Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concerns.

Warfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly overrepresented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.

Nasal bone shape is under complex epistatic genetic control in mouse interspecific recombinant congenic strains.

BACKGROUND: Genetic determinism of cranial morphology in the mouse is still largely unknown, despite the localization of putative QTLs and the identification of genes associated with Mendelian skull malformations. To approach the dissection of this multigenic control, we have used a set of interspecific recombinant congenic strains (IRCS) produced between C57BL/6 and mice of the distant species Mus spretus (SEG/Pas). Each strain has inherited 1.3% of its genome from SEG/Pas under the form of few, small-sized, chromosomal segments. RESULTS: The shape of the nasal bone was studied using outline analysis combined with Fourier descriptors, and differential features were identified between IRCS BcG-66H and C57BL/6. An F2 cross between BcG-66H and C57BL/6 revealed that, out of the three SEG/Pas-derived chromosomal regions present in BcG-66H, two were involved. Segments on chromosomes 1 (∼32 Mb) and 18 (∼13 Mb) showed additive effect on nasal bone shape. The three chromosomal regions present in BcG-66H were isolated in congenic strains to study their individual effect. Epistatic interactions were assessed in bicongenic strains. CONCLUSIONS: Our results show that, besides a strong individual effect, the QTL on chromosome 1 interacts with genes on chromosomes 13 and 18. This study demonstrates that nasal bone shape is under complex genetic control but can be efficiently dissected in the mouse using appropriate genetic tools and shape descriptors.

Requirement for Twist1 in frontonasal and skull vault development in the mouse embryo.

Using a Cre-mediated conditional deletion approach, we have dissected the function of Twist1 in the morphogenesis of the craniofacial skeleton. Loss of Twist1 in neural crest cells and their derivatives impairs skeletogenic differentiation and leads to the loss of bones of the snout, upper face and skull vault. While no anatomically recognizable maxilla is formed, a malformed mandible is present. Since Twist1 is expressed in the tissues of the maxillary eminence and the mandibular arch, this finding suggests that the requirement for Twist1 is not the same in all neural crest derivatives. The effect of the loss of Twist1 function is not restricted to neural crest-derived bones, since the predominantly mesoderm-derived parietal and interparietal bones are also affected, presumably as a consequence of lost interactions with neural crest-derived tissues. In contrast, the formation of other mesodermal skeletal derivatives such as the occipital bones and most of the chondrocranium are not affected by the loss of Twist1 in the neural crest cells.

Novel skeletogenic patterning roles for the olfactory pit.

The position of the olfactory placodes suggests that these epithelial thickenings might provide morphogenetic information to the adjacent facial mesenchyme. To test this, we performed in ovo manipulations of the nasal placode in the avian embryo. Extirpation of placodal epithelium or placement of barriers on the lateral side of the placode revealed that the main influence is on the lateral nasal, not the frontonasal, mesenchyme. These early effects were consistent with the subsequent deletion of lateral nasal skeletal derivatives. We then showed in rescue experiments that FGFs are required for nasal capsule morphogenesis. The instructive capacity of the nasal pit epithelium was tested in a series of grafts to the face and trunk. Here, we showed for the first time that nasal pits are capable of inducing bone, cartilage and ectopic PAX7 expression, but these effects were only observed in the facial grafts. Facial mesenchyme also supported the initial projection of the olfactory nerve and differentiation of the olfactory epithelium. Thus, the nasal placode has two roles: as a signaling center for the lateral nasal skeleton and as a source of olfactory neurons and sensory epithelium.

Frontal nasal prominence expression driven by Tcfap2a relies on a conserved binding site for STAT proteins.

The AP-2 transcription factor family is linked with development of the head and limbs in both vertebrate and invertebrate species. Recent evidence has also implicated this gene family in the evolution of the neural crest in chordates, a critical step that allowed the development and elaboration of the vertebrate craniofacial skeleton. In mice, the inappropriate embryonic expression of one particular AP-2 gene, Tcfap2a, encoding AP-2alpha, results in multiple developmental abnormalities, including craniofacial and limb defects. Thus, Tcfap2a provides a valuable genetic resource to analyze the regulatory hierarchy responsible for the evolution and development of the face and limbs. Previous studies have identified a 2-kilobase intronic region of both the mouse and human AP-2alpha locus that directs expression of a linked LacZ transgene to the facial processes and the distal mesenchyme of the limb bud in transgenic mice. Further analysis identified two highly conserved regions of approximately 200-400 bp within this tissue-specific enhancer. We have now initiated a transgenic and biochemical analysis of the most important of these highly conserved regions. Our analysis indicates that although the sequences regulating face and limb expression have been integrated into a single enhancer, different cis-acting sequences ultimately control these two expression domains. Moreover, these studies demonstrate that a conserved STAT binding site provides a major contribution to the expression of Tcfap2a in the facial prominences.

Is osteoid osteoma an iodophilic lesion?: pathologically proved osteoid osteoma of nasal bone first seen on whole-body iodine-131 scan.

The authors present a case of pathologically proved osteoid osteoma that was visualized randomly on an I-131 whole-body scan. Search of the medical literature did not reveal any mention of radioactive iodine uptake by osteoid osteomas. Therefore, the authors concluded that this pathology must be included in the differential diagnostic list of positive findings on I-131 scans.

Expression patterns of connexin43 protein during facial development in the chick embryo: associates with outgrowth, attachment, and closure of the midfacial primordia.

BACKGROUND: In a prior report, evidence was presented for the presence of gap junction proteins [connexin32 and connexin43 (Cx43)] in embryonic facial primordia. The purpose of the present study was, first, to examine in detail the patterns of distribution of Cx43 protein in embryonic chick facial primordia and, second, to consider the possible roles played by this protein during midfacial development. METHODS: Chick embryo heads were serially sectioned and processed for immunofluorescent localization of Cx43. The developmental stages examined encompassed the period of formation, enlargement, and union of the facial primordia. Western blot analysis of the facial primordia was also performed. RESULTS: Analysis of serial sections revealed the presence of signal in both epithelium and mesenchyme at sites of attachment in each of the midfacial primordia (i.e., the medial nasal, lateral nasal, and maxillary processes). Furthermore, although signal was concentrated in mesenchyme in the distal tips of the primordia at sites of attachment, immunoreactivity was absent, sparse, or less intense outside the areas of attachment. In some cases (i.e., the maxillary process), immunoreactive signal in mesenchyme did not appear in the distal tip until the primordia approximated each other or contact of the primordia was initiated. Most significantly, signal was also found between the facial primordia in nonprimordial epithelium and mesenchyme at sites where the primordia were joined. CONCLUSIONS: These data suggest that the expression of Cx43 protein is spatially and temporally regulated in the facial primordia and that the patterns of expression that were observed are significant to the cascade of events that ultimately lead to the attachment and union of the primordia that form the midface.

Migration of hydroxyapatite onlays into the mandible and nasal bone and local bone turnover in growing rabbits.

To determine the effects of local bone turnover on the migration of macroporous hydroxyapatite onlays in the nasal bone and mandibular ramus, we performed histomorphometric analyses of the underlying bone area in 41 New Zealand White rabbits from the age of 4 weeks. The hydroxyapatite implants were placed under the periosteum of the right nasal bone (a depository bone onto its periosteal surface and endosteal resorptive) and the mandibular ramus (resorptive onto its outer surface). The corresponding left sides were sham operated. Following fluorescence bone labeling, composite specimens of the hydroxyapatite block including both sides of the nasal bone and mandible were removed at 0 (n = 1), 3, 6, 9, 12, and 16 weeks postoperatively (n = 8, respectively) and processed to yield undecalcified sections. Bone-bone marrow interfaces in the entire area within 200 microns beneath the base of the hydroxyapatite and in the counter-area on the sham-operated side were measured under a light microscope. In all grafted specimens, the hydroxyapatite matrix was directly united with the underlying tissue by bone ingrowth. However, the sinking of the hydroxyapatite graft in the nasal bone was significant at 3 weeks postoperatively and gradually increased thereafter. In the mandible, the sinking became significant at 6 weeks. In the nasal bone, the bone area density beneath the graft showed a time-dependent decrease during the experimental period, but in the mandibular bone, the value was initially decreased at 3 weeks and then recovered to baseline level. In both bones, parameters of bone resorption, such as osteoclast number and osteoclast surface, were significantly increased from 3 weeks. While the parameters of bone formation, such as osteoblast surface and mineralizing surface, were significantly decreased from 3 weeks in the nasal bone, they were significantly increased in the mandible. Mineral apposition rate showed a significant decrease in both bones. Our data indicate that while the bone area density beneath the hydroxyapatite seemed to depend on bone formation, increased bone resorption would be more critical for the remodeling of underlying bony architecture in the migration of the hydroxyapatite graft.

Increased turnover of small proteoglycans synthesized by human osteoblasts during cultivation with ascorbate and beta-glycerophosphate.

The small proteoglycan decorin had been localized previously at the d-band in the gap zone of collagen fibrils in nonmineralizing tissues. In bone matrix this zone is proteoglycan free and is at least in some species the place where mineralization along collagen fibrils starts. To study the metabolism of the small proteoglycans decorin and biglycan under mineralizing conditions, osteoblasts from human nasal bone were cultured for several weeks in the presence or absence of beta-glycerophosphate and ascorbate. An immediate consequence of the treatment was a reduced expression of decorin, as judged by immune precipitation, whereas the biosynthesis of biglycan was not affected. Pulse-chase experiments were performed with osteoblasts embedded in floating type I collagen gels. In the presence of beta-glycerophosphate and ascorbate, a more rapid turnover of both proteoglycans was noted; the one of biglycan reached statistical significance. Indirect evidence for an enhanced rate of proteoglycan endocytosis was obtained. This effect was not seen in cultured skin fibroblasts. Thus, osteoblasts respond rapidly to mineralizing conditions with alterations of small proteoglycan biosynthesis and turnover.

Concentrations of ofloxacin in ear and nasal tissues.

The results of two kinetic studies examining soft tissue, cartilage and bone after uptake of oral ofloxacin [administered as 200 mg twice daily (study I/nose) or 400 mg once daily (study II/ear)] show that antibiotic concentrations lie within the therapeutic range. Findings demonstrate that 400 mg ofloxacin daily is a compliance-enhancing and effective approach to the treatment of ENT-related infections, in particular those caused by problem organisms such as Pseudomonas aeruginosa.

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro.

Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.