Decreased proliferation and change in physical properties of mutant optic vesicle.

<p>(A) Immunohistochemical analysis for the presence of BrdU-labelled cells in the prospective neural retina on a coronal section of an E10.5 control embryo (left panel) and an <i>Lhx2-Cre:β-catenin^flox/flox</i> embryo (right panel). The part of the prospective neural retina that was analysed for BrdU⁺ cells in the control and mutant embryos are outlined. (B) Immunohistochemical analysis for the presence apoptotic cells as revealed by the presence of activated Casp-3⁺ cells on coronal section of an E10.5 control embryo (left panel) and a <i>Lhx2-Cre:β-catenin^flox/flox</i> embryo (right panel). (C) Immunohistochemical analyses for the presence of phosphorylated myosin light chain 2 (pMLC2) on a coronal section of an E10.5 control embryo. (D) Immunohistochemical analyses for the presence of pMLC2 on a coronal section on an E10.5 <i>Lhx2-Cre:β-catenin^flox/flox</i> embryo. Insets in C and D are a magnification of the areas indicated by the squares. Scale bars: (A, B and C, D) 100 µm.</p

Dorsal shift in expression of retina-specific eye field transcription factors in the mutant optic vesicle.

<p>(A–E) <i>In situ</i> hybridization analyses on coronal sections of E10.5 optic cups from control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> mutant embryos (right panels). Arrows indicate the dorsal shift in expression of the retina-specific transcription factors <i>Six3</i>, <i>Six6</i> and <i>Rx</i> in mutant embryos (C–E). Scale bar: 100 µm.</p

The few RPE cells that develop in the mutant embryos are derived from eye committed progenitor cells in the anterior neural plate that escape <i>β-catenin</i> inactivation.

<p>(A) Immunohistochemical analyses of Mitf (green) and β-catenin (red) on coronal sections of an E13.5 optic rudiment in an <i>Lhx2-Cre:β-catenin^flox/</i>⁻ embryo. RPE cells are identified by pigment (pseudocoloured blue) and Mitf (green). (B) Immunohistochemical analysis of β-catenin and DAPI labelling of the same coronal section as in (A) to reveal that both β-catenin⁺ and β-catenin⁻ cells are present in the optic rudiment that develops in mutant embryos. * indicates the area of β-catenin⁻ non-RPE cells and the arrow head indicates the area of β-catenin⁺ non-RPE cells in the optic rudiment. (C–E) is a magnification of the area indicated by a rectangle in (A). (C) and (D) are merged in (E) to show co-localisation of β-catenin, Mitf and pigment. (F) Immunohistochemical analysis of Mitf (green) and β-catenin (red) on a coronal section of an E12.5 optic rudiment in a <i>Lhx2-Cre:β-catenin^flox/flox:ROSA26R</i> embryo. (G) Immunohistochemical analysis of Mitf (green) and β-Gal (red) on a serial section following (F) revealing that all cells in the optic rudiment, including the RPE cells, are β-Gal⁺ and hence derived from Cre⁺ progenitor cells in the anterior neural plate. (H–J) is a magnification of the area indicated by a rectangle in (G). (H) and (I) are merged in (J) to show co-localisation of Mitf, β-Gal and pigment. Scale bars: (A, B, and H–J) 50 µm. (C–E) 25 µm. (F, G) 100 µm.</p

β-catenin remains associated with N-cadherin and F-actin at the apical side of the cells in the mutant optic vesicle.

<p>(A–D) Immunohistochemical analyses for cellular localisation of the indicated proteins on coronal sections of E10 (ss30–31) optic vesicles from control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (C) is a merge of (A) and (B) to show co-localisation (yellow) in the apical side of the cells in the optic vesicle on both control and mutant embryos (arrows). (D) A serial section of the same embryo as in (A–C) revealing F-actin protein at the apical side of the cells in both control and mutant optic vesicles (arrows). Scale bars: (A–C and D) 50 µm.</p

β-catenin is important for maintenance of dorsoventral patterning of the retina.

<p>(A–D) <i>In situ</i> hybridization analyses (A, C, D) and immunohistochemical analysis (B) on coronal sections of E9.5 (ss25–26) optic vesicles from control (left panels) and on <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (E–H) In situ hybridization analyses (E, G, H) and immunohistochemical analysis (F) on coronal sections of E10.5 (ss32–34) optic cups from control (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). Black arrow heads in A and E, and white arrow heads in B and F, indicate the boundaries of the area where β-catenin has been inactivated. (I, J) <i>In situ</i> hybridization analyses on coronal sections of E10.5 (ss32–34) optic cups from control (left panel) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (K, L) <i>In situ</i> hybridization analyses on coronal sections of an eye from E12.5 control (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos. Dorsal-ventral (D–V) orientation of all panels is indicated in A. Scale bars: (A–E, G–J, F and K, L) 100 µm.</p

Canonical Wnt/β-catenin signalling is required for RPE cell commitment.

<p>(A–C) <i>In situ</i> hybridization analyses of the indicated genes on coronal sections of E9.25 somite stage 21–23 (ss21–23) control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (D–I) <i>In situ</i> hybridization analyses of the indicated genes on coronal sections of E9.75 (ss26–28) control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). (J–O) <i>In situ</i> hybridization analyses (K, M–O) or immunohistochemical analyses (J, L) for gene expression of the indicated genes on coronal sections of E10.5 (ss32–34) control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels). β-catenin protein is indicated by red labelling in J and L while Mitf protein is indicated by green labelling in L. Arrows indicate the area where <i>β-catenin</i> has been inactivated in A, D and J. Arrow heads indicate the area of Axin2 expression in E and K. Dorsal-ventral (D–V) orientation for all panels is indicated in A. Scale bars: (A–C and D–O) 100 µm.</p

β-catenin mutant embryos are anophthalmic.

<p>(A–C) The left panels show lateral views of E16.5 embryos of control (A), <i>Lhx2-Cre:β-catenin^flox/flox</i> (β-cat^f/f) (B) and <i>Lhx2-Cre:β-catenin^flox/</i>⁻ (<i>β-cat^f/</i>⁻) (C) embryos. Arrow heads indicate where the eye should be located in the mutant embryos. The right panels show hematoxylin/eosin staining of coronal tissue sections of the same embryos. Black arrows indicate the optic rudiment surrounded by RPE cells (red arrow heads) that can develop in the mutant embryos. NR: neural retina. L: lens. RPE: retinal pigment epithelium. (D) Relative size of the optic vesicle-derived structure in the <i>Lhx2-Cre:β-catenin^flox/flox</i> (<i>f/f</i>) embryos and <i>Lhx2-Cre:β-catenin^flox/</i>⁻ (<i>f/−</i>) embryos compared with control embryos (c) at the indicated embryonic age and postnatal day 1 (P1). To get an approximate value of the relative eye size for the different genotypes, the largest diameter of the control eyes and the mutant optic rudiment at different ages were measured and compared. The diameter of the eye in control embryos at each developmental stage is arbitrarily defined as 1.0. The number of eyes analysed for each genotype at the respective age is indicated for each group. SD is indicated when applicable. Scale bar: 200 µm.</p

<i>β-catenin</i> mutant optic vesicles do not form an optic cup.

<p>(A) <i>In situ</i> hybridization analyses of <i>β-catenin</i> expression on coronal sections of optic vesicles of an E9.5 control embryo (left panel) and an <i>Lhx2-Cre:β-catenin^flox/flox</i> embryo (right panel). The arrow heads indicate the boundaries of the area in the optic vesicle where <i>β-catenin</i> has been inactivated. (B–E) <i>In situ</i> hybridization analyses for gene expression of the indicated genes on coronal sections of control embryos (left panels) and <i>Lhx2-Cre:β-catenin^flox/flox</i> embryos (right panels) at E11.5. Arrows indicate the lens pit where the early lens ectoderm remains attached to the epidermal ectoderm in mutants. Arrow head in (B) indicate the area where the <i>β-catenin</i> gene has been inactivated. Scale bar: 100 µm.</p

Efficiency in generating DoxHPC lines from individual colonies in clonal assays of EB cells

*<p>(Number of DoxHPC lines generated/Number of colonies picked)</p><p>ND, Not done.</p

Lhx2 expression in intact EBs transiently induce expansion of definitive hematopoietic progenitor cells in the absence of exogenously added growth factors.

<p>A. Gene expression analysis by real-time PCR on day 6 EB generated from the iLhx2 ES cells comparing control EBs (−Dox) to those where Lhx2 expression was turned on at day 4 of differentiation (+Dox). B. Frequency of definitive CFCs responding to SCF/IL-6/epo in clonal assays of day 6 EBs in the presence (+Dox) or absence (−Dox) of dox, if dox was added at day 3 or 4 of differentiation, or if dox was present between day 3 and 5 of differentiation. Control is day 6 EBs generated without dox. C. Frequency of definitive CFCs responding to SCF/IL-6/epo in clonal assays of day 6, 7 and 8 EBs in the presence (−Dox) of absence (+Dox) of dox if dox was added or not at day 4 of differentiation. Hence, the following combinations were analysed: −− no dox added to the ES cells differentiation or to clonal assays of EB cells, −+ dox was added to clonal assays of day 6, 7 and 8 EB cells. +− dox was added to day 4 EB and no dox was added to clonal assays of the day 6, 7 and 8 EB cells. ++ dox was added to day 4 EBs and to the clonal assays of the day 6, 7 and 8 EB cells. Asterisks indicate where the difference in CFC between clonal assays performed in the absence or presence of dox is statistically significant, *p<0,005, **p<0,05.</p