The retina is a striking example of architectural polarity. At its vitreal surface, it is bordered by the retinal-ganglion cells, and sclerad by the retinal pigmented epithelium (RPE). Adjacent to the RPE, photoreceptor nuclei comprise the outer nuclear layer; a variety of interneurons and Müller glia make up the inner nuclear layer, which is adjacent to the ganglion-cell layer. The ganglion and RPE cell layers, which are the first layers to differentiate, establish the polarity of the remaining layers during development. In some pathological states, this ordered arrangement is disrupted resulting in "retinal dysplasia", which is characterized by tubular structures known as "rosettes". Such rosettes are reminiscent of the Flexner-Wintersteiner rosettes and fleurettes of retinoblastoma. However, unlike their malignant counterpart, dysplastic rosettes do not represent a neoplastic, or even pre-neoplastic process. Little is known about the histogenesis of dyplastic rosettes. Cyclopia provides an ideal setting to study retinal dysplasia as rosettes are common in this condition.

Cyclopia may be associated with holoprosencephaly, the most common developmental defect of the forebrain with an incidence of 1:250 during embryogenesis. Due to intrauterine lethality, live born prevalence of holoprosencephaly is 1:16,000. It can range from major brain and facial anomalies to clinically normal individuals having only a single fused central incisor [reviewed in 12345]. Holoprosencephaly is a malformation sequence in which impaired midline cleavage of the embryonic forebrain is a fundamental feature 67891011. The prosencephalon fails to cleave sagitally into cerebral hemispheres, and transversely into telencephalon and diencephalons. Given the complexity of the cellular interactions occurring during forebrain development, it is not surprising that a variety of genes and teratogens have been implicated in the pathogenesis of holoprosencephaly 2367111213141516171819202122.

The study of ocular development is providing insight into the pathogenesis of holosproencephaly. It is now appreciated that during development, a single eye field is bisected by signals from the underlying prechordal mesoderm. The early eye field therefore represents a larger area of neural ectoderm that is competent to respond to the inducer signal to form an eye. If the competence of the central eye field is not inhibited when induction begins, the entire field will respond with the formation of a single eye. This inhibition appears to be mediated by Sonic Hedgehog (Shh). Mutational inactivation of Shh, or its receptor Ptch, can result in holoproscencephaly with cyclopia 79121315182223242526.

Although cyclopia is well documented clinically, detailed understanding of the consequences of cyclopia to ocular development is limited 27. Here, we hope to extend our understanding of human cyclopia in the setting of holoprosencephaly with particular attention to the histogenesis of retinal dysplasia, a poorly understood yet common ophthalmic pathological finding 28.

A common thread in the constellation of features found in this case is the failure of structures normally bilaterally positioned in the term neonate to separate from the midline during development. This is seen, for example, here in the failure of the brain to establish lateral hemispheres and in the globes to become separate eyes. The retained fusion of bilateral structures may be regarded as a defect in the establishment of polarity. In view of the increasing appreciation of embryonic induction and Hedgehog signaling in these processes, we wanted to explore whether such mechanisms could be extended to the tissue level allowing for an explanation of the ocular changes seen in human cyclopia.

The autopsy findings are consistent with the diagnosis of Patau's syndrome. This conclusion is confirmed by the cytogenetic demonstration of trisomy 13 in the patient. The etiology of trisomy 13 is unknown. The clinical presentation is typical except for the young age of the mother (19 years). Trisomies tend to occur with greater frequency with advanced age 31. The age effect is not as great with trisomy 13 compared to 18 and 21 32. Trisomy 13 is more common in females 33. It should be noted that in contrast to trisomy 21, the risk for recurrence of trisomies 18 and 13 is considered to be very low 34. The phenotype in this case represents a more severe end of the spectrum with the manifestation of holosproencephaly and cyclopia.

In theory, cyclopia could result from the fusion of two originally separate eyes, or from the failure of a single primordium to separate during development. Microdissection studies have shown that removal of the prechordal mesoderm leads to the formation of a single retina in chick embryos and Xenopus explants 3536. For example, removal of the prechordal plate results in fusion of the forebrain as well as the retina 36. Furthermore, the prechordal plate expresses Shh and was able to rescue the cyclopic phenotype in transplantation studies. Interestingly, transplantation of the prechordal plate to the vicinity of the optic cup suppresses the expression of pax6 in the retina. We can conclude that there is a single retinal morphogenetic field that resolves into two retina primordial. This is accomplished by a prechordal-plate signal that suppresses retinal formation in the medial region of the field 36. This model is consistent with that suggested more than 75 years ago by Adelmann (1929) 37.

In the present case, a disruption of the normal sequence of development appears to have occurred after lens induction, but before the formation of the cornea and angle structures, suggesting an interruption of neural crest migration. Normally, the corneal stroma, endothelium, anterior iris, and trabecular meshwork are formed by neural crest cells entering the optic-cup lip. Although the reason for the defective neural crest migration is unknown, it is interesting that inhibition of Shh in vivo results in neural crest cell death 10. The absence of the corneal endothelium, which promotes formation of the anterior chamber, could explain the lack of separation of the lens from the cornea 38.

Insight into the role that abnormal Hedgehog signaling may have in the histogenesis of retinal dysplasia has come from studies of Ptch. Ptch mutations cause Gorlin syndrome, an autosomal dominant disorder characterized by dental, skeletal and radiographic abnormalities including falx calcification, bifid/fused ribs and altered vertebral segmentation, and a predisposition to tumor development including early-onset basal cell carcinomas 394041424344. Ocular abnormalities are often present as were in the first patient described by Dr Gorlin 40. These findings include microphthalmia, coloboma, cataract, inappropriate retinal myelination, and retinoschisis 454647484950. It is therefore intriguing that the Hedgehog pathway has not only a role in establishing separate eyes, but also in the development of the retina itself. This is further supported by studies in zebrafish indicating that signals from the prechordal mesoderm can effect retinal neurogenesis 515253.

Our finding that Shh is found in the cytoplasm of the retinal-ganglion cells in humans is consistent with recent studies showing that its mRNA is expressed in the ganglion cells of other vertebrates 52545556. Our finding of Shh positive cells in the inner nuclear layer is consistent with its known expression in zebra fish amacrine cells 55. The significance of these observations is that Shh secretion by the ganglion cells appears to be critical to orchestrating the normal organization of the developing neural retina 5254555758596061. This notion is supported by the fact that ptch and gli are expressed in retinal neuroblasts, and astrocyte precursor cells in the optic nerve 6263. Retinal-ganglion cell derived Shh expression is required for Hedgehog target gene induction in the retina and optic nerve 565758.

Notwithstanding the potential importance of the ganglion cells in these processes, it should be pointed out that the system is complex with other local sources and targets of Hh signaling 6465. In mouse, Ihh is expressed by RPE, and recombinant Hh protein promotes photoreceptor differentiation in vitro 57. In zebrafish, Shh and Twhh are both expressed by RPE, and Hh signaling knockdown results in reduced photoreceptor differentiation, and retinal cell death 6566. Finally, the RPE itself may need Hh signaling in order to properly differentiate 67.

To address the question of the role of the Hedgehog pathway and the ptch receptor in particular on the development of the mammalian retina, Black et al (2004) 68 studied ocular development in mice heterozygotic for disruption of the ptch gene 69707172. The retinas of PtchlacZ +/- mice exhibit abnormal cell cycle regulation, culminating in photoreceptor dysplasia and Müller cell gliosis. Interestingly, the PtchlacZ +/- mice also show vitreoretinal abnormalities resembling those found in patients with Gorlin syndrome and dysplastic rosette formation.

The histopathology of cyclopia appears to be more complex than what may have been previously appreciated. In fact, the terms "cyclopia" and "synophthalmia" do not reflect the histopathology or pathophysiology. True cyclopia, i.e., a single eye, is vanishingly rare, with most cases showing two fused eyes (synophthalmia). However, synophthalmia does not accurately reflect the mechanism resulting in the fusion of the eyes. That is, the fusion is not the result of two eyes coming together, but rather the failure of the eyes to form separately during development. According to the animal studies reviewed above, the defect is a failure of the Shh pathway to inhibit competence in the central eye field. The result is an incomplete separation of the eyes during development. At the tissue level, the complexity extends to a failure of the angle structures to form, and the retina to achieve its orientation toward the RPE.

Dysplastic rosettes are laminated retina failing to establish a polarized orientation toward the RPE resulting is the formation of tubules. The finding, which is not considered to be neoplastic or preneoplastic, is often associated with trisomy 13, but may be seen in other clinical 28 and experimental settings. The rosettes should be distinguished from the various rosettes seen in retinoblastoma which do not contain Müller cells 307374. The rosettes found in the dysplastic retina are fundamentally different than those of retinoblastoma, in that unlike their neoplastic counterpart they are composed of a variety of differentiated cell types.

The mechanism responsible for the histogenesis of retinal dysplasia is far from clear. The application of the term "retinal dysplasia" to describe a "maldevelopment of the retina" and early descriptions of its rosettes have been reviewed by Lahav et al. (1973) 28. It has been proposed that the rosettes may represent an abortive attempt at regeneration 75, or an abnormality in programmed cell death 73. Although the mechanism for the formation of the rosettes remains unknown, it is interesting that retinal dysplasia including rosette-like formations are found in Patch +/- mice and in conditional ablation of Shh 6168. In the present case, offering a mechanism for the histogenesis of the retinal dysplasia should be done with caution. That is, we cannot with certainty establish a cause-effect relationship between the absence of Shh expression and the formation of the rosettes. For example, it is possible that the lack of Shh staining could simply be a secondary effect due, for example, to the absence of the cells producing the protein. Thus, although alternative models should be taken into account, our findings, taken together with the available literature, suggest the possibility that the dysplastic rosettes in human cyclopia result from a direct or indirect failure of Hedgehog signaling.

IRBP: Interphotoreceptor retinoid-binding protein;

CRALBP: Cellular retinaldehyde binding protein;

RPE: Retinal pigmented epithelium;

Shh: Sonic hedgehog.

The author(s) declare that they have no competing interests.

AC and RH performed the autopsy. SL followed the patient clinically. The ocular histopathological studies were carried out in FGF's laboratory. AC and FGF conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.