Loss of lysyl oxidase-like 3 causes cleft palate and spinal deformity in mice.

In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.

Morphometric study of the T6 vertebra and its three ossification centers in the human fetus.

PURPOSE: Knowledge on the normative growth of the spine is critical in the prenatal detection of its abnormalities. We aimed to study the size of T6 vertebra in human fetuses with the crown-rump length of 115-265 mm. MATERIALS AND METHODS: Using the methods of computed tomography (Biograph mCT), digital image analysis (Osirix 3.9) and statistics, the normative growth of the T6 vertebral body and the three ossification centers of T6 vertebra in 55 spontaneously aborted human fetuses (27 males, 28 females) aged 17-30 weeks were studied. RESULTS: Neither male-female nor right-left significant differences were found. The height, transverse, and sagittal diameters of the T6 vertebral body followed natural logarithmic functions as y = -4.972 + 2.732 × ln(age) ± 0.253 (R (2) = 0.72), y = -14.862 + 6.426 × ln(age) ± 0.456 (R (2) = 0.82), and y = -10.990 + 4.982 × ln(age) ± 0.278 (R (2) = 0.89), respectively. Its cross-sectional area (CSA) rose proportionately as y = -19.909 + 1.664 × age ± 2.033 (R (2) = 0.89), whereas its volumetric growth followed the four-degree polynomial function y = 19.158 + 0.0002 × age(4) ± 7.942 (R (2) = 0.93). The T6 body ossification center grew logarithmically in both transverse and sagittal diameters as y = -14.784 + 6.115 × ln(age) ± 0.458 (R (2) = 0.81) and y = -12.065 + 5.019 × ln(age) ± 0.315 (R (2) = 0.87), and proportionately in both CSA and volume like y = -15.591 + 1.200 × age ± 1.470 (R (2) = 0.90) and y = -22.120 + 1.663 × age ± 1.869 (R (2) = 0.91), respectively. The ossification center-to-vertebral body volume ratio was gradually decreasing with age. On the right and left, the neural ossification centers revealed the following models: y = -15.188 + 6.332 × ln(age) ± 0.629 (R (2) = 0.72) and y = -15.991 + 6.600 × ln(age) ± 0.629 (R (2) = 0.74) for length, y = -6.716 + 2.814 × ln(age) ± 0.362 (R (2) = 0.61) and y = -7.058 + 2.976 × ln(age) ± 0.323 (R (2) = 0.67) for width, y = -5.665 + 0.591 × age ± 1.251 (R (2) = 0.86) and y = -11.281 + 0.853 × age ± 1.653 (R (2) = 0.78) for CSA, and y = -9.279 + 0.849 × age ± 2.302 (R (2) = 0.65) and y = -16.117 + 1.155 × age ± 1.832 (R (2) = 0.84) for volume, respectively. CONCLUSIONS: Neither sex nor laterality differences are found in the morphometric parameters of evolving T6 vertebra and its three ossification centers. The growth dynamics of the T6 vertebral body follow logarithmically for its height, and both sagittal and transverse diameters, linearly for its CSA, and four-degree polynomially for its volume. The three ossification centers of T6 vertebra increase logarithmically in both transverse and sagittal diameters, and linearly in both CSA and volume. The age-specific reference intervals for evolving T6 vertebra present the normative values of potential relevance in the diagnosis of congenital spinal defects.

Early development of the costovertebral joints.

Researches on the development of the vertebral column were focused mainly on the growth of lumbar and cervical regions. However, none of them were related to the development of costovertebral joints. To our knowledge, the relationship between ribs and thoracic vertebrae has never been addressed. The aims of the presented study are: (1) to trace the formation of processes of vertebral arches and articular surfaces of the vertebral bodies, and (2) to trace the development of the ribs, particularly their joint surfaces. Study subjects are staged human embryos (from the 5th week) from the collection of the Department of Anatomy, Poznan University of Medical Sciences in Poland. Serial sections were stained according to methods allowed for tracing the development of investigated structures. The primordia of costovertebral joints are interzones from which differentiate all structures of synovial joints such as joint surfaces, capsule and cavity. Based on the result of this study, these interzones appeared in the embryonic stages 17 and 18 with approximately 40 to 42 postfertilizational days, and the cavitation within the interzone was observed in some embryos from stage 19 with approximately postfertilizational 44 days.

Fetal anatomy of the lower cervical and upper thoracic fasciae with special reference to the prevertebral fascial structures including the suprapleural membrane.

The aim of this study was to find basic rules governing the fetal anatomy of the deep cervical fasciae and their connections to the mediastinal fasciae. We examined the histology of paraffin-embedded preparations of 18 mid-term fetuses (5 between 9 and 12 weeks of gestation, 3 between 15 and 18 weeks, and 10 between 20 and 25 weeks). The prevertebral lamina of the deep cervical fasciae (PLDCF) developed as an intermediate aponeurosis for the bilateral bellies of the longus colli muscles. In contrast, the alar fascia developed as a connecting band between the bilateral adventitiae of the common carotid artery. The retropharyngeal fascia became evident much later than the latter two fasciae. The fascia covering the thymus was thicker than the fascia for the strap muscles (the pretracheal lamina of the cervical fascia). The primitive suprapleural membrane, or Sibson's fascia, contained veins and fatty tissues, and was composed of the alar fascia rather than the PLDCF, tranversalis fascia, or endothoracic fascia. The prevertebral two-laminar configuration was rather evident in the early stages of development because, in the later stages, the fasciae together provided a multilaminar structure, especially in the lateral area in front of the longus colli, which suspended the cupula pleurae. To consider a continuation from the base of the neck to the upper mediastinum, the alar fascia seems to be a key structure for connecting the vascular sheath to the parietal pleura.

From fetus to adult--an allometric analysis of the giraffe vertebral column.

As mammalian cervical vertebral count is almost always limited to seven, the vertebral column of the giraffe (Giraffa camelopardalis) provides an interesting study on scaling and adaptation to shape in light of these constraints. We have defined and described the growth rates of the lengths, widths, and heights of the vertebrae from fetal through neonatal life to maturity. We found that the disproportionate elongation of the cervical vertebrae is not a fetal process but occurs after birth, and that each cervical (C2-C7) vertebrae elongates at the same rate. C7 is able to specialize toward elongation as its function has been shifted to T1. We concluded that T1 is a transitional vertebra whose scaling exponent and length is between that of the cervical and thoracic series. Despite its transitional nature, T1 is still regarded as thoracic, as it possesses an articulating rib that attaches to the sternum. The other dimensions taken (width, height, and spinous process length) show that giraffe vertebral morphology exhibit adaptations to biomechanical strain, and we have underlined the importance of the thoracic spinous processes in supporting the head and neck.

Transgenic over-expression of growth differentiation factor 11 propeptide in skeleton results in transformation of the seventh cervical vertebra into a thoracic vertebra.

Growth differentiation factor 11 (GDF11) is one of the significant genes that control skeletal formation. Knockout of GDF11 function causes abnormal patterning of the anterior/posterior axial skeleton. The mRNA of GDF11 is initially translated to a precursor protein that undergoes a proteolytic cleavage to generate the C-terminal peptide or mature GDF11, and the N-terminal peptide named GDF11 propeptide. The propeptide can antagonize GDF11 activity in vitro. To investigate the effects of GDF11 propeptide on GDF11 function in vivo, we generated transgenic mice that over-express the propeptide cDNA in skeletal tissue. The transgenic mice showed formation of extra ribs on the seventh cervical vertebra (C7) as a result of transformation of the C7 vertebra into a thoracic vertebra. The GDF11 propeptide transgene mRNA was detected in tail tissue in embryos and was highly expressed in tail and calvaria bones after birth. A high frequency of C7 rib formation was noticed in the transgenic mouse line with a high level of transgene expression. The anterior boundaries of Hoxa-4 and Hoxa-5 mRNA in situ expressions showed cranial shifts from their normal prevertebra locations in transgenic embryos. These results demonstrated significant effects of GDF11 propeptide transgene on vertebral formation, which are likely occurring through depressing GDF11 function and altered locations of Hoxa-4 and Hoxa-5 expression.

Formation of the vertebral arches of the cervical, thoracic and lumbar vertebrae in early human foetuses.

Fusion of the neural arches was studied in 6 serially sectioned human foetuses aged 9 and 10 weeks. In foetuses of 9 weeks, the completion of arches was observed in the cervical, upper thoracic, and middle thoracic regions of the vertebral column. During the 10th week of development, fusion of neural processes progresses in the lower thoracic and upper three lumbar vertebrae.

The first appearance of sympathetic ganglia in human embryos at stage 13.

The primordia of the sympathetic trunk ganglia were traced on serial sections of 10 embryos at stage 13 (32 postovulatory days). It was found that in all embryos, these primordia were present in the thoracic level T4 to T9 and they appeared as scattered aggregates of cells lying dorsally and laterally to the dorsal aortae.

Innervation of the human cervical and thoracic vertebrae at eight postovulatory weeks.

The nerves to the cervical and thoracic vertebrae were traced in 10 serially sectioned human embryos. It was found that the vertebral bodies receive nerve fibres from the trunks of the spinal nerves, anterior branches and meningeal branches of the spinal nerves, and from the sympathetic trunks. Slender twigs from the trunk of the spinal nerve arise close to the spinal ganglion and terminate in the posterior and lateral surfaces of the vertebrae. Fibres from the anterior branches of the spinal nerves terminate in the lateral and anterior surfaces of the vertebrae. Thin rami from the sympathetic trunk reach the anterior surface of the vertebrae.

Conservation in the Hox code during morphological evolution.

The expression domains in paraxial mesoderm of the chicken embryo are described for Hoxb-3, a-4 and c-6 genes, and these are compared with published expression data for the corresponding genes in the mouse. In both species, it is found that the anterior limits of Hoxb-3 and a-4 expression lie in the upper cervical region, and the anterior limits of Hoxc-6 expression lie in the upper thoracic region. This finding is remarkable because the cervical region, or neck, of the chicken (with fourteen cervical vertebrae) is much longer than that of the mouse (seven cervical vertebrae). The results suggest that the Hox code, at least in the development of homologous axial structures, is conserved between species (Hoxb-3 and a-4, for example, being associated with an anterior cervical phenotype; Hoxc-6 being associated with an anterior thoracic phenotype). The results also suggest that an evolutionary change in body proportions is accomplished by a shift in the relative positions of Hox expression domains during embryonic development.

Ultrasonographic assessment of fetal growth in miniature "Shiba" goats (Capra hircus).

The aim of the present study was to monitor fetal growth in relation to gestational stage to generate formulae which could be used to estimate fetal age in goats. Eight miniature Shiba goats (Capra hircus) were examined weekly by transrectal and transabdominal ultrasound scanning during the gestation period between Day 21 and 126 days of gestation. For accurate judgment, all fetometric parameters were measured at least three times per one examination for each animal. Quantification of the growth of the fetus allowed the development of a number of predictors of fetal age. Low correlations were associated with measurement of the chest diameter (R(2)=0.869), trunk diameter (R(2)=0.8969), tibia length (R(2)=0.8662) and placentome diameter (R(2)=0.8999). Moderate correlation was assessed by calculation of the length of six successive lumbar vertebrae (R(2)=0.9296), femur length (R(2)=0.9278), heart axis length (R(2)=0.9382 and 0.9589; for the longitudinal and transverse axis, respectively), occipitonasal length (R(2)=0.9527), umbilical cord diameter (R(2)=0.9119) and orbit diameter (R(2)=0.9239). A high correlation was estimated in investigating the length of six successive thoracic vertebrae (R(2)=0.9674), braincase diameter (R(2)=0.9831) and crown rump length (R(2)=0.9848). In conclusion, the intrauterine fetal biometry estimation through ultrasound might be useful to predict the accurate gestational age in miniature goats.

Abnormal development of vertebrae in paralyzed chick embryos.

Ventral bars, cartilaginous projections from the ventral aspect of the synsacrum that contact and form a joint with the ilium, were found in all normal chick embryos of age E9 and older. Bars were absent in a number of embryos which had been paralyzed from age E4 by the use of the acetylcholine receptor blocker alpha-bungarotoxin. They were also absent in some embryos that had been paralyzed between ages E4 and E10 but allowed to move thereafter. The bars, already formed, remained present in a third group of embryos in which paralysis was initiated age E10. Apparently, normal embryonic movements induce the formation of bars. In support of this conclusion is the observation that two of three embryos which had had their hindlimb buds amputated at age E3 lacked bars. In these embryos with amputations, the ilium was present at least in part, but the forces exerted on the region where the bars develop would have been greatly reduced because of the lack of hind limb musculature. It is concluded that the bars, which form part of the iliosynsacral joint, are induced epigenetically by normal embryonic movements.

Innervation and functional characteristics of connective tissues, especially elastic fibers, in human fetal thoracic intervertebral articular capsule and its surroundings.

The articular capsules between the thoracic vertebrae, which have physiologically different functions from those of other levels of the vertebrae, have yet to be subjected to neuro-anatomical and fine structural analysis. In the present study, we analyzed serial frozen sections of decalcified thoracic vertebrae in human fetuses, and identified the articular capsule tissue with its unique distribution of elastic fibers. The fine structure of the elastic fibers was studied by transmission electron microscopy. In the early-stage fetus, the fibrous membrane forming the lateral intervertebral articular capsule contained abundant thin elastic fibers consisting of microfibrils. In the late-stage fetus, the lateral capsule of fibrous membrane was occupied by thick elastic fibers. A medial articular capsule, namely the ligamenta flava, contained numerous thick elastic fibers in both early and late-stage fetuses. The distributional differences in nerve fibers between early and late-stage fetuses were determined by immunostaining, using antibodies raised against protein gene product 9.5 (PGP 9.5; ubiquitin carboxyl-terminal hydrolase). Innervation by PGP 9.5 immunoreactive fibers was limited to the areas of the articular capsules near the blood vessels, which may indicate their functional relation with blood flow. No PGP 9.5 immunoreactive fibers were found in the ligamenta flava of the late-stage fetus. Innervation might be directly involved in the development of the intervertebral articular capsules in normal human fetuses.

An immunohistochemical study of protein kinase C distribution in fetal mouse vertebral column.

Using polyclonal antibodies we have studied the distribution of protein kinase C in fetal mouse low thoracic vertebrae. By means of a pan protein kinase C antiserum recognizing the catalytic domain of the enzyme, we show that protein kinase C is markedly expressed in chondrocytes before birth. The enzyme seems to be very abundant in the more mature cells that are close to ossification centres as well as the periphery of the intervertebral disc, although it can also be detected in chondrocytes. In order to establish which protein kinase C isoenzyme(s) the chondrocytes produce, we employed polyclonal isoenzyme-specific antisera developed against three calcium-dependent isoforms (alpha, beta, gamma) and three calcium-independent isoforms (delta, epsilon, zeta). Secondary antibody conjugated to alkaline phosphatase revealed that chondrocytes markedly express the beta-isoform. Cells were also weakly stained by the anti-epsilon serum. The immunostaining was completely abolished by pre-incubating primary antibodies with the peptide antigens to which they were raised. These results suggest that protein kinase C (and particularly the beta isoform) could play an important role in mouse fetal chondrogenesis of the vertebral column.

A quantitative study on the spatial and temporal ossification patterns of vertebral centra and neural arches and their relationship to the fetal age.

A double-staining technique on 37 human embryos and fetuses (crown-rump length, CRL, between 38 and 116 mm) has been performed to study the ossification patterns of the vertebral column. Different growth sequences for centra and neural arches were observed. The survey of ossified centers suggested it was possible to relate significantly their appearance with the CRL. On the basis of already known data defining the developmental age in relationship to the latter parameter, we suggest their numerical evaluation as a further parameter for the assessment of the fetal age. Therefore, we have worked out a table that may be used either to determine the normal fetal growth, or when other parameters cannot be relied upon (i.e. in morphological diseases) for this aim.

Development of the spinous process of the second thoracic vertebra of the mouse in the late fetal and early postnatal period.

The bony spinous process of T2 in certain inbred strains of the mouse is variable in size or in some cases absent. The development of this process has been investigated in histological sections of CBA, C57BL and tk/tk mice between birth and 14 days. The spinous process is shown to be modified in shape and size late in development.

Skeletopy of the brachiocephalic trunk and the common carotid arteries in human fetuses.

By means of anatomical and radiological methods and with help of the Computer Digital Image Analysis System the brachiocephalic trunk and the common carotid arteries in relation to the vertebral column were studied in 60 human fetuses. The vessels were found to be between the upper borders: of the third thoracic vertebra and the first cervical vertebra (Th3s-C1s). In the 6th month of the ontogenetic development the vessels descended by one vertebra and established their location in the next prenatal compartment (8-9) between the lower borders: of these same vertebra (Th3i-C1i). We have counted the skeletopie age correlation coefficients of these vessels and found the diminuation of their values. Sexual skeletopic dimorphism have not been observed. These investigations have clinical implications.

Dorsolumbar spine duplication.

We report a case of dorsolumbar spine duplication in a female patient. She had several vertebral anomalies, such as fused vertebrae, hemivertebrae and butterfly vertebrae, together with duplication of the spinal column in the lower thoracic and the whole lumbar tract. Clinically, she had no control of the sphincters, but her gait was only slightly affected. This is probably the third reported case of this rare anomaly.

Loss of NAC1 expression is associated with defective bony patterning in the murine vertebral axis.

NAC1 encoded by NACC1 is a member of the BTB/POZ family of proteins and participates in several pathobiological processes. However, its function during tissue development has not been elucidated. In this study, we compared homozygous null mutant Nacc1(-/-) and wild type Nacc1(+/+) mice to determine the consequences of diminished NAC1 expression. The most remarkable change in Nacc1(-/-) mice was a vertebral patterning defect in which most knockout animals exhibited a morphological transformation of the sixth lumbar vertebra (L6) into a sacral identity; thus, the total number of pre-sacral vertebrae was decreased by one (to 25) in Nacc1(-/-) mice. Heterozygous Nacc1(+/-) mice had an increased tendency to adopt an intermediate phenotype in which L6 underwent partial sacralization. Nacc1(-/-) mice also exhibited non-closure of the dorsal aspects of thoracic vertebrae T10-T12. Chondrocytes from Nacc1(+/+) mice expressed abundant NAC1 while Nacc1(-/-) chondrocytes had undetectable levels. Loss of NAC1 in Nacc1(-/-) mice was associated with significantly reduced chondrocyte migratory potential as well as decreased expression of matrilin-3 and matrilin-4, two cartilage-associated extracellular matrix proteins with roles in the development and homeostasis of cartilage and bone. These data suggest that NAC1 participates in the motility and differentiation of developing chondrocytes and cartilaginous tissues, and its expression is necessary to maintain normal axial patterning of murine skeleton.

Comparative immunolocalization of the elastin fiber-associated proteins fibrillin-1, LTBP-2, and MAGP-1 with components of the collagenous and proteoglycan matrix of the fetal human intervertebral disc.

STUDY DESIGN: A comparative immunolocalization study of elastin-associated proteins and established intervertebral disc (IVD) extracellular matrix (ECM) components. OBJECTIVE: To localize for the first time, elastic fiber­associated proteins with structural fibrillar components in the annulus fibrosus (AF) of the fetal IVD. SUMMARY OF BACKGROUND DATA: Elastin has been identified histochemically in adult bovine, human, and immature rat IVDs, and in fetal human IVDs using electron microscopy; however, no immunolocalization studies have been undertaken for associated components in human fetal IVDs. METHODS: En-bloc fixation of thoracolumbar spinal segments in formalin and Histochoice followed by standard histochemical processing, paraffin embedding, microtome sectioning, and identification of IVD ECM components using a range of specific mono- and polyclonal antibodies and bright-field and laser scanning confocal microscopy. RESULTS: The elastic fiber-associated proteins fibrillin-1, LTBP-2, and MAGP-1 were prominently immunolocalized in the outer lamellar layers of the AF of the human fetal IVD. Dual localization of selected components by confocal microscopy demonstrated that versican and LTBP-2 were colocalized with fibrillin-1 microfibrils in the AF lamellae with a similar distribution to the elastin fibers. LTBP-2 was also associated with pericellular perlecan in the outer AF. These interconnections between elastin-associated proteins resulted in an elastic network, which connected the AF cells with the adjacent cartilaginous vertebral bodies. CONCLUSION: Specific immunolocalization of fibrillin-1, MAGP-1, and versican with elastin in the outer AF of the fetal human IVD has been demonstrated. We deduce from the established distributions of the elastin-associated proteins and their known interactivities with matrix components that these stabilize and aid in the integration of the elastic fibers in the annular lamellae and may be responsible for the generation of tensional forces in the outer AF, which direct the assembly of this tissue.