliferative capacity of chondrocyte. Thus, Runx2 is obligatory for both proliferation and differentiation of chondrocytes.

Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis.

The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H(+)-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

Sprengel's deformity and spinal dysraphism: connecting the shoulder and the spine.

BACKGROUND AND PURPOSE: Sprengel's deformity, a rare congenital malformation of the scapula, may be observed in combination with spinal dysraphism. The co-occurrence of these malformations suggests an unknown shared etiology. Therefore, we reviewed the medical records of eight children presenting with both malformations and performed a review of the literature. PATIENTS AND METHODS: Databases from four university medical centers were searched for children presenting between 1992 and 2012 with spinal dysraphism and a Sprengel's deformity. CONCLUSION: The combination of spinal dysraphism and Sprengel's deformity is rare, and is associated with segmentation defects of the spine and ribs. Although the etiology of both spinal dysraphism and Sprengel's deformity remains unclear, all deformities of the spine, ribs, and shoulder might result from a common genetic defect affecting somitogenesis.