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

Abstract

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. Developmental Dynamics 237: 3240 -3251, 2008.

Optimized <em>Ex-ovo</em> Culturing of Chick Embryos to Advanced Stages of Development

“…In general, survivability of the embryo in ex ovo culture is high up to HH 35-36 (90-100%), but it does drop in the more advanced stages of development (about 40% survive to HH 40-41). Additionally, although it has been shown that development appears normal at these late stages and patterning of the skeleton is unaffected 4,15 , we have observed that the limbs are bent at these late stages suggesting that the degree or timing of ossification may be affected. This is not surprising considering the need for calcium from the shell for ossification.…”

“…Previous research has shown that culturing the embryos ex ovo does not negatively affect patterning 4,15 . For example, we do not see any changes in the patterning or induction of the skeleton despite the absence of the eggshell.…”

“…This is often done using affigel or heparin beads (Figure 2A). For example, bone morphogenetic proteins (BMPs) can be locally inhibited during the induction of neural crest derived bone 4,15 , or in the developing limb bud 13 . After a bead is implanted, the embryo can be returned to the incubator and it will continue to develop.…”

“…After a bead is implanted, the embryo can be returned to the incubator and it will continue to develop. This allows researchers to view the downstream effects of the inhibition of a particular gene (in this case BMP), such as the inhibition of scleral ossicle formation in the sclerotic ring (Figure 2B and 2C) 15 . Similarly, barriers can easily be inserted between tissue layers in order to study tissue-to-tissue signaling and the microinjection of dyes can also easily be conducted.…”

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

“…The first papilla forms at Hamburger and Hamilton stage 30 (HH 30) directly over the 66 ciliary artery, and is designated papilla #12. This is followed by the remainder of the temporal 67 group (#10 -14), the nasal group (#2 -6), the dorsal group (#7-9) and finally a ventral papilla (#1) 68 forms directly over the choroid fissure (Coulombre et al, 1962;Franz-Odendaal, 2008; Fig. 1A).…”