Surface ectoderm

Abstract: The Pax6 transcription factor plays a key role in ocular development of vertebrates and invertebrates. Homozygosity of the Pax6 null mutation in human and mice results in arrest of optic vesicle development and failure to initiate lens formation. This phenotype obscures the understanding of autonomous function of Pax6 in these tissue components and during later developmental stages. We employed the Cre/loxP approach to inactivate Pax6 specifically in the eye surface ectoderm concomitantly with lens induction. Although lens induction occurred in the mutant, as indicated by Sox2 up-regulation in the surface ectoderm, further development of the lens was arrested. Hence, Pax6 activity was found to be essential in the specified ectoderm for lens placode formation. Furthermore, this mutant model allowed us for the first time to address in vivo the development of a completely normal retina in the absence of early lens structures. Remarkably, several independent, fully differentiated neuroretinas developed in a single optic vesicle in the absence of a lens, demonstrating that the developing lens is not necessary to instruct the differentiation of the neuroretina but is, rather, required for the correct placement of a single retina in the eye.

Abstract: Vertebrate eye development proceeds by a series of reciprocal tissue interactions between derivatives of the head surface ectoderm and the forebrain neuroectoderm. In particular, the process of lens induction has been studied as a model system to explore general mechanisms underlying embryonic induction (for review, see Jacobson and Sater 1988). Soon after the turn of this century, a few classical experiments using amphibian embryos led to the idea that the optic vesicle has an instructive role in inducing lens formation in the overlying surface ectoderm (Spemann 1901; Lewis 1904, 1907a,b). According to this original model, the optic vesicle is potentially able to induce a lens from ectoderm anywhere on the embryo. Subsequently, however, other studies using different species and experimental conditions raised doubts about this hypothesis, and rather supported the idea that the optic vesicle is not essential for lens formation (for review, see Saha et al. 1989). Furthermore, a key experiment using a host–donor cell-marking technique revealed that the optic vesicle is insufficient to induce lens in ectopic ectoderm taken from outside the lens field; the induced lens in the recombinants was derived from the optic rudiment, rather than from the grafted ectopic ectoderm (Grainger et al. 1988). The current model, based mainly on experiments using Xenopus embryos, proposes that lens induction proceeds through multiple intermediate states, starting early in development from the gastrula stage (for review, see Grainger 1992). Evidence from many in vivo and in vitro studies indicates that the lens can be formed in the absence of the optic vesicle in several vertebrate species (for review, see Jacobson and Sater 1988; Saha et al. 1989; Henry and Grainger 1990). The role of the optic vesicle during lens induction has, therefore, been considered minor—merely to establish the precise location of the lens within the head ectoderm (Grainger 1992). Several lines of evidence, however, suggest that, in vivo, there is apparent species specificity in the extent to which lens formation is dependent on the optic vesicle. Ablation of prospective retinal neuroectoderm in chick embryos abolishes lens formation (Li et al. 1994; Kamachi et al. 1998). Furthermore, lens formation is completely absent in mouse embryos lacking a functional Lhx2 gene, which encodes a LIM-homeodomain protein and is expressed in the forebrain, including the forming optic vesicle neuroectoderm. In these embryos, impairment of optic vesicle development results in failure of the optic vesicle to contact the surface ectoderm (Porter et al. 1997). Therefore, it appears that lens formation in higher vertebrates requires the presence of the optic vesicle in vivo. Like many other embryonic induction processes, secreted signaling molecules are likely to have critical roles during lens induction. In spite of the extensive experimental studies in the amphibian system described above, however, no actual signaling molecules have yet been identified. Likewise, although a number of genes have been implicated in mammalian eye development by molecular and genetic approaches (for review, see Graw 1996; Oliver and Gruss 1997), characterization of their precise in vivo function in many cases awaits further studies. One exception is the Small eye mutant in mouse (Pax6Sey) and rat (rSey), which have been studied extensively in relation to lens induction, as homozygous mutant embryos completely lack lens formation (Hill et al. 1991; Matsuo et al. 1993). Mutations in the Pax6 gene, which encodes a paired-type homeodomain protein, have also been associated with congenital eye defects in humans (Jordan et al. 1992; Glaser et al. 1994; Hanson et al. 1994). Tissue recombination experiments using rSey mutant embryos have revealed that homozygous mutant ectoderm does not form a lens when recombined with a wild-type optic vesicle, whereas the reciprocal recombination allows lens formation in wild-type ectoderm (Fujiwara et al. 1994). This clearly implies a requirement for PAX6 in the head ectoderm for competence to respond to the optic vesicle signal, and indicates that the optic vesicle retains lens inducing activity in the absence of PAX6 function. The underlying mechanisms of PAX6 function in the ectoderm and inductive signaling factors involved in lens induction are still unknown. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily of secretory signaling molecules, have been implicated in many aspects of embryonic tissue interactions (for review, see Hogan 1996). Several BMP family members have been reported to be expressed during mouse eye development (Dudley and Robertson 1997). Furthermore, Bmp7 is required for normal embryonic eye development in the mouse (Dudley et al. 1995; Luo et al. 1995). Here, we report that another member of the Bmp gene family, Bmp4, has critical roles during the lens induction process. We show that the optic vesicle is the major source of BMP4 in the early developing eye, and that it is required for lens induction. A series of explant culture experiments suggest that BMP4 is an essential factor to manifest lens inducing activity of the optic vesicle. We also show that BMP4 regulates specific gene expression in the optic vesicle neuroectoderm. We discuss a model in which BMP4 has multiple roles during mammalian lens induction.