Mouse Skull Research Articles

Spatio-temporal analysis of molecular delivery through the blood–brain barrier using focused ultrasound

The deposition of gadolinium through ultrasound-induced blood–brain barrier (BBB) openings in the murine hippocampus was investigated. First, wave propagation simulations through the intact mouse skull revealed minimal beam distortion while thermal deposition simulations, at the same sonication parameters used to induce BBB opening in vivo, revealed temperature increases lower than 0.5 °C. The simulation results were validated experimentally in ex vivo skulls (m = 6) and in vitro tissue specimens. Then, in vivo mice (n = 9) were injected with microbubbles (Optison™; 25–50 µl) and sonicated (frequency: 1.525 MHz, pressure amplitudes: 0.5–1.1 MPa, burst duration: 20 ms, duty cycle: 20%, durations: 2–4 shots, 30 s per shot, 30 s interval) at the left hippocampus, through intact skin and skull. Sequential, high-resolution, T1-weighted MRI (9.4 Tesla, in-plane resolution: 75 µm, scan time: 45–180 min) with gadolinium (Omniscan™; 0.5 ml) injected intraperitoneally revealed a threshold of the BBB opening at 0.67 MPa and BBB closing within 28 h from opening. The contrast-enhancement area and gadolinium deposition path were monitored over time and the influence of vessel density, size and location was determined. Sonicated arteries, or their immediate surroundings, depicted greater contrast enhancement than sonicated homogeneous brain tissue regions. In conclusion, gadolinium was delivered through a transiently opened BBB and contained to a specific brain region (i.e., the hippocampus) using a single-element focused ultrasound transducer. It was also found that the amount of gadolinium deposited in the hippocampal region increased with the acoustic pressure and that the spatial distribution of the BBB opening was determined not only by the ultrasound beam, but also by the vasculature of the targeted brain region.

Computational mouse atlases and their application to automatic assessment of craniofacial dysmorphology caused by the Crouzon mutation Fgfr2C342Y

Crouzon syndrome is characterized by premature fusion of sutures and synchondroses. Recently, the first mouse model of the syndrome was generated, having the mutation Cys342Tyr in Fgfr2c, equivalent to the most common human Crouzon/Pfeiffer syndrome mutation. In this study, a set of micro-computed tomography (CT) scannings of the skulls of wild-type mice and Crouzon mice were analysed with respect to the dysmorphology caused by Crouzon syndrome. A computational craniofacial atlas was built automatically from the set of wild-type mouse micro-CT volumes using (1) affine and (2) non-rigid image registration. Subsequently, the atlas was deformed to match each subject from the two groups of mice. The accuracy of these registrations was measured by a comparison of manually placed landmarks from two different observers and automatically assessed landmarks. Both of the automatic approaches were within the interobserver accuracy for normal specimens, and the non-rigid approach was within the interobserver accuracy for the Crouzon specimens. Four linear measurements, skull length, height and width and interorbital distance, were carried out automatically using the two different approaches. Both automatic approaches assessed the skull length, width and height accurately for both groups of mice. The non-rigid approach measured the interorbital distance accurately for both groups while the affine approach failed to assess this parameter for both groups. Using the full capability of the non-rigid approach, local displacements obtained when registering the non-rigid wild-type atlas to a non-rigid Crouzon mouse atlas were determined on the surface of the wild-type atlas. This revealed a 0.6-mm bending in the nasal region and a 0.8-mm shortening of the zygoma, which are similar to characteristics previously reported in humans. The most striking finding of this analysis was an angulation of approximately 0.6 mm of the cranial base, which has not been reported in humans. Comparing the two different methodologies, it is concluded that the non-rigid approach is the best way to assess linear skull parameters automatically. Furthermore, the non-rigid approach is essential when it comes to analysing local, non-linear shape differences.

Scanning electron microscope studies of bone samples: Influence of simulated microgravity

A scanning electron microscope (SEM) with backscatter and secondary electron emission detectors plus a Si(Li) detector for photon yield measurements was used to study bone samples from skull and leg of mice and rats. These animals were either suspended by their tail to induce simulated microgravity, characterized as hind-limb suspension (HLS) or not suspended (control). Analyses of the SEM images and energy dispersive spectrometer (EDS) spectra using Si(Li) detector indicate variation in the lattice structures, and in intensities of the characteristics X-rays, produced from the exposed bone surface due to its interaction with the electron beam. Using Flame software, the X-ray spectra were analyzed and normalized ratios of the elements determined. The elemental analysis indicated a variation in the density of calcium, potassium, and oxygen near the knee joints and near the sutures in the skull bones. The comparison of simulated microgravity subjected samples of the rat skull bones with that of the control samples revealed that in the suture region there was a large increase in the ratio of calcium, and to some degree for phosphorus, suggesting simulated microgravity affects distribution of these elements. Elemental composition for control samples with depth (within the cross section of the leg bones) revealed decrease of oxygen and increase of calcium in the first millimeter of the bone depth after which the relative percentage of elements stayed constant.

Role of annexin 1 gene expression in mouse craniofacial bone development

Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system.

Inhibiting Osteoclast Formation

Hyaluronic acid (hyaluronan, HA) is a component of the extracellular matrix and is also used clinically to treat joint and cartilage diseases such as osteoarthritis. Chang et al . show that high molecular mass HA (HMM-HA), but not low molecular mass HA, inhibited osteoclast differentiation of bone marrow-derived macrophages in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). HMM-HA did not block RANKL-stimulated osteoclastogenesis of RAW264.7 cells, which is independent of M-CSF. Although CD44 is one known receptor for HA, antibodies that block CD44 had no effect on the inhibition of osteoclast formation by HA. However, function-blocking antibodies against Toll-like receptor 4 (TLR4) blocked the inhibitory effect of HA, suggesting that HA was acting through the TLR4. TLR4-transfected cells bound labeled HA based on fluorescence-activated cell sorting analysis, and osteoclastogenesis of cells from mice with a nonfunctional TLR4 were not inhibited by HA. HA decreased signaling pathways activated by M-CSF but did not inhibit those activated by RANKL unless the cells were pretreated with HA for 2 days before RANKL exposure. HA decreased the production of reactive oxygen species stimulated by the receptor tyrosine kinase activated by M-CSF and also inhibited activation of Rac and downstream kinases of the mitogen-activated protein kinase family (p38, ERK, and JNK). HA reduced the abundance of RANK and appeared to mediate this effect by its inhibition of M-CSF signaling, because M-CSF leads to the phosphorylation and activation of two transcription factors for which binding sites exist in the RANK promoter. Thus, M-CSF may stimulate osteoclastogenesis by increasing the responsiveness to RANKL through increased abundance of the receptor RANK, and HA blocks this pathway. In a mouse model for bone erosion, implantation of collagen films containing RANKL in the presence or absence of HA onto mouse skulls showed that HA decreased the RANKL-induced bone loss. These results show a possible mechanism by which HA is clinically useful, expand the role of innate immune receptors to regulation of bone metabolism, and provide a potential connection between bone metabolism and the innate immune system. E.-J. Chang, H. J. Kim. J. Ha, H. J. Kim, J. Ryu, K.-H. Park, U.-H. Kim, Z. H. Lee, H.-M. Kim, D. E. Fisher, H.-H. Kim, Hyaluronan inhibits osteoclast differentiation via Toll-like receptor 4. J. Cell Sci. 120 , 166-176 (2007). [Abstract] [Full Text]

Heparan sulfate Ndst1 gene function variably regulates multiple signaling pathways during mouse development

Disruption of heparan sulfate (HS) synthesis in vertebrate development causes malformations that are composites of those caused by mutations of multiple HS binding growth factors and morphogens. We previously reported severe developmental defects of the forebrain and the skull in mutant mice bearing a targeted disruption of the heparan sulfate-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (Ndst1). Here, we further characterize the molecular mechanisms leading to frontonasal dysplasia in Ndst1 mutant embryos and describe additional malformations, including impaired spinal and cranial neural tube fusion and skeletal abnormalities. Of the numerous proteins that bind HS, we show that impaired fibroblast growth factor, Hedgehog, and Wnt function may contribute to some of these phenotypes. Our findings, therefore, suggest that defects in HS synthesis may contribute to multifactor types of congenital developmental defects in humans, including neural tube defects.

A Novel Real-Time In Vivo Homing Model of Multiple Myeloma.

Background: Multiple myeloma (MM) is characterized by widespread involvement of the bone marrow (BM) at diagnosis, implying a continuous (re) circulation of the MM cells in the peripheral blood and (re) entrance into the BM. The normal process of B cell homing is regulated by cytokines and receptors such as SDF-1, CXCR4, VLA4, LFA-1, VCAM-1 and ICAM-1. In order to better understand the role of homing in MM, we developed an in vivo model which allows the continuous real-time imaging of MM cells as they home and adhere to the BM, as well as quantifying the numbers of cells in the circulation.Methods: MM.1S (2 ×106/ml) were fluorescently labeled by incubation with the dialkylcarbocyanine membrane dye “DiD” (Molecular Probes) 1uM dye for 15 min at 37°C. Cells were i.v injected in Balb/c mice. Appropriate arterioles in the ear pinnae of the mice were chosen for obtaining measurements, and the fluorescence signal on the MM cells was excited as the labeled cells passed through a slit of light (from a 632 nm He:Ne laser) focused across the vessel. Cell counts were obtained every 5 min from the time of injection. MM cell homing to bone marrow vasculature of the skull was analyzed using fluorescence confocal microscopy. A small incision was made in the scalp so as to expose the underlying dorsal skull surface. The mouse was placed on a warmed microscope stage. Imaging duration was 1–3 hours per session. DiD was excited with a 635 nm diode laser. High-resolution images with cellular details were obtained through the intact mouse skull at depths of up to 250 um from the surface of the skull. Images from several depths were obtained and z-stacking was performed to merge the images. Quantitative evaluation was made by dividing the bone marrow into pre-determined quadrants (areas 1 to 4) and counting numbers of fluorescent cells per field. To demonstrate that that this new model identifies changes in homing of MM cells, we used the CXCR4 inhibitor AMD3100 and anti-VLA-4 antibody to inhibit homing to the BM. MM cells were pre-incubated with AMD3100 (50 uM overnight, Sigma, MO) or anti-VLA-4 antibody (1hr incubation with 10 ug/ml, BD Pharmingen, CA) or control PBS under the same conditions. In vivo flow cytometry and confocal imaging were then performed on control and AMD3100 treated mice.Results: The number of cells in the control group decreased dramatically (86% decrease) after 1 hour indicating homing, whereas there was only a 47% reduction in the cells at 1 hour in the AMD3100 treated cohort, (p=0.002). Similarly, we demonstrated that the number of cells present in the perivascular bone marrow niches of the skull was significantly higher in the control mice as compared to the AMD3100-treated group at 1 hour after injection. The mean cell count in the AMD3100 treated mice decreased to 38% as compared to controls, p=0.01 Likewise, the use of anti-VLA-4 antibody demonstrated significant retention of MM cells in the circulation 1 hr after injection (4% reduction in circulating cell count vs 82% for control).Conclusion: We describe a new model that detects in vivo real-time homing of MM cells from the peripheral circulation into BM niches, which can be used to study the trafficking of MM cells into and out of the BM, as well as the effect of novel agents on this dynamic process.

Effects of developmental and functional interactions on mouse cranial variability through late ontogeny

The mammalian skull performs a variety of functions and its growth and development mirrors this complexity. Cranial growth and development have been actively studied for many years. Despite this interest, the variation in the patterns and processes of skull growth has attracted little attention. An important and unanswered question is the extent to which patterns of cranial covariation and variation are dynamically reworked throughout postnatal growth. To address this question, we examine patterns of variability in random-bred mouse skulls aged 35, 90, and 150 days. Using a battery of both Procrustes coordinate and Euclidean distance-based methods, we measure mean shape, canalization, developmental stability, and morphological integration in these skulls. We predict that the patterns of variability are dynamic, particularly between the youngest and the two oldest age groups due to the influence of functional effects such as postweaning mastication. We also hypothesize that patterns of variability are structured by the same functional and developmental factors that have been shown to influence cranial growth in primates. Our results indicate that contrary to our predictions, patterns of canalization, developmental stability, and morphological integration are stabilized before 35 days. The mean shape, however, changed significantly with growth. We found that only the facial region showed significant integration as predicted by the functional matrix model used in other studies of integration. These results indicate that phenotypic integration in these mice does not closely match those found for primate species, suggesting that comparisons between species should be made with care.

Focused ultrasound-induced opening of the blood-brain barrier in mice

The feasibility of blood-brain barrier (BBB) opening in the hippocampus of mice using transcranial focused ultrasound (FUS) was investigated on simulations and in vivo experiments. A micro-CT scan of an excised mouse skull was performed (resolution: 0.01 mm) and mappings of porosity, speed of sound, density, and attenuation were calculated. A 1.5-MHz wave propagating through the skull mappings was simulated, revealing a well-formed beam, minimal attenuation, and minute focus displacement. These results were verified with needle hydrophone measurements through five excised mouse skulls. In experiments in vivo, three mice were intravenously injected with a bolus of 0.01-mL ultrasound contrast agents (Optison) and the brains were sonicated transcranially. Ultrasound sonications (frequency: 1.5 MHz; burst length: 20 ms; duty cycle: 20%; acoustic pressure range: 2.0 to 2.7 MPa) were applied five times for 30 s per shot with a 30-s delay between shots. High-resolution (9.4 tesla), contrast-enhanced MR imaging revealed BBB opening and slow permeation of gadolinium from the posterior cerebral artery (10-min postinjection) through the surrounding tissue (35-min postinjection) and to the entire left hippocampus (95-min postinjection). These results demonstrate the feasibility of localized, noninvasive BBB opening in mice using transcranial FUS for potential precise drug delivery.

Aspects of achondroplasia in the skulls of dwarf transgenic mice: A cephalometric study

Achondroplasia, the most common short-limbed dwarfism in humans, results from a single nucleotide substitution in the gene for fibroblast growth factor receptor 3 (FGFR3). FGFR3 regulates bone growth in part via the mitogen-activated protein kinase pathway (MAPK). To examine the role of this pathway in chondrocyte differentiation, a transgenic mouse was generated that expresses a constitutively active mutant of MEK1 in chondrocytes and exhibits dwarfing characteristics typical of human achondroplasia, i.e., shortened axial and appendicular skeletons, mid-facial hypoplasia, and dome-shaped cranium. In this study, cephalometrics of the MEK1 mutant skulls were assessed to determine if the MEK1 mice are a good model of achondroplasia. Skull length, arc of the cranial vault, and area, maximum and minimum diameters of the brain case were measured on digitized radiographs of skulls of MEK1 and control mice. Cranial base and nasal bone length and foramen magnum diameter were measured on midsagittal micro-CT sections. Data were normalized by dividing by the cube root of each animal's weight. Transgenic mice exhibited a domed skull, deficient midface, and (relatively) prognathic mandible and had a shorter cranial base and nasal bone than the wild-type. Skull length was significantly less in transgenic mice, but cranial arc was significantly greater. The brain case was larger and more circular and minimum diameter of the brain case was significantly greater in transgenic mice. The foramen magnum was displaced anteriorly but not narrowed. MEK1 mouse cephalometrics confirm these mice as a model for achondroplasia, demonstrating that the MAP kinase signaling pathway is involved in FGF signaling in skeletal development.

Anatomical phenotyping in the brain and skull of a mutant mouse by magnetic resonance imaging and computed tomography

Since genetically modified mice have become more common in biomedical research as models of human disease, a need has also grown for efficient and quantitative methods to assess mouse phenotype. One powerful means of phenotyping is characterization of anatomy in mutant vs. normal populations. Anatomical phenotyping requires visualization of structures in situ, quantification of complex shape differences between mouse populations, and detection of subtle or diffuse abnormalities during high-throughput survey work. These aims can be achieved with imaging techniques adapted from clinical radiology, such as magnetic resonance imaging and computed tomography. These imaging technologies provide an excellent nondestructive method for visualization of anatomy in live individuals or specimens. The computer-based analysis of these images then allows thorough anatomical characterizations. We present an automated method for analyzing multiple-image data sets. This method uses image registration to identify corresponding anatomy between control and mutant groups. Within- and between-group shape differences are used to map regions of significantly differing anatomy. These regions are highlighted and represented quantitatively by displacements and volume changes. This methodology is demonstrated for a partially characterized mouse mutation generated by N-ethyl-N-nitrosourea mutagenesis that is a putative model of the human syndrome oculodentodigital dysplasia, caused by point mutations in the gene encoding connexin 43.

Effects of anoxia on serum immunoglobulin and albumin leakage through blood–brain barrier in mouse cerebellum as revealed by cryotechniques

The purpose of this study was to examine time-dependent topographical changes of leaking proteins from blood vessels in the mouse cerebellum to assess the effect of normal blood circulation on the blood–brain barrier (BBB). The distribution of leaking serum proteins was immunohistochemically examined by various cryotechniques including our “in vivo cryotechnique”. The cryofixed cerebellar tissues were processed for the freeze-substitution method, and finally embedded in the common paraffin wax. Serial de-paraffinized sections were immunostained by anti-mouse immunoglobulin-G (IgG) or albumin antibody. By combination of the “in vivo cryotechnique”, in which normal blood flow into the cerebellum was always kept in vivo, with the freeze-substitution method, serum IgG and albumin were clearly localized inside of cerebellar blood vessels. To examine abnormal leakage of blood vessels as a model of anoxia, some cerebellar tissues were partially removed from brains in the mouse skull and quickly frozen in the isopentane–propane within a minute. In such resected cerebellar tissues, serum IgG and albumin were diffusely immunostained in large areas around the blood capillaries, probably because of easy leakage of the serum components through the immediately changed BBB. To the contrary, no serum protein could be identified outside blood capillaries under living conditions of the anesthetized mice. The present combination method, both “in vivo cryotechnique” and freeze-substitution, for immunohistochemistry enabled us to examine the in vivo localization of serum components in mouse brains due to alteration of the BBB.

Thermal assessment of 40-MHz ultrasound at soft tissue-bone interfaces

Tissue exposure to diagnostic ultrasound (US) can cause significant temperature rises. However, little has been reported on thermal effects of high-frequency US, and guidelines for the use of US do not necessarily apply to higher frequencies. Temperature rise induced by US biomicroscopy (UBM) was measured in phantoms containing mouse skulls and in anesthetized mice and mice post mortem, with a 50-μm K-type thermocouple. The operating frequency was 40 MHz with a free field I SPTA of 2.6 mW/cm 2 (B-mode) and 11.9 W/cm 2 (Doppler). Peak negative pressures were 5.22 MPa (B mode) and 7.32 MPa (Doppler), resulting in a mechanical index (MI) of 0.83 (B-mode) and 1.05 (Doppler mode). In Doppler mode, mean temperature rises of 1.80°C and 1.73°C were measured for proximal and distal skull phantom surfaces after a 3-min insonation. In vivo, the proximal mouse skull surface showed a mean temperature rise of 2.1°C, with no statistically significant differences post mortem. Our results indicate temperature rise from insonation of bone interfaces using similar exposure parameters should not cause adverse bioeffects. (E-mail: aduckett@sten.sunnybrook.utoronto.ca)

Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration.

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast--mineralized matrix complex. Histologically, at this time point a tissue structure with a "connective tissue"-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of immunodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.

Earliest mineral and matrix changes in force-induced musculoskeletal disease as revealed by Raman microspectroscopic imaging.

Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common human birth defect in the skull. Raman microspectroscopy was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Raman imaging revealed decreased relative mineral content in skulls undergoing craniosynostosis compared with unloaded specimens. Raman microspectroscopy, a nondestructive vibrational spectroscopic technique, was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common birth defect in the face and skull. The calvaria, or flat bones that comprise the top of the skull, are most often affected, and craniosynostosis is a feature of over 100 human syndromes and conditions. Raman images of the suture, the tips immediately adjacent to the suture (osteogenic fronts), and mature parietal bones of loaded and unloaded calvaria were acquired. Images were acquired at 2.6 x 2.6 microm spatial resolution and ranged in a field of view from 180 x 210 microm to 180 x 325 microm. This study found that osteogenic fronts subjected to uniaxial compression had decreased relative mineral content compared with unloaded osteogenic fronts, presumably because of new and incomplete mineral deposition. Increased matrix production in osteogenic fronts undergoing craniosynostosis was observed. Understanding how force affects the composition, relative amounts, and location of the mineral and matrix provides insight into musculoskeletal disease in general and craniosynostosis in particular. This is the first report in which Raman microspectroscopy was used to study musculoskeletal disease. These data show how Raman microspectroscopy can be used to study subtle changes that occur in disease.

From Lysosomes to the Plasma Membrane: LOCALIZATION OF VACUOLAR TYPE H+-ATPase WITH THE a3 ISOFORM DURING OSTEOCLAST DIFFERENTIATION

Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification is carried out by vacuolar type H+-ATPase (V-ATPase) localized in the plasma membrane. We have shown that a3, one of the four subunit a isoforms (a1, a2, a3, and a4), is a component of the plasma membrane V-ATPase (Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760-8765). To establish the unique localization of V-ATPase, we have used a murine macrophage cell line, RAW 264.7, that can differentiate into multinuclear osteoclast-like cells on stimulation with RANKL (receptor activator of nuclear factor kappaB ligand). The V-ATPase with the a3 isoform was localized to late endosomes and lysosomes, whereas those with the a1 and a2 isoforms were localized to organelles other than lysosomes. After stimulation, the V-ATPase with the a3 isoform was immunochemically colocalized with lysosome marker lamp2 and was detected in acidic organelles. These organelles were also colocalized with microtubules, and the signals of lamp2 and a3 were dispersed by nocodazole, a microtubule depolymerizer. In RAW-derived osteoclasts cultured on mouse skull pieces, the a3 isoform was transported to the plasma membrane facing the bone and accumulated inside podosome rings. These findings indicate that V-ATPases with the a3 isoform localized in late endosomes/lysosomes are transported to the cell periphery during differentiation and finally assembled into the plasma membrane of mature osteoclasts.

Molecular mechanisms in calvarial bone and suture development, and their relation to craniosynostosis.

The development and growth of the skull is a co-ordinated process involving many different tissues that interact with each other to form a complex end result. When normal development is disrupted, debilitating pathological conditions, such as craniosynostosis (premature calvarial suture fusion) and cleidocranial dysplasia (delayed suture closure), can result. It is known that mutations in the fibroblast growth factor receptors 1, 2, and 3(FGFR1, 2, and 3), as well as the transcription factors MSX2 and TWIST cause craniosynostosis, and that mutations in the transcription factor RUNX2 (CBFA1) cause cleidocranial dysplasia. However, relatively little is known about the development of the calvaria: where and when these genes are active during normal calvarial development, how these genes may interact in the developing calvaria, and the disturbances that may occur to cause these disorders. In this work an attempt has been made to address some of these questions from a basic biological perspective. The expression patterns of the above-mentioned genes in the developing mouse skull are detailed. The microdissection and in vitro culture techniques have begun the task of identifying Fgfrs, Msx2, and Twist interacting in intricate signalling pathways that if disrupted could lead to craniosynostosis.

Cortical imaging through the intact mouse skull using two-photon excitation laser scanning microscopy.

Two-photon excitation scanning microscopy has several advantages for imaging brain cells and other anatomical features in whole animals (Denk et al., 1994). These advantages stem from three main factors. The first is the quadratic dependence of optical absorption, so that optical sectioning is performed solely by the incident light. The second is the use of infrared light, which scatters less than visible light and hence increases the depth of focal penetration. The third is the use of a point-scanning system to optimize spatial resolution. The application of two-photon microscopy for in vivo imaging has typically utilized a surgically-prepared “cortical window” in which a section of the skull has been replaced by a agarose plug that is sealed with a glass coverslip (Svoboda et al., 1997). While this method adequately controls for motion due to cardiac and respiratory rhythms, the “cortical window” preparation renders the brain susceptible to fluctuations in temperature and pressure. In contrast, intrinsic optical imaging techniques are performed through thinned, intact skull, thus protecting the brain from external influences (Masino et al., 1993). However, the spatial resolution of intrinsic optical imaging is insufficient to visualize fine features like single cells or capillaries. In an effort to image the brain with subcellular spatial resolution and without the “cortical window” procedure, a method was devised to image directly through thinned mouse skull using two-photon excitation microscopy. The use of mice for these studies was motivated on two fronts. First of all, the murine dura mater is of negligible thickness and does not impede cellular imaging deep in the brain. Secondly, since mouse cortex is only 70 ‐75% as thick as rat cortex, more cortical anatomy may be imaged for a given depth of penetration. This is a salient point, as the attainable cortical penetration is reduced by the depth of the thinned skull.

Correction for Debat et al. , Independence between developmental stability and canalization in the skull of the house mouse

Correction for ‘Independence between developmental stability and canalization in the skull of the house mouse’ by V. Debat, P. Alibert, P. David, E. Paradis and J.-C. Auffray (Proc. R. Soc. Lond. B 267 , 423–430. (doi: 10.1098/rspb.2000.1017)). There were multiple errors in the values in table 1 (p. 426). A corrected version of this table is presented below. Consequences on F -tests are also presented.

Premature vertebral endplate ossification and mild disc degeneration in mice after inactivation of one allele belonging to the Col2a1 gene for Type II collagen.

Skeletal tissues of mice with an inactivated allele of the Col2a1 gene for Type II collagen ("heterozygous knockout") were studied. To determine whether a heterozygous inactivation of the Col2a1 gene has a role in the etiology of spine disorders such as disc degeneration. Mutations in the COL2A1, COL11A1, COL11A2, and COL9A2 genes have been linked to spine disorders. However, the mechanism by which genetic factors lead to disc degeneration still are largely unknown. Spine tissues were studied using radiograph analyses; conventional, quantitative, and polarized light microscopy; immunohistochemistry for the major extracellular components, and in situ hybridization for procollagens alpha1(I) and alpha1(II). Voluntary running activity also was monitored in half of the mice. As the findings showed, 1-month-old heterozygous knockout mice had shorter limb bones, skulls, and spines, as well as thicker and more irregular vertebral endplates, which calcified earlier than in the control mice. They also had a lower concentration of glycosaminoglycans in the anulus fibrosus, in the endplates, and in the vertebral bone than the controls. These features in the heterozygous knockout mice were compensated by the age of 15 months. However, the long bones and skulls of the mature heterozygous mice remained shorter than those of the controls. Gene-deficient mice used the running wheel less. However, physical exercise did not induce any marked structural changes in the skeleton. Mice with heterozygous knockout of Col2a1 show subtle early skeletal manifestations that bear some resemblance to those of human spine disorders.