Scleral Ossicles Research Articles

The Role of the Neural Crest Derived Periocular Mesenchyme in the Development of the Conjunctival Papillae

The periocular mesenchyme that surrounds the optic cup gives rise to the sclera including the scleral cartilage and scleral ossicles of the eye. The scleral ossicles are induced by overlying thickenings in the epithelium known as conjunctival papillae (CP). The CP are induced from Hamburger and Hamilton (HH) stages 30 to 34 in a defined sequence and are transient structures, disappearing after inducing osteogenesis in the underlying mesenchyme. To investigate the involvement of neural crest cells (NCC), we performed surgical ablations of signaling centers as well as DiI labeling of NCC streams. Our results show that surgical ablation of the otic vesicle between HH 16-18 does not affect the patterning of the CP (n=5). However when NCC streams are DiI labeled between the otic vesicle and the eye we find DiI in some CP (n=12). Similarly, when the nasal pit epithelium is ablated between HH 16-19, there is an effect on the patterning of the CP (n=2); this is supported by our DiI injections (n=2). These results indicate that NC streams contribute to the scleral mesenchyme throughout HH stages 16-23 and it is these NCCs that are important in the development of the sclerotic bones. Further research will be required to understand how the NCCs contribute to the development and patterning of the scleral ossicle system. This research was primarily funded by the Natural Sciences and Engineering Research Council of Canada and the Killam Trusts.

Vascular Endothelial‐Derived Growth Factor is Required for Proper Formation and Patterning of Intramembranous Bones

Bone development is regulated by extensive crosstalk between osteoblasts and vascular endothelial cells. Vascular endothelial growth factor (Vegf), encoded by the vegfa gene, plays a key role in coupling bone and vascular development during the formation of the mesoderm-derived endochondral bones of the limb skeleton. Despite the importance of Vegf in endochondral ossification, little work has been done to better understand the role of Vegf during the development of the neural crest-derived intramembranous bones of the skull. Using the scleral ossicles of the embryonic chick as a model, we examine the spatiotemporal expression pattern of vegfa throughout the course of scleral ossicle development from induction to mineralization and determine the effect of Vegf inhibition of ossicle development. Vegfa is strongly expressed at the time of ossicle induction and is down regulated thereafter. Vegf inhibition via implantation of beads soaked in a soluble, inactive form of the Vegf receptor 1 resulted in severe mispatterning of the scleral ossicles, including changes in ossicle size and shape, as well as loss of boundaries between neighboring ossicles. Our results suggest that Vegf plays a previously unidentified role in the earliest phases of intramembranous ossification. This work was funded by the Natural Sciences and Engineering Research Council of Canada and the Killam Trusts.

The Chick Scleral Ossicle System: Unravelling the Induction and Patterning of the Conjunctival Papillae

The conjunctival papillae (CP) are epithelial thickenings on the surface of the eye and are required for the induction of the underlying neural crest-derived, intramembranous scleral ossicles. However, little research has been conducted on the mechanisms underlying the induction and patterning of the CP. In order to investigate their development, we used both surgical manipulations and a candidate gene approach. Our results show that a single CP, when removed, is able to regenerate and induce the underlying condensation, demonstrating that the developmental window for CP induction is wider than was previously reported. We are also investigating a list of candidate genes to characterize their involvement in the development of the CP, including such genes as gsc, eya1, dlx5, and wnt1. This is the first investigation into the molecular factors controlling the induction and patterning of these CP. This study will further our understanding of the permissive role played by the epithelium in controlling the patterning of intramembranous bone. Additionally, as the CP share many characteristics with non-neurogenic placodes, understanding their development may provide further insights into the factors that may be controlling the development of other non-neurogenic placodes. This research was primarily funded by the Natural Sciences and Engineering Research Council of Canada and the Killam Trusts.

Sclerotic rings in mosasaurs (Squamata: Mosasauridae): structures and taxonomic diversity.

Mosasaurs (Squamata: Mosasauridae) were a highly diverse, globally distributed group of aquatic lizards in the Late Cretaceous (98–66 million years ago) that exhibited a high degree of adaptation to life in water. To date, despite their rich fossil record, the anatomy of complete mosasaur sclerotic rings, embedded in the sclera of the eyeball, has not been thoroughly investigated. We here describe and compare sclerotic rings of four mosasaur genera, Tylosaurus, Platecarpus, Clidastes, and Mosasaurus, for the first time. Two specimens of Tylosaurus and Platecarpus share an exact scleral ossicle arrangement, excepting the missing portion in the specimen of Platecarpus. Furthermore, the exact arrangement and the total count of 14 ossicles per ring are shared between Tylosaurus and numerous living terrestrial lizard taxa, pertaining to both Iguania and Scleroglossa. In contrast, two species of Mosasaurus share the identical count of 12 ossicles and the arrangement with each other, while no living lizard taxa share exactly the same arrangement. Such a mosaic distribution of these traits both among squamates globally and among obligatorily aquatic mosasaurs specifically suggests that neither the ossicle count nor their arrangement played major roles in the aquatic adaptation in mosasaur eyes. All the mosasaur sclerotic rings examined consistently exhibit aperture eccentricity and the scleral ossicles with gently convex outer side. Hitherto unknown to any squamate taxa, one specimen of Platecarpus unexpectedly shows a raised, concentric band of roughened surface on the inner surface of the sclerotic ring. It is possible that one or both of these latter features may have related to adaptation towards aquatic vision in mosasaurs, but further quantitative study of extant reptilian clades containing both terrestrial and aquatic taxa is critical and necessary in order to understand possible adaptive significances of such osteological features.

The embryology of the retinal pigmented epithelium in dwarf geckos (Gekkota: Sphaerodactylinae): a unique developmental pattern.

BackgroundThe retinal pigmented epithelium (RPE) is a rounded shaped structure in almost all lizards. In the New World dwarf geckos, this structure shows an unusual morphology. In addition to this ocular character, we describe notable differences in the development of these geckos in comparison with available developmental staging tables for other geckos and squamate reptiles.ResultsWe identified two main patterns of development of the RPE for squamates. These patterns were mapped onto a metatree of concordant hypotheses of squamates based on molecular data. During post-ovopositional stages the representative species of sphaerodactyls exhibit a RPE layer that transforms gradually from an ovoid form into the generalized spherical form. Sphaerodactyls are the only group of squamates in which this pattern is known.ConclusionsThis transition might be circumstantial evidence that the accessory RPE plays a role in providing additional protection for their apomorphic concaviclivate temporal fovea. We also report the presence of conjunctival papillae in a developmental stage prior to the formation of scleral ossicles. This developmental progression is similar to that of birds and turtles.

Investigation of an outbreak of craniofacial deformity in yellow-eyed penguin (Megadyptes antipodes) chicks

AIM: To investigate an outbreak of severe craniofacial deformity in yellow-eyed penguin (Megadyptes antipodes, hōiho) chicks at a single breeding site on the Otago Peninsula in the South Island of New Zealand.METHODS: Morbidity and mortality of yellow-eyed penguins breeding on the coastal regions of Otago was monitored from November 2008 to March 2009. Dead chicks and unhatched eggs were recovered and examined. Between October and December 2008 32 eggs were recorded at 17 nests in the Okia Reserve. Eleven chicks survived to about 90 days of age, of which eight were found to have moderate to severe craniofacial deformity. The six most severe chicks were subject to euthanasia and examined in detail at necropsy, and the remaining two affected chicks were released to the wild after a period of care in a rehabilitation centre. Post-mortem samples were analysed for inorganic and organic toxins.RESULTS: The six deformed chicks all had severe shortening of the mandible and maxilla by 20−50 mm. The rostral and caudal regions of the skull were approximately 40 and 80% of normal length, respectively. Other, more variable lesions included crossbill deformity, malformed bill keratin, microphthalmia with misshapen scleral ossicles and oral soft tissue excess thought to be secondary to bony malformations. During the same year, mild sporadic bill deformities were also reported in 10 unrelated chicks from >167 chicks at other breeding sites on the southern Otago coast. Concentrations of organic toxins and heavy metals in body tissues from affected chicks were apparently similar to those in unaffected chicks on other beaches.CONCLUSIONS: No cause of this outbreak of craniofacial deformity could be established although the high prevalence at a single site suggests that it was due to an unidentified local teratogen.

An investigation of cellular dynamics during the development of intramembranous bones: the scleral ossicles.

The development of intramembranous bone is a dynamic and complex process requiring highly coordinated cellular activities. Although the literature describes the detailed cellular dynamics of early mesoderm-derived endochondral bone, studies regarding neural crest-derived intramembranous bone have failed to keep pace. We analyzed the development of chick scleral ossicles from the onset of osteoid deposition to mineralization at morphological, histological, and ultrastructural levels. We find that the mesenchymal condensations from which ossicles develop change their shape from ellipsoidal to trapezoidal concurrent with an increase in size. Furthermore, the size of an ossicle is dependent upon its time of induction. Our histological analyses of condensation growth reveal cell migration and osteoid secretion as key cellular processes determining condensation size; these processes occur concomitantly to increase both the area and thickness of condensations. We also describe the formation of the zone of overlap between ossicles and conclude that the process is similar to that of cranial suture formation. Finally, transmission electron microscopy of early condensations demonstrates that early osteoblasts secrete collagen parallel to the long axis of the condensation. This study elucidates fundamental mechanisms of intramembranous bone development at the cellular level, furthering our knowledge of this important process among vertebrates.

Comparison of Computed Tomographic Images of Birds Obtained With Sedation vs General Anesthesia

Computed tomography (CT) is an increasingly available and valuable imaging modality for the diagnosis of companion avian pets. Previously, CT studies of birds required general anesthesia with inhalant anesthetics. Owing to the risks associated with general anesthesia, the authors of this article investigated the effect of sedation on birds during a CT examination. In this study, 10 psittacine birds were imaged using a 40-slice helical CT scanner. Birds were sedated with midazolam and butorphanol and placed in a positioning device. Following the initial study, birds were removed from the device and placed under general anesthesia with isoflurane. A second study was then performed. Two radiologists, blinded to the identity of the birds and partially blinded to the conditions of the study, reviewed the images. Studies were evaluated using a questionnaire consisting of 18 questions. Each question was scored on a Likert scale. A Wilcoxon signed rank test compared scores of sedated and anesthetized birds. A significant difference (P = 0.05) between sedated and anesthetized studies was found for 2 of 18 (11.1%) questions for radiologist 1 and 1 of 18 (5.5%) questions for radiologist 2, with differences identified in the scleral ossicles and the femoral heads. Interrater agreement for all questions using a linearly weighted κ was 0.334 and 0.311 for sedated and anesthetized birds, respectively, indicating fair agreement. The interrater agreement, excluding the head and musculoskeletal system, was 0.381 for sedated animals, indicating fair agreement, and 0.404 for anesthetized birds, indicating moderate agreement. Based on our results, performing CT studies in birds with sedation is a viable alternative to studies performed under general anesthesia.

Refining the timeline of conjunctival papillae induction in the chick scleral ossicle system

The conjunctival papillae, epithelial thickenings on the surface of the eye, are required for the induction of the underlying neural crest‐derived, intramembranous bones (the scleral ossicles). However, very little research has been conducted in the past 50 years on the mechanisms underlying the induction and patterning of these papillae. In order to further investigate this question, single papillae were surgically removed using fine tungsten needles at various time points during their development and the embryos were cultured for 1 to 5 days post‐surgery. Our results show that papilla removal had no effect on the patterning of the adjacent conjunctival papillae, and surprisingly, a normal patterned scleral ossicle develops under the site of surgery. This research demonstrates that the developmental timeline of the conjunctival papillae is much more flexible than previously noted. Our results also suggest that there is some compensatory mechanism taking place at the epithelial level for the induction of a complete ring of scleral ossicles. Thus, this study provides new insights into the mechanisms patterning the epithelium involved in epithelial‐mesenchymal induction of intramembranous bones. This research was primarily funded by the Natural Sciences and Engineering Research Council of Canada.Grant Funding Source: Natural Sciences and Engineering Research Council of Canada

A survey of skeletal elements in the orbit of Pseudosuchia and the origin of the crocodylian palpebral

ABSTRACTOssified skeletal elements within the orbit, such as scleral ossicles, palpebrals, supraorbitals and sesamoids, are widespread across Reptilia, including extant members of Crocodylia, Aves, Squamata and Chelonia. Extant crocodylians lack scleral ossicles, but have a unique palpebral that has recently been shown, through developmental studies, to be an osteoderm in the upper eyelid. Here, we examine the diversity and disparity of the crocodylian palpebral in extant members of Crocodylia and, through the fossil record, trace the origin and evolutionary history of the element throughout Pseudosuchia (all archosaurs more closely related to crocodylians than avians). We show that the crocodylian palpebral originated outside of Crocodylomorpha in early pseudosuchian groups (e.g. Aetosauria, Loricata) and that scleral ossicles are lost in nearly all groups of pseudosuchians, but appear in poposauroids, at least one early crocodylomorph, and some partially or fully marine Crocodyliformes (e.g. thalattosuchians). The morphology and number of palpebrals differs across Crocodyliformes; the presence of two palpebrals is plesiomorphic, but this is reduced to one by Crocodylia. We further recommend the restriction of the term palpebral to the structure in crocodylians and their homologues, but not to the structure in ornithischian dinosaurs.

Mesenchymal condensation sets the stage for intramembranous bone development

Mesenchymal condensation is one of the earliest, and most critical, stages in the formation of intramembranous (directly ossifying) bone. It is during the condensation phase that key skeletogenic genes are first upregulated and during which collagen I, the scaffold on which the inorganic portion of bone will be deposited, is first established. Thus, morphogenesis and tissue specific gene expression occur concurrently. Previously, we used microarray analysis and in situ hybridisation to determine which genes are important and at what stage of development these genes act during intramembranous ossification in chick and zebrafish. In the current study, we utilize a combination of light and electron microscopy to better understand the patterning and polarization of cells in early skeletogenic condensations. We compare neural crest derived intramembranous bones, specifically the scleral ossicles of the chick (Gallus gallus) and the opercula of the zebrafish (Danio rerio). While our preliminary results in the chick suggest osteoblasts are polarized toward the center of the early condensations, more data is required to make a solid conclusion. Data collection in zebrafish is also ongoing. Combined, these studies provide a means to correlate gene expression and morphogenesis during intramembranous bone formation.Grant Funding Source : Nova Scotia Health Research Foundation (Canada) ; Natural Sciences and Engineering Research Council of Canada

BMP and Hedgehog signaling during the development of scleral ossicles

Bone development is a complex process, involving multiple tissues and hierarchical inductive interactions. The study of skeletal development has largely focused on endochondral bones while intramembranous bones, such as the scleral ossicles within the avian eye, have received less attention. Our previous research directly demonstrated the involvement of sonic hedgehog and suggested the involvement of bmp2 and 4 during the development of scleral ossicles. The bones of the sclerotic ring are induced by overlying conjunctival papillae at HH 35 and 36. Here, we examine the spatial and temporal expression patterns of ptc1, ihh, bmp2, bmp4 and bmp7. We show that the cells of conjunctival papillae express ptc1, ihh and bmp2 at these stages; coincident with shh expression previously described. Interestingly, both ihh and ptc1 are also expressed in the mesenchyme underlying the papillae unlike shh and bmp2. Bmp4 and bmp7 are not expressed in these regions at any stages examined. Furthermore, using Noggin soaked beads implanted adjacent to papillae, we provide direct evidence that the BMP family of genes are important factors in the development of scleral ossicles. Localized inhibition of BMPs in this way causes a reduced expression of ihh in the surrounding tissue demonstrating that the BMP and Hedgehog pathways interact. Our data also demonstrates that the sclerotic ring has an intrinsic ability to compensate for missing elements. The scleral ossicle system provides a unique opportunity to investigate the epithelial–mesenchymal induction of intramembranous bones of the vertebrate skull.

Vasculogenesis and the Induction of Skeletogenic Condensations in the Avian Eye

Blood vessels form via two distinct mechanisms: vasculogenesis, the formation of new blood vessels; and angiogenesis, the remodeling of preexisting blood vessels to form mature vasculature. Little research, however, focuses on the relationship between blood vessels and skeletogenic condensations, a key step in bone formation. Here, the development of the scleral ossicles in the chick begins with the induction of a neural crest-derived condensation at HH Stages 35 and 36 by overlying papillae in a 1:1 pattern. These papillae, which are epithelial thickenings of the conjunctiva, begin to form at HH Stage 30, following a distinct pattern. Nothing is currently known about their induction, or patterning. As the first papilla always forms above the ciliary artery, we mapped blood vessel development in the eye between HH Stages 28 and 36.5 using camera lucida drawings, fluorescence microscopy, and histology. Our results show that a blood vessel meshwork begins to form de novo once the ring of conjunctival papillae is complete (HH Stages 34 through 36) suggesting no direct correlation between these two events. We also observe an avascular zone beneath each conjunctival papilla, which is first visible at HH Stage 35, coinciding with the onset of induction of the skeletogenic condensations. Importantly, our findings suggest that remodeling of the vasculature and development of the avascular zones occurs at the same time as induction, but prior to the presence of the skeletogenic condensations of the intramembranous bones; this process is dissimilar to that documented for endochondral ossification in avian limb buds.

Osteocyte Apoptosis and Absence of Bone Remodeling in Human Auditory Ossicles and Scleral Ossicles of Lower Vertebrates: A Mere Coincidence or Linked Processes?

Considering the pivotal role as bone mechanosensors ascribed to osteocytes in bone adaptation to mechanical strains, the present study analyzed whether a correlation exists between osteocyte apoptosis and bone remodeling in peculiar bones, such as human auditory ossicles and scleral ossicles of lower vertebrates, which have been shown to undergo substantial osteocyte death and trivial or no bone turnover after cessation of growth. The investigation was performed with a morphological approach under LM (by means of an in situ end-labeling technique) and TEM. The results show that a large amount of osteocyte apoptosis takes place in both auditory and scleral ossicles after they reach their final size. Additionally, no morphological signs of bone remodeling were observed. These facts suggest that (1) bone remodeling is not necessarily triggered by osteocyte death, at least in these ossicles, and (2) bone remodeling does not need to mechanically adapt auditory and scleral ossicles since they appear to be continuously submitted to stereotyped stresses and strains; on the contrary, during the resorption phase, bone remodeling might severely impair the mechanical resistance of extremely small bony segments. Thus, osteocyte apoptosis could represent a programmed process devoted to make stable, when needed, bone structure and mechanical resistance.

Role of osteocyte apoptosis in peculiar ossicles of the hearing sense organ: preliminary observations on hearing loss and osteoporosis

Starting point of the present study is the osteocyte role in bone remodelling that allows bone adaptation to mechanical load [1-3]. Bone remodelling has been investigated in relation to the occurrence of apoptosis [4] to understand if and how the process of programmed cell death interferes with bone turnover. In 1998, in a study on human middle ear, Marotti et al. [5] demonstrated that: 1) over 40% of osteocytes are dead within the 2nd year of age (but the authors were not able to demonstrate if osteocyte death occurred by degeneration or apoptosis); 2) bone remodelling occurs only occasionally. Recently [6], we showed that: 1) osteocytes of human auditory ossicles die by apoptosis; 2) also osteocytes located inside scleral ossicles of lower vertebrate eye (reptiles and birds) phylogenetically so far from human auditory ossicles are widely affected by apoptosis (about 60%); 3) in scleral ossicles bone turnover never occur. It is to be noted that both auditory ossicles of human ear and scleral ossicles of vertebrate eye are peculiar bony segments continuously submitted to stereotyped stresses and strains, with specialized functions: the first are involved in sound wave transmission and the latter protect the eyeball against deformation during the movement and have a role in visual accomodation, providing attachment for the ciliary muscles. In both cases, bone remodelling might severely impair, by resorption, the mechanical resistance of these extremely small specialized bony segments. Thus, we suggested that in auditory and scleral ossicles, submitted to stereotyped loading for all life, bone mechanical adaptation is not needed and osteocyte programmed death could represent the mechanism to avoid bone remodelling and to make stable, when necessary, bone structure and mechanical resistance. More recently, to confirm this hypothesis, clinical data were collected from a cohort of patients aged 55-85 years affected by hearing loss. The main target of the present study is to exclude any correlation between hearing loss and osteoporosis. During osteoporosis, unbalanced bone turnover causes the bone depletion in skeletal segments; such condition, in the peculiar ossicles of human middle ear, should imply hearing impairment. Our preliminary observations indicate, instead, that osteoporotic patients do not show higher percentage of hearing loss with respect to non osteoporotic ones. This evidence is ascribable to osteocyte apoptosis of auditory ossicles that avoid bone remodelling, thus assuring the integrity of such bony segments also in osteoporotic conditions.

CASE REPORT: Intraocular osteosarcoma in an Umbrella Cockatoo (Cacatua alba)

AbstractAn adult, presumed intact female Umbrella Cockatoo (Cacatua alba), presented with acute hemorrhage from an intraocular mass that perforated through the right cornea. Computed tomography scanning revealed a large soft tissue mass in the right orbit, invading and displacing the globe laterally, and destroying the scleral ossicles. There was no evidence of bony changes of the orbit or extension of the mass into the optic nerve or brain. Exenteration and mass removal were performed, and osteosarcoma was diagnosed via histopathology. Radiotherapy was delivered with an orthovoltage unit to a total dose of 68 Gray delivered in 17 fractions over 6 weeks. The bird recovered well from treatment, but died 2 months after the last radiation session with neurological signs. Necropsy was not performed. To our knowledge, this is the first case of an intraocular osteosarcoma reported in a bird, and the first case of attempted treatment of osteosarcoma in a bird by a combination of surgery and radiation therapy.

The zebrafish mutant bumper shows a hyperproliferation of lens epithelial cells and fibre cell degeneration leading to functional blindness

The development of the eye lens is one of the classical paradigms of induction during embryonic development in vertebrates. But while there have been numerous studies aimed at discovering the genetic networks controlling early lens development, comparatively little is known about later stages, including the differentiation of secondary lens fibre cells. The analysis of mutant zebrafish isolated in forward genetic screens is an important way to investigate the roles of genes in embryogenesis. In this study we describe the zebrafish mutant bumper ( bum), which shows a transient, tumour-like hyperproliferation of the lens epithelium as well as a progressively stronger defect in secondary fibre cell differentiation, which results in a significantly reduced lens size and ectopic location of the lens within the neural retina. Interestingly, the initial hyperproliferation of the lens epithelium in bum spontaneously regresses, suggesting this mutant as a valuable model to study the molecular control of tumour progression/suppression. Behavioural analyses demonstrate that, despite a morphologically normal retina, larval and adult bum −/− zebrafish are functionally blind. We further show that these fish have defects in their craniofacial skeleton with normal but delayed formation of the scleral ossicles within the eye, several reduced craniofacial bones resulting in an abnormal skull shape, and asymmetric ectopic bone formation within the mandible. Genetic mapping located the mutation in bum to a 4 cM interval on chromosome 7 with the closest markers located at 0.2 and 0 cM, respectively.

Use of skeletochronological analysis to estimate the age of leatherback sea turtles Dermochelys coriacea in the western North Atlantic

Although growth rate and age data are essential for leatherback management, esti- mates of these demographic parameters remain speculative due to the cryptic life history of this endangered species. Skeletochronological analysis of scleral ossicles obtained from 8 captive, known-age and 33 wild leatherbacks originating from the western North Atlantic was conducted to characterize the ossicles and the growth marks within them. Ages were accurately estimated for the known-age turtles, and their growth mark attributes were used to calibrate growth mark counts for the ossicles from wild specimens. Due to growth mark compaction and resorption, the number of marks visible at ossicle section tips was consistently and significantly greater than the number visible along the lateral edges, demonstrating that growth mark counts should be performed at the tips so that age is not underestimated. A correction factor protocol that incorporated the trajectory of early growth increments was used to estimate the number of missing marks in those ossicles exhibiting resorption, which was then added to the number of observed marks to obtain an age estimate for each turtle. A generalized smoothing spline model, von Bertalanffy growth curve, and size-at-age function were used to obtain estimates of age at maturity for leatherbacks in the western North Atlantic. Results of these analyses suggest that median age at maturation for leatherbacks in this part of the world may range from 24.5 to 29 yr. These age estimates are much greater than those proposed in previous studies and have significant implications for population management and recovery.

Analyses of the craniofacial skeleton of a zebrafish lens degeneration mutant

The zebrafish mutant, bumper, is characterized as having a lens defect, which begins at 4dpf. Previous work has shown that in the blind morph of Astyanax mexicanus, which also has a lens defect, the ocular skeleton is affected. The ocular skeleton of sighted Astyanax mexicanus and wildtype Danio rerio consists of two scleral ossicles joined by scleral cartilage. In the blind tetra morph, however, the scleral cartilage is persistent and is not replaced by bone. In this study, homozygous and heterozygous bumper mutants of Danio rerio were analyzed. Whole‐mount analyses show that the ocular skeleton is unaffected in this mutant, unlike in blind tetras. Surprisingly, however, several other regions of the craniofacial skeleton are affected; these include the jaws and skull vault. The significance of these results is discussed with respect to the nature and timing of eye degeneration in the two species analyzed. Funding: NSERC, CanadaGrant Funding SourceNSERC, Canada

Toward understanding the development of scleral ossicles in the chicken, Gallus gallus

Scleral ossicles are dermal bones that are present in the eye of many vertebrates. Despite this, little is understood about their development. This study investigates the cellular dynamics during and after induction, and attempts to identify inducing factors. Both cell death and proliferation were found to play limited roles in mesenchymal condensation formation, but are involved in development of the inducing epithelium overlying the presumptive ossicle. Real-time reverse transcriptase polymerase chain reaction of candidate genes identified significant increases in sonic hedgehog (SHH) expression. In situ hybridization confirmed that SHH is exclusively expressed in the conjunctival (scleral) papillae and not in the mesenchyme. Direct localized inhibition of Hedgehog signaling, by means of cyclopamine, supports the finding that SHH may play a role in scleral ossicle induction. In addition, a nonfluctuating asymmetry with respect to the number of ossicles per eye was found. This study provides significant insight into understanding the development of the neural crest derived dermal bones.