Frizzled 3 acts upstream of Alcam during embryonic eye development

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Frizzled 3 acts upstream of Alcam during embryonic eye development Franziska A. Seigfrieda,b,c,1, Wiebke Cizelskya,b,1, Astrid S. Pfistera, Petra Dietmanna, ⁎ Paul Waltherd, Michael Kühla, Susanne J. Kühla, a

Institute of Biochemistry and Molecular Biology, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany International Graduate School in Molecular Medicine Ulm, 89081 Ulm, Germany Tissue Homeostasis Joint-PhD-Programme in Cooperation with the University of Oulu, Finland d Central Facility for Electron Microscopy, Ulm University, Albert-Einstein-Allee 11, D-89081 Ulm, Germany b c

A R T I C L E I N F O

A BS T RAC T

Keywords: Frizzled 3 Alcam CD166, DM-GRASP Eye development Xenopus laevis Wnt/JNK Pax2

Formation of a functional eye during vertebrate embryogenesis requires different processes such as cell differentiation, cell migration, cell-cell interactions as well as intracellular signalling processes. It was previously shown that the non-canonical Wnt receptor Frizzled 3 (Fzd3) is required for proper eye formation, however, the underlying mechanism is poorly understood. Here we demonstrate that loss of Fzd3 induces severe malformations of the developing eye and that this defect is phenocopied by loss of the activated leukocyte cell adhesion molecule (Alcam). Promoter analysis revealed the presence of a Fzd3 responsive element within the alcam promoter, which is responsible for alcam expression during anterior neural development. In-depth analysis identified the jun N-terminal protein kinase 1 (JNK1) and the transcription factor paired box 2 (Pax2) to be important for the activation of alcam expression. Altogether our study reveals that alcam is activated through non-canonical Wnt signalling during embryonic eye development in Xenopus laevis and shows that this pathway plays a similar role in different tissues.

1. Introduction Vertebrate eye development starts during gastrulation with the induction of the crescent-shaped eye field in the anterior neural plate (Donner et al., 2006). Different homeobox transcription factors such as Rax (retina and anterior neural fold homeobox 1), Six3 (six homeobox 3), Otx2 (orthodentricle homeobox 2), and Pax6 (paired box 6) are required during this early phase (Callaerts et al., 1997; Hirsch and Harris, 1997; Mathers et al., 1997). During neurulation, the eye field splits into two distinct eye anlagen by the influence of shh (sonic hedgehog), which is expressed in the underlying axial mesoderm (Li et al., 1997; Macdonald et al., 1995). At the end of neurulation, the eye vesicles evaginate bilaterally from the diencephalon towards the ectoderm in which the lens placode is induced (Hyer et al., 1998). Subsequently, the optic vesicles start to invaginate thereby forming the bilayered optic cups consisting of the outer layer, the retinal-pigmented

epithelium (RPE), and the inner layer, the neural retina (MartinezMorales et al., 2003). Later on, the neural retina differentiates into seven distinct cell types organized in a multi-layered structure (Jean et al., 1998). Wingless-type MMTV integration site family member (Wnt) pathways are known to have important roles during development and are classified in the canonical/β-catenin-dependent and the non-canonical/β-catenin-independent Wnt signalling cascades (Rao and Kuhl, 2010). In both cases, Frizzled (Fzd) proteins act as receptors of Wnt ligands. It is well known that Wnt signalling is important for different processes during neural development such as patterning the neural plate, neural tube closure or axonal pathfinding (Liu and Nathans, 2008; Lyuksyutova et al., 2003; Wang et al., 2006). Both Wnt signalling branches are also essential during eye development. β-catenin signalling is required, for example, for retinal lamination and differentiation of the RPE (Fu et al., 2006;

List of symbols and abbreviations: Alcam, activated leukocyte cell adhesion molecule; Arr3, arrestin 3; ATF2, activating transcription factor 2; BS, binding site; caJNK1, constitutively activated JNK1; Cryba1, crystallin beta A1; Celf1, CUGBP elav-like family member 1; CMZ, Ciliary marginal zone; Dsh, dishevelled; Fzd, frizzled; Gfp, green fluorescent protein; ICS, intercellular space; Ig, immunoglobulin; JNK1, jun N-terminal protein kinase 1; MO, morpholino oligonucleotide; Otx2, orthodentricle homeobox 2; Pax2, paired box 2; Pax6, paired box 6; Pou4f1, POU class IV homeobox 1; Prox1, prospero homeobox protein 1; Rho, rhodopsin; RPE, retinal-pigmented epithelium; Rax, retina and anterior neural fold homeobox 1; Shh, sonic hedgehog; Six3, six homeobox 3; Sox3, sex determining region Y-box 3; TEM, transmission electron microscopy; Vsx1, visual system homeobox 1; WMISH, whole mount in situ hybridization; Wnt, wingless-type MMTV integration site family member ⁎ Corresponding author. E-mail address: [email protected] (S.J. Kühl). 1 Equal first authors. http://dx.doi.org/10.1016/j.ydbio.2017.04.004 Received 22 November 2016; Received in revised form 9 February 2017; Accepted 14 April 2017 0012-1606/ © 2017 Published by Elsevier Inc.

Please cite this article as: Seigfried, F.A., Developmental Biology (2017), http://dx.doi.org/10.1016/j.ydbio.2017.04.004

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CCC TCC -3′; ΔPax2-rev.: P-5′-TTT TTG TGA AGT GCT GGA AAG GGG-3′; ΔPax2BS2-forw.: P-5′-TCC TCC TGT TTA TCA CAC CCC TCC −3′; ΔPax2BS2-rev.: 5′-CCA TTG ACA TAG GGG GCC TTC C-3′. For the generation of the -2.7kb-ΔPax2BS1-luc construct a mutagenesis PCR was performed using the Pfu Ultra II DNA Polymerase (Agilent, USA), the -2.7kb-luc plasmid as template and the following primers: ΔPax2BS1-forw.: 5′- GCT AGC CCG GGC TCG AGA TCT -3′; ΔPax2BS1-rev: 5′-GGC CTT CCC GGG TAG TTT TTG TGA AG -3′. The amplified DNA as well as the empty luc vector were digested with Sma I (NEB, Germany) and ligated using the Ligate-IT Ligation Kit (Affymetrix USB, USA). All constructs were verified by sequence analyses.

Westenskow et al., 2009). The canonical Wnt receptors Fzd5 and Fzd8 regulate optical fissure/disk formation and progenitor expansion (Liu et al., 2012). In contrast to canonical Wnt signalling, which regulates late eye development, non-canonical Wnt signalling has been shown to be required for early eye development (Bugner et al., 2011; Gessert et al., 2007; Maurus et al., 2005). In particular Wnt4, a ligand known to activate non-canonical Wnt signalling, is already required for the specification of the eye field (Maurus et al., 2005). Overexpression of a dominant-negative Fzd3 construct, a potential Wnt4 receptor (Lyuksyutova et al., 2003; Maurus et al., 2005), leads to an inhibition of eye-specific marker genes resulting in deformed eyes (Rasmussen et al., 2001). Multiple reports indicate that Fzd3 preferentially activates β-catenin independent Wnt signalling (Kuhl et al., 2000; Maurus et al., 2005; Rasmussen et al., 2001). It remains unclear, however, how Fzd3 signalling regulates eye development. The activated leukocyte cell adhesion molecule (Alcam) belongs to the neuronal immunoglobulin (Ig)-domain superfamily of cell adhesion molecules. Alcam contains five extracellular Ig domains and a single transmembrane domain (Burns et al., 1991) and has been implicated to act downstream of non-canonical Wnt signalling (Choudhry et al., 2011; Gessert et al., 2008; Ruiz-Villalba et al., 2016). Additionally, we recently reported that alcam is a direct target gene of β-catenin independent Fzd3 signalling during embryonic kidney development (Cizelsky et al., 2014). Furthermore, Neurolin-a, the zebrafish ortholog of Alcam, has been shown to be necessary for retinal ganglion cell differentiation (Diekmann and Stuermer, 2009). In Xenopus laevis, tissue-specific expression of alcam starts at developmental stage 13 in the anterior neural plate and persists in the developing eye (Gessert et al., 2008) suggesting it has a possible role in anterior neural development, such as in the eye. Thus, we aimed to shed more light onto the mechanism of how noncanonical Wnt signalling regulates vertebrate eye development. For this purpose, we used Xenopus laevis as our model organism. We performed antisense morpholino oligonucleotide (MO) based knock down approaches, which do not cause genetic compensation, as has been observed upon deleterious mutations (Blum et al., 2015; Rossi et al., 2015). We demonstrate that the cell adhesion molecule Alcam mediates, at least in part, the effect of Fzd3 during eye development. Moreover, we show that alcam is regulated by non-canonical Wnt signalling, involving jun N-terminal protein kinase 1 (JNK1) and paired box 2 (Pax2). These results show that alcam expression is regulated in a similar manner as previously shown in the embryonic kidney (Cizelsky et al., 2014).

2.3. Morpholino oligonucleotide (MO) injections, RNA injections, animal cap experiments

2. Materials and methods

All MOs were obtained from Gene Tools, LLC, OR and solved in DEPC-treated water. For knockdown approaches, we injected 25– 30 ng of Fzd3 MO (5′- CGC AAA GCC ACA TGC ACC TCT TGA A3′); 25–30 ng of Alcam MO (5′-CAC TTG CTT CCA TAG CCC ACG ATC C-3′) or 30 ng of Pax2 MO (5′-GGT CTG CCT TGC AGT GCA TAT CCA T-3′) (Deardorff et al., 2001; Gessert et al., 2008; Koenig et al., 2010). For control injection experiments, the standard Control MO of Gene Tools was used in corresponding amounts. To target anterior neural tissue including the eye, unilateral injections were performed into one dorso-animal blastomere of 8-cell stage Xenopus embryos (Moody and Kline, 1990). The un-injected side served as an internal control. gfp mRNA was coinjected as a lineage tracer (Fig. S1). Each injected embryo was controlled under a fluorescence microscope (MVX10; Olympus, Japan) and only correctly injected embryos were further analyzed. For rescue experiments, the following mRNA amounts were injected: 1 ng alcam, 100 pg dhsΔDIX or dshΔDEP and 1 ng caJNK1. For Luciferase assays, embryos were injected at 2-cell stage with 100 pg pGL3 basic (Promega, USA), -2.7kb-luc, -2.7kb-ΔATF2/Pax2-luc, -2.7kb-ΔPax2-luc, -2.7kb-ΔPax2BS1-luc or -2.7kb-ΔPax2BS2-luc together with 5 pg phRL-TK (Promega), and 600 pg noggin mRNA to induce neural tissue (Lamb et al., 1993). Animal cap explants were dissected at stage 9, cultivated in 1x MBSH (10 mH HEPES, 88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3)+50 U/ml penicillin+50 μg/ml streptomycin at 14 °C and fixed at stages between 21 and 23 at −70 °C. Frozen animal caps were lysed in 100 µl 1x Passive Lysis Buffer (Promega, USA). Luciferase activity was determined using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol and measured with a luminometer (Lumat LB 9507; EG & G BERTHOLD, Germany).

2.1. Xenopus laevis

2.4. Whole mount in situ hybridizations (WMISH)

Xenopus laevis embryos were obtained and cultured according to general protocols and staged as described previously (Nieuwkoop, 1956). For egg generation, healthy, pigmented and mature female frogs were used (LM00535MX, Nasco, USA). Testis was taken from healthy and mature male frogs (LM00715MX, Nasco, USA). All experiments were performed in agreement with the German law and registered at the Regierungspraesidium Tuebingen.

Wildtype or MO injected embryos were fixed at indicated stages with MEMFA buffer (0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO4, 4% fomaldehyde) over night at 4 °C. WMISH was performed following standard protocols (Hemmati-Brivanlou et al., 1990) using DIG-labeled antisense RNA probes and BM Purple (Roche, Swiss) or NBP/BCIP (Roche, Swiss) as substrates. After staining, the embryos were refixed in MEMFA and bleached in 30% H2O2. For histology, embryos were embedded in gelatin/BSA and vibratome sections of stained embryos were performed with a thickness of 25–30 µm using a vibratome (Vibratome series 1500; The vibratome company).

2.2. Cloning for alcam promoter analysis To analyze the 5′UTR upstream regulatory region of alcam, previously described -2.7kb-luc as well as -2.7kb-ΔATF2/Pax2-luc (formerly named -2.7kbΔ-luc) plasmid were used (Cizelsky et al., 2014). Further deletion constructs (-2.7kb-ΔPax2-luc, -2.7kbΔPax2BS2-luc) were cloned, using the Quickchange II Site Directed Mutagenesis Kit (Agilent, USA), the -2.7kb-luc plasmid as a template and following primers: ΔPax2-forw.: P-5′-TCC TCC TGT TTA TCA CAC

2.5. Cryosectioning and immunohistochemistry For cryostat sections with a thickness of 10 µm, embryos at stage 42 were fixed in 4% PFA over night at 4 °C. After equilibrating in 80% methanol/20% DMSO overnight at −20 °C, embryos were prepared for sectioning by cryostat (Jung Frigocut 2800; Leica) as described earlier 2

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Fig. 1. Fzd3 loss of function results in an abnormal eye development. A. Spatial expression pattern of fzd3. Fzd3 transcripts are found in the neural tube (black arrowheads), migrating neural crest cells (white arrows) and the developing eye (white arrowheads) at indicated stages. B. Loss of Fzd3 led to smaller and deformed eyes on the injected side (white arrows) in comparison to the un-injected or Control MO injected side. Furthermore, Fzd3 inhibition resulted in disturbed retinal pigmented epithelium (RPE, red arrows). Representative embryos are shown. Scale bar (sections): 100 µm. C. Quantitative representation of the data shown in B. Note, that also the data from Fig. 5B are incorporated into the statistics. D. Detailed views of Fzd3 MO and Control MO injected eyes. Dashed red lines indicate measured eye areas. Red lines indicate the eye fissure angle (°fis.) measured. Quantitative representations of eye area and eye fissure angle (°fis.) measurements are given. E. Cross sections of Fzd3-depleted embryos after WMISH using marker genes against different retinal cell types. Loss of Fzd3 led to disorganized retinal cell layers including an invagination of the photoreceptor layer into inner retina layers (black arrows) and the formation of rosette-like structures by ectopic photoreceptor cells (red arrowheads). The ratios of Fzd3 MO injected embryos showing retinal lamination defects versus all investigated embryos are indicated. Scale bar: 100 µm. F. Lens specific markers celf1 and cryba1 are shown by WMISH. Marker gene expression was unaffected upon Fzd3 depletion although some lenses were smaller (the ratios of Fzd3 MO injected embryos showing smaller lenses versus all investigated embryos are indicated). Scale bar: 100 µm. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear cell layer; n, number of independent experiments; N, total number of analyzed embryos; ONL, outer nuclear cell layer; st, stage. Error bars indicate standard error of the mean (s.e.m.). **** p≤0.0001. p values were calculated by non-parametric Mann–Whitney rank sum test.

used a previously described and functional Fzd3 MO (Cizelsky et al., 2014; Deardorff et al., 2001). In all experiments, we performed unilateral injections into one animal-dorsal blastomere of 8-cell stage Xenopus embryos to target anterior neural tissues (Moody, 1987a, 1987b; Moody and Kline, 1990). The un-injected side of the embryo served as an internal control whereas injecting a standard Control MO was used as an injection control. In addition, 0.5 ng of green fluorescent protein (gfp) mRNA was co-injected in all experiments to verify proper injections (Fig. S1). Interfering with Fzd3 function in the early embryo resulted in the formation of abnormal eyes (Fig. 1B andC). More specifically, eyes were significantly smaller in size on the Fzd3 MO injected side compared to the un-injected side or compared to Control MO injected embryos (Fig. 1D). By measuring the optic fissure angle, we revealed a coloboma phenotype as a common eye defect (Fig. 1D). In some cases, part of the RPE was missing in addition to the coloboma phenotype (Fig. 1B, red arrows). Vibratome transversal sections confirmed the microphthalmia phenotype including smaller lenses and indicated a disturbance in retinal lamination upon Fzd3 depletion (Fig. 1B lower row). To further characterize retinal differentiation and lamination in Fzd3-depleted embryos, marker genes specific for different retinal cell types were analyzed by WMISH (Cizelsky et al., 2014). We used the following marker genes to label different cell populations in the neural retina: arrestin3 (arr3) and rhodopsin (rho) to determine photoreceptor cells (Chang and Harris, 1998), pax6 to visualize amacrine and ganglion cells (Hitchcock et al., 1996), visual system homeobox 1 (vsx1) for bipolar cells (Hayashi et al., 2000), prospero homeobox protein 1 (prox1) to show horizontal cells (Dyer et al., 2003) and POU class IV homeobox 1 (pou4f1) to stain for ganglion cells (Liu et al., 2000) (Fig. 1E). These experiments showed that the differentiation of retinal cell types was not affected upon Fzd3 depletion as all analyzed marker genes were expressed. In contrast, retinal lamination was severely impaired upon Fzd3 deficiency. This was observed especially for the photoreceptor specific staining, which was often displaced forming rosette-like structures that disturbed other inner retinal cell layers (Fig. 1E, red arrows). To investigate lens development on a molecular level, we made use of two lens-specific marker genes, CUGBP elav-like family member 1 (celf1) to determine mature lens fiber cells in the center of the lens and crystallin beta A1 (cryba1) to visualize epithelial stem cells in the anterior lens pole (Day and Beck, 2011; Rothe et al., 2017). Upon Fzd3 depletion, some lenses were smaller when compared to the un-injected side, but neither celf1 nor cryba1 expression were down-regulated by the absence of Fzd3 (Fig. 1F). In summary, these experiments confirmed and extended earlier findings implicating Fzd3 in eye development in Xenopus laevis. Loss of Fzd3 led to microphthalmia, coloboma and disturbed retinal lamination.

(Fagotto et al., 1999). For immunohistochemistry, slides were successively washed in acetone, 1x PBS and PBT (0.1% Triton X-100 in 1x PBS). Sections were then blocked in 10% goat serum in PBT for 1 h. Incubation with the anti-alcam antibody (1:100, Sigma, HPA010926) was performed over night at 4 °C. Sections were washed three times with 1x PBS and incubated with an anti rabbit secondary antibody conjugated to alkaline phosphatase (1:1000, Sigma, A3937) for 1 h at room temperature. After washing with 1x PBS, slides were incubated in AP-buffer (0.2 M NaCl, 0.1 mM MgCl2, 0.1% in 0.1 M Tris buffer) and stained with BM Purple. Slides were washed in 1x PBS and mounted with glycerol. 2.6. Transmission electron microscopy Xenopus embryos at stage 42 were fixed with 2.5% glutaraldehyde (Fluka, USA) containing 1% saccharose (Roche, Swiss) in phosphate buffer (pH 7.3) washed 5 times with 0.01 M PBS buffer and postfixed in 2% aqueous osmium tetroxide (Fluka, USA) (Gessert et al., 2008). Embryos were then dehydrated in graded series of 1-propanol, block stained in 1% of uranyl acetate and embedded in Epon (Fluka, USA). Ultra-thin sections (80 nm) were contrasted with 0.3% lead citrate for 1 min and imaged in a Zeiss TEM 109 or in a Jeol TEM 1400 using magnifications of 250x or 4000x. 2.7. Statistics Data were obtained from at least three independent experiments and analyzed with the statistical program Prism (Prism, Version 5.0d, Irvine, USA). Only tadpole experiments with a survival rate of more than 50% of injected embryos and a final survival number of more than 20 individuals per group were considered for statistical evaluation. If the control group (either control MO injected or un-injected controls) showed more than 20% phenotype, being indicative for a bad batch of embryos, the experiment was excluded from evaluation. The nonparametric Mann-Whitney rank sum test was used to determine statistical differences. A P value of ≤0,05 was considered to be significant. In all figures, statistical significances are indicated as: *P≤0.05, **P≤0.01, ***P≤0.001 and ****P≤0.0001. 3. Results 3.1. Fzd3 is required for Xenopus eye development Previous studies have implicated Fzd3 in Xenopus eye development: using a dominant negative Fzd2 construct (Rasmussen et al., 2001). Here, we aimed to expand these earlier findings and further characterized Fzd3 expression and function in eye development including the eye field induction and retinal lamination. Using whole mount in situ hybridization (WMISH) of Xenopus laevis embryos, we detected fzd3 transcripts in the evaginating eye vesicles and during subsequent ocular development (Fig. 1A). Next, we interfered with Fzd3 function using the powerful antisense MO based knock down approach (Blum et al., 2015). To this end, we

3.2. Loss of Alcam function phenocopies loss of Fzd3 We previously demonstrated that alcam is a direct target gene of βCatenin independent Wnt/Fzd3 signalling during embryonic kidney development (Cizelsky et al., 2014). Moreover, alcam expression in 4

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Fig. 2. Alcam deficiency results in abnormal eye development. A. The spatial expression pattern of alcam by WMISH shows alcam expression in the developing eye (white arrowheads) and the neural tube (black arrowheads) at indicated stages. At stage 42, alcam RNA was predominantly found in the ganglion cell layer (lower left panel), the lens epithelium (LE) and the cornea epithelium (CE) as shown by WMISH. Antibody staining demonstrated that the Alcam protein is mainly localized in the ciliary marginal zone (CMZ) and inner synaptic and nuclear layer of the retina (lower right panel). B. Loss of Alcam led to smaller and deformed eyes on the injected side (white arrows) in comparison to the un-injected side or Control MO injected embryos. Furthermore, Alcam depletion results in a disorganized retinal lamination as well as absent RPE (red arrows). Representative embryos are shown. Scale bar (sections): 100 µm. C. Quantitative representation of the data shown in B. Loss of Alcam led to disturbed eye morphology in a MO-dose dependent manner. Co-injection of alcam RNA significantly rescued the Alcam MO-induced eye phenotype. D. Detailed views of Control MO and Alcam MO injected embryos. Dashed red lines indicate measured eye areas. Red lines indicate the eye fissure angle (°fis.) measured. Quantitative representations of eye area and eye fissure angle (°fis.) measurements are given. E. Cross sections of Alcam-depleted embryos after WMISH. In contrast to the un-injected side, loss of Alcam led to a disorganization of the retinal cell layers due to ectopic formation of rosette-like structures of photoreceptor cells (red arrowheads). The ratios of Alcam MO injected embryos showing retinal lamination defects versus all investigated embryos are indicated in each marker gene picture respectively. Scale bar: 100 µm. F. Lens specific markers celf1 and cryba1 are shown by WMISH. Marker gene expression was unaffected upon Alcam depletion although some lenses were smaller (the ratios of Alcam MO injected embryos showing smaller lenses versus all investigated embryos are indicated). Scale bar: 100 µm. Abbreviations: CE, cornea epithelium; CMZ, ciliary marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; inj., injected; ISL, inner synaptic layer; LE, lens epithelium; n, number of independent experiments; N, total number of analyzed embryos; ONL, outer nuclear layer; RPE, retinal pigmented epithelium; st, stage. Error bars indicate standard error of the mean (s.e.m.). ** p≤0.01, *** p≤0.001, **** p≤0.0001. p values were calculated by non-parametric Mann–Whitney rank sum test.

pigmented epithelium layer). Depending on the location, the RPE was thinner, thicker (Fig. 3A and B, retinal pigmented epithelium layer) or even absent. In Control MO injected embryos, photoreceptor cells were normally embedded in the RPE (Fig. 3A and B, photoreceptor cell layer). Upon loss of Fzd3 or Alcam, however, photoreceptors were detached from the RPE (Fig. 3B, photoreceptor cell layer). Hence, we confirmed that the formation of the rosette-like structures in the inner retinal layers was caused by misplaced photoreceptor cells (Fig. 3A and B, nuclear layer). Intriguingly, Fzd3 as well as Alcam depletion resulted in a increase of intercellular spaces (ICS) in all analyzed retinal layers indicating a disturbance in cell adhesion (Fig. 3A and B). Cell differentiation in the ganglion cell layer was unaffected (Fig. 3A and B). The lens epithelium, which constitutes the stem cell niche of lens fiber cells, as well as the mature lens fiber cells were also normally developed in Fzd3 and Alcam morphants (Fig. S2A and B). In summary, TEM approaches showed disturbed retinal lamination and cell adhesion in the retina as well as an accumulation of ectopic photoreceptor cells in inner retinal layers upon Fzd3 or Alcam deficiency.

cardiac tissue was also shown to be regulated by non-canonical Wnt signalling (Choudhry et al., 2011; Gessert et al., 2008). Beside its expression in the embryonic kidney and heart, alcam is also expressed in the developing eye (Cizelsky et al., 2014; Gessert et al., 2008). Here, we examined alcam expression during eye development in more detail by WMISH (Fig. 2A, white arrowheads) and antibody staining (Fig. 2A, lower row, right panel). At stage 16, alcam transcripts were prominently detected in the anterior neural plate including the eye anlage. Later during development, alcam was found in the evaginating optic vesicles at stage 23. alcam mRNA expression persisted in the eye until stage 42, where it was mainly detected in the ganglion cell layer as well as the lens and the cornea epithelium (Fig. 2A, lower row, left panel). On the protein level, Alcam expression was visualized in the ciliary marginal zone (CMZ) and the inner synaptic and nuclear retinal layers (Fig. 2A, lower row, right panel). According to this specific alcam expression in the developing eye, loss of function experiments were performed using a previously well characterized antisense MO targeting endogenous alcam mRNA (Gessert et al., 2008). At stage 42, we observed a similar phenotype to what we saw upon loss of Fzd3. Eyes on the Alcam MO injected side were significantly smaller in size and showed a deformed morphology including coloboma (Fig. 2B, red arrows; C; D). Additionally, rescue experiments with the Alcam MO injected together with an alcam RNA that is not targeted by the Alcam MO (Gessert et al., 2008) showed a significant rescue of the eye phenotype. This result demonstrates that the Alcam MO-induced eye phenotype is caused specifically by the Alcam knockdown. Next, retinal lamination was examined on vibratome transversal sections of WMISH stained embryos using proper retina-specific marker genes. As was seen upon loss of Fzd3, ectopic photoreceptor cells were detected, forming rosette-like structures in the Alcam MO injected retinas (Fig. 2E, red arrowheads). In contrast, retinal lamination of the eyes on the un-injected side remained unchanged. The examination of lenses in Alcam morphants revealed a phenotype similar to Fzd3 depletion. Lens marker gene expression was unchanged although some specimens exhibited smaller lenses when compared to the un-injected side (Fig. 2F). Taken together, Alcam depletion resulted in an eye phenotype comparable to the loss of Fzd3 phenotype.

3.4. Fzd3 and Alcam are required for eye field induction and differentiation Next we aimed to elucidate the onset of the observed eye phenotype upon Fzd3 or Alcam deficiency during early development. To this end, Fzd3 or Alcam MOs were injected unilaterally and the expression of marker genes was analyzed at stages 13 (eye field induction) and 23 (differentiation of visual cells) (Cizelsky et al., 2013). At stage 13, the expression of the three eye-field specific marker genes rax, pax6 and otx2 (Zuber et al., 2003), as well as the pan-neural marker gene sox3 (sex determining region Y-box 3; (Bylund et al., 2003)) were analyzed. While otx2 and sox3 did not reveal any changes in expression after loss of either gene, rax and pax6 expression was slightly but significantly reduced in some of the embryos (Fig. 4A and B). Additionally, injection of Fzd3 or Alcam MO at stage 23 reduced the expression domain of all eye specific marker genes rax, pax6 and otx2 compared to the un-injected side or Control MO injected embryos (Fig. 4C and D). Moreover, vibratome transversal sections of rax stained embryos revealed smaller optic vesicles in Fzd3 and Alcam MO injected embryos compared to the un-injected or Control MO injected side (Fig. S3A and B). Note that the intensity of rax expression is reduced in the smaller Alcam-deficient optic vesicle (Fig. S3B).

3.3. Depletion of Fzd3 or Alcam results in disturbed cell lamination and adhesion in the retina To investigate the eye phenotype upon Fzd3 and Alcam depletion on a cellular level, we performed transmission electron microscopy (TEM) of Control, Fzd3 and Alcam MO injected embryos. In the retina, all retina-specific cell types were found in Fzd3 and Alcam morphant eyes confirming normal cell differentiation as was observed by WMISH (Fig. 3A and B). In contrast, the RPE structure was affected upon Fzd3 and Alcam depletion as its width was not uniform compared to Control MO injected eyes (Fig. 3A and B, retinal

3.5. Fzd3 regulates alcam expression via JNK1 During embryonic kidney development, Fzd3 activates alcam expression via β-Catenin independent Wnt/JNK signalling (Cizelsky et al., 2014). To test whether this mechanism is similar in different tissues, alcam expression was analyzed in the developing eye in Fzd3-depleted embryos at stage 21 by WMISH. Indeed, Fzd3 deficiency led to a 6

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Fig. 3. Loss of Fzd3 as well as Alcam leads to defects in retinal lamination and cell adhesion. Transmission electron microscopy of Control MO (A-C, upper rows), Fzd3 MO (A, lower row), Alcam MO (B, lower row) and Pax2 MO (C, lower row) injected Xenopus eyes at stage 42. Specific cells are highlighted by dotted circles. Overview: Overviews of the eyes including the location of close-ups in the retinal pigmented epithelial layer (a), photoreceptor cell layer (b), nuclear layer (c) and ganglion cell layer (d). Scale bar: 100 µm. Retinal pigmented epithelial layer (a): Fzd3, Alcam or Pax2 deficient eyes show thicker RPE layers. Photoreceptor cell layer (b): In Control MO injected embryos, the photoreceptor cell layer mainly includes photoreceptor cells, which are embedded in the retinal-pigmented epithelium (RPE). RPE cells contain melanin granuli (mg), photoreceptors specific membrane stacks (ms). Selected photoreceptor cells are highlighted by dotted circles. Upon Fzd3, Alcam or Pax2 depletion, intercellular spaces (ICS) are increased leading to detachment of photoreceptors (red arrowhead). Nuclear layer (c): The natural nuclear cell layer (NCL) of Control MO injected eyes is characterized by retina-specific cell nuclei such as nuclei of bipolar (BC), amacrine (AC) and photoreceptor cells (PRN). In contrast, the NCL of Fzd3, Alcam and Pax2 morphants was disorganized by the formation of rosette-like structures characterized by membrane stacks (ms) indicating ectopic photoreceptor cells. Moreover, Fzd3 and Alcam MO injection leads to an increase of intercellular spaces (ICS), an indication for cell adhesion defects. Ganglion cell layer (d): Ganglions are normally differentiated in Control, Fzd3, Alcam or Pax2 MO injected retinas. Scale bar: 5000 nm. Abbreviations: AC, amacrine cell; BC, bipolar cell; ICS, intercellular space; NF, neuronal fiber; RPE, retinal pigmented epithelium; mg, melanin granula; ms, membrane stacks; PRN, photoreceptor nucleus. Number of embryos analyzed by TEM: Control MO, 9; Fzd3 MO, 4; Alcam MO, 4; Pax2 MO, 4.

Fig. 4. Eye marker gene expression upon Fzd3 and Alcam deficiency. After depletion of Fzd3 (A) or Alcam (B), rax and pax6 expression in the eye field was slightly but significantly reduced at stage 13 compared to Control MO injected sides as shown by WMISH. In contrast, otx2 and sox3 were unaffected upon Alcam or Fzd3 MO injection. At stage 23, expression of rax, pax6, and otx2 in the eye region was reduced after loss of either Fzd3 (C) or Alcam (D) in comparison to Control MO injected embryos. Quantitative representations are given. Abbreviations: inj., injected; n, number of independent experiments; N, total number of analyzed embryos; n.s., not significant. Error bars indicate standard error of the mean (s.e.m.). * p≤0.05, ** p≤0.01. p values were calculated by a non-parametric Mann–Whitney rank sum test.

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Fig. 5. Fzd3 regulates alcam expression during eye development through JNK1. A. After injection of Fzd3 MO, alcam expression in the eye region was significantly reduced on the injected side of the embryo (black arrow) at stage 21 as shown by WMISH. A quantitative representation is given. B. The eye phenotype upon loss of Fzd3 could be reverted by coinjection of alcam RNA. A quantitative representation is given. C. Loss of Fzd3 led to a downregulation of alcam expression in the eye anlage at stage 20 (black arrow). Co-injection of dshΔDEP mRNA could not rescue the phenotype (black arrow), whereas co-injection of dshΔDIX or caJNK1 mRNA could significantly restore the alcam expression upon Fzd3 depletion. Abbreviations: inj., injected; n, number of independent experiments; N, total number of analyzed embryos; n.s., not significant. Error bars indicate the standard error of the mean (s.e.m.); n.s., not significant p≥0.05, * p≤0.05, ** p≤0.01. p values were calculated by a non-parametric Mann-Whitney rank sum test.

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Fig. 6. One Pax2 binding site is sufficient to activate alcam promoter activity in neural animal caps. A. A schematic drawing of different deletion constructs of the alcam promoter region. In the native promoter region, seven ATF2 binding sites (blue) and two Pax2 binding sites (yellow) were found. The different regulatory alcam promoter regions were cloned in front of the luciferase reporter gene. See Fig. S4 for more details. B. Promoter activity of different alcam promoter constructs in neuralized animal caps by co-injection of 600 pg noggin mRNA. C. Loss of Fzd3 led to a reduction of the promoter activity of −2.7kb-luc, whereas −2.7kb-ΔATF2/Pax2-luc was not sensitive to a reduction of Fzd3. D. Promoter activity of different promoter constructs as illustrated in A. in neuralized animal caps. Alcam promoter activity was lost in the absence of both Pax2 binding sites (blue bar). Alcam promoter activity was rescued by the presence of at least one Pax2 binding site (yellow bars). Abbreviations: n, number of independent experiment; n.s., not significant; RLU, relative light units. Error bars indicate standard error of the mean (s.e.m.); * p≤0.05. p values were calculated by a non-parametric Mann–Whitney rank sum test.

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Fig. 7. Loss of Pax2 phenocopies loss of Alcam and Fzd3. A. Loss of Pax2 led to smaller and deformed eyes on the injected side (white arrows) in comparison to the un-injected or Control MO side. Pax2 inhibition resulted in a disturbed retinal pigmented epithelium (RPE, red arrows). Representative embryos are shown. Scale bar (sections): 100 µm. B. Quantitative representation of the data shown in A is given. C. Detailed views of Control MO and Pax2 MO injected embryos. Dashed red lines indicate measured eye areas. Red lines indicate the eye fissure angles (°fis.) measured. Quantitative representation of eye area and eye fissure angle (°fis.) measurements are demonstrated. D. Transversal vibratome sections of Pax2 depleted embryos after WMISH with markers for different retinal cell types. Loss of Pax2 led to disorganized retinal cell layers by the formation of rosette-like structures by ectopic photoreceptor cells (red arrowheads). Ratios of Fzd3 MO injected embryos showing retinal lamination defects versus all investigated embryos are indicated in marker gene pictures respectively. Scale bar: 100 µm. E. Lens specific markers celf1 and cryba1 are shown by WMISH. Marker gene expression was unaffected upon Fzd3 depletion although some lenses were smaller (the ratios of Fzd3 MO injected embryos showing smaller lenses versus all investigated embryos are indicated). Scale bar: 100 µm. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear cell layer; n, number of independent experiments; N, total number of analyzed embryos; ONL, outer nuclear cell layer. Error bars indicate standard error of the mean (s.e.m.); ** p≤0.01, *** p≤0.001. p values were calculated by a non-parametric Mann–Whitney rank sum test. 11

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Fig. 8. Fzd3 and Pax2 are upstream of alcam. A. After injection of Pax2 MO, alcam expression was reduced on the injected side of the embryo at stage 21 (black arrow). A quantitative representation is given. B. After injection of Fzd3 MO, pax2 expression was mildly, but significantly reduced on the injected side of the embryo at stage 21 (black arrow). C. Schematic drawing. Fzd3 regulates alcam expression via the β-catenin independent Wnt/JNK pathway and Pax2. Abbreviations: n, number of independent experiment; N, total number of analyzed embryos. Error bars indicate standard error of the mean (s.e.m.); ** p≤0.01. p values were calculated by a non-parametric Mann–Whitney rank sum test.

activity indeed depended on Fzd3 function, whereas the −2.7kbΔATF2/Pax2-luc construct was not sensitive to the loss of Fzd3. These findings clearly support the hypothesis that Fzd3 regulates alcam expression in multiple tissues (Cizelsky et al., 2014). To investigate the Fzd3 responsive element in more detail, the following additional deletion constructs were generated: −2.7kbΔPax2-luc (Pax2 binding site 1 (BS1) and BS2 as well as ATF2 BS3 and BS4 were deleted), −2.7kb-ΔPax2BS1-luc (only Pax2 BS1 was deleted) and −2.7kb-ΔPax2BS2-luc (Pax2 BS2 and ATF2 BS4 were deleted) (Fig. 6A; Fig. S4). Luciferase experiments demonstrated that the depletion of two ATF2 and both Pax2 sites (−2.7kb-ΔPax2-luc) leads to a severe decrease of alcam promoter activity. Interestingly, promoter activation is fully restored by co-expressing constructs containing either only the first (−2.7kb-ΔPax2BS2-luc) or the second (−2,7kb-ΔPax2BS1-luc) Pax2 binding site. These data indicate that a single Pax2 binding site is sufficient to activate the alcam promoter in neuralized animal caps and implicate Pax2 as crucial regulator of alcam in the developing neural tissue.

significant down-regulation of alcam expression in the eye region (Fig. 5A). Note that alcam expression in the cranial placodes was not affected upon Fzd3 MO injection suggesting a specific effect of Fzd3 knockdown in the eye (Fig. 5A). This result was supported by rescue experiments injecting Fzd3 MO together with alcam RNA. At stage 42, the eye phenotype observed after loss of Fzd3 was reverted by coinjection of alcam (Fig. 5B). Taken together, these data indicate alcam to be downstream of Fzd3 in the developing eye. To test whether Fzd3 activates alcam through a β-Catenin independent Wnt/JNK signalling pathway, as it does during renal development, we next performed rescue experiments using two dishevelled (dsh) deletion constructs, namely dshΔDIX and dshΔDEP. These constructs activate either non-canonical (dshΔDIX) or canonical (dshΔDEP) Wnt signalling, respectively (Cizelsky et al., 2014; Kishida et al., 1999; Li et al., 1999). Co-injection of Fzd3 MO with dshΔDIX mRNA significantly rescued alcam expression in the eye anlage at stage 21, whereas dshΔDEP mRNA was not able to do so (Fig. 5C) suggesting non-canonical Wnt signalling to be important in this context. It is well known that dshΔDIX can activate the non-canonical Wnt mediator jun N-terminal kinase (JNK) (Boutros et al., 1998). Therefore, we next performed rescue experiments by injecting Fzd3 MO together with a constitutively activated JNK1 (caJNK1) construct (Lei et al., 2002). Indeed, upon co-injection of Fzd3 MO and caJNK1, we observed a reactivation of alcam expression (Fig. 5C). These observations support the idea that alcam is a target of Fzd3 via the non-canonical Wnt/JNK pathway during eye development, which is similar to what we have observed during embryonic kidney development (Cizelsky et al., 2014).

3.7. Pax2 acts upstream of alcam during Xenopus eye development Pax2 has previously been reported to be involved in eye development in several species including Drosophila, zebrafish, mouse and human (Cai et al., 2013; Fu and Noll, 1997; Torres et al., 1996; Viringipurampeer et al., 2012). In addition, pax2 is strongly expressed in the developing Xenopus eye ((Heller and Brandli, 1997); Fig. 8B). To investigate Pax2 function during Xenopus eye development, we performed loss of function experiments using a well-described antisense MO targeting Pax2 (Koenig et al., 2010). At stage 42, we observed an eye phenotype comparable with the phenotype observed upon loss of Fzd3 or Alcam (Fig. 7A and B). Pax2 morphants showed microphthalmia and a coloboma phenotype (Fig. 7C). WMISH using retina cell type specific marker gene also revealed lamination defects in Pax2 morphants and no changes in lens specific marker genes (Fig. 7D). In addition, TEM investigation of Pax2 depleted embryos revealed a similar retinal phenotype to Fzd3 or Alcam depleted embryos (Fig. 3C). Lens differentiation was not affected (Fig. 7E, Fig. S2C). Moreover, the evaginating eye vesicle is smaller in size in Pax2deficient embryos (Fig. S3C). These results raised the hypothesis that Fzd3, Pax2 and Alcam act in one signalling pathway with Fzd3 and Pax2 acting upstream of Alcam. If so, depletion of Pax2 should result in a loss of alcam expression. Depletion of Pax2 indeed led to a significant downregulation of alcam expression at stage 20 (Fig. 8A), confirming that Pax2 is upstream of alcam during Xenopus eye development. In line

3.6. One Pax2 binding site of the Fzd3 responsive element is sufficient to trigger alcam expression in the neural tissue To examine the regulation of alcam by Fzd3 in more detail, we used the previously described 5′ upstream regulatory region of alcam (with a length of 2.7kb) that was cloned in front of the luciferase reporter gene (-2.7kb-luc; (Cizelsky et al., 2014)) (Fig. 6A). The alcam promoter (-2.7 kb-luc) was significantly activated in neuralized animal cap cells compared to the empty control vector (Fig. 6B). In contrast, the −2.7kb-ΔATF2/Pax2-luc construct that did not contain a previously identified Fzd3 responsive element (Fzd3RE) consisting of two Pax2 and seven ATF2 (activating transcription factor 2) binding sites (BS) (Cizelsky et al., 2014; formerly named -2.7kbΔ-luc), shows a significantly reduced activation in neuralized caps (Fig. 6B). We next measured luciferase activity in neuralized animal caps after injecting Fzd3 MO (Fig. 6C). We found that the −2.7kb-luc promoter 12

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depleted retinas. We already demonstrated earlier that Alcam is involved in forming proper cell-cell contacts in the myocardium of the developing heart (Gessert et al., 2008) arguing for a conserved function of Alcam in the development of different tissues. Furthermore, it is known that Alcam can participate in homophilic as well as heterophilic interactions, e.g. to N-CAM (DeBernardo and Chang, 1995; van Kempen et al., 2001). As disrupted cell-cell contacts can lead to reduced adhesion, altered cell polarity and shape as well as cytoskeletal rearrangements, it is tempting to speculate that Alcam is involved in these processes to regulate vertebrate eye development. Indeed, an interaction of Alcam with the cytoskeleton has already been observed by several independent studies supporting this hypothesis (Nelissen et al., 2000; Tudor et al., 2014; Zimmerman et al., 2004). Beside its role in cell adhesion, we showed that Alcam is involved in the induction of the eye field and differentiation of visual cells. In detail, Alcam depletion led to a down-regulation of eye-specific marker genes such as rax, pax6 and otx2. These findings are in line with our previous results in the developing heart where Alcam deficiency resulted in a reduced cardiac differentiation (Gessert et al., 2008). Also in zebrafish, alcam has been shown to be required for cellular differentiation in the eye (Diekmann and Stuermer, 2009) as well as in the heart (Choudhry et al., 2011). In summary, these data indicate that Alcam has an essential function during the differentiation of various cell types. As described by others, loss of any of these genes such as rax, pax6 and otx2 results in severe defects during eye development (Bailey et al., 2004; Viczian et al., 2003; Zuber et al., 2003). Interestingly, mutations in RAX, OTX2 as well as PAX6 have been linked to human patients showing eye phenotypes such as microphthalmia (smaller eyes; (Deml et al., 2016; London et al., 2009; Ragge et al., 2005; Zhang et al., 2009)), anophthalmia (one missing eye; (Deml et al., 2016; Lequeux et al., 2008)) and coloboma (non closure of the optic fissure; (Deml et al., 2016; London et al., 2009; Zhang et al., 2009)). Therefore, it would be of high interest to investigate whether ALCAM is also mutated in human patients showing microphthalmia or coloboma.

with these results, Fzd3 inhibition led to a mild but significant downregulation of pax2 expression at stage 23 (Fig. 8B). In summary, our data reveal that alcam expression is regulated by the Wnt/JNK pathway via the transcription factor Pax2 (Fig. 8C). 4. Discussion In this study, we expanded our knowledge of how non-canonical Wnt signalling regulates early embryonic eye development. Specifically, we showed that I) loss of Alcam function phenocopies loss of Fzd3 during vertebrate eye development, II) Fdz3 regulates alcam expression through JNK1, III) the transcription factor Pax2 is upstream of alcam and IV) one Pax2 binding site in the Fzd3 responsive element is sufficient to trigger alcam expression. Furthermore, the mechanism of alcam activation shown in this study is in line with a former study on alcam regulation in the embryonic kidney (Cizelsky et al., 2014), strongly indicating that Fzd3 acts through a similar mechanism in multiple tissues. 4.1. Fzd3 during vertebrate eye development Previous studies identified the potential non-canonical Wnt receptor, Fzd3, to be expressed and to have important functions during Xenopus ocular development and regeneration (Dawes et al., 2013; Hamilton et al., 2016; Rasmussen et al., 2001). We confirmed the data on the role of Fzd3 in ocular development and expanded those findings by describing the role of Fzd3 during eye field induction and retinal lamination. We demonstrated that Fzd3 deficiency leads to a mild, but significant phenotype in eye field induction and to a disturbed retinal lamination. This is in accordance with several earlier studies, which showed that non-canonical Wnt is required for early vertebrate eye development (Bugner et al., 2011; Gessert et al., 2007; Maurus et al., 2005). 4.2. Alcam acts downstream of Fzd3 during eye development By detailed analyses, we further showed that the cell adhesion molecule Alcam is required for Xenopus eye development and that the loss of function phenotype fully phenocopies loss of Fzd3. Alcamdepleted embryos showed microphthalmia (smaller eyes), coloboma (incomplete closure of the optic fissure) as well as defects in retinal lamination. Electron microscopy demonstrated that loss of Alcam results in ectopic photoreceptor cells in the developing retina. This is in line with previous studies showing showed abnormalities in the retina of Alcam-deficient mice including evaginated or invaginated regions of the retina (Weiner et al., 2004) and photoreceptor ectopia (Buhusi et al., 2009). In line with these findings, blocking of N-CAM, a heterophilic interaction partner of Alcam, alters retinal cell layer formation (Crossin and Krushel, 2000). In addition, Alcam function in the retinal ganglion cells has been previously examined in detail (Paschke et al., 1992). It has been shown in fish that Neurolin-a, the ortholog of Alcam, is highly expressed in the developing retinal ganglion cells but repressed during adulthood (Fournier-Thibault et al., 1999; Paschke et al., 1992). Knock down of Neurolin-a leads to misguidance of the new RGCs on their way to the optic disk (Ott et al., 1998). Also in Alcam-/- mice, the motor and retina ganglion cell axons are only poorly fasciculated and occasionally misdirected. Taken together our data complement these earlier findings and argue for a critical role of Alcam for proper formation of different retinal cell types and retinal architecture.

4.4. Alcam as a direct target gene of the non-canonical Wnt/JNK/ Pax2 branch We and others showed earlier that alcam is regulated by noncanonical Wnt11a (also known as Wnt11R), as knocking down of Wnt11a led to reduced alcam expression in the developing heart and Alcam is able to rescue the cardiac phenotype induced by loss of Wnt11a (Choudhry et al., 2011; Gessert et al., 2008). In another study, focusing on the embryonic kidney, we showed alcam to be transcriptionally regulated by non-canonical Wnt/JNK involving ATF2 and Pax2 in a direct manner (Cizelsky et al., 2014). In the present study, we confirmed this mechanism in the developing neural tissue indicating a conserved mechanism of regulation across different tissues. This is also supported by the observation from others showing an activation of gene expression induced by the phosphorylation of Pax2 by JNK1 (Cai et al., 2002). The rescue experiments performed here demonstrated that the reduction of alcam expression by Fzd3 down-regulation can be rescued by overexpressing dshΔDIX or JNK1. These rescue experiments support the assumption that alcam is regulated by non-canonical Wnt signalling during eye development. This is also in line with studies that showed JNK to be required for proper eye development (Maurus et al., 2005) including the closure of the optic fissure (Weston et al., 2003). Furthermore, we could narrow down the alcam promoter to one Pax2 binding site as one Pax2 binding site is sufficient to induce alcam expression in neuralized Xenopus animal caps. This is supported by the observation that Pax2 depletion results in a reduced alcam expression in the anterior neural tissue and that loss of Pax2 phenocopies the loss of Alcam phenotype in the developing eye. This is in accordance with

4.3. Alcam during cell adhesion and differentiation By electron microscopy we showed that Alcam is required for cell adhesion in the developing Xenopus retina. Intercellular spaces are enlarged and photoreceptor cells are detached from the RPE in Alcam13

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