The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm

In the current analysis, we have investigated both the cytoskeletal and signaling roles of beta-catenin during the early phases of lens development using conditional loss- and gain-of-function strategies. Conditional loss of beta-catenin in the presumptive lens does not perturb the normal sequential appearance of lens fate markers but results in a dramatic failure of the coordinated epithelial cell behavior that constitutes lens morphogenesis. Similarly, loss-of-function for Lrp6, the Wnt pathway coreceptor expressed in the eye primordium, does not prevent expression of lens induction markers. Surprisingly, conditional deletion of beta-catenin in periocular ectoderm results in the formation of Prox-1 and beta-crystallin-positive ectopic lentoid bodies. Combined with the observation that the Wnt pathway reporter TOPGAL is expressed in nasal periocular ectoderm, these data suggest that, in this location, the canonical Wnt signaling pathway normally suppresses lens fate in favor of other structures. Consistent with this proposal, a dominant-active form of beta-catenin causes a loss of lens fate and a complete absence of lens development when expressed in the presumptive lens ectoderm.     

Abnormal lens morphogenesis and ectopic lens formation in the absence of beta-catenin function

beta-Catenin plays a key role in cadherin-mediated cell adhesion as well as in canonical Wnt signaling. To study the role of beta-catenin during eye development, we used conditional Cre/loxP system in mouse to inactivate beta-catenin in developing lens and retina. Inactivation of beta-catenin does not suppress lens fate, but instead results in abnormal morphogenesis of the lens. Using BAT-gal reporter mice, we show that beta-catenin-mediated Wnt signaling is notably absent from lens and neuroretina throughout eye development. The observed defect is therefore likely due to the cytoskeletal role of beta-catenin, and is accompanied by impaired epithelial cell adhesion. In contrast, inactivation of beta-catenin in the nasal ectoderm, an area with active Wnt signaling, results in formation of crystallin-positive ectopic lentoid bodies. These data suggest that, outside of the normal lens, beta-catenin functions as a coactivator of canonical Wnt signaling to suppress lens fate.     

Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development

Recent studies indicate a role for Wnt signaling in regulating lens cell differentiation (Stump et al., 2003). Here we investigated expression patterns of Wnt receptors, the Frizzleds (Fzs) and the Wnt signaling regulators, the secreted frizzled-related proteins (Sfrps), during rodent lens development. RT-PCR showed that Fz receptors, Fz1-Fz8 are expressed in lens. In situ hybridization showed that all the Fz genes examined have similar expression patterns. Fzs are expressed throughout the early lens primordium. At embryonic day 14.5 (E14.5), Fz gene expression is predominantly localized to the epithelium and elongating cells at the lens equator. Fz expression is absent from lens fibers. This pattern of Fz gene expression continues throughout early postnatal development. Immunolocalization studies showed that Fz protein distribution closely follows that of the mRNAs. In addition, epithelial cells in FGF-treated explants show strongest Fz reactivity in cellular protrusions as they migrate and elongate. Sfrp1- Sfrp5 are expressed and all, except Sfrp2, have similar patterns of expression to each other and to the Fzs during lens development. Sfrp2 is strongly expressed in all lens pit cells but becomes restricted to the presumptive epithelial cells of the lens vesicle. By E14.5, Sfrp2 is only present in a few cells above the lens equator. Sfrp2 is not detected in the lens at E18.5 or at later stages. This study shows that multiple Fz and Sfrp genes are expressed during lens morphogenesis and differentiation. This is consistent with a role for Wnt-Fz signaling during both embryonic and postnatal lens development.     

Canonical Wnt signaling is required for ophthalmic trigeminal placode cell fate determination and maintenance

Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral sensory nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a useful model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the signaling pathways governing opV placode development have not. Here we tested in chick whether the canonical Wnt signaling pathway regulates opV placode development. By introducing a Wnt reporter into embryonic chick head ectoderm, we show that the canonical pathway is active in Pax3+ opV placode cells as, or shortly after, they are induced to express Pax3. Blocking the canonical Wnt pathway resulted in the failure of targeted cells to adopt or maintain an opV fate, as assayed by the expression of various markers including Pax3, FGFR4, Eya2, and the neuronal differentiation markers Islet1, neurofilament, and NeuN, although, surprisingly, it led to upregulation of Neurogenin2, both in the opV placode and elsewhere in the ectoderm. Activating the canonical Wnt signaling pathway, however, was not sufficient to induce Pax3, the earliest specific marker of the opV placode. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells toward an opV placode fate.     

Six3 activation of Pax6 expression is essential for mammalian lens induction and specification

The homeobox gene Six3 regulates forebrain development. Here we show that Six3 is also crucial for lens formation. Conditional deletion of mouse Six3 in the presumptive lens ectoderm (PLE) disrupted lens formation. In the most severe cases, lens induction and specification were defective, and the lens placode and lens were absent. In Six3-mutant embryos, Pax6 was downregulated, and Sox2 was absent in the lens preplacodal ectoderm. Using ChIP, electrophoretic mobility shift assay, and luciferase reporter assays, we determined that Six3 activates Pax6 and Sox2 expression. Misexpression of mouse Six3 into chick embryos promoted the ectopic expansion of the ectodermal Pax6 expression domain. Our results position Six3 at the top of the regulatory pathway leading to lens formation. We conclude that Six3 directly activates Pax6 and probably also Sox2 in the PLE and regulates cell autonomously the earliest stages of mammalian lens induction.     

Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm

A number of regulatory genes have been implicated in neural crest development. However, the molecular mechanism of how neural crest determination is initiated in the exact ectodermal location still remains elusive. Here, we show that the cooperative function of Pax3 and Zic1 determines the neural crest fate in the amphibian ectoderm. Pax3 and Zic1 are expressed in an overlapping manner in the presumptive neural crest area of the Xenopus gastrula, even prior to the onset of the expression of the early bona fide neural crest marker genes Foxd3 and Slug. Misexpression of both Pax3 and Zic1 together efficiently induces ectopic neural crest differentiation in the ventral ectoderm, whereas overexpression of either one of them only expands the expression of neural crest markers within the dorsolateral ectoderm. The induction of neural crest differentiation by Pax3 and Zic1 requires Wnt signaling. Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. Thus, co-activation of Pax3 and Zic1, in concert with Wnt, plays a decisive role for early neural crest determination in the correct place of the Xenopus ectoderm.     

Sfrp1a and Sfrp5 function as positive regulators of Wnt and BMP signaling during early retinal development

Axial patterning of the developing eye is critically important for proper axonal pathfinding as well as for key morphogenetic events, such as closure of the optic fissure. The dorsal retina is initially specified by the actions of Bone Morphogenetic Protein (BMP) signaling, with such identity subsequently maintained by the Wnt-β catenin pathway. Using zebrafish as a model system, we demonstrate that Secreted frizzled-related protein 1a (Sfrp1a) and Sfrp5 work cooperatively to pattern the retina along the dorso-ventral axis. Sfrp1a/5 depleted embryos display a reduction in dorsal marker gene expression that is consistent with defects in BMP- and Wnt-dependent dorsal retina identity. In accord with this finding, we observe a marked reduction in transgenic reporters of BMP and Wnt signaling within the dorsal retina of Sfrp1a/5 depleted embryos. In contrast to studies in which canonical Wnt signaling is blocked, we note an increase in BMP ligand expression in Sfrp1a/5 depleted embryos, a phenotype similar to that seen in embryos with inhibited BMP signaling. Overexpression of a low dose of sfrp5 mRNA causes an increase in dorsal retina marker gene expression. We propose a model in which Sfrp proteins function as facilitators of both BMP and Wnt signaling within the dorsal retina.     

Sfrp1 and Sfrp2 are not involved in Wnt/β-catenin signal silencing during lens induction but are required for maintenance of Wnt/β-catenin signaling in lens epithelial cells

During eye lens development, regulation of Wnt/β-catenin signaling is critical for two major processes: initially it must be silent in the lens placode for lens development to proceed, but subsequently it is required for maintenance of the lens epithelium. It is not known how these different phases of Wnt/β-catenin activity/inactivity are regulated. Secreted frizzled related protein-2 (Sfrp2), a putative Wnt-Fz antagonist, is expressed in lens placode and in lens epithelial cells and has been put forward as a candidate for regional Wnt/β-catenin pathway regulation. Here we show its closely-related isoform, Sfrp1, has a complimentary pattern of expression in the lens, being absent from the placode and epithelium but expressed in the fibers. As mice with single knockouts of Sfrp1 or Sfrp2 had no defects in lens formation, we examined lenses of Sfrp1 and Sfrp2 double knockout (DKO) mice and showed that they formed lens placode and subsequent lens structures. Consistent with this we did not observe ectopic TCF/Lef activity in lens placode of DKOs. This indicates that Sfrp1 and Sfrp2 individually, or together, do not constitute the putative negative regulator that blocks Wnt/β-catenin signaling during lens induction. In contrast, Sfrp1 and Sfrp2 appear to have a positive regulatory function because Wnt/β-catenin signaling in lens epithelial cells was reduced in Sfrp1 and Sfrp2 DKO mice. Lenses that formed in DKO mice were smaller than controls and exhibited a deficient epithelium. Thus Sfrps play a role in lens development, at least in part, by regulating aspects of Wnt/β-catenin signaling in lens epithelial cells.     

Secreted frizzled related protein 1 (Sfrp1) and Wnt signaling in innervated and denervated skeletal muscle

Wnts are secreted proteins with functions in differentiation, development and cell proliferation. Wnt signaling has also been implicated in neuromuscular junction formation and may function in synaptic plasticity in the adult as well. Secreted frizzled-related proteins (Sfrps) such as Sfrp1 can function as inhibitors of Wnt signaling. In the present study a potential role of Wnt signaling in denervation was examined by comparing the expression levels of Sfrp1 and key proteins in the canonical Wnt pathway, Dishevelled, glycogen synthase kinase 3β and β-catenin, in innervated and denervated rodent skeletal muscle. Sfrp1 mRNA and immunoreactivity were found to be up-regulated in mouse hemidiaphragm muscle following denervation. Immunoreactivity, detected by Western blots, and mRNA, detected by Northern blots, were both expressed in extrasynaptic as well as perisynaptic parts of the denervated muscle. Immunoreactivity on tissue sections was, however, found to be concentrated postsynaptically at neuromuscular junctions. Using β-catenin levels as a readout for canonical Wnt signaling no evidence for decreased canonical Wnt signaling was obtained in denervated muscle. A role for Sfrp1 in denervated muscle, other than interfering with canonical Wnt signaling, is discussed.     

Methylation‐reprogrammed Wnt/β‐catenin signalling mediated prenatal hypoxia‐induced brain injury in foetal and offspring rats

Prenatal hypoxia (PH) is a common pregnancy complication, harmful to brain development. This study investigated whether and how PH affected Wnt pathway in the brain. Pregnant rats were exposed to hypoxia (10.5% O₂) or normoxia (21% O₂; Control). Foetal brain weight and body weight were decreased in the PH group, the ratio of brain weight to body weight was increased significantly. Prenatal hypoxia increased mRNA expression of Wnt3a, Wnt7a, Wnt7b and Fzd4, but not Lrp6. Activated β‐catenin protein and Fosl1 expression were also significantly up‐regulated. Increased Hif1a expression was found in the PH group associated with the higher Wnt signalling. Among 5 members of the Sfrp family, Sfrp4 was down‐regulated. In the methylation‐regulating genes, higher mRNA expressions of Dnmt1 and Dnmt3b were found in the PH group. Sodium bisulphite and sequencing revealed hyper‐methylation in the promoter region of Sfrp4 gene in the foetal brain, accounting for its decreased expression and contributing to the activation of the Wnt‐Catenin signalling. The study of PC12 cells treated with 5‐aza further approved that decreased methylation could result in the higher Sfrp4 expression. In the offspring hippocampus, protein levels of Hif1a and mRNA expression of Sfrp4 were unchanged, whereas Wnt signal pathway was inhibited. The data demonstrated that PH activated the Wnt pathway in the foetal brain, related to the hyper‐methylation of Sfrp4 as well as Hif1a signalling. Activated Wnt signalling might play acute protective roles to the foetal brain in response to hypoxia, also would result in disadvantageous influence on the offspring in long‐term.     

BMP7 acts in murine lens placode development

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.     

Dominant inhibition of lens placode formation in mice

The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and αA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.     

Secreted frizzled-related proteins are required for Wnt/β-catenin signalling activation in the vertebrate optic cup

Secreted frizzled-related proteins (Sfrps) are considered Wnt signalling antagonists but recent studies have shown that specific family members enhance Wnt diffusion and thus positively modulate Wnt signalling. Whether this is a general and physiological property of all Sfrps remains unexplored. It is equally unclear whether disruption of Sfrp expression interferes with developmental events mediated by Wnt signalling activation. Here, we have addressed these questions by investigating the functional consequences of Sfrp disruption in the canonical Wnt signalling-dependent specification of the mouse optic cup periphery. We show that compound genetic inactivation of Sfrp1 and Sfrp2 prevents Wnt/β-catenin signalling activation in this structure, which fails to be specified and acquires neural retina characteristics. Consistent with a positive role of Sfrps in signalling activation, Wnt spreading is impaired in the retina of Sfrp1(-/-);Sfrp2(-/-) mice. Conversely, forced expression of Sfrp1 in the wing imaginal disc of Drosophila, the only species in which the endogenous Wnt distribution can be detected, flattens the Wg gradient, suppresses the expression of high-Wg target genes but expands those typically activated by low Wg concentrations. Collectively, these data demonstrate that, in vivo, the levels of Wnt signalling activation strongly depend on the tissue distribution of Sfrps, which should be viewed as multifunctional regulators of Wnt signalling.     

Growth and differentiation of the retina and the optic tectum in the medaka fish requires olSfrp5

Secreted Frizzled‐Related Proteins (SFRPs) are extracellular modulators of Wnt and Bmp signaling. Previous studies in birds and fishes have shown that Sfrp1, a member of this family, is strongly expressed throughout the development of the eye contributing to the specification of the eye field, retina neurogenesis and providing guidance information to retina ganglion cell axons. Here, we report that in medaka fish (Oryzias latipes) the expression of olSfrp5, which is closely related to olSfrp1, largely overlaps with that of olSfrp1 in the eye, but is additionally expressed in the developing midbrain and gut primordium. Morpholino‐based interference with olSfrp5 expression causes microphthalmia and reduction of the tectum size associated with an increase in apoptotic cell death in these structures. Furthermore, interference with the levels of olSfrp5 expression impairs the patterning of the ventral portion of the optic cup, leading in some cases to a fissure coloboma. These early defects are followed by an abnormal retinal and tectal neurogenesis. In particular, only reduced numbers of photoreceptor and RGC were generated in olSfrp5 morphants retinas. The results point to an important role of olSfrp5 in visual system formation and indicate that olSfrp1 and olSfrp5, despite their overlapping expression, have only partially redundant function during eye development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009