The physical contact of optic vesicle with head surface ectoderm is an initial event triggering eye morphogenesis. This interaction leads to lens specification followed by coordinated invagination of the lens placode and optic vesicle, resulting in formation of the lens, retina and retinal pigmented epithelium. Although the role of Pax6 in early lens development has been well documented, its role in optic vesicle neuroepithelium and early retinal progenitors is poorly understood. Here we show that conditional inactivation of Pax6 at distinct time points of mouse neuroretina development has a different impact on early eye morphogenesis. When Pax6 is eliminated in the retina at E10.5 using an mRx-Cre transgene, after a sufficient contact between the optic vesicle and surface ectoderm has occurred, the lens develops normally but the pool of retinal progenitor cells gradually fails to expand. Furthermore, a normal differentiation program is not initiated, leading to almost complete disappearance of the retina after birth. By contrast, when Pax6 was inactivated at the onset of contact between the optic vesicle and surface ectoderm in Pax6Sey/flox embryos, expression of lens-specific genes was not initiated and neither the lens nor the retina formed. Our data show that Pax6 in the optic vesicle is important not only for proper retina development, but also for lens formation in a non-cell-autonomous manner.

Proper eye development is dependent on the coordinated formation of two main tissues in the eye: the retina and the lens. Vertebrate eye development begins with invagination of the optic vesicle (OV) toward the lens-competent head surface ectoderm (SE). As OV contacts SE, a series of reciprocal inductive signals elicit formation of the lens placode (LP) and subsequent invagination of both LP and OV to form a two-layered optic cup (OC), with retinal pigmented epithelium (RPE) surrounding the retina (reviewed by Fuhrmann, 2010; Chow and Lang, 2001; Ogino et al., 2012). Genetic studies have identified multiple transcription factors and signaling pathways interacting in a complex network orchestrating early eye development (reviewed by Fuhrmann, 2010; Chow and Lang, 2001; Ogino et al., 2012; Xie and Cvekl, 2011). Among the signaling pathways, BMP (Furuta and Hogan, 1998; Rajagopal et al., 2009; Sjödal et al., 2007; Wawersik et al., 1999) and FGF (Faber et al., 2001; Garcia et al., 2011; Gotoh et al., 2004; Pan et al., 2006) were found to be essential for lens induction and coordinated OV-to-OC transition, as severe eye defects are associated with their inactivation.

At the time the LP is formed, the dorsal region of the OV becomes specified to the retina and is populated with mitotically active retinal progenitor cells (RPCs) (Fuhrmann, 2010; Levine and Green, 2004). Lineage-tracing studies have shown that RPCs are multipotent, with a single progenitor cell competent to give rise to all retinal neuron and glia cell types (Holt et al., 1988; Turner and Cepko, 1987; Turner et al., 1990). The defining feature of RPCs is co-expression of the transcription factors Rx (Rax), Pax6, Lhx2, Six3/Six6, Chx10 (Vsx2) and Hes1, which are expressed prior to activation of the neurogenic program and contribute to the proliferative and retinogenic potential of RPCs (Burmeister et al., 1996; Grindley et al., 1995; Jean et al., 1999; Li et al., 2002; Liu et al., 2010; Marquardt et al., 2001; Mathers et al., 1997; Oliver et al., 1995; Porter et al., 1997; Tomita et al., 1996; Walther and Gruss, 1991). In a defined birth order, RPCs differentiate into seven retinal cell types: retinal ganglion cells, horizontal cells and cone photoreceptors differentiate first, followed by amacrine cells and rod photoreceptors, bipolar cells, and finally Muller glia cells (Young, 1985). As retinogenesis proceeds, RPCs are exposed to the changing environment of extrinsic cues (Cepko, 1999). These, in cooperation with intrinsic factors represented by transcription factors, most prominently of the basic helix-loop-helix (bHLH) and homeodomain class, regulate progenitor proliferation and operate to direct the bias towards particular cell types (Brown et al., 1998; Cepko, 1999; Hatakeyama and Kageyama, 2004; Inoue et al., 2002; Lillien, 1995; Morrow et al., 1999; Perron and Harris, 2000; Tomita et al., 1996).

At the time of neuronal differentiation, a subpopulation of progenitors undergoes a transition from the proliferative stage toward the lineage-restricted neurogenic stage, upon which they withdraw from the cell cycle and take up a neuronal or glial fate. Accordingly, a proper balance between cell cycle exit and re-entry is required to ensure the temporal generation of all retinal cell types (reviewed by Agathocleous and Harris, 2009). It is in cell cycle phase G1 that growth-promoting and growth-inhibiting signals determine whether a progenitor cell will exit or re-enter the cell cycle. In mammalian retina, the KIP proteins p57Kip2 (Cdkn1c) and p27Kip1 (Cdkn1b) and cyclin D1 have been implicated in direct regulation of progenitor proliferative potential (Das et al., 2009; Dyer and Cepko, 2000; Dyer and Cepko, 2001; Geng et al., 2001; Levine et al., 2000; Levine and Green, 2004), promoting either cell cycle exit or progression. The observation that some neurogenic factors promote both neuronal fate determination and cell cycle withdrawal implies that the processes of cell type specification and cell cycle exit are tightly coupled (Farah et al., 2000; Ochocinska and Hitchcock, 2009). However, the mechanism that orchestrates these complex events remains largely elusive.

The paired and homeodomain transcription factor Pax6 plays a pivotal role in both vertebrate and invertebrate eye development (Kozmik, 2005). Since Pax6-deficient (Pax6-/-) mice are anophthalmic (Hill et al., 1991; Hogan et al., 1986), with eye development arrested at the OV stage, much attention has been paid to elucidation of Pax6 function in the development of individual ocular structures (reviewed by Shaham et al., 2012). Pax6 is expressed from very early stages of eye formation, in SE and OV, and later in developing lens, RPE and all mitotically active RPCs of the retina (Walther and Gruss, 1991). Conditional ablation of Pax6 revealed its autonomous requirement for lens development (Ashery-Padan et al., 2000; Shaham et al., 2009) as well as for later retinal neurogenesis (Marquardt et al., 2001; Oron-Karni et al., 2008). Nevertheless, the autonomous role of Pax6 in the progenitors of the OV and newly formed OC remains unresolved. Here we employed the Cre-loxP system to conditionally inactivate Pax6 specifically in retina-committed eye progenitors.

Pax6 deletion in early RPCs results in strongly hypocellular retinae

Pax6 is expressed in both the OV and SE of the developing eye (Walther and Gruss, 1991). In Pax6-/- (Sey/Sey) mouse embryos, eye development is arrested at the OV stage and no eyes are formed (Hill et al., 1991; Hogan et al., 1986). Although Pax6 expression in SE was found to be essential for lens induction (Ashery-Padan et al., 2000), the role of Pax6 in the OV is less well understood. We used the mRx-Cre transgenic mouse line (Klimova et al., 2013) to conditionally inactivate Pax6 in retina-committed progenitor cells selectively. Expression of Cre recombinase in the mRx-Cre line is controlled by regulatory sequences of the mouse Rx gene (supplementary material Fig. S1B). To monitor mRx-Cre, the ROSA26R reporter line was employed in which Cre-expressing cells can be traced by X-gal staining for lacZ expression (β-galactosidase) after Cre-mediated recombination (Soriano, 1999). Consistent with the expression pattern of the endogenous Rx gene (Mathers et al., 1997), strong Cre activity was observed in the OVs (Fig. 1A-C) of mRx-Cre/ROSA26R embryos at E9.0 and later in the neuroretina, RPE and optic stalk (Fig. 1D-F). Next, mRx-Cre mice were crossed to Pax6fl/fl mice to inactivate Pax6 in the OV. Pax6fl/fl/mRx-Cre mice were viable and fertile. The eyes of Pax6fl/fl/mRx-Cre (i.e. Pax6 loss-of-function) mutants were analyzed for the presence of Pax6 protein by immunohistochemistry (Fig. 1G-J). At E9.5, we observed decreased Pax6 levels in OV neuroepithelium of Pax6fl/fl/mRx-Cre embryos, whereas Pax6 protein expression was very high in the SE and OV of Pax6fl/fl control embryos (compare Fig. 1G with 1H). One day later, very few Pax6+ cells were detected in the neuroretina and optic stalk of Pax6fl/fl/mRx-Cre (Fig. 1J). Importantly, the Pax6 protein levels remained unchanged in the SE and lens pit upon OV-specific Pax6 elimination (Fig. 1H,J).

Fig. 1.

mRx-Cre-mediated Pax6 inactivation in early retinal progenitors. (A-F) mRx-Cre activity was assessed using the ROSA26R reporter mouse line. Whole-mounts or coronal sections were stained with X-gal at the indicated stages to show Cre activity in eye primordium. Side (A) and frontal (B) view of the embryo at E9.0. (C) Transverse section showing Cre activity in the optic vesicle. Side (D) and frontal (E) view of the embryo at E11.5. (F) Transverse section showing strong Cre activity in developing neuroretina, RPE and optic stalk. (G-J) Confocal images of transverse sections of wild-type (Pax6fl/fl) (G,I) and Pax6 loss-of-function mutant (Pax6fl/fl/mRx-Cre) (H,J) eye primordium stained with Pax6 antibody at the indicated stages. The RPC population with inactivated Pax6 at E10.5 is indicated by the dashed line (J). NR, neuroretina; OV, optic vesicle; SE, surface ectoderm; LPi, lens pit; RPE, retinal pigmented epithelium; OS, optic stalk; le, lens. Scale bars: 50 μm.

Fig. 1.

mRx-Cre-mediated Pax6 inactivation in early retinal progenitors. (A-F) mRx-Cre activity was assessed using the ROSA26R reporter mouse line. Whole-mounts or coronal sections were stained with X-gal at the indicated stages to show Cre activity in eye primordium. Side (A) and frontal (B) view of the embryo at E9.0. (C) Transverse section showing Cre activity in the optic vesicle. Side (D) and frontal (E) view of the embryo at E11.5. (F) Transverse section showing strong Cre activity in developing neuroretina, RPE and optic stalk. (G-J) Confocal images of transverse sections of wild-type (Pax6fl/fl) (G,I) and Pax6 loss-of-function mutant (Pax6fl/fl/mRx-Cre) (H,J) eye primordium stained with Pax6 antibody at the indicated stages. The RPC population with inactivated Pax6 at E10.5 is indicated by the dashed line (J). NR, neuroretina; OV, optic vesicle; SE, surface ectoderm; LPi, lens pit; RPE, retinal pigmented epithelium; OS, optic stalk; le, lens. Scale bars: 50 μm.

The consequences of Pax6 inactivation in retina-committed progenitor cells of Pax6fl/fl/mRx-Cre embryos were first investigated at the histological level. The first manifestation of abnormal retina development was observed at E10.5. Already at this stage, the retina was thinner than in wild type (compare Fig. 2A with 2B), suggesting a decreased number of RPCs. We counted DAPI-stained (DAPI+) cells per retinal section and found that the number of RPCs was decreased by 44±4.8% (± s.d.) in Pax6-deficient retinae (Fig. 2I). Hypocellularity became more obvious at E14.5, when the number of retinal cells reached only 19.7±3.1% of wild-type levels (Fig. 2C,D,I). At E16.5, Pax6-deficient retinae became progressively smaller (Fig. 2E-F′) and, whereas the retina in wild-type newborns was properly laminated (Fig. 2G), Pax6-deficient retinae reproducibly formed a thin layer around the lens with no sign of lamination (Fig. 2H,H′, red arrowheads). At postnatal stages, the eye was generally smaller with hardly distinguishable retinal cells (data not shown).

Fig. 2.

Morphological consequences of Pax6 inactivation in early retinal progenitors. (A-H′) Coronal sections of wild-type (Pax6fl/fl) (A,C,E,G) and Pax6-deficient (Pax6fl/fl/mRx-Cre) (B,D,F,H) eye were stained with Hematoxylin-Eosin at the indicated stages. Position of the retina is indicated with arrows for E16.5 and P0 (F,H) and with red arrowheads for magnifications (F′,H′). The double-headed arrows in A and B indicate thickness of the retina. (I) The growth of wild-type (wt) and Pax6-deficient (mut) retinae, assessed by the number of DAPI+ cells per retinal section from three different retinae. Error bars indicate s.d. le, lens; re, retina.

Fig. 2.

Morphological consequences of Pax6 inactivation in early retinal progenitors. (A-H′) Coronal sections of wild-type (Pax6fl/fl) (A,C,E,G) and Pax6-deficient (Pax6fl/fl/mRx-Cre) (B,D,F,H) eye were stained with Hematoxylin-Eosin at the indicated stages. Position of the retina is indicated with arrows for E16.5 and P0 (F,H) and with red arrowheads for magnifications (F′,H′). The double-headed arrows in A and B indicate thickness of the retina. (I) The growth of wild-type (wt) and Pax6-deficient (mut) retinae, assessed by the number of DAPI+ cells per retinal section from three different retinae. Error bars indicate s.d. le, lens; re, retina.

Altered cell cycle length and disrupted balance between progenitor proliferation and cell cycle exit reduce the RPC population size

Tissue hypocellularity could be due to a decreased proliferation rate, premature cell cycle exit or cell death. To address this issue in Pax6-deficient retina, we first analyzed the proliferation potential of RPCs. The proportion of actively proliferating cells (BrdU+/DAPI+ per retinal section) was counted at several stages of embryonic development using incorporation of BrdU applied 1 hour prior to analysis. Despite the fact that Pax6-deficient retinae appeared strongly hypocellular, we did not observe a dramatic difference in the proportion of BrdU+ cells relative to all retinal cells between E10.5 and E13.0 (Fig. 3A-D,I). However, at E14.5 a rapid decrease of BrdU+ retinal cells was observed (Fig. 3E,F,I). Only 9.1±2% of RPCs were BrdU+, compared with 34±6.2% BrdU+ cells in wild-type retinae (Fig. 3I). Although BrdU+ cells were localized in the neuroblastic layer (NBL) throughout the whole retina of the wild-type OC (Fig. 3E), in Pax6-deficient retinae cycling cells were localized mostly in the central part of the retina with almost no BrdU+ cells localized peripherally (Fig. 3F). From E16.5 onwards, almost no dividing BrdU+ cells were found in Pax6-deficient retinae (Fig. 3H,I).

Fig. 3.

Cell cycle dynamics is affected in Pax6-deficient retinae. (A-I) Following a 1 hour BrdU pulse, eye sections of wild-type (Pax6fl/fl) and Pax6-deficient (Pax6fl/fl/mRx-Cre) retinae were stained for BrdU incorporation at the indicated stages. Retina is indicated by the dashed line. The solid red line indicates the interface between the differentiating and neuroblastic layers in wild-type retina (E,G). (I) The proportion of BrdU+ cells relative to all retinal (DAPI+) cells in wild-type (wt) and Pax6-deficient (mut) retinae at the indicated stages. n=8 sections from four retinae (E10.5); n=4 sections from three retinae (E13, E14.5, E16.5). (J-K′) Cell cycle length determined by BrdU, EdU and Pcna triple staining. Pregnant females were injected 2.5 hours and 0.5 hours prior to dissection with BrdU and EdU, respectively. (J) Single fields of eye sections stained with BrdU, EdU and for Pcna. Beneath are the formulae for cell cycle (Tc), S phase (Ts) and G1+G2+M phase (Tc-Ts) length determination. (K) Quantification of average Tc, Ts and G1+G2+M phase time in wild-type (wt) and Pax6-deficient (mut) retinae at E11.5 and E13.0. (K′) The proportions of EdU+ cells relative to proliferating progenitor (Pcna+) cells in wild-type (wt) and Pax6-deficient (mut) retinae at E11.5 and E13.0. n=9 sections from three retinae (E11.5) and n=3 sections from three retinae (E13.0) were used. (L-Q) Sections stained with antibodies against cyclin D1, p27Kip1 and p57Kip2 at E14.5. (R,S) p57kip2 mRNA expression at E14.5. (T-W) Sections stained with antibodies against cyclin D1 and p57Kip2 at E16.5. Areas with changed expression are indicated by arrowheads. Error bars indicate s.d. P-values are by Student’s t-test. NBL, neuroblastic layer; le, lens. Scale bars: 100 μm.

Fig. 3.

Cell cycle dynamics is affected in Pax6-deficient retinae. (A-I) Following a 1 hour BrdU pulse, eye sections of wild-type (Pax6fl/fl) and Pax6-deficient (Pax6fl/fl/mRx-Cre) retinae were stained for BrdU incorporation at the indicated stages. Retina is indicated by the dashed line. The solid red line indicates the interface between the differentiating and neuroblastic layers in wild-type retina (E,G). (I) The proportion of BrdU+ cells relative to all retinal (DAPI+) cells in wild-type (wt) and Pax6-deficient (mut) retinae at the indicated stages. n=8 sections from four retinae (E10.5); n=4 sections from three retinae (E13, E14.5, E16.5). (J-K′) Cell cycle length determined by BrdU, EdU and Pcna triple staining. Pregnant females were injected 2.5 hours and 0.5 hours prior to dissection with BrdU and EdU, respectively. (J) Single fields of eye sections stained with BrdU, EdU and for Pcna. Beneath are the formulae for cell cycle (Tc), S phase (Ts) and G1+G2+M phase (Tc-Ts) length determination. (K) Quantification of average Tc, Ts and G1+G2+M phase time in wild-type (wt) and Pax6-deficient (mut) retinae at E11.5 and E13.0. (K′) The proportions of EdU+ cells relative to proliferating progenitor (Pcna+) cells in wild-type (wt) and Pax6-deficient (mut) retinae at E11.5 and E13.0. n=9 sections from three retinae (E11.5) and n=3 sections from three retinae (E13.0) were used. (L-Q) Sections stained with antibodies against cyclin D1, p27Kip1 and p57Kip2 at E14.5. (R,S) p57kip2 mRNA expression at E14.5. (T-W) Sections stained with antibodies against cyclin D1 and p57Kip2 at E16.5. Areas with changed expression are indicated by arrowheads. Error bars indicate s.d. P-values are by Student’s t-test. NBL, neuroblastic layer; le, lens. Scale bars: 100 μm.

Cell death, as a possible cause of hypocellularity, was analyzed on retinal sections between E10.5 and E15.5 using an antibody against cleaved caspase 3 (supplementary material Fig. S2A-J). Although we revealed no significant difference between Pax6-deficient and wild-type retinae at E10.5-E12.5, increased cell death was observed at E13.5 and E14.5 (supplementary material Fig. S2K).

Because the BrdU incorporation assay between E10.5 and E13.0 did not indicate perturbed S-phase re-entry and since no increase in cell death was observed in Pax6-deficient RPCs of these stages, we next analyzed a potential M-phase arrest and the length of RPC cell cycle as potential contributors to the phenotype. For M-phase arrest, staining for phosphorylated histone H3 (PH3) was performed at E10.5, E11.5 and E14.5 (supplementary material Fig. S3A). No difference in the proportion of PH3+ cells was observed at E10.5 and E11.5. However, a decreased proportion of PH3+ cells was observed in Pax6-deficient retinae at E14.5 (supplementary material Fig. S3B). This decrease corresponds to the decreased proliferation rate at E14.5 observed in the BrdU incorporation assay (Fig. 3I). To measure the length of the cell cycle, we used window labeling based on two thymidine analogs that can be differentially detected (Burns and Kuan, 2005; Das et al., 2009). Retinal sections of E11.5 and E13.0 embryos were co-stained for BrdU, EdU and Pcna (Fig. 3J; data not shown) and the lengths of the whole cell cycle (Tc), S phase (Ts) and G1+G2+M phase (Tc-Ts) were determined as previously described (Das et al., 2009). At both stages analyzed, Tc of Pax6-deficient RPCs was significantly prolonged compared with wild-type littermates (Fig. 3K). The prolonged Tc was not caused by a lengthened S phase, as Ts was unchanged, but instead Tc-Ts was increased (Fig. 3K). Furthermore, quantification of EdU+ cells relative to Pcna+ proliferating progenitors showed that overall progenitor proliferation was also affected at E11.5 and E13.0 (Fig. 3K′), at stages when no significant difference in the proportion of BrdU+ cells relative to all retinal cells was detected in Pax6-deficient retinae (Fig. 3I). This difference can be attributed to neurogenesis in wild-type retinae, which decreases the fraction of BrdU+ cells relative to all (DAPI+) retinal cells. Taken together, these data indicate that Pax6 positively regulates progression through the RPC cell cycle.

During development, decreased proliferation usually coincides with cell cycle exit and subsequent differentiation. To address whether premature cell cycle exit might contribute to the phenotype observed in Pax6-deficient retina, we analyzed the expression of cyclin D1 and of the cyclin-dependent kinase inhibitors p27Kip1 and p57Kip2, which are known regulators of RPC proliferation. At E14.5, the stage when the decrease in BrdU+ cells was noted (Fig. 3F), we observed a decreased level of cyclin D1 and p27Kip1 and elevated expression of p57Kip2 (Fig. 3L-S). For cyclin D1 and p57Kip2, expression changes were obvious mostly in the distal parts of retinae (Fig. 3M,Q,S), in a pattern complementary to that of BrdU staining (Fig. 3F). This suggested that peripherally located cells have just left the cell cycle, as they were p57Kip2+, cyclin D1- and BrdU-. To address the possible gradual peripheral-to-central progression of this phenomenon, we analyzed the retinae of E16.5 embryos. Whereas no p57Kip2+ cells were detected in any wild-type retina, p57Kip2 was detected in the majority of Pax6-deficient retinal cells (Fig. 3V,W). At the same time, cyclin D1 was downregulated in Pax6-deficient retinae (compare Fig. 3T with 3U). These results indicate that Pax6 depletion from early retinal progenitors dramatically restricted their proliferation potential and shifted RPCs to forced cell cycle exit.

Pax6-deficient RPCs maintain RPC characteristics but are unable to proceed through the general differentiation program

As Pax6-deficient retinae exhibit severe proliferation defects, we next analyzed whether retinal progenitor characteristics were maintained in mutants. We assessed the expression of known markers such as Rx, Lhx2, Chx10, Sox2, Six3, Hes1 and cyclin D1 at E10.5, when the cell number was already decreased. However, the expression of none of these factors was significantly changed (Fig. 4A-G′).

Fig. 4.

Retinal progenitor characteristics are maintained but the differentiation program is not initiated in Pax6-deficient retina. (A-G′) Expression of the indicated genes (mRNA, A,A′) and proteins (B-G′) involved in RPC pool maintenance analyzed at E10.5. (H-L′) Expression of the indicated genes (mRNA) at E14.5. (M-N′) Sections stained with antibody against the pan-neuronal marker Tuj1 at E14.5 (M,M′) and E16.5 (N,N′). Tuj1+ cells in Pax6-deficient retina are indicated by red arrowheads. The solid red line indicates the interface between differentiating and neuroblastic cell layers. Retina is indicated with a dashed line. le, lens. Scale bars: 100 μm.

Fig. 4.

Retinal progenitor characteristics are maintained but the differentiation program is not initiated in Pax6-deficient retina. (A-G′) Expression of the indicated genes (mRNA, A,A′) and proteins (B-G′) involved in RPC pool maintenance analyzed at E10.5. (H-L′) Expression of the indicated genes (mRNA) at E14.5. (M-N′) Sections stained with antibody against the pan-neuronal marker Tuj1 at E14.5 (M,M′) and E16.5 (N,N′). Tuj1+ cells in Pax6-deficient retina are indicated by red arrowheads. The solid red line indicates the interface between differentiating and neuroblastic cell layers. Retina is indicated with a dashed line. le, lens. Scale bars: 100 μm.

It has been reported that conditional inactivation of Pax6 in the distal parts of the OC at later stages (E12) of retinogenesis using α-Cre leads to exclusive generation of amacrine cells (Marquardt et al., 2001; Oron-Karni et al., 2008). In accordance, generation of amacrine cells was observed in Pax6fl/fl/α-Cre mice, as documented by staining for the amacrine cell marker syntaxin and Vc1.1 (HNK-1) immunoreactivity in the distal part of Pax6fl/fl/α-Cre retinae (supplementary material Fig. S1C). We therefore tested the differentiation potential of RPCs in which Pax6 is absent from E10.5 in the whole retina. We used RNA in situ hybridization to analyze the expression of the pro-neural bHLH transcription factors Atoh7, Ngn2 (Neurog2), Neurod1, Mash1 (Ascl1) and Math3 (Neurod4), which that have been shown to initiate the differentiation program and exert bias towards particular cell fates. The expression of these bHLH factors was not initiated at E14.5 (Fig. 4H′-L′), suggesting that not only proliferation but also the retina-specific differentiation program was severely affected in the absence of Pax6.

To test whether general neuronal differentiation took place in the mutants, retinae were stained with antibody against the pan-neuronal marker acetylated beta III tubulin (Tuj1, also known as Tubb3), which marks differentiating cells and reveals formation of the differentiated cell layer (DCL) (Sharma and Netland, 2007; Sigulinsky et al., 2008). Although strong Tuj1 staining was observed in the DCL of wild-type retinae at E14.5 and E16.5 (Fig. 4M,N), only a very few Tuj1+ cells were detected in Pax6-deficient retinae, as DCL was not established at all (Fig. 4M′,N′, arrowheads). In addition, it should be noted that staining for the early amacrine cell-specific factor bHLHb5 (Bhlhe22) and Vc1.1 immunoreactivity at E15.5 revealed no appearance of amacrine cells in the Pax6fl/fl/mRx-Cre retina (supplementary material Fig. S4A-D). Taken together, these results indicate that the overall differentiation potential of Pax6-deficient early progenitors is severely compromised.

Pax6-deficient RPCs transiently upregulate Crx expression but do not accomplish photoreceptor differentiation

Previous studies have shown that Pax6 inactivation in RPCs located in the most peripheral region of the OC leads to premature activation of the photoreceptor differentiation program (Oron-Karni et al., 2008). This process is accompanied by upregulation of cone-rod homeobox protein (Crx), which is the earliest expressed photoreceptor determinant (Furukawa et al., 1997; Chen et al., 1997). We therefore analyzed early RPCs in Pax6fl/fl/mRx-Cre for the presence of Crx transcripts. Already at E10.5, following Pax6 protein elimination, Crx mRNA was detected throughout the invaginating Pax6-deficient retina (Fig. 5B), whereas in wild-type controls Crx expression was not detectable (Fig. 5A). In E13.5 control retina, Crx protein was immunohistochemically detected in a few photoreceptor-committed cells of the OC (Fig. 5C). Strikingly, in Pax6-deficient retina of the same stage, Crx protein was produced by virtually all RPCs (Fig. 5D). At the protein level, elevated Crx expression was reproducibly detected between E11.5 and E14.5 in Pax6-deficient RPCs (supplementary material Fig. S4F,H).

Fig. 5.

Expression of photoreceptor-specific factors in Pax6-deficient retina. (A,B) Crx mRNA expression at E10.5 showing elevated levels in Pax6-deficient (Pax6fl/fl/mRx-Cre) retina (B). (C-F) Confocal images showing Crx (C,D) and Otx2 (E,F) protein expression using Crx/Otx2 and Otx2 only antibody at E13.5. (G,H) Trβ2 mRNA expression at E14.5. (I-N) Protein expression of Otx2 (I,J), Blimp1 (K,L) and Rxrγ (M,N) at E15.5. (O,P) Nr2e3 protein expression at E18.5. Retina is indicated with dashed line. le, lens; RPE, retinal pigmented epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 100 μm.

Fig. 5.

Expression of photoreceptor-specific factors in Pax6-deficient retina. (A,B) Crx mRNA expression at E10.5 showing elevated levels in Pax6-deficient (Pax6fl/fl/mRx-Cre) retina (B). (C-F) Confocal images showing Crx (C,D) and Otx2 (E,F) protein expression using Crx/Otx2 and Otx2 only antibody at E13.5. (G,H) Trβ2 mRNA expression at E14.5. (I-N) Protein expression of Otx2 (I,J), Blimp1 (K,L) and Rxrγ (M,N) at E15.5. (O,P) Nr2e3 protein expression at E18.5. Retina is indicated with dashed line. le, lens; RPE, retinal pigmented epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 100 μm.

Crx protein is known to enhance the expression of photoreceptor-specific genes (Hennig et al., 2008; Chen et al., 1997; Mitton et al., 2000; Peng and Chen, 2005); however, Crx alone does not determine the specific photoreceptor cell fate and is supposed to activate transcription in cooperation with other transcription factors (Akagi et al., 2005; Furukawa et al., 1999; Hennig et al., 2008). To further test the ability of Crx to induce photoreceptor differentiation, we analyzed Pax6-deficient RPCs for the expression of Otx2, a key regulator of the photoreceptor lineage (Nishida et al., 2003). Otx2 expression failed to be activated, with the exception of a few cells in the most distal part of the OC (Fig. 5F). To rule out a possible delay of Otx2 expression in Pax6-deficient retinae, the expression of Otx2 and its photoreceptor-specific target Blimp1 (Prdm1) was analyzed at E15.5. Although the expression of both Otx2 and Blimp1 was apparent in the outer layer, where differentiating photoreceptors reside in wild-type retinae (Fig. 5I,K), their expression was not initiated in Pax6fl/fl/mRx-Cre (Fig. 5J,L). We observed elevated levels of the cone-specifying nuclear receptor retinoid X receptor-γ (Rxrγ) (Fig. 5N); however, other factors essential for cone lineage specification, such as thyroid hormone nuclear receptor TRβ2 (Thrb), or for rod specification, such as Nr2e3, were not expressed upon Pax6 inactivation (Fig. 5H,P). In summary, although Crx expression was efficiently induced by all Pax6-deficient RPCs, these cells were not able to fully accomplish the photoreceptor differentiation program, as they failed to express other factors indispensable for this process.

The absence of Pax6 in the OV during OV-SE tissue interaction leads to lens development arrest

Despite the fact that Pax6fl/fl/mRx-Cre animals did not exhibit severe lens defects in general (Fig. 2), we occasionally observed morphological abnormalities interfering with lens pit/OC formation in these mutants (supplementary material Fig. S5A,B′). Since SE and OV continuous interaction is required for lens and OC morphogenesis, our observation led us to hypothesize that, at the OV stage, OV-expressed Pax6 might also play a role in lens formation. To test whether earlier removal of Pax6 protein in the OV enhances the lens phenotype, we introduced a single Sey allele (Pax6Sey/+) into the mRx-Cre/Pax6fl background. Under these conditions, only one allele of Pax6 has to be recombined since the second allele is genetically inactive in Sey. Although there are several lens phenotypes associated with the inactivation of one Pax6 allele, including small lens size or its incomplete separation from the overlying ectoderm, the lens is always formed (Hogan et al., 1986). This genetic combination resulted in downregulation of Pax6 protein levels in the OV neuroepithelium of Pax6Sey/fl/mRx-Cre embryos before its transition to the OC (Fig. 6G). Note that this effect can be partially attributed to slightly delayed OC/lens pit morphogenesis in Pax6Sey/fl embryos (Fig. 6F). At E11.0, Pax6Sey/fl/mRx-Cre embryos reproducibly exhibited defective lens/OC formation (Fig. 6B,B′). In all embryos analyzed, the lens was completely missing, and the OV either remained arrested or occasionally showed invagination into a small OC-like structure (Fig. 6D; supplementary material Fig. S5D,D′). The arrest of eye development can also be observed when Pax6 protein is eliminated specifically in the OV neuroepithelium already at E9.5 using an earlier deleting founder of mRx-Cre (EmRx-Cre) (supplementary material Fig. S5E-H′). To further test whether the lens fate was established in Pax6Sey/fl/mRx-Cre embryos, we analyzed expression of the LP-specific transcription factors Pax6, Six3 and Sox2. Although their expression in the SE was maintained (Fig. 6G-G′), indicating that the LP was initially formed, expression of the lens differentiation genes Foxe3 and Prox1 was not initiated and the LP did not invaginate to form the lens vesicle (Fig. 6J,J′).

Fig. 6.

When Pax6 is eliminated from OV before its transition to the OC then the lens is not formed. (A-D) Morphological consequences of OV-specific Pax6 inactivation. Heads with the eye region indicated (arrowheads) are shown for control (Pax6Sey/fl) (A) and mutant (Pax6Sey/fl/mRx-Cre) (B) embryos at E11. Hematoxylin-Eosin-stained sections of E11.5 (A′,B′) and E14.5 (C,D) eyes showing the absence of lens development upon OV-specific Pax6 elimination. (E-G′) Confocal images showing expression of Pax6, Sox2 and Six3 protein in control (Pax6fl/+ and Pax6Sey/fl) and mutant (Pax6Sey/fl/mRx-Cre) E10.5 eyes. Lens pit/lens placode are indicated with a dashed line. Decrease of the Pax6 protein level in OV neuroepithelium of Pax6Sey/fl/mRx-Cre eyes is indicated by arrowheads (G). (H-J′) Expression of Prox1 and Foxe3 protein at E11.0. The lens or the corresponding region is indicated with a dashed line. Insets (J,J′) are magnifications of the SE area. Scale bars: 100 μm.

Fig. 6.

When Pax6 is eliminated from OV before its transition to the OC then the lens is not formed. (A-D) Morphological consequences of OV-specific Pax6 inactivation. Heads with the eye region indicated (arrowheads) are shown for control (Pax6Sey/fl) (A) and mutant (Pax6Sey/fl/mRx-Cre) (B) embryos at E11. Hematoxylin-Eosin-stained sections of E11.5 (A′,B′) and E14.5 (C,D) eyes showing the absence of lens development upon OV-specific Pax6 elimination. (E-G′) Confocal images showing expression of Pax6, Sox2 and Six3 protein in control (Pax6fl/+ and Pax6Sey/fl) and mutant (Pax6Sey/fl/mRx-Cre) E10.5 eyes. Lens pit/lens placode are indicated with a dashed line. Decrease of the Pax6 protein level in OV neuroepithelium of Pax6Sey/fl/mRx-Cre eyes is indicated by arrowheads (G). (H-J′) Expression of Prox1 and Foxe3 protein at E11.0. The lens or the corresponding region is indicated with a dashed line. Insets (J,J′) are magnifications of the SE area. Scale bars: 100 μm.

Since mRx-Cre-mediated gene manipulation was performed in the OV and not in the SE, a non-cell-autonomous process is likely to regulate lens development in a fashion dependent on OV-expressed Pax6. The BMP and FGF signaling pathways are known to play an important role in the lens-inductive ability of OV. Using antibody staining we examined the intracellular mediators of these pathways: phosphorylated Erk proteins (pErk1/2; also known as pMapk3/1) for FGF signaling and phosphorylated Smad proteins (pSmad1/5) for BMP signaling in Pax6Sey/fl/mRx-Cre LP. In both wild type and the Pax6Sey/fl/mRx-Cre mutant, strong pErk1/2 staining was observed in the LP/lens (Fig. 7A), indicating that FGF signaling was unaffected even when lens formation was disrupted. Similarly, we did not observe any significant difference in pSmad1/5 levels in the LP/lens between wild-type and Pax6Sey/fl/mRx-Cre mutant eyes (Fig. 7B). Furthermore, the expression of BMP ligands essential for eye development, Bmp4 and Bmp7, was not abolished, indicating that BMP signaling was not grossly affected upon OV-specific Pax6 inactivation (Fig. 7C).

Fig. 7.

The Pax6-dependent lens-inductive ability of OV neuroepithelium is not dependent on FGF, BMP or Wnt/β-catenin signaling. (A) pErk1/2 detection in control (Pax6fl/+ and Pax6Sey/fl) and mutant (Pax6Sey/fl/mRx-Cre) E11.0 eyes of littermate embryos. (B) pSmad1/5 detection in E10.5 eyes of littermate embryos. (C) Bmp4 (left) and Bmp7 mRNA expression on whole-mount littermate embryos. Insets are magnifications of the eye region (white arrowheads). The right-hand Bmp4 panel shows coronal sections of the same samples (black arrowheads indicate the area with Bmp4 expression). (D) Activity of Wnt/β-catenin signaling at E10.5 as assessed using a BAT-gal reporter mouse (top) and expression of the Wnt/β-catenin target Lef1 (bottom). Arrowheads in Pax6 mutants indicate the position of the region corresponding to lens placode (A,B,D). Dashed lines (D) indicate retina and RPE (black) and area corresponding to developing lens (white and yellow). Scale bars: 100 μm.

Fig. 7.

The Pax6-dependent lens-inductive ability of OV neuroepithelium is not dependent on FGF, BMP or Wnt/β-catenin signaling. (A) pErk1/2 detection in control (Pax6fl/+ and Pax6Sey/fl) and mutant (Pax6Sey/fl/mRx-Cre) E11.0 eyes of littermate embryos. (B) pSmad1/5 detection in E10.5 eyes of littermate embryos. (C) Bmp4 (left) and Bmp7 mRNA expression on whole-mount littermate embryos. Insets are magnifications of the eye region (white arrowheads). The right-hand Bmp4 panel shows coronal sections of the same samples (black arrowheads indicate the area with Bmp4 expression). (D) Activity of Wnt/β-catenin signaling at E10.5 as assessed using a BAT-gal reporter mouse (top) and expression of the Wnt/β-catenin target Lef1 (bottom). Arrowheads in Pax6 mutants indicate the position of the region corresponding to lens placode (A,B,D). Dashed lines (D) indicate retina and RPE (black) and area corresponding to developing lens (white and yellow). Scale bars: 100 μm.

Unlike BMP and FGF, activation of the Wnt/β-catenin pathway has been shown to inhibit lens fate since stabilization of β-catenin in lens primordium prevents lens formation (Kreslova et al., 2007; Machon et al., 2010; Smith et al., 2005). Activation of the Wnt/β-catenin pathway results in the accumulation of β-catenin in the nucleus, which allows the TCF/Lef family of transcription factors to activate downstream target genes. We therefore assayed the activity of the canonical Wnt/β-catenin pathway using the BAT-gal reporter mouse line carrying lacZ driven by multimerized TCF/Lef binding sites (Maretto et al., 2003). Although we observed decreased mRNA expression of known Wnt/β-catenin inhibitors Sfrp1 and Sfrp2 in Pax6Sey/fl/mRx-Cre E10.5 eyes (supplementary material Fig. S6A-F), expression of another Wnt inhibitor, Dkk1, remained unchanged (supplementary material Fig. S6G-I), and BAT-gal reporter mice did not exhibit overall aberrant Wnt activation in the LP (Fig. 7D, arrowheads). Since the BAT-gal reporter does not always display sufficient sensitivity (Barolo, 2006), we used staining for Lef1 as a target of Wnt signaling (Planutiene et al., 2011; Wu et al., 2012). As with BAT-gal reporter activity, Lef1 was not aberrantly expressed in the LP of Pax6Sey/fl/mRx-Cre mutants (Fig. 7D), indicating no activation of Wnt signaling. By contrast, aberrant Wnt signaling activity was observed in the OV. However, this is unlikely to cause the lens development arrest, since intentional activation of the Wnt/β-catenin pathway in OV neuroepithelium in Catnblox(ex3) mice (Harada et al., 1999) did not interfere with lens formation (supplementary material Fig. S6K). In summary, our findings suggest that, although OV-expressed Pax6 is essential for lens formation, this process is independent of FGF, BMP and Wnt/β-catenin signaling.

In our study, we focused on Pax6 function in retina-committed eye progenitors of the OV and early OC. The current model assumes that prospective retina-expressed Pax6 is dispensable for lens and OC development (Fujiwara et al., 1994). In addition it is known that, at the stage of neurogenesis, Pax6 loss leads to the exclusive generation of amacrine interneurons, indicating Pax6 requirement for progenitor cell multipotency (Marquardt et al., 2001). Here we show that, in prospective retina, Pax6 is required for three crucial processes: lens induction, propagation of early retinal progenitors, and initiation of the retinal differentiation program.

The role of Pax6 in proliferation of early retinal progenitors

Although Pax6 has been found to be involved in neural progenitor proliferation, the response to Pax6 loss seems to be dependent on the developmental context. Pax6-/- mutants display an increased number of early cortical progenitors in S phase (Estivill-Torrus et al., 2002; Götz et al., 1998; Warren et al., 1999), but a reduction in proliferation was observed in the diencephalon and optic rudiment (Philips et al., 2005; Warren and Price, 1997). Conditional inactivation of Pax6 in RPCs of the peripheral OC at E12 results in hypocellularity accompanied by a decreased proportion of cells in S phase (Marquardt et al., 2001; Oron-Karni et al., 2008), indicating a pro-proliferative effect of Pax6 in RPCs. The molecular mechanism of how Pax6 regulates cell proliferation remains elusive. One possibility includes the regulated expression or function of general components of the cell cycle machinery either directly by Pax6 or indirectly by some of its targets (Cvekl et al., 1999; Estivill-Torrus et al., 2002; Farah et al., 2000; Holm et al., 2007; Ochocinska and Hitchcock, 2009). Here, we show that after Pax6 inactivation the cyclin-dependent kinase inhibitor p57Kip2 exhibits aberrant accumulation. This process was accompanied by RPC incompetence to re-enter S phase and by downregulation of cyclin D1. Although cyclin D1 is normally expressed by cycling RPCs, promoting progression through the cell cycle, its expression is rapidly downregulated in emerging postmitotic cells (Barton and Levine, 2008; Das et al., 2009; Dyer and Cepko, 2001). By contrast, the expression of p57Kip2 is upregulated in a small subset of RPCs between E14.5 and E17.5 as they exit the cell cycle (Dyer and Cepko, 2000). Loss-of-function and overexpression studies performed in the mouse retina demonstrated that p57Kip2 is both necessary and sufficient to induce cell cycle exit (Dyer and Cepko, 2000). Thus, the pro-proliferative effect of Pax6 in the retina might be mediated, at least in part, by the inhibition of premature cell cycle exit through regulation of p57Kip2 protein levels. As we also observed upregulation of p57Kip2 mRNA, the negative control appears to occur at the transcriptional level. The mechanism by which p57Kip2 mediates cell cycle exit in Pax6-deficient RPCs might include blocking of phosphorylation of the retinoblastoma protein (reviewed by Sherr and Roberts, 1995).

It is worth noting that, before Pax6-deficient RPCs exit the cell cycle, a p57Kip2/cyclin D1-independent mechanism regulates the proliferation rate. The cell cycle length of the Pax6-deficient RPC population is significantly increased relative to that of the wild-type RPC population at E11.5 and E13. In contrast to Pax6-deficient cortical progenitors manifesting prolonged S phase (Estivill-Torrus et al., 2002), the cumulative time spent in the G1, G2 and M phases was increased in Pax6-deficient RPCs, indicating Pax6 function in these phases of the cell cycle.

The role of Pax6 in differentiation into multiple retinal cell types

Once the OV starts to invaginate to form the OC, the population of RPCs is established. Previous studies have indicated that some retinal progenitor characteristics are maintained in the arrested OV rudiment of germline Pax6-/- embryos (Bäumer et al., 2003; Bernier et al., 2001). The mRx-Cre line allows inactivation of Pax6 precisely at the time when the RPC population is being established and before the differentiation program has been initiated (at E10). Our analysis shows that Pax6 is absolutely essential for the generation of all retinal cell types, since no sign of general neuronal differentiation was observed upon Pax6 inactivation, pointing out a specific Pax6 role in the maintenance of RPC multipotency. This can be explained by the ability of Pax6 to activate the expression of proneurogenic bHLH factors, including Atoh7, Mash1, Math3, Ngn2 and Neurod1 (Hatakeyama and Kageyama, 2004; Marquardt et al., 2001; Oron-Karni et al., 2008; Riesenberg et al., 2009) (this study).

Our observation that Pax6 is indispensable for neuronal differentiation in the retina is seemingly inconsistent with previous studies. Marquardt and colleagues (Marquardt et al., 2001) showed that inactivation of Pax6 at the OC stage using α-Cre leads to the exclusive generation of amacrine interneurons. Further detailed analysis revealed two populations of RPCs that differentially responded to Pax6 loss: whereas progenitors located more centrally in the OC adopted amacrine cell fate, those located peripherally activated expression of Crx (Oron-Karni et al., 2008). Nevertheless, our data show that Pax6 is also indispensable for amacrine cell genesis, as Neurod1, Math3, Atoh7 and other amacrine cell-specific factors are not expressed in the absence of Pax6. This difference can be attributed to the timing of Pax6 inactivation. When using mRx-Cre, Pax6 is completely eliminated before the differentiation program is initiated (E10) (this study); for α-Cre (Marquardt et al., 2001; Oron-Karni et al., 2008), Pax6 is eliminated 2 days later (E12) (Riesenberg et al., 2009) (our observation). At E12 the differentiation program has already been initiated, as some proneurogenic factors, including Neurod1 and Atoh7, are expressed (reviewed by Hatakeyama and Kageyama, 2004). The amacrine cell genesis is likely to be the result of biphasic inactivation of Pax6 by α-Cre with respect to the onset of neurogenesis. Since progenitors located in the central OC differentiate earlier, the presence of two populations of RPCs in the OC of α-Cre/Pax6fl/fl conditional mutants, with the amacrine cell population located more centrally, then apparently reflects the different degree of neuronal differentiation along the central-to-peripheral axis (Oron-Karni et al., 2008).

The role of Pax6 in the lens-inductive ability of the OV

In Pax6-/- embryos eye development is arrested at the OV stage and neither lens nor OC is formed (Grindley et al., 1995; Hill et al., 1991; Hogan et al., 1986). Since Pax6 is expressed in both SE and OV (Walther and Gruss, 1991), it was not clear which component is the source of the defect. Several studies indicated that SE-expressed Pax6 might be responsible (Collinson et al., 2000; Fujiwara et al., 1994; Grindley et al., 1995; Quinn et al., 1996), leading to a general acceptance of the notion that Pax6 activity in the OV is, by and large, not required for lens formation (reviewed by Ashery-Padan and Gruss, 2001; Lang, 2004; Mathers and Jamrich, 2000; Ogino and Yasuda, 2000). However, such a conclusion has not been tested genetically. Experiments in which anti-Pax6 morpholinos were electroporated into chick embryo OV indicated that OV-expressed Pax6 might play an essential role in retina and lens formation as well (Canto-Soler and Adler, 2006). Conditional inactivation of Pax6 in the SE revealed that SE-expressed Pax6 is autonomously required for LP/lens but not retina formation (Ashery-Padan et al., 2000). In this study we present evidence that early expression of Pax6 in the OV is indispensable for the development of both tissue components: cell-autonomously for OC/retina development and non-cell-autonomously for lens formation. In OV Pax6 mutants, eye development was arrested at the OV stage in a manner morphologically reminiscent of the Pax6-/- (Sey) phenotype. Thus, in Pax6-/- embryos, the defect in eye formation is apparently attributable to Pax6 function in both OV and SE (this study) (Canto-Soler and Adler, 2006), in sharp contrast to the current, prevailing view (Ashery-Padan and Gruss, 2001; Ogino and Yasuda, 2000). Interestingly, Pax6 is required for lens formation only before the OV-to-OC transition. Once the lens pit starts to emerge from the LP, lens development is no longer dependent on OC-expressed Pax6. This accords with the idea that lens development becomes independent of OV/OC when the lens has reached a certain developmental stage (Adler and Canto-Soler, 2007; Lang, 2004).

How Pax6 regulates the ability of OV to induce lens formation remains elusive. It has been demonstrated that lens formation is dependent on the deposition of molecules of the extracellular matrix between the LP and OV, and that this process is dependent on Pax6 expression (Huang et al., 2011). There is good evidence that signaling from the OV is essential for the activation of lens-specific expression in the SE and subsequent lens formation (Faber et al., 2001; Furuta and Hogan, 1998; Kamachi et al., 1998; Wawersik et al., 1999; Yun et al., 2009). We have not been able to detect conspicuous changes in components of the BMP, FGF and Wnt/β-catenin pathways. It remains possible that the causal changes in Pax6Sey/fl/mRx-Cre are transient or too subtle to be detected in our analysis. Alternatively, additional, as yet ill-defined signals might be involved in lens induction. The identification of further molecules acting downstream of Pax6 in the OV might help uncover the molecular details of this process.

Mouse lines

A mouse with floxed Pax6 alleles (Pax6fl/fl) was generated by homologous recombination in embryonic stem cells, with loxP sites flanking exons 3 and 6 (supplementary material Fig. S1A). ROSA26R (Soriano, 1999), ROSA26R-EYFP [R-EYFP (Soriano, 1999; Srinivas et al., 2001)], Sey1Neu (Hill et al., 1991), BAT-gal reporter line (Maretto et al., 2003), Catnblox(ex3) (Harada et al., 1999) and mRx-Cre (Klimova et al., 2013) (supplementary material Fig. S1B) mice have been described previously.

Tissue collections and histology

Mouse embryos were harvested from timed pregnant females. The morning of vaginal plug was considered embryonic day (E) 0.5. Embryos were fixed in 4% (w/v) paraformaldehyde (PFA), washed with PBS, cryopreserved in 30% (w/w) sucrose, frozen in OCT (Tissue Tek, Sekura Finetek) and sectioned.

Immunohistochemistry, in situ hybridization and β-galactosidase assay

Cryosections for immunohistochemistry were permeabilized with PBT (PBS with 0.1% Tween 20), blocked with 10% BSA in PBT and incubated with primary antibody overnight at 4°C. Primary antibodies are listed in supplementary material Table S1. Sections were washed with PBT, incubated with secondary antibody (Molecular Probes) coupled to Alexa fluorophore, counterstained with DAPI and mounted in Mowiol (Sigma). To detect phosphorylated Erk (pErk), the TSA Indirect Tyramide Signal Amplification Kit (Perkin Elmer) was used. To detect phosphorylated Smad1/5 (pSmad1/5), the Vectastain ABC Kit (Vector Labs) was used. Antisense mRNA probes for in situ hybridization were synthesized using RNA polymerase and digoxigenin-labeled nucleotides (Roche) following the manufacturer’s instructions (for a list of probes see supplementary material Table S1). RNA in situ hybridization and X-gal staining were carried out as previously described (Fujimura et al., 2009).

BrdU incorporation assay and measurement of cell cycle phase length

For analysis of all proliferation markers, a minimum of three animals from two individual litters were used. Wild-type littermates were used as control. To determine the proportion of actively proliferating RPCs, timed pregnant females were injected 1 hour prior to dissection with BrdU (0.1 mg/g body weight). Embryos were fixed with 4% PFA, cryopreserved in 30% sucrose, embedded in OCT and sectioned. Antigen retrieval was performed by microwave heating in 10 mM sodium citrate (pH 6.5) followed by incubation in 2 M HCl and neutralization (0.1 M borate buffer pH 8.3). Sections were blocked in 10% BSA and incubated overnight at 4°C with anti-BrdU antibody (Abcam, ab6326; 1:100). The cell proliferation rate was always calculated from at least two central sections per individual eye as the ratio of BrdU+ cells versus DAPI+ cells and statistical significance analyzed by Student’s t-test.

To determine the cell cycle rate at E11.5 and E13, two thymidine analogs were used as previously described (Das et al., 2009). Pregnant females were injected 2.5 hours and 0.5 hours prior to dissection with BrdU and EdU (5-ethynyl-2′-deoxyuridine), respectively. BrdU was detected using anti-BrdU antibody, EdU using the Click-it Reaction (Molecular Probes), and Pcna staining was used to identify all cycling RPCs. The length of the cell cycle (Tc) and of S phase (Ts) in hours (h) was determined by: Tc=2h(Pcna+ cells/BrdU+ only cells); Ts=2h(EdU+ cells/BrdU+ only cells). The combined length of G1, G2 and M phase was calculated as: Tc-Ts. Cell counts were performed from a single field of central sections of the eye (as depicted in Fig. 3J). Three fields per individual eye were counted. Tc and Ts for individual fields were determined and analyzed by Student’s t-test.

We thank J. Lachova, A. Zitova and V. Noskova for technical assistance; Drs N. Brown, P. Bovolenta, B. Hogan, S. Chen, C. Craft, P. Carlsson, J. Rubenstein, S. Pleasure, M. Taketo, J. Favor and H. Edlund for reagents and mice; and O. Machon for reading the manuscript.

Author contributions

L.K. and Z.K. designed experiments, analyzed data and wrote the manuscript. L.K. performed experiments.

Funding

This study was supported by the Grant Agency of the Czech Republic [P305/11/2198]; by the Ministry of Education, Youth and Sports of the Czech Republic [OP RDI CZ.1.05/1.1.00/02.0109, Project BIOCEV]; and by the Academy of Sciences of the Czech Republic [RVO68378050 and IAA500520908].

Adler
R.
,
Canto-Soler
M. V.
(
2007
).
Molecular mechanisms of optic vesicle development: complexities, ambiguities and controversies
.
Dev. Biol.
305
,
1
13
.
Agathocleous
M.
,
Harris
W. A.
(
2009
).
From progenitors to differentiated cells in the vertebrate retina
.
Annu. Rev. Cell Dev. Biol.
25
,
45
69
.
Akagi
T.
,
Akita
J.
,
Haruta
M.
,
Suzuki
T.
,
Honda
Y.
,
Inoue
T.
,
Yoshiura
S.
,
Kageyama
R.
,
Yatsu
T.
,
Yamada
M.
, et al. 
. (
2005
).
Iris-derived cells from adult rodents and primates adopt photoreceptor-specific phenotypes
.
Invest. Ophthalmol. Vis. Sci.
46
,
3411
3419
.
Ashery-Padan
R.
,
Gruss
P.
(
2001
).
Pax6 lights-up the way for eye development
.
Curr. Opin. Cell Biol.
13
,
706
714
.
Ashery-Padan
R.
,
Marquardt
T.
,
Zhou
X.
,
Gruss
P.
(
2000
).
Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye
.
Genes Dev.
14
,
2701
2711
.
Barolo
S.
(
2006
).
Transgenic Wnt/TCF pathway reporters: all you need is Lef?
Oncogene
25
,
7505
7511
.
Barton
K. M.
,
Levine
E. M.
(
2008
).
Expression patterns and cell cycle profiles of PCNA, MCM6, cyclin D1, cyclin A2, cyclin B1, and phosphorylated histone H3 in the developing mouse retina
.
Dev. Dyn.
237
,
672
682
.
Bäumer
N.
,
Marquardt
T.
,
Stoykova
A.
,
Spieler
D.
,
Treichel
D.
,
Ashery-Padan
R.
,
Gruss
P.
(
2003
).
Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6
.
Development
130
,
2903
2915
.
Bernier
G.
,
Vukovich
W.
,
Neidhardt
L.
,
Herrmann
B. G.
,
Gruss
P.
(
2001
).
Isolation and characterization of a downstream target of Pax6 in the mammalian retinal primordium
.
Development
128
,
3987
3994
.
Blixt
A.
,
Landgren
H.
,
Johansson
B. R.
,
Carlsson
P.
(
2007
).
Foxe3 is required for morphogenesis and differentiation of the anterior segment of the eye and is sensitive to Pax6 gene dosage
.
Dev. Biol.
302
,
218
229
.
Brown
N. L.
,
Kanekar
S.
,
Vetter
M. L.
,
Tucker
P. K.
,
Gemza
D. L.
,
Glaser
T.
(
1998
).
Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis
.
Development
125
,
4821
4833
.
Burmeister
M.
,
Novak
J.
,
Liang
M. Y.
,
Basu
S.
,
Ploder
L.
,
Hawes
N. L.
,
Vidgen
D.
,
Hoover
F.
,
Goldman
D.
,
Kalnins
V. I.
, et al. 
. (
1996
).
Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation
.
Nat. Genet.
12
,
376
384
.
Burns
K. A.
,
Kuan
C. Y.
(
2005
).
Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus
.
Eur. J. Neurosci.
21
,
803
807
.
Canto-Soler
M. V.
,
Adler
R.
(
2006
).
Optic cup and lens development requires Pax6 expression in the early optic vesicle during a narrow time window
.
Dev. Biol.
294
,
119
132
.
Cepko
C. L.
(
1999
).
The roles of intrinsic and extrinsic cues and bHLH genes in the determination of retinal cell fates
.
Curr. Opin. Neurobiol.
9
,
37
46
.
Chen
S.
,
Wang
Q. L.
,
Nie
Z.
,
Sun
H.
,
Lennon
G.
,
Copeland
N. G.
,
Gilbert
D. J.
,
Jenkins
N. A.
,
Zack
D. J.
(
1997
).
Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes
.
Neuron
19
,
1017
1030
.
Chen
J.
,
Rattner
A.
,
Nathans
J.
(
2005
).
The rod photoreceptor-specific nuclear receptor Nr2e3 represses transcription of multiple cone-specific genes
.
J. Neurosci.
25
,
118
129
.
Chow
R. L.
,
Lang
R. A.
(
2001
).
Early eye development in vertebrates
.
Annu. Rev. Cell Dev. Biol.
17
,
255
296
.
Collinson
J. M.
,
Hill
R. E.
,
West
J. D.
(
2000
).
Different roles for Pax6 in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye
.
Development
127
,
945
956
.
Cvekl
A.
,
Kashanchi
F.
,
Brady
J. N.
,
Piatigorsky
J.
(
1999
).
Pax-6 interactions with TATA-box-binding protein and retinoblastoma protein
.
Invest. Ophthalmol. Vis. Sci.
40
,
1343
1350
.
Das
G.
,
Choi
Y.
,
Sicinski
P.
,
Levine
E. M.
(
2009
).
Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells
.
Neural Dev.
4
,
15
.
Diep
D. B.
,
Hoen
N.
,
Backman
M.
,
Machon
O.
,
Krauss
S.
(
2004
).
Characterisation of the Wnt antagonists and their response to conditionally activated Wnt signalling in the developing mouse forebrain
.
Brain Res. Dev. Brain Res.
153
,
261
270
.
Dyer
M. A.
,
Cepko
C. L.
(
2000
).
p57(Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina
.
Development
127
,
3593
3605
.
Dyer
M. A.
,
Cepko
C. L.
(
2001
).
p27Kip1 and p57Kip2 regulate proliferation in distinct retinal progenitor cell populations
.
J. Neurosci.
21
,
4259
4271
.
Estivill-Torrus
G.
,
Pearson
H.
,
van Heyningen
V.
,
Price
D. J.
,
Rashbass
P.
(
2002
).
Pax6 is required to regulate the cell cycle and the rate of progression from symmetrical to asymmetrical division in mammalian cortical progenitors
.
Development
129
,
455
466
.
Faber
S. C.
,
Dimanlig
P.
,
Makarenkova
H. P.
,
Shirke
S.
,
Ko
K.
,
Lang
R. A.
(
2001
).
Fgf receptor signaling plays a role in lens induction
.
Development
128
,
4425
4438
.
Farah
M. H.
,
Olson
J. M.
,
Sucic
H. B.
,
Hume
R. I.
,
Tapscott
S. J.
,
Turner
D. L.
(
2000
).
Generation of neurons by transient expression of neural bHLH proteins in mammalian cells
.
Development
127
,
693
702
.
Fuhrmann
S.
(
2010
).
Eye morphogenesis and patterning of the optic vesicle
.
Curr. Top. Dev. Biol.
93
,
61
84
.
Fujimura
N.
,
Taketo
M. M.
,
Mori
M.
,
Korinek
V.
,
Kozmik
Z.
(
2009
).
Spatial and temporal regulation of Wnt/beta-catenin signaling is essential for development of the retinal pigment epithelium
.
Dev. Biol.
334
,
31
45
.
Fujiwara
M.
,
Uchida
T.
,
Osumi-Yamashita
N.
,
Eto
K.
(
1994
).
Uchida rat (rSey): a new mutant rat with craniofacial abnormalities resembling those of the mouse Sey mutant
.
Differentiation
57
,
31
38
.
Furukawa
T.
,
Morrow
E. M.
,
Cepko
C. L.
(
1997
).
Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation
.
Cell
91
,
531
541
.
Furukawa
T.
,
Morrow
E. M.
,
Li
T.
,
Davis
F. C.
,
Cepko
C. L.
(
1999
).
Retinopathy and attenuated circadian entrainment in Crx-deficient mice
.
Nat. Genet.
23
,
466
470
.
Furuta
Y.
,
Hogan
B. L.
(
1998
).
BMP4 is essential for lens induction in the mouse embryo
.
Genes Dev.
12
,
3764
3775
.
Garcia
C. M.
,
Huang
J.
,
Madakashira
B. P.
,
Liu
Y.
,
Rajagopal
R.
,
Dattilo
L.
,
Robinson
M. L.
,
Beebe
D. C.
(
2011
).
The function of FGF signaling in the lens placode
.
Dev. Biol.
351
,
176
185
.
Geng
Y.
,
Yu
Q.
,
Sicinska
E.
,
Das
M.
,
Bronson
R. T.
,
Sicinski
P.
(
2001
).
Deletion of the p27Kip1 gene restores normal development in cyclin D1-deficient mice
.
Proc. Natl. Acad. Sci. USA
98
,
194
199
.
Gotoh
N.
,
Ito
M.
,
Yamamoto
S.
,
Yoshino
I.
,
Song
N.
,
Wang
Y.
,
Lax
I.
,
Schlessinger
J.
,
Shibuya
M.
,
Lang
R. A.
(
2004
).
Tyrosine phosphorylation sites on FRS2alpha responsible for Shp2 recruitment are critical for induction of lens and retina
.
Proc. Natl. Acad. Sci. USA
101
,
17144
17149
.
Götz
M.
,
Stoykova
A.
,
Gruss
P.
(
1998
).
Pax6 controls radial glia differentiation in the cerebral cortex
.
Neuron
21
,
1031
1044
.
Grindley
J. C.
,
Davidson
D. R.
,
Hill
R. E.
(
1995
).
The role of Pax-6 in eye and nasal development
.
Development
121
,
1433
1442
.
Harada
N.
,
Tamai
Y.
,
Ishikawa
T.
,
Sauer
B.
,
Takaku
K.
,
Oshima
M.
,
Taketo
M. M.
(
1999
).
Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene
.
EMBO J.
18
,
5931
5942
.
Hatakeyama
J.
,
Kageyama
R.
(
2004
).
Retinal cell fate determination and bHLH factors
.
Semin. Cell Dev. Biol.
15
,
83
89
.
Hennig
A. K.
,
Peng
G. H.
,
Chen
S.
(
2008
).
Regulation of photoreceptor gene expression by Crx-associated transcription factor network
.
Brain Res.
1192
,
114
133
.
Hill
R. E.
,
Favor
J.
,
Hogan
B. L.
,
Ton
C. C.
,
Saunders
G. F.
,
Hanson
I. M.
,
Prosser
J.
,
Jordan
T.
,
Hastie
N. D.
,
van Heyningen
V.
(
1991
).
Mouse small eye results from mutations in a paired-like homeobox-containing gene
.
Nature
354
,
522
525
.
Hogan
B. L.
,
Horsburgh
G.
,
Cohen
J.
,
Hetherington
C. M.
,
Fisher
G.
,
Lyon
M. F.
(
1986
).
Small eyes (Sey): a homozygous lethal mutation on chromosome 2 which affects the differentiation of both lens and nasal placodes in the mouse
.
J. Embryol. Exp. Morphol.
97
,
95
110
.
Holm
P. C.
,
Mader
M. T.
,
Haubst
N.
,
Wizenmann
A.
,
Sigvardsson
M.
,
Götz
M.
(
2007
).
Loss- and gain-of-function analyses reveal targets of Pax6 in the developing mouse telencephalon
.
Mol. Cell. Neurosci.
34
,
99
119
.
Holt
C. E.
,
Bertsch
T. W.
,
Ellis
H. M.
,
Harris
W. A.
(
1988
).
Cellular determination in the Xenopus retina is independent of lineage and birth date
.
Neuron
1
,
15
26
.
Huang
J.
,
Rajagopal
R.
,
Liu
Y.
,
Dattilo
L. K.
,
Shaham
O.
,
Ashery-Padan
R.
,
Beebe
D. C.
(
2011
).
The mechanism of lens placode formation: a case of matrix-mediated morphogenesis
.
Dev. Biol.
355
,
32
42
.
Inoue
T.
,
Hojo
M.
,
Bessho
Y.
,
Tano
Y.
,
Lee
J. E.
,
Kageyama
R.
(
2002
).
Math3 and NeuroD regulate amacrine cell fate specification in the retina
.
Development
129
,
831
842
.
Jean
D.
,
Bernier
G.
,
Gruss
P.
(
1999
).
Six6 (Optx2) is a novel murine Six3-related homeobox gene that demarcates the presumptive pituitary/hypothalamic axis and the ventral optic stalk
.
Mech. Dev.
84
,
31
40
.
Jones
C. M.
,
Lyons
K. M.
,
Hogan
B. L.
(
1991
).
Involvement of bone morphogenetic protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse
.
Development
111
,
531
542
.
Kamachi
Y.
,
Uchikawa
M.
,
Collignon
J.
,
Lovell-Badge
R.
,
Kondoh
H.
(
1998
).
Involvement of Sox1, 2 and 3 in the early and subsequent molecular events of lens induction
.
Development
125
,
2521
2532
.
Klimova
L.
,
Lachova
J.
,
Machon
O.
,
Sedlacek
R.
,
Kozmik
Z.
(
2013
).
Generation of mRx-Cre transgenic mouse line for efficient conditional gene deletion in early retinal progenitors
.
PLoS ONE
8
,
e63029
.
Kozmik
Z.
(
2005
).
Pax genes in eye development and evolution
.
Curr. Opin. Genet. Dev.
15
,
430
438
.
Kreslova
J.
,
Machon
O.
,
Ruzickova
J.
,
Lachova
J.
,
Wawrousek
E. F.
,
Kemler
R.
,
Krauss
S.
,
Piatigorsky
J.
,
Kozmik
Z.
(
2007
).
Abnormal lens morphogenesis and ectopic lens formation in the absence of beta-catenin function
.
Genesis
45
,
157
168
.
Lang
R. A.
(
2004
).
Pathways regulating lens induction in the mouse
.
Int. J. Dev. Biol.
48
,
783
791
.
Lee
H. Y.
,
Wroblewski
E.
,
Philips
G. T.
,
Stair
C. N.
,
Conley
K.
,
Reedy
M.
,
Mastick
G. S.
,
Brown
N. L.
(
2005
).
Multiple requirements for Hes 1 during early eye formation
.
Dev. Biol.
284
,
464
478
.
Levine
E. M.
,
Green
E. S.
(
2004
).
Cell-intrinsic regulators of proliferation in vertebrate retinal progenitors
.
Semin. Cell Dev. Biol.
15
,
63
74
.
Levine
E. M.
,
Close
J.
,
Fero
M.
,
Ostrovsky
A.
,
Reh
T. A.
(
2000
).
p27(Kip1) regulates cell cycle withdrawal of late multipotent progenitor cells in the mammalian retina
.
Dev. Biol.
219
,
299
314
.
Li
X.
,
Perissi
V.
,
Liu
F.
,
Rose
D. W.
,
Rosenfeld
M. G.
(
2002
).
Tissue-specific regulation of retinal and pituitary precursor cell proliferation
.
Science
297
,
1180
1183
.
Lillien
L.
(
1995
).
Changes in retinal cell fate induced by overexpression of EGF receptor
.
Nature
377
,
158
161
.
Liu
W.
,
Lagutin
O.
,
Swindell
E.
,
Jamrich
M.
,
Oliver
G.
(
2010
).
Neuroretina specification in mouse embryos requires Six3-mediated suppression of Wnt8b in the anterior neural plate
.
J. Clin. Invest.
120
,
3568
3577
.
Lyons
K. M.
,
Hogan
B. L.
,
Robertson
E. J.
(
1995
).
Colocalization of BMP 7 and BMP 2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development
.
Mech. Dev.
50
,
71
83
.
Machon
O.
,
Kreslova
J.
,
Ruzickova
J.
,
Vacik
T.
,
Klimova
L.
,
Fujimura
N.
,
Lachova
J.
,
Kozmik
Z.
(
2010
).
Lens morphogenesis is dependent on Pax6-mediated inhibition of the canonical Wnt/beta-catenin signaling in the lens surface ectoderm
.
Genesis
48
,
86
95
.
Maretto
S.
,
Cordenonsi
M.
,
Dupont
S.
,
Braghetta
P.
,
Broccoli
V.
,
Hassan
A. B.
,
Volpin
D.
,
Bressan
G. M.
,
Piccolo
S.
(
2003
).
Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors
.
Proc. Natl. Acad. Sci. USA
100
,
3299
3304
.
Marquardt
T.
,
Ashery-Padan
R.
,
Andrejewski
N.
,
Scardigli
R.
,
Guillemot
F.
,
Gruss
P.
(
2001
).
Pax6 is required for the multipotent state of retinal progenitor cells
.
Cell
105
,
43
55
.
Mathers
P. H.
,
Jamrich
M.
(
2000
).
Regulation of eye formation by the Rx and pax6 homeobox genes
.
Cell. Mol. Life Sci.
57
,
186
194
.
Mathers
P. H.
,
Grinberg
A.
,
Mahon
K. A.
,
Jamrich
M.
(
1997
).
The Rx homeobox gene is essential for vertebrate eye development
.
Nature
387
,
603
607
.
Mitton
K. P.
,
Swain
P. K.
,
Chen
S.
,
Xu
S.
,
Zack
D. J.
,
Swaroop
A.
(
2000
).
The leucine zipper of NRL interacts with the CRX homeodomain. A possible mechanism of transcriptional synergy in rhodopsin regulation
.
J. Biol. Chem.
275
,
29794
29799
.
Morrow
E. M.
,
Furukawa
T.
,
Lee
J. E.
,
Cepko
C. L.
(
1999
).
NeuroD regulates multiple functions in the developing neural retina in rodent
.
Development
126
,
23
36
.
Nishida
A.
,
Furukawa
A.
,
Koike
C.
,
Tano
Y.
,
Aizawa
S.
,
Matsuo
I.
,
Furukawa
T.
(
2003
).
Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development
.
Nat. Neurosci.
6
,
1255
1263
.
Ochocinska
M. J.
,
Hitchcock
P. F.
(
2009
).
NeuroD regulates proliferation of photoreceptor progenitors in the retina of the zebrafish
.
Mech. Dev.
126
,
128
141
.
Ogino
H.
,
Yasuda
K.
(
2000
).
Sequential activation of transcription factors in lens induction
.
Dev. Growth Differ.
42
,
437
448
.
Ogino
H.
,
Ochi
H.
,
Reza
H. M.
,
Yasuda
K.
(
2012
).
Transcription factors involved in lens development from the preplacodal ectoderm
.
Dev. Biol.
363
,
333
347
.
Oliver
G.
,
Mailhos
A.
,
Wehr
R.
,
Copeland
N. G.
,
Jenkins
N. A.
,
Gruss
P.
(
1995
).
Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development
.
Development
121
,
4045
4055
.
Oron-Karni
V.
,
Farhy
C.
,
Elgart
M.
,
Marquardt
T.
,
Remizova
L.
,
Yaron
O.
,
Xie
Q.
,
Cvekl
A.
,
Ashery-Padan
R.
(
2008
).
Dual requirement for Pax6 in retinal progenitor cells
.
Development
135
,
4037
4047
.
Pan
Y.
,
Woodbury
A.
,
Esko
J. D.
,
Grobe
K.
,
Zhang
X.
(
2006
).
Heparan sulfate biosynthetic gene Ndst1 is required for FGF signaling in early lens development
.
Development
133
,
4933
4944
.
Peng
G. H.
,
Chen
S.
(
2005
).
Chromatin immunoprecipitation identifies photoreceptor transcription factor targets in mouse models of retinal degeneration: new findings and challenges
.
Vis. Neurosci.
22
,
575
586
.
Perron
M.
,
Harris
W. A.
(
2000
).
Determination of vertebrate retinal progenitor cell fate by the Notch pathway and basic helix-loop-helix transcription factors
.
Cell. Mol. Life Sci.
57
,
215
223
.
Philips
G. T.
,
Stair
C. N.
,
Young Lee
H.
,
Wroblewski
E.
,
Berberoglu
M. A.
,
Brown
N. L.
,
Mastick
G. S.
(
2005
).
Precocious retinal neurons: Pax6 controls timing of differentiation and determination of cell type
.
Dev. Biol.
279
,
308
321
.
Planutiene
M.
,
Planutis
K.
,
Holcombe
R. F.
(
2011
).
Lymphoid enhancer-binding factor 1, a representative of vertebrate-specific Lef1/Tcf1 sub-family, is a Wnt-beta-catenin pathway target gene in human endothelial cells which regulates matrix metalloproteinase-2 expression and promotes endothelial cell invasion
.
Vasc. Cell
3
,
28
.
Porter
F. D.
,
Drago
J.
,
Xu
Y.
,
Cheema
S. S.
,
Wassif
C.
,
Huang
S. P.
,
Lee
E.
,
Grinberg
A.
,
Massalas
J. S.
,
Bodine
D.
, et al. 
. (
1997
).
Lhx2, a LIM homeobox gene, is required for eye, forebrain, and definitive erythrocyte development
.
Development
124
,
2935
2944
.
Quinn
J. C.
,
West
J. D.
,
Hill
R. E.
(
1996
).
Multiple functions for Pax6 in mouse eye and nasal development
.
Genes Dev.
10
,
435
446
.
Rajagopal
R.
,
Huang
J.
,
Dattilo
L. K.
,
Kaartinen
V.
,
Mishina
Y.
,
Deng
C. X.
,
Umans
L.
,
Zwijsen
A.
,
Roberts
A. B.
,
Beebe
D. C.
(
2009
).
The type I BMP receptors, Bmpr1a and Acvr1, activate multiple signaling pathways to regulate lens formation
.
Dev. Biol.
335
,
305
316
.
Rattner
A.
,
Hsieh
J. C.
,
Smallwood
P. M.
,
Gilbert
D. J.
,
Copeland
N. G.
,
Jenkins
N. A.
,
Nathans
J.
(
1997
).
A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors
.
Proc. Natl. Acad. Sci. USA
94
,
2859
2863
.
Riesenberg
A. N.
,
Le
T. T.
,
Willardsen
M. I.
,
Blackburn
D. C.
,
Vetter
M. L.
,
Brown
N. L.
(
2009
).
Pax6 regulation of Math5 during mouse retinal neurogenesis
.
Genesis
47
,
175
187
.
Shaham
O.
,
Smith
A. N.
,
Robinson
M. L.
,
Taketo
M. M.
,
Lang
R. A.
,
Ashery-Padan
R.
(
2009
).
Pax6 is essential for lens fiber cell differentiation
.
Development
136
,
2567
2578
.
Shaham
O.
,
Menuchin
Y.
,
Farhy
C.
,
Ashery-Padan
R.
(
2012
).
Pax6: a multi-level regulator of ocular development
.
Prog. Retin. Eye Res.
31
,
351
376
.
Sharma
R. K.
,
Netland
P. A.
(
2007
).
Early born lineage of retinal neurons express class III beta-tubulin isotype
.
Brain Res.
1176
,
11
17
.
Sherr
C. J.
,
Roberts
J. M.
(
1995
).
Inhibitors of mammalian G1 cyclin-dependent kinases
.
Genes Dev.
9
,
1149
1163
.
Sigulinsky
C. L.
,
Green
E. S.
,
Clark
A. M.
,
Levine
E. M.
(
2008
).
Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina
.
Dev. Biol.
317
,
560
575
.
Sjödal
M.
,
Edlund
T.
,
Gunhaga
L.
(
2007
).
Time of exposure to BMP signals plays a key role in the specification of the olfactory and lens placodes ex vivo
.
Dev. Cell
13
,
141
149
.
Smith
A. N.
,
Miller
L. A.
,
Song
N.
,
Taketo
M. M.
,
Lang
R. A.
(
2005
).
The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm
.
Dev. Biol.
285
,
477
489
.
Sommer
L.
,
Ma
Q.
,
Anderson
D. J.
(
1996
).
neurogenins, a novel family of atonal-related bHLH transcription factors, are putative mammalian neuronal determination genes that reveal progenitor cell heterogeneity in the developing CNS and PNS
.
Mol. Cell. Neurosci.
8
,
221
241
.
Soriano
P.
(
1999
).
Generalized lacZ expression with the ROSA26 Cre reporter strain
.
Nat. Genet.
21
,
70
71
.
Srinivas
S.
,
Watanabe
T.
,
Lin
C. S.
,
William
C. M.
,
Tanabe
Y.
,
Jessell
T. M.
,
Costantini
F.
(
2001
).
Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus
.
BMC Dev. Biol.
1
,
4
.
Tomita
K.
,
Ishibashi
M.
,
Nakahara
K.
,
Ang
S. L.
,
Nakanishi
S.
,
Guillemot
F.
,
Kageyama
R.
(
1996
).
Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis
.
Neuron
16
,
723
734
.
Turner
D. L.
,
Cepko
C. L.
(
1987
).
A common progenitor for neurons and glia persists in rat retina late in development
.
Nature
328
,
131
136
.
Turner
D. L.
,
Snyder
E. Y.
,
Cepko
C. L.
(
1990
).
Lineage-independent determination of cell type in the embryonic mouse retina
.
Neuron
4
,
833
845
.
Walther
C.
,
Gruss
P.
(
1991
).
Pax-6, a murine paired box gene, is expressed in the developing CNS
.
Development
113
,
1435
1449
.
Warren
N.
,
Price
D. J.
(
1997
).
Roles of Pax-6 in murine diencephalic development
.
Development
124
,
1573
1582
.
Warren
N.
,
Caric
D.
,
Pratt
T.
,
Clausen
J. A.
,
Asavaritikrai
P.
,
Mason
J. O.
,
Hill
R. E.
,
Price
D. J.
(
1999
).
The transcription factor, Pax6, is required for cell proliferation and differentiation in the developing cerebral cortex
.
Cereb. Cortex
9
,
627
635
.
Wawersik
S.
,
Purcell
P.
,
Rauchman
M.
,
Dudley
A. T.
,
Robertson
E. J.
,
Maas
R.
(
1999
).
BMP7 acts in murine lens placode development
.
Dev. Biol.
207
,
176
188
.
Wu
C. I.
,
Hoffman
J. A.
,
Shy
B. R.
,
Ford
E. M.
,
Fuchs
E.
,
Nguyen
H.
,
Merrill
B. J.
(
2012
).
Function of Wnt/β-catenin in counteracting Tcf3 repression through the Tcf3-β-catenin interaction
.
Development
139
,
2118
2129
.
Xie
Q.
,
Cvekl
A.
(
2011
).
The orchestration of mammalian tissue morphogenesis through a series of coherent feed-forward loops
.
J. Biol. Chem.
286
,
43259
43271
.
Young
R. W.
(
1985
).
Cell differentiation in the retina of the mouse
.
Anat. Rec.
212
,
199
205
.
Yun
S.
,
Saijoh
Y.
,
Hirokawa
K. E.
,
Kopinke
D.
,
Murtaugh
L. C.
,
Monuki
E. S.
,
Levine
E. M.
(
2009
).
Lhx2 links the intrinsic and extrinsic factors that control optic cup formation
.
Development
136
,
3895
3906
.
Zhu
X.
,
Craft
C. M.
(
2000
).
Modulation of CRX transactivation activity by phosducin isoforms
.
Mol. Cell. Biol.
20
,
5216
5226
.

Competing interests

The authors declare no competing financial interests.

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