Lens morphogenesis is dependent on Pax6‐mediated inhibition of the canonical Wnt/beta‐catenin signaling in the lens surface ectoderm

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens‐specific gene expression in the surface‐ectoderm. Supression of canonical Wnt/β‐catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down‐regulation of canonical Wnt signaling in the presumptive lens ectoderm. genesis 48:86–95, 2010. © 2009 Wiley‐Liss, Inc.     

Aphakia (ak), a mouse mutation affecting early eye development: Fine mapping,consideration of candidate genes and altered Pax6 and Six3 gene expression pattern

The homozygous mouse mutant aphakia (ak) has been characterized by bilaterally aphakic eyes without a pupil [Varnum DS, Stevens, LC (1968): J Hered 59:147–150]. The mutation was mapped to chromosome 19 [Varnum DS, Stevens, LC (1975): Mouse News Lett 53:35]. Our linkage studies yielded a precise localization of the ak gene 0.6 ± 0.3 cM proximal to the microsatellite marker D19Mit10 and 0.7 ± 0.4 cM distal to D19Mit4 and D19Mit91. No recombination was found with the marker D19Mit9 among 418 backcross offspring tested. The developmental control gene Pax2 mapped 11.0 ± 3.5 cM proximal to ak and is excluded as a candidate gene. Sequence analysis of Fgf8 and Chuk1, which are localized close to the marker D19Mit10, detected no mutations in the ak/ak mutants. Histological analysis of homozygous mutants suggested the arrest of lens development at the lens stalk stage, a transient morphological structure during the formation of the lens vesicle. In the lens remnants, Pax6 and Six3 are expressed, whereas in the persisting lens stalk only Pax6 was detected. The expression pattern of Pax2 appeared normal; Cryaa expression could not be detected. As a consequence of the arrested lens development, other ocular tissues that require for their development information from the intact lens, such as iris, ciliary muscle, retina, and vitreous body, are absent or formed abnormally. Dev. Genet. 23:299–316, 1998. © 1998 Wiley-Liss, Inc.     

Determinative roles of FGF and Wnt signals in iris-derived lens regeneration in newt eye

Total regeneration of experimentally excised lens from the dorsal part of the iris-pigmented epithelium of newts has been a key model of tissue regeneration via cells originating from a foreign tissue. Due to the strict spatial restriction of the lens origin in the newt iris, it has often been assumed that only the dorsal iris cells are endowed with an intrinsic potential to give rise to lens tissues. However, our reinvestigation of the process revealed completely different mechanisms underlying lens regeneration and its spatial restriction, comprising the following two steps: (i) Fibroblast growth factor (FGF) 2-dependent proliferation of iris-pigmented epithelium and activation of early lens genes ( Pax6, Sox2, MafB ) over the entire circumference of the iris; and (ii) dorsal iris-restricted activation of the canonical Wnt signals (involving Wnt2b and Frizzeld4) that leads to localized expression of late lens genes ( Prox1, Sox1, β-crystallin ). Injection of FGF2 into normal eyes specifically elicited the second lens development from the dorsal iris, and the administration of recombinant Wnt3a to the cultured iris-pigmented epithelium caused even ventral iris-derived lens development. Thus, it is concluded that the regulation of FGF2 and Wnt signals is a determinative of the iris-derived lens regeneration in the newt eye.     

Drosophila Pax‐6/eyeless is essential for normal adult brain structure and function

A role for the Pax‐6 homologue eyeless in adult Drosophila brain development and function is described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of Pax‐6/eyeless that result in truncated proteins. Consistent with the morphological lesions, we observe defective walking behavior for these eyeless mutants. The implications of these data for understanding postembryonic brain development and function in Drosophila are discussed. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 73–88, 2001     

The homeodomain of Eyeless regulates cell growth and antagonizes the paired domain-dependent retinal differentiation function

Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.     

Activation of Six1 target genes is required for sensory placode formation

In vertebrates, cranial placodes form crucial parts of the sensory nervous system in the head. All cranial placodes arise from a common territory, the preplacodal region, and are identified by the expression of Six1/4 and Eya1/2 genes, which control different aspects of sensory development in invertebrates as well as vertebrates. While So and Eya can induce ectopic eyes in Drosophila, the ability of their vertebrate homologues to induce placodes in non-placodal ectoderm has not been explored. Here we show that Six1 and Eya2 are involved in ectodermal patterning and cooperate to induce preplacodal gene expression, while repressing neural plate and neural crest fates. However, they are not sufficient to induce ectopic sensory placodes in future epidermis. Activation of Six1 target genes is required for expression of preplacodal genes, for normal placode morphology and for placode-specific Pax protein expression. These findings suggest that unlike in the fly where the Pax6 homologue Eyeless acts upstream of Six and Eya, the regulatory relationships between these genes are reversed in early vertebrate placode development.     

Hindbrain-derived Wnt and Fgf signals cooperate to specify the otic placode in Xenopus

Induction of the otic placode, the rudiment of the inner ear, is believed to depend on signals derived from surrounding tissues, the head mesoderm and the prospective hindbrain. Here we report the first attempt to define the specific contribution of the neuroectoderm to this inductive process in Xenopus. To this end we tested the ability of segments of the neural plate (NP), isolated from different axial levels, to induce the otic marker Pax8 when recombined with blastula stage animal caps. We found that one single domain of the NP, corresponding to the prospective anterior hindbrain, had Pax8-inducing activity in this assay. Surprisingly, more than half of these recombinants formed otic vesicle-like structures. Lineage tracing experiments indicate that these vesicle-like structures are entirely derived from the animal cap and express several pan-otic markers. Pax8 activation in these recombinants requires active Fgf and canonical Wnt signaling, as interference with either pathway blocks Pax8 induction. Furthermore, we demonstrate that Fgf and canonical Wnt signaling cooperate to activate Pax8 expression in isolated animal caps. We propose that in the absence of mesoderm cues the combined activity of hindbrain-derived Wnt and Fgf signals specifies the otic placode in Xenopus, and promotes its morphogenesis into an otocyst.