Development of scleral ossicles in Podocnemis expansa (Testudines: Podocnemididae) embryos exposed to atrazine.

Understanding the effects of atrazine exposure on embryo development in oviparous animals may provide important data regarding the impacts of agrochemical use on wildlife and the ecosystem. This study set out to determine the effects of embryonic atrazine exposure on the development of osseous and cartilaginous components of scleral ossicles in Podocnemis expansa. Eggs were collected at the Environmental Protection Area Meandros do Rio Araguaia, Brazil, and artificially incubated in sand treated with solutions containing 2, 20 or 200 µg/L of atrazine. Sixty embryos were collected per treatment throughout the incubation period. Embryos were diaphanized with potassium hydroxide (KOH) and stained with Alizarin Red S and Alcian blue (bone and cartilage tissue respectively). Scleral ossicles were then counted and examined for skeletal abnormalities at different stages of embryonic development. Scleral ossicle counts were significantly reduced in P. expansa embryos treated with 200 µg/L atrazine solution. Rudimentary ossicles and gaps were also noted in embryos exposed to atrazine concentrations of 2 µg/L or 200 µg/L. Findings of this study emphasize the relevance of ecotoxicological investigations in determining the impacts of agrochemicals on native fauna.

Quantitative gene expression dynamics of key placode signalling factors in the embryonic chicken scleral ossicle system.

Development of the scleral ossicles, a ring of bony elements within the sclera, is directed by a series of papillae that arise from placodes in the conjunctival epithelium over a 1.5-day induction period in the chicken embryo. The regular spacing of the papillae around the corneal-scleral limbus suggests that their induction may be regulated by a reaction-diffusion mechanism, similar to other epithelial appendages. Some key placode signalling molecules, including ß-catenin, are known to be expressed throughout the induction period. However, others have been studied only at certain stages or have not been successfully detected. Here we use qPCR to study the gene expression patterns of the wingless integration (WNT)/ß-catenin, bone morphogenetic protein (BMP), ectodysplasin (EDA), fibroblast growth factor (FGF) and hedgehog (HH) signalling families in discrete regions of the eye throughout the complete conjunctival placode and papillae induction period. This comprehensive analysis revealed a variable level of gene expression within specific eye regions, with some genes exhibiting high, moderate or low changes. Most genes exhibited an initial increase in gene expression, followed by a decrease or plateau as development proceeded, suggesting that some genes are important for a brief initial period whilst the sustained elevated expression level of other genes is needed for developmental progression. The timing or magnitude of these changes, and/or the overall gene expression trend differed in the temporal, nasal and/or dorsal eye regions for some, but not all genes, demonstrating that gene expression may vary across different eye regions. Temporal and nasal EDA receptor (EDAR) had the greatest number of strong correlations (r > 0.700) with other genes and ß-catenin had the greatest number of moderate correlations (r = 0.400-0.700), while EDA had the greatest range in correlation strengths. Among the strongly correlated genes, two distinct signalling modules were identified, connected by some intermediate genes. The dynamic gene expression patterns of the five signalling pathways studied here from conjunctival placode formation through to papillae development is consistent with other epithelial appendages and confirms the presence of a conserved induction and patterning signalling network. Two unique gene expression patterns and corresponding gene interaction modules suggest functionally distinct roles throughout placode development. Furthermore, spatial differences in gene expression patterns among the temporal, nasal and dorsal regions of the eye may indicate that the expression of certain genes is influenced by mechanical forces exerted throughout development. Therefore, this study identifies key placode signalling factors and their interactions, as well as some potential region-specific features of gene expression in the scleral ossicle system and provides a basis for further exploration of the spatial expression of these genes and the patterning mechanism(s) active throughout conjunctival placode and papillae formation.

The lymphangiogenic and hemangiogenic privilege of the human sclera.

PURPOSE: Most organs of the human body are supplied with a dense network of blood and lymphatic vessels. However, some tissues are either hypovascular or completely devoid of vessels for proper function, such as the ocular tissues sclera and cornea, cartilage and tendons. Since many pathological conditions are affecting the human sclera, this review is focussing on the lymphangiogenic and hemangiogenic privilege in the human sclera. METHODS: This article gives an overview of the current literature based on a PubMed search as well as observations and experience from clinical practice. RESULTS: The healthy human sclera is the outer covering layer of the eye globe consisting mainly of collagenous extracellular matrix and fibroblasts. Physiologically, the sclera shows only a superficial network of blood vessels and a lack of lymphatic vessels. This vascular privilege is actively regulated by balancing anti- and proangiogenic factors expressed by cells within the sclera. In pathological situations, such as open globe injuries or ciliary body melanomas with extraocular extension, lymphatic vessels can secondarily invade the sclera and the inner eye. This mechanism most likely is important for tumor cell metastasis, wound healing, immunologic defense against intruding microorganism, and autoimmune reactions against intraocular antigens. CONCLUSIONS: The human sclera is characterized by a tightly regulated vascular network that can be compromised in pathological situations, such as injuries or intraocular tumors affecting healing outcomes Therefore, the molecular and cellular mechanisms underlying wound healing following surgical interventions deserve further attention, in order to devise more effective therapeutic strategies.

An overlooked placode: Recharacterizing the papillae in the embryonic eye of reptilia.

The papillae in the chicken embryonic eye, described as scleral papillae in the well-known Hamburger and Hamilton (1951) staging table, are one of the key anatomical features used to stage reptilian (including bird) embryos from HH30-36. These papillae are epithelial thickenings of the conjunctiva and are situated above the mesenchymal sclera. Here, we present evidence that the conjunctival papillae, which are required for the induction and patterning of the underlying scleral ossicles, require epithelial pre-patterning and have a placodal stage similar to other placode systems. We also suggest modifications to the Hamburger Hamilton staging criteria that incorporate this change in terminology (from "scleral" to "conjunctival" papillae) and provide a more detailed description of this anatomical feature that includes its placode stage. This enables a more complete and accurate description of chick embryo staging. The acknowledgment of a placode phase, which shares molecular and morphological features with other cutaneous placodes, will direct future research into the early inductive events leading to scleral ossicle formation.

Skeletons of the Eye: An Evolutionary and Developmental Perspective.

The ocular skeleton, composed of the scleral cartilage and scleral ossicles, is present in many vertebrates. The morphology of the scleral cartilage and ossicles varies within different extant reptiles (including birds) and also varies dramatically from the morphology in extant teleosts. This incredible range of diverse morphologies is the result of millions of years of evolution. Both the position of these elements within the eye and the timing of development vary amongst different vertebrates. While the development of both the scleral cartilage and scleral ossicles is somewhat understood in reptiles and in teleosts, the functional advantage of these elements is still debated. Most reptiles have a multi-component scleral ossicle ring composed of a series of flat bone plates and a scleral cartilage cup lining the retina, some sharks have calcified cartilage plates, and some teleosts have two bones while most others only have a ring of scleral cartilage. The data presented shows that different vertebrates have adapted to similar selective pressures in different ways. However, the reason why sarcopterygians have a series of overlapping bones in the sclera remains unclear. A better understanding of the ocular skeletal diversity in Reptilia as well as a better understanding of the mechanisms of vision within different environments (i.e., air vs. water) and that used by secondarily aquatic organisms is needed. This review discusses the observed variation in morphology and development of the ocular skeleton in the context of evolution and highlights our knowledge gaps in these areas. Anat Rec, 2018. © 2018 American Association for Anatomy.

Localization of Tfap2ß, Casq2, Penk, Zic1, and Zic3 Expression in the Developing Retina, Muscle, and Sclera of the Embryonic Mouse Eye.

Optic development involves sequential interactions between several different tissue types, including the overlying ectoderm, adjacent mesoderm, and neural crest mesenchyme and the neuroectoderm. In an ongoing expression screen, we identified that Tfap2ß, Casq2, Penk, Zic1, and Zic3 are expressed in unique cell types in and around the developing eye. Tfap2ß, Zic1, and Zic3 are transcription factors, Casq2 is a calcium binding protein and Penk is a neurotransmitter. Tfap2ß, Zic1, and Zic3 have reported roles in brain and craniofacial development, while Casq2 and Penk have unknown roles. These five genes are expressed in the major tissue types in the eye, including the muscles, nerves, cornea, and sclera. Penk expression is found in the sclera and perichondrium. At E12.5 and E15.5, the extra-ocular muscles express Casq2, the entire neural retina expresses Zic1, and Zic3 is expressed in the optic disk and lip of the optic cup. The expression of Tfap2ß expanded from corneal epithelium to the neural retina between E12.5 to E15.5. These genes are expressed in similar domains as Hedgehog (Gli1, and Ptch1) and the Wnt (Lef1) pathways. The expression patterns of these five genes warrant further study to determine their role in eye morphogenesis.

Towards understanding the dose and timing effect of hydrocortisone treatment on the scleral ossicle system within the chicken eye.

Previous work, almost four decades ago, showed that hydrocortisone (HC) treatment reduces the number of skeletogenic condensations that give rise to the scleral ossicles in the chicken eye. The scleral ossicles are a ring of overlapping intramembranous bones, the sclerotic ring, and are present in most reptiles, including birds. The scleral condensations that give rise to the scleral ossicles are induced by a series of overlying thickenings (or papillae) of the conjunctival epithelium. Here, we further explore the effects of altering the dosage and timing of HC treatment on the morphology and number of skeletogenic condensations and conjunctival papillae. We show that high doses can completely obliterate the entire sclerotic ring. Significantly, the reduction in papillae number we observed was less extreme than that of the scleral condensations, indicating that additional factors contribute to the observed skeletogenic condensation loss. Via immunohistochemical analyses, we show that HC treatment alters the spatial expression pattern of several extracellular matrix components (tenascin-C, decorin and procollagen I) and also alters the vasculature network within the sclera. This research provides important insights into understanding the role of the scleral tissue components in ossicle development within the vertebrate eye.

Development of the New Zealand White Rabbit Eye: I. Pre- and Postnatal Development of Eye Tunics.

The New Zealand white (NZW) rabbit has been and is right now regularly utilized in ophthalmic surgery evaluation. Inside NZW rabbit eye, the visibility of ocular structures throughout surgical procedure is fantastic. Younger rabbits are used in different ages for the evaluation of ophthalmic surgery. Complete studies of ocular development in the NZW rabbits have not been reported previously. The aim of the present investigation was to describe the major landmarks and the time course of the pre- and post-natal development of the complete eye tunics of the NZW rabbit to give a superb model as well as a fruitful area for further ophthalmological investigations. Serial histological sections of NZW rabbit prenatal (E13-E28) and post-natal (P1-P14) stages were examined, respectively. The eye of the NZW rabbit developed in a similar manner to that of the human and domestic animals eyes; the principal differences were at the time of occurrence of certain developmental events, absence of pigmentation which represent an exploited benefit for ophthalmic surgery, remarkable Bowman's membrane at E25, poor developed ciliary stroma and juvenile retinal layer until P9. In human, the basic morphogenetic processes of the development of eye tunics are completed towards the end of the first half of gestation period. However, the latter represents the beginning stage of the development of eye tunics in the rabbit. Thus, allowing various extensive ophthalmic researches to be performed.

Effects of Glucose Administration on Development of Sclera in Chick Embryos.

OBJECTIVE: To determine the effect of glucose administration on the development of sclera in the chick embryo Gallus domesticus. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Anatomy Department, CPSPRegional Centre, Islamabad, from January 2013 to January 2014. METHODOLOGY: The study was carried out in two main groups, control Aand experimental B, which were subdivided into three subgroups comprising 30 eggs each. The group Awas injected with normal saline (0.3 ml) in the egg albumen. The group B was injected with 0.3 ml of 5% w/v solution of glucose equivalent to 15 mg of glucose. Subgroups A1 and B1 were opened on day 10 of incubation. Subgroups A2 and B2 were sacrificed on day 12 of incubation. Eggs from subgroups A3 and B3 were opened on day 15 of incubation. Experimental subgroups were compared with matched control subgroups and quantitative data was analysed statistically. RESULTS: Administration of glucose resulted in changes in thickness of sclera. The mean thickness (µm) of sclera at day 10 of incubation was 43.54 ±2.45 in control subgroup and 43.03 ±5.86 in the experimental subgroup (p=0.673). The mean thickness (µm) of sclera at day 15 of incubation 77.48 ±8.32 in control subgroup and 73.99 ±8.62 in experimental subgroup (p=0.145). The mean number of chondrocytes/unit area of hyaline cartilage of sclera in day 10 was 17.40 ±1.44 control subgroup and 14.57 ±1.87 in the experimental subgroup (p < 0.001). The mean number of chondrocytes/unit area of hyaline cartilage of sclera on day 15 was 10.02 ±0.86 in the control subgroup and 9.54 ±0.59 in the experimental subgroup (p=0.025). There was disrupted ossicular formation indicating adverse effects on the development of bony sclera as well. CONCLUSION: Administration of glucose caused alteration in the histology of sclera in developing chick embryos.

Puerarin, an isoflavone compound extracted from Gegen (Radix Puerariae Lobatae), modulates sclera remodeling caused by extremely low frequency electromagnetic fields.

OBJECTIVE: To evaluate the protective effect of puerarin [an isoflavone compound extracted from Gegen (Radix Puerariae Lobatae)] in scleral remodeling induced by extremely low frequency electromagnetic fields (ELF-EMFs). METHODS: Human fetal scleral fibroblasts (HFSFs) were divided into 5 groups: (a) untreated controls; (b) cells treated with ELF-EMFs; (c) cells treated with ELF-EMFs and puerarin 0.1 µM; (d) cells treated with ELF-EMFs and puerarin 1 µM; (e) cells treated with ELF-EMFs and puerarin 10 µM. Cell proliferation activity was measured by the cell-counting kit-8 assay. Matrix metalloproteinase-2 (MMP-2) activity was measured by gelatin enzymography. MMP-2 and collagenⅠ(COL1A1) mRNA, protein expression were measured by Real-Time polymerase chain reaction , Western blot analysis, respectively. RESULTS: Puerarin reduced the inhibition in cell proliferation, MMP-2 activity, mRNA, protein expression of HFSFs exposed to ELF-EMFs and enhanced the COL1A1 mRNA and protein expression. CONCLUSION: Puerarin was found to participate in the matrix remodeling process. It might be a potential agent for the treatment of extracellular matrix degradation of sclera associated with ocular conditions.

Vascular endothelial growth factor signaling affects both angiogenesis and osteogenesis during the development of scleral ossicles.

Intramembranous ossification is a complex multi-step process which relies on extensive interactions among bone cells and surrounding tissues. The embryonic vasculature is essential in regulating endochondral ossification; however, its role during intramembranous ossification remains poorly understood, and in vivo studies are lacking. Previous research from our lab on the development of the intramembranous scleral ossicles has demonstrated an intriguing pattern of vascular development in which the areas of future osteogenesis remain avascular until after bone induction has occurred. Such avascular zones are located directly beneath each of the conjunctival papillae, epithelial structures which provide osteogenic signals to the underlying mesenchyme. Here we provide a high-resolution map of the developing vasculature from the time of ossicle induction to mineralization using a novel technique. We show that vegfa is expressed by the papillae and nearby mesenchymal tissue throughout HH 34-37, when vascular growth is taking place, and is down-regulated thereafter. Localized inhibition of Vegf results in expansion of the avascular zone surrounding the implanted papilla and mispatterning of the scleral ossicles. These results demonstrate that Vegf signaling could provide important insights into the complex relationship between bone and vasculature during intramembranous bone development.

Absence of lymphatic vessels in the developing human sclera.

The adult sclera is free of lymphatic vessels, but contains a net of blood vessels. Whether and when this selectively lymphangiogenic privilege is achieved during embryologic development is not known yet. Therefore, we investigated the developing human sclera for blood- and lymphatic vessels in 34 abortions/stillborns (12-38 weeks of gestation). The probes were subdivided into three groups (group 1: 12-18 weeks of gestation, n = 10; group 2: 19-23 weeks of gestation, n = 13; group 3: 24-38 weeks of gestation, n = 11), and prepared for paraffin sections followed by immunohistochemistry against CD31 to detect blood vessels, and against lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1)/podoplanin to detect lymphatic vessels. We could show, that in the human episclera distinct CD31 + blood vessels are present as early as week of gestation 13. Their amount increased during pregnancy, whereas stromal CD31 + blood vessels were elevated in early pregnancy and regressed with ongoing pregnancy. In the lamina fusca CD31 + blood vessels were absent at any time point investigated. Single LYVE1 + cells were identified primarily in the episclera; their amount decreased significantly with increasing gestational ages (group 1 compared to group 3: p < 0.01). However, LYVE1+/podoplanin + lymphatic vessels were not detectable in the sclera at any gestational ages analyzed. In contrast to the conjunctiva where LYVE1+/podoplanin + lymphatic vessels were detectable as early as week 17, the amount of LYVE1 + cells in the sclera was highest in early pregnancy (group 1), with a significant decrease during continuing pregnancy (p < 0.001). These findings are the first evidence for a fetal lymphangiogenic privilege of the sclera and show, that the fetal human sclera contains CD31 + blood vessels, but is primarily alymphatic. Our findings suggest a strong expression of selectively antilymphangiogenic factors, making the developing sclera a potential model to discern antilymphangiogenic mechanisms.

Scleral micro-RNA signatures in adult and fetal eyes.

INTRODUCTION: In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses. METHODS: Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods. RESULTS: Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10(11)). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples. CONCLUSION: For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.

Whole genome expression profiling of normal human fetal and adult ocular tissues.

To study growth and development of ocular tissues, gene expression patterns in normal human fetal versus adult eyes were compared. Human retina/retinal pigment epithelium, choroid, sclera, optic nerve* and cornea* tissues were dissected from fetal (24 week gestational age) (N = 9; *N = 6), and adult (N = 6) normal donor eyes. The Illumina(®) whole genome expression microarray platform was used to assess differential expression. Statistical significance for all comparisons was determined using the Benjamin and Hochberg False Discovery Rate (FDR, 5%). Significant gene expression fold changes > 1.5 were found in adult versus fetal retina/RPE (N = 1185), choroid (N = 6446), sclera (N = 1349), and cornea (N = 3872), but not optic nerve. Genes showing differential expression were assessed using Ingenuity Pathway Analysis (IPA) for enriched functions and canonical pathways. In all tissues, development, cell death/growth, cancer functions, and signaling canonical pathways were enriched. There was also a general trend of down-regulation of collagen genes in adult tissues.

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.

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.

Development and mineralization of embryonic avian scleral ossicles.

PURPOSE: To investigate the development and mineralization of avian scleral ossicles using fluorescence microscopy in combination with field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS). METHODS: The anterior halves of whole eyeballs from chickens on embryonic (E) days E10 to E21 and Japanese quail on embryonic days E8 to E17 were fixed in 100% methanol for 1 min, stained with Giemsa solution for 5 min, destained with distilled water for 30 min, and then viewed by epifluorescence. Propidium iodide (PI) was used to detect the nuclei of osteocytes in scleral ossicles. FESEM and EDS were then used to show areas of mineralization and to identify differences in the elemental composition of different regions of the ossicles. RESULTS: Using Giemsa as a fluorescence stain, it was possible to observe the detailed morphology and development of both chicken and quail scleral ossicles. In chickens, bone microporosities first became visible at E15. Each microporosity contained a single nucleus, likely that of an osteocyte. The amount of carbon in ossicles steadily decreased during embryogenesis and post-hatching, while the concentration of oxygen showed a distinct increase over this time period. Calcium and phosphate levels in the ossicles increased gradually during embryonic and post-hatching stages. CONCLUSIONS: A novel approach to study the development and mineralization of avian scleral ossicles during embryogenesis is presented. This methodology was validated by studying two different species, both important models for avian developmental research.

The retinal pigment epithelium of the eye regulates the development of scleral cartilage.

The majority of vertebrate species have a layer of hyaline cartilage within the fibrous sclera giving an extra degree of support to the eyeball. In chicks, this is seen as a cuplike structure throughout the scleral layer. However, the mechanisms that control the development of scleral cartilage are largely unknown. Here we have studied the phases of scleral cartilage development and characterised expression profiles of genes activated during the cartilage differentiation programme. CART1 and SOX9, the earliest markers of pre-committed cartilage, are expressed in the mesenchyme surrounding the optic cup. Later AGGRECAN, a matrix protein expressed during chondrocyte differentiation, is also expressed. The expression of these genes is lost following early removal of the optic cup, suggesting a role for this tissue in inducing scleral cartilage. By grafting young retinal pigment epithelium (RPE) and retina into cranial mesenchyme in vivo, it was found that RPE alone has the ability to induce cartilage formation. There are some exceptions within the vertebrates where scleral cartilage is not present; one such example is the placental mammals. However, we found that the cartilage differentiation pathway is initiated in mice as seen by the expression of Cart1 and Sox9, but expression of the later cartilage marker Aggrecan is weak. Furthermore, cartilage forms in mouse peri-ocular mesenchyme micromass culture. This suggests that the process halts in vivo before full differentiation into cartilage, but that murine scleral mesenchyme has retained the potential to make cartilage in vitro. RA, Wnts and Bmps have been linked to the cartilage development process and are expressed within the developing RPE. We find that RA may have a role in early scleral cartilage development but is not likely to be the main factor involved. These data reveal the course of scleral cartilage formation and highlight the key role that the optic cup plays in this process. The driving element within the optic cup is almost certainly the retinal pigmented epithelium.

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.

Indian hedgehog signaling from endothelial cells is required for sclera and retinal pigment epithelium development in the mouse eye.

The development of extraocular orbital structures, in particular the choroid and sclera, is regulated by a complex series of interactions between neuroectoderm, neural crest and mesoderm derivatives, although in many instances the signals that mediate these interactions are not known. In this study we have investigated the function of Indian hedgehog (Ihh) in the developing mammalian eye. We show that Ihh is expressed in a population of non-pigmented cells located in the developing choroid adjacent to the RPE. The analysis of Hh mutant mice demonstrates that the RPE and developing scleral mesenchyme are direct targets of Ihh signaling and that Ihh is required for the normal pigmentation pattern of the RPE and the condensation of mesenchymal cells to form the sclera. Our findings also indicate that Ihh signals indirectly to promote proliferation and photoreceptor specification in the neural retina. This study identifies Ihh as a novel choroid-derived signal that regulates RPE, sclera and neural retina development.