335 (2009) 305–316

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Developmental Biology

journal homepage: www.elsevier.com/developmentalbiology

The type I BMP receptors, Bmpr1a and Acvr1, activate multiple signaling pathways to regulate formation

Ramya Rajagopal a, Jie Huang a, Lisa K. Dattilo a, Vesa Kaartinen b, Yuji Mishina c, Chu-Xia Deng d, Lieve Umans e,f, An Zwijsen e,f, Anita B. Roberts g, David C. Beebe a,h,⁎ a Departments of Ophthalmology and Visual Sciences, Washington University, St. Louis, MO, USA b Developmental Biology Program, Childrens Hospital Los Angeles, Departments of Pathology and Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA c Molecular Developmental Biology Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA d Genetics of Development and Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA e Laboratory of Molecular Biology (Celgen), Department for Molecular and Developmental Genetics, VIB, Leuven, Belgium f Laboratory of Molecular Biology (Celgen), Center for Human Genetics, K.U. Leuven, Leuven, Belgium g Laboratory of Cell Regulation and Carcinogenesis, NCI, NIH, Bethesda, MD, USA h Cell Biology and Physiology, Washington University, St. Louis, MO, USA article info abstract

Article history: BMPs play multiple roles in development and BMP signaling is essential for lens formation. However, the Received for publication 9 April 2009 mechanisms by which BMP receptors function in vertebrate development are incompletely understood. To Revised 18 June 2009 determine the downstream effectors of BMP signaling and their functions in the ectoderm that will form the Accepted 25 August 2009 lens, we deleted the genes encoding the type I BMP receptors, Bmpr1a and Acvr1, and the canonical Available online 3 September 2009 transducers of BMP signaling, Smad4, Smad1 and Smad5. Bmpr1a and Acvr1 regulated cell survival and proliferation, respectively. Absence of both receptors interfered with the expression of proteins involved in Keywords: Lens formation normal lens development and prevented lens formation, demonstrating that BMPs induce lens formation by Bone morphogenetic proteins acting directly on the prospective lens ectoderm. Remarkably, the canonical Smad signaling pathway was not Smads needed for most of these processes. Lens formation, placode cell proliferation, the expression of FoxE3, a lens-specific transcription factor, and the lens protein, αA-crystallin were regulated by BMP receptors in a Smad-independent manner. Placode cell survival was promoted by R-Smad signaling, but in a manner that did not involve Smad4. Of the responses tested, only maintaining a high level of Sox2 protein, a transcription factor expressed early in placode formation, required the canonical Smad pathway. A key function of Smad- independent BMP receptor signaling may be reorganization of actin cytoskeleton to drive lens invagination. © 2009 Elsevier Inc. All rights reserved.

Introduction the lens vesicle withdraw from the cell cycle and elongate to form the primary fiber cells on E11, occluding the lumen of the vesicle (Fig. 1E). Lens formation is a classical example of embryonic induction, A fully formed lens consists of an anterior sheet of proliferating depending on tissue interactions that begin during gastrulation epithelial cells covering a posterior mass of post-mitotic fiber cells (Grainger, 1992). The morphogenesis of the lens commences on (Fig. 1F). In most species, failure of the to contact the embryonic day 9 (E9) in mice after the optic vesicle, an outpocketing prevents lens formation. of the ventral diencephalon, comes in close contact with the surface Lens formation involves signaling by two members of the bone ectoderm. The apposed tissues become tightly adherent (Fig. 1A), morphogenetic protein family of morphogens, BMP4 and BMP7 followed by thickening of the ectoderm in contact with the optic (Dudley et al., 1995; Furuta and Hogan, 1998; Jena et al., 1997; Luo vesicle to form the lens placode (Fig. 1B). On E10, the placode et al., 1995; Wawersik et al., 1999). As members of the TGFβ invaginates, together with the optic vesicle, giving rise to the lens pit superfamily, BMPs activate a heteromeric complex of type I and type II and (Fig. 1C). As the lens pit separates from the surface receptors. The receptors signal by phosphorylating cytoplasmic Smad ectoderm, it forms the lens vesicle (Fig. 1D). Cells in the posterior of proteins (R-Smads), which then associate with the co-Smad, Smad4. The R-Smad/Smad4 complex accumulates in the nucleus to modulate transcription (Heldin et al., 1997). There is increasing evidence that ⁎ Corresponding author. Department of Ophthalmology and Visual Sciences, TGFβ superfamily receptors also activate pathways that do not Washington University, 660 S. Euclid Ave., Campus Box 8096, Rm. 101 McMillan, St. Louis, MO 63110, USA. depend on the canonical Smad pathway (Derynck and Zhang, 2003; E-mail address: [email protected] (D.C. Beebe). Heldin and Moustakas, 2006; Moustakas and Heldin, 2005).

0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2009.08.027 306 R. Rajagopal et al. / Developmental Biology 335 (2009) 305–316

signaling pathways and cellular processes that orchestrate lens formation by BMP signaling are not understood. (Furuta and Hogan, 1998; Wawersik et al., 1999). The early embryonic lethality of the Bmp4 knockout mice and the variability in the phenotype of the Bmp7 null animals complicate attempts to address these issues. To better understand the cellular and molecular mechanisms underlying lens induction, we used a Pax6-Cre transgene (LeCre) that is expressed in the early lens placode (Ashery-Padan et al., 2000)to inactivate one or both of the two type I BMP receptors that are expressed in the lens-forming ectoderm. We found that Bmpr1a contributed to the survival of placode cells, while Acvr1 promoted their proliferation. Although neither receptor was required for lens formation, conditional deletion of both from the surface ectoderm reduced placode thickening and prevented lens invagination, leading to eyes that lacked a lens. These results demonstrate that BMPs signal directly to the lens forming ectoderm to promote lens formation. To investigate the downstream pathways that mediate BMP signaling, we also deleted the R-Smads, Smad1 and Smad5, or the co- Smad, Smad4 from the prospective lens placode. Among the several aspects of lens induction that were regulated by BMP receptor signaling, a few were mediated by the canonical Smad signaling pathway, but most were not. Lens formation, placode cell proliferation, the expression of FoxE3, a transcription factor required for later lens development, and expression of the abundant lens protein, αA-crystallin were regulated by BMP receptors in a Smad-independent manner. Placode cell survival depended on R-Smad signaling, but was independent of Smad4. Of the aspects of lens formation studied, only full expression of Sox2, a transcription factor expressed early in placode formation, was mediated by BMP signaling through the canonical Smad pathway. The Bmpr1a; Acvr1DCKO (double conditional knockout) lens ectoderm cells failed to reorganize the actin cytoskeleton to their apical ends at the onset of invagination. Based on this observation, we Fig. 1. A schematic diagram representing different stages in lens formation. (A) The propose that the reorganization of the actin cytoskeleton, which optic vesicle arises as an evagination of the forebrain and comes in contact with the drives the invagination of the lens placode, is an essential function of head surface ectoderm. (B) Surface ectoderm in contact with optic vesicle thickens to BMP signaling leading to lens formation. form the lens placode. (C) The lens placode and optic vesicle invaginate resulting in a lens pit and optic cup, respectively. (D) The lens pit separates from the surface ectoderm giving rise to a lens vesicle. (E) The cells in the posterior of the lens vesicle Materials and methods elongate to form the primary lens fiber cells. (F) A fully formed lens consists of an fi anterior layer of epithelial cells covering a posterior mass of ber cells. Mice and genotyping

BMP7 and the type I BMP receptors Acvr1 (originally called Alk2) Mice expressing Cre recombinase under the control of Pax6 P0 and Bmpr1a (Alk3) are expressed in the mouse lens placode (Dudley enhancer/promoter (Le-Cre) were described previously (Ashery-Padan and Robertson, 1997; Furuta and Hogan, 1998; Wawersik et al., 1999; et al., 2000). Mice carrying the Cre transgene or the floxed alleles used in Yoshikawa et al., 2000). In contrast, expression of the third type I BMP this study ((Acvr1fx(exon7) (Dudas et al., 2004)andBmpr1afx(exon2) receptor, Bmpr1b (Alk6), appears to be limited to a part of the future (Gaussin et al., 2002), Smad4fx(exon8) (Yang et al., 2002), Smad1fx(exon2) retina and the head mesenchyme in the developing eye (Furuta and (Huang et al., 2002)andSmad5fx(exon2) (Umans et al., 2003)) were Hogan, 1998). Although BMP4 expression is initially seen in both optic genotyped by PCR. Genomic DNA from embryonic tail tissue was vesicle and the overlying ectoderm, it becomes restricted to the optic extracted using the HotSHOT method (Truett et al., 2000). PCR vesicle during placode formation (Furuta and Hogan, 1998). Germline conditions were selected according to the Universal PCR protocol deletion of Bmp4 or Bmp7 prevented lens formation in most (Bmp7) (Stratman et al., 2003). Mice that were homozygous floxed for BMP or all cases (Bmp4)(Dudley et al., 1995; Furuta and Hogan, 1998; Jena receptor or Smad genes, one of which was Cre-positive, were mated to et al., 1997; Luo et al., 1995; Wawersik et al., 1999). Expression generate 50% Cre-positive (conditional knockout, CKO) and 50% Cre- pattern, tissue recombination and other rescue experiments in the negative offspring (WT). Cre-positive animals were always mated to Bmp4 null background showed that BMP4 is required for the optic Cre-negative animals, assuring that Cre-positive offspring inherited vesicle to manifest its lens-inducing activity (Furuta and Hogan, only one copy of the Cre transgene. 1998). However, BMP4 was not able to induce lens formation from the ectoderm unless the optic vesicle was also present. This result raised Histology, antibodies and immunostaining the possibility that BMP4 functions in lens formation by promoting the production of an inducer by the optic cup, rather than by acting Embryos or post-natal heads were fixed in 10% formalin overnight directly on the ectoderm. The expression pattern of BMP7 suggests at room temperature, embedded in 5% agarose, processed and that it functions predominantly in the lens placode to regulate lens embedded in paraffin and sectioned 4 μm. For morphological studies, induction (Wawersik et al., 1999). However, because BMP4 and BMP7 sections were stained with hematoxylin and eosin (Surgipath, null mice lack the ligands in the lens placode, the optic vesicle and in Richmond, IL). For antibody staining, the slices were deparaffinized the periocular mesenchyme, conclusively determining the functions and rehydrated. Endogenous peroxidase activity was inactivated with of BMP signaling in these mutually interacting tissues is difficult. A 3% H2O2 in methanol for 30 min at room temperature for those few of the genes downstream of BMP signaling are known, but the samples that would be treated for horseradish peroxidase (HRP). R. Rajagopal et al. / Developmental Biology 335 (2009) 305–316 307

Epitope retrieval was performed in 0.01 M citrate buffer (pH 6.0) Imaging either at 100 °C for 20 min using a water bath or by placing slides in a Decloaking Chamber (Biocare Medical, Walnut Creek, CA) for 3 min All the brightfield images were taken using the Olympus BX60 Slides were then incubated in blocking solution containing 20% microscope (Olympus, Melville, NY) and Spot camera (Spot Diagnos- inactivated normal donkey serum for 30 min at room temperature tic Instruments, Sterling Heights, MI). The fluorescent images were followed by incubation in primary antibodies overnight at 4 °C. The taken either using the Olympus BX51 with Spot camera or the Zeiss primary antibodies used were anti-αA crystallin at 1:1000 dilution (a 510 confocal microscope (Carl Zeiss, Thornburgh, NY). gift from Dr. Usha Andley), anti-Smad4 (Epitomics, Burlingame, CA) at 1:100 dilution, anti-Pax6 at 1:500 dilution (Developmental Studies Determination of thickness in the lens placode and extent of ectodermal Hybridoma Bank, Iowa City, IA) and anti-Sox2 at 1:1000 dilution contact with the optic vesicle (Chemicon, Temecula, CA). Slides were then incubated for 1 h at room temperature either with Alexa-Fluor-labeled secondary antibodies To analyze the thickness of the placode, 5 equidistant points were (Molecular Probes, Eugene, OR) or biotinylated secondary antibodies marked across the length of the placode on the images of H&E stained (Vector Laboratories, Burlingame, CA). Slides incubated with biotiny- embryo heads sections and the height of the tissue was measured at lated secondary antibodies were treated with the ABC-peroxidase those points using Spot camera software. The extent of contact reagent from Vectastain Elite ABC Kit (Vector Laboratories, Burlin- between the surface ectoderm and the optic vesicle was also game, CA) followed by treatment with diaminobenzidine (DAB) measured using the Spot camera software.

(Sigma, St. Louis, MO) and H2O2. The slides were washed with PBS, and counterstained with hematoxylin (Surgipath, Richmond, IL). Statistical tests To compare the levels of nuclear Pax6 in the knockouts, 12 nuclei were picked at random from the placode and 12 from the optic vesicle. For statistical analysis of two groups of samples, an unpaired t-test After outlining the nuclei, the staining intensities were measured using was performed using GraphPad InStat, Version 3.05 (GraphPad ImageJ software, version 1.36b (National Institutes of Health, Software, San Diego, CA). Error bars are ±S.E.M. Bethesda, MD). To compare the levels of nuclear Sox2 in the knockouts, 12 nuclei were picked at random, using TOPRO as the nuclear marker, Results from the placode and optic vesicle. Nuclei were outlined and intensities of Sox2 immunostaining were measured using ImageJ. The type I BMP receptors Bmpr1a and Acvr1 mediate different effects Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyur- on the cells of the lens placode idine triphosphate nick end-labeling (TUNEL) was done with an Apoptag kit (Chemicon, Temecula, CA). The deparaffinized slides were Previous studies have shown that the BMP ligands BMP4 and treated with 3% H2O2 in methanol for 30 min, followed by proteinase BMP7 are required for lens formation (Dudley et al., 1995; Furuta and K treatment (20 μg/ml) for 15 min. Slides were incubated with TdT Hogan, 1998; Jena et al., 1997; Luo et al., 1995; Wawersik et al., 1999). enzyme in equilibration buffer for 1 h at 37 °C. The reaction was To determine the roles of different BMP receptors in the formation of terminated with wash buffer provided by the manufacturer for 10 min the lens, we inactivated the genes for the type I BMP receptors Bmpr1a at room temperature. Anti-digoxigenin-peroxidase conjugate was or Acvr1 in the surface ectoderm using Cre recombinase driven by the added for 30 min at room temperature, followed by DAB+H202 Pax6 P0 promoter/enhancer (Le-Cre). In these transgenic mice, Cre is treatment. Slides were counterstained with hematoxylin. expressed in the prospective lens placode by E9.0 (Ashery-Padan et For BrdU staining, pregnant females or post-natal day 3 (P3) mice al., 2000). were injected with 50 mg/kg of body weight of a mixture of 10 mM Loss of Bmpr1a did not prevent lens formation (Fig. 2B). BrdU (Roche, Indianapolis, IN) and 1 mM 5-fluoro-5-deoxyuridine Bmpr1aCKO lenses were reduced in size with defects in fiber cell (Sigma, St. Louis, MO) and sacrificed after 1 h. A monoclonal anti-BrdU differentiation (Beebe et al., 2004). In the lens placode, loss of Bmpr1a antibody (1:250) (Dako, Carpinteria, CA) was used with a Vectastain resulted in a more than two-fold increase in the TUNEL labeling index Elite Mouse IgG ABC kit as described above. Sections were counter- (pb0.0001) (Fig. 2E), with no significant alteration of the BrdU stained with hematoxylin. labeling index (Fig. 2J). Some embryos were fixed in 10% formalin for an hour. The heads Deletion of Acvr1 also resulted in the formation of lenses that were were cut in half, embedded in 5% agarose and sectioned into 100 μm reduced in size (Fig. 2C), with several defects that appeared later in thick sections using a vibrating tissue slicer (EM Sciences, Hatfield, lens formation (Rajagopal et al., 2008). Acvr1CKO placodes showed a PA). The sections were permeabilized and blocked in PBS supple- significant decrease in BrdU labeling compared to placodes from wild- mented with 0.5% Triton X-100 and 5% goat serum and labeled type littermate (pb0.01) (Fig. 2K), but with no difference in the overnight with antibodies specific for pSmad1/5/8 (Cell Signaling TUNEL-labeling index (Fig. 2F). Although the two type I BMP receptors Technology, Danvers, MA), FoxE3 (a gift from Dr. Peter Carlsson), αA separately maintained normal levels of cell proliferation and survival crystallin (from Dr. Usha Andley), E-cadherin (Cell Signaling Tech- during the formation of the lens placode, neither was required for lens nology, Danvers, MA), ZO-1 (Invitrogen, Carlsbad, CA), laminin α1 formation. (generated by Dr. Dale Abrahamson), laminin α5 (generated by Dr. Jeffery Miner), laminin γ1 (Chemicon, Temecula, CA) and entactin Bmpr1a; Acvr1DCKO ectoderm does not form a lens (Chemicon, Temecula, CA). All these antibodies were used at a dilution of 1:250. The sections were then washed using PBS with 0.5% To test whether the type1 BMP receptors act redundantly to Tween 20, incubated in the second secondary antibody for 2 h, regulate lens formation, we generated Bmpr1a; Acvr1DCKO mice. The washed again in PBS with Tween 20 and mounted using a 1:1 dilution double receptor knockout ectoderm displayed a fully penetrant of VectaShield (Vector Laboratories, Burlingame, CA) in PBS. Some of phenotype of failure of lens formation (Fig. 2D). At E10.5, the wild- the sections were incubated with fluorescent labeled phalloidin, type lens vesicle expressed the lens protein, αA-crystallin (Fig. 2H). In TOPRO or TOTO-1 along with the secondary antibodies. Alexa-Fluor the Bmpr1a; Acvr1DCKO eye, αA-crystallin-positive cells were not labeled secondary antibodies (Molecular Probes, Eugene, OR) and detected (Fig. 2I). Consistent with the expression of BMP ligands and Alexa-Fluor labeled phalloidin (Molecular Probes, Eugene, OR) were receptors in the lens placode (Dudley and Robertson, 1997; Furuta used at 1:1000 dilution. TOPRO or TOTO-1 (Molecular Probes, Eugene, and Hogan, 1998; Wawersik et al., 1999; Yoshikawa et al., 2000), OR) were used at a dilution of 1:10,000. wild-type placode cells stained with an antibody against the 308 .Rjgple l eeomna ilg 3 20)305 (2009) 335 Biology Developmental / al. et Rajagopal R. – 316

Fig. 2. BMP receptors, Bmpr1a and Acvr1, perform redundant functions in lens formation, but play non-redundant roles in stimulating placode cell proliferation and survival. (A–D) Hematoxylin and eosin-stained sections of wild-type, Bmpr1aCKO , Acvr1CKO and Bmpr1a; Acvr1DCKO P3 eyes. (E–G) Quantification and comparison of TUNEL indices in E9.5 Bmpr1aCKO , Acvr1CKO and Bmpr1a; Acvr1DCKO lens placodes with their respective wild-type littermate controls. (H, I) Sections of E10.5 wild-type and Bmpr1a; Acvr1DCKO embryo heads labeled with an αA-crystallin antibody. (J–L) Quantification and comparison of BrdU indices in E9.5 Bmpr1aCKO , Acvr1CKO and Bmpr1a; Acvr1DCKO lens placodes with their respective wild-type littermate controls. (M, N) E9.5 wild-type and Bmpr1a; Acvr1DCKO embryo head sections labeled with anti-phospho Smad 1/5/8. (⁎pb0.05, ⁎⁎pb0.01, ⁎⁎⁎⁎pb0.0001, NS—not significant, arrowheads—loss of phospho-Smad1/5/ 8 staining specifically in the knockout ectoderm, OV—optic vesicle). .Rjgple l eeomna ilg 3 20)305 (2009) 335 Biology Developmental / al. et Rajagopal R. – 316 Fig. 3. Lens formation and placode cell proliferation do not require signals from the canonical Smad pathway. However, placode cell survival is mediated by the R-Smads. (A) Hematoxylin and eosin-stained section of Smad4CKO P3 eye. (B, C) Sections of E9.5 Smad4WT and Smad4CKO embryo heads labeled with an antibody against Smad4. (D, E) Quantification of TUNEL and BrdU indices in E9.5 Smad4WT and Smad4CKO lens placodes. (F) Hematoxylin and eosin-stained section of Smad1; Smad5DCKO P3 eye. (G, H) E9.5 Smad1; Smad5WT and Smad1; Smad5DCKO embryo head sections labeled with anti-phospho Smad 1/5/8. (I, J) Quantification of TUNEL and BrdU indices in E9.5 Smad1; Smad5WT and Smad1; Smad5DCKO lens placodes. (⁎pb0.05, NS—not significant, arrows—loss of nuclear Smad4 staining in the ectoderm within the Pax6-Cre expression domain, arrowhead in C—presence of Smad4 staining in ectoderm cells outside the Pax6-Cre expression domain, OC—optic cup, arrowheads in H—loss of phospho-Smad1/5/8 staining specifically in the knockout ectoderm, OV—optic vesicle). 309 310 R. Rajagopal et al. / Developmental Biology 335 (2009) 305–316 phosphorylated form of the BMP-activated Smads, Smad1/5/ knockouts (not shown). Therefore, Bmpr1a signals through Smads 1 8 (pSmad1/5/8) (Fig. 2M). In contrast, the Bmpr1a; Acvr1DCKO and 5 to promote cell survival, while Acvr1 promotes lens cell ectoderm showed greatly reduced staining for pSmad1/5/8 (Fig. proliferation by a Smad-independent mechanism. 2N). In the knockout embryos, pSmad1/5/8 levels were maintained in the optic vesicle, where Cre is not expressed. The double receptor Inactivation of Bmpr1a and Acvr1 in the surface ectoderm alters the knockout ectoderm displayed increased cell death (pb0.05) (Fig. 2G) level of Sox2, but not Pax6, in a Smad-dependent manner and decreased proliferation (pb0.05) (Fig. 2L), similar to the single receptor knockouts. Pax6, a transcription factor with paired and homeobox domains, is required in the surface ectoderm for lens formation (Ashery-Padan et BMP receptors do not require Smad4 to promote the survival or al., 2000). Pax6 expression is gradually lost in the Bmp7 null placodal proliferation of lens placode cells or for lens formation ectoderm, which fails to form a lens (Wawersik et al., 1999). Since Bmpr1a; Acvr1DCKO ectoderm does not form a lens, we tested the Since lenses fail to form upon inactivation of Bmpr1a and Acvr1, possibility that Pax6 levels might be altered in the knock-out we determined whether these receptors signal through the down- ectoderm. Pax6 levels were indistinguishable in wild-type placodes stream co-Smad, Smad4, to promote lens formation. Conditional and in double receptor knockout surface ectoderm (Figs. 4A and B). As deletion of Smad4 did not inhibit lens formation (Fig. 3A). Smad4 expected, the levels of Pax6 were also unaltered in Smad4CKO and antibodies stained the cytoplasm and nuclei of cells in wild type lens Smad1; Smad5DCKO placodes (Figs. 4C and D). Quantification con- placodes and optic vesicles (Fig. 3B). However, Smad4 staining was firmed the similar levels of Pax6 protein in the wild type, the double undetectable in all but a few of the placode cell nuclei in Smad4CKO receptor knockout ectoderm and in Smad1; Smad5DCKO and Smad4CKO embryos (Fig. 3C). The increased cell death and decreased prolifer- placodes (Figs. 4A′–D′). Expression of Sox2, a HMG box-containing ation observed in the Bmpr1a; Acvr1DCKO surface ectoderm was also transcription factor, is reduced in the lens-forming ectoderm of Bmp4 not seen in Smad4CKO lens placodes (Figs. 3D and E). Therefore, Smad4 null mice and lost in the ectoderm of Bmp7 knockout mice (Furuta is not required for lens formation or promotion of cell survival or and Hogan, 1998; Wawersik et al., 1999). Quantification confirmed proliferation in the lens placode. that Sox2 protein was present at a lower level than wild type in the double receptor knockout ectoderm and in Smad1; Smad5DCKO and CKO BMP receptors do not require the R-Smads, Smad1 or Smad5, to Smad4 placodes (Figs. 4E–H and E′–H′). Therefore, maximal levels promote lens formation of Sox2 depend on BMP receptor signaling through the canonical R- Smad–Smad4 pathway. In the canonical Smad signaling pathway, R-Smads interact with Smad4 to regulate gene expression in response to ligands of the TGFβ Smad-independent BMP signaling regulates the expression of FoxE3 superfamily (Moustakas et al., 2001). Since lens formation did not and αA-crystallin in the surface ectoderm require Smad4, we determined whether it required the R-Smads that are downstream of BMP receptors, Smad1 and 5. The third BMP- FoxE3 is a lens-specific member of the forkhead family of specific R-Smad, Smad8, is not expressed in the surface ectoderm transcription factors. It is initially detected late in placode formation during lens formation (Arnold et al., 2006). Inactivation of Smad1 and (Blixt et al., 2000). Although lenses form in mice mutant for Foxe3, Smad5 did not prevent lens formation (Fig. 3F). Greatly reduced severe defects in lens cell proliferation and survival appear soon after staining for pSmad1/5/8 in the Smad1; Smad5DCKO placode (Fig. 3H) invagination (Blixt et al., 2000; Medina-Martinez et al., 2005). Wild- demonstrated that the R-Smads had been efficiently deleted. The type placode cells at E10 (33 somites) showed nuclear FoxE3 staining antibody to pSmad1/5/8 binds to a phosphopeptide that is nearly (Fig. 4I), but no specific staining was detected in the surface ectoderm identical in the three proteins. Therefore, our data suggest that Smad8 of Bmpr1a; Acvr1DCKO littermates (Fig. 4J). However, FoxE3 expression expression did not increase to compensate for the absence of Smad1 was unaltered in Smad4CKO and Smad1; Smad5DCKO embryos at a and 5. Microarray analysis of transcripts from wild type and Bmpr1a; similar stage (Figs. 4K and L). Expression of αA-crystallin commenced Acvr1DCKO lens placodes confirmed that Smad8 mRNA was not in a few cells at the 30-somite stage (early lens invagination) (Fig. detectable in either genotype (not shown). Thus, signaling by BMP4 4M). In 36-somite embryos (the lens vesicle stage), αA-crystallin was and BMP7 through Bmpr1a and Acvr1 activates Smad-independent present in all lens cells (Fig. 4M′). Immunostaining for αA-crystallin pathways to promote lens formation. was not detected in Bmpr1a; Acvr1DCKO prospective lens ectoderm in 33-somite embryos (Fig. 4N), but was present in Smad4CKO and DCKO Cell survival, but not proliferation, is mediated by R-Smads Smad1; Smad5 lens pits (Figs. 4O and P). Therefore, Bmpr1a and Acvr1 signaling initiates the expression of FoxE3 and αA-crystallin in Although the R-Smads, Smad1 and Smad5 were not required for the lens placode in a Smad-independent fashion. lens formation, we determined whether they might promote cell survival or proliferation in response to BMP signaling. Smad1; Bmp1a; Acvr1DCKO ectoderm loses contact with the optic vesicle and Smad5DCKO placodes showed a significant increase in TUNEL-positive fails to fully thicken into a placode cells compared to the wild type littermates (pb0.05) (Fig. 3I). Lens placodes lacking either Smad1 or Smad5 also had significantly more During the formation of the wild-type lens placode, the surface TUNEL labeling (not shown). The BrdU labeling index was unaffected ectoderm and the underlying optic vesicle remain in close contact, in the R-Smad double knockouts (Fig. 3J) and in each of the single only separating during the invagination of the lens and optic vesicle

Fig. 4. Bmpr1a and Acvr1 mediate full expression of Sox2, but not of Pax6, through the canonical Smad pathway, whereas expression of FoxE3 and αA-crystallin by Bmpr1a and Acvr1 does not require the canonical Smads. (A–D) E9.5 wild-type, Bmpr1a; Acvr1DCKO, Smad4CKO and Smad1; Smad5DCKO embryo head sections labeled with an antibody against Pax6. (A′– D′) Quantification of nuclear Pax6 staining intensities in the lens placode and optic vesicle of the wild-type, Bmpr1a; Acvr1DCKO, Smad4CKO and Smad1; Smad5DCKO embryos. (E–H) E9.5 embryo head sections of wild-type, Bmpr1a; Acvr1DCKO, Smad4CKO and Smad1; Smad5DCKO labeled with an antibody against Sox2. (E′–H′) Quantification of nuclear Sox2 staining intensities in the lens placode and optic vesicle of the wild-type, Bmpr1a; Acvr1DCKO, Smad4CKO and Smad1; Smad5DCKO embryos. (I–L) 33 somite wild-type, Bmpr1a; Acvr1DCKO, Smad4CKO and Smad1; Smad5DCKO embryo head sections labeled with anti-FoxE3. (M, M′) 30 and 36 somite wild-type embryo head sections labeled with an antibody against αA- crystallin. (N–P) 33 somite Bmpr1a; Acvr1DCKO, Smad4CKO and Smad1; Smad5DCKO embryo head sections labeled with an antibody against αA-crystallin. Slight staining seen in the Bmpr1a; Acvr1DCKO embryos labeled with antibody to αA-crystallin was due to the secondary, anti-mouse antibody (⁎⁎pb0.01, ⁎⁎⁎⁎pb0.0001, NS—not significant). R. Rajagopal et al. / Developmental Biology 335 (2009) 305–316 311 312 .Rjgple l eeomna ilg 3 20)305 (2009) 335 Biology Developmental / al. et Rajagopal R. – 316 Fig. 5. Lens ectoderm lacking BMP receptors, Bmpr1a and Acvr1, fail to maintain contact with the underlying optic vesicle and fail to thicken like wild-type placodes. (A, B) Hematoxylin and eosin-stained sections of 24 somite Bmpr1a; Acvr1WT and Bmpr1a; Acvr1DCKO embryos heads. (A′,B′) Hematoxylin and eosin stained sections of 28 somite Bmpr1a; Acvr1WT and Bmpr1a; Acvr1DCKO embryos heads. (C, C′) Extent of contact between lens ectoderm and optic vesicle measured in 24 and 28 somite stage Bmpr1a; Acvr1WT and Bmpr1a; Acvr1DCKO embryos. (D, D′) Thickness of lens ectoderm/placode measured in 24 and 28 somite stage Bmpr1a; Acvr1WT and Bmpr1a; Acvr1DCKO embryos. (⁎pb0.05, ⁎⁎pb0.01, ⁎⁎⁎⁎pb0.0001). R. Rajagopal et al. / Developmental Biology 335 (2009) 305–316 313

(Figs. 5A and A′). The surface ectoderm and the underlying optic than wild type and did not fully elongate or remain adherent to the vesicle were in close contact in wild type and Bmpr1a; Acvr1DCKO, 24 optic vesicle, we determined whether these cells showed altered somite embryos (Figs. 5B, C). However, at the late placode stage (28 expression or distribution of markers of cell polarity. The expression somites) the contact area between these tissues decreased and distribution of the adherens junction marker, E-cadherin, and the (pb0.0001; Figs. 5B′,C′). Although wild-type and Bmpr1a; Acvr1DCKO tight junction marker, ZO-1, appeared similar in wild-type and double lens-forming ectoderm were of similar thickness early in placode knockout ectoderm cells (Fig. S1). This suggested that the cell polarity formation (Fig. 5D), the Bmpr1a; Acvr1DCKO ectoderm failed to thicken was not grossly affected in the Bmpr1a; Acvr1DCKO cells. as much as wild type during placode formation (Fig. 5D′). The ventral The lens placode and the optic vesicle are held in contact by a zone region of the placode, where separation from the optic vesicle first of extracellular matrix (ECM), which is secreted by both tissues occurred, was thinner than the dorsal region. Thus, signaling through (Hendrix and Zwaan, 1975; Silver and Wakely, 1974). Since Bmpr1a; Bmpr1a and Acvr1 may contribute to placode formation by main- Acvr1DCKO ectoderm had reduced contact with the underlying optic taining contact between the surface ectoderm and the optic vesicle vesicle, we determined whether any defects were apparent in the (Hendrix and Zwaan, 1975). basal laminae of the two epithelia or in the ECM between them. The Since the polarized distribution of plasma membrane proteins is overall composition and individual components of the basal lamina important for cell organization and the function of the cytoskeleton and ECM were not obviously affected in the double BMP receptor (Knust, 2000), defects in cell polarity could hinder cell elongation or knockouts (Fig. S2). prevent the basal secretion of basement membrane components. Because Bmpr1a; Acvr1DCKO ectoderm cells appeared less organized Deletion of Bmpr1a and Acvr1 prevents the reorganization of the actin cytoskeleton that normally occurs during lens invagination

One proposed mechanisms of lens invagination involves the contraction of actin microfilaments at the apical ends of placode cells (Wrenn and Wessells, 1969). Prior to lens invagination, staining with fluorescently labeled phalloidin showed that filamentous actin (F-actin) was distributed around the apical, basal and lateral surfaces of wild type lens placode cells (Fig. 6A). As the placode began to invaginate to form the lens pit at E10.0, phalloidin staining decreased along the lateral surfaces of the cells and increased at their apical ends (Fig. 6C). In contrast to the discontinuous distribution seen at the placode stage (Fig. S1E), the apical distribution of ZO-1 appeared continuous as wild-type placode cells began to invaginate (Fig. 6E). This suggested that contraction of apical actin filaments draws the apical ends of the placode cells together to cause bending of the placode and formation of the lens pit. In Bmpr1a; Acvr1DCKO ectoderm cells, F-actin did not accumulate at the apical ends of the cells, remaining uniformly distributed around the cell periphery (Fig. 6B, D). At the same time, the ZO-1 distribution remained discontinuous at the apical ends of the cells (Fig. 6F), implying failure of apical contraction. The apical redistribution of F-actin occurred normally in Smad4CKO and Smad1; Smad5DCKO embryos (Figs. 6G and H). We conclude that BMP signaling through Bmpr1a and Acvr1 promotes reorganization of the actin cytoskeleton prior to lens invagination in a Smad-independent manner, thereby facilitating lens morphogenesis.

Discussion

Germline knockout of Bmp4 or Bmp7 in mice demonstrated that BMP signaling is essential for lens induction, although these studies did not identify the cellular mechanisms underlying this process (Dudley and Robertson, 1997; Furuta and Hogan, 1998; Jena et al., 1997; Luo et al., 1995; Wawersik et al., 1999). Here, we provide evidence that the type I BMP receptors, Bmpr1a and Acvr1, act redundantly and in a Smad-independent manner to mediate lens formation. Although their most important functions do not require Smads, some of their actions depend on receptor-activated Smads, independent of Smad4, while others use the canonical R-Smad– Smad4 pathway. Each receptor activates one or more Smad- independent mechanisms that redistribute the actin cytoskeleton to the apical ends of lens placode cells, a process that is likely to drive lens invagination. Fig. 6. Bmpr1a and Acvr1 mediate apical redistribution of F-actin during lens invagination in a Smad-independent manner. (A, B) E9.5 Bmpr1a; Acvr1WT and Either Bmpr1a or Acvr1 is sufficient for lens formation Bmpr1a; Acvr1DCKO embryo head sections stained with phalloidin. (C, D) E10 Bmpr1a; Acvr1WT and Bmpr1a; Acvr1DCKO embryo head sections stained with phalloidin. (E, F) Our results provide genetic evidence that Bmpr1a and Acvr1 play Sections of E10 Bmpr1a; Acvr1WT and Bmpr1a; Acvr1DCKO embryo heads labeled with an CKO fi antibody against ZO-1 and TOPRO. (G, H) Phalloidin labeled sections of Smad4 and redundant roles; either receptor is suf cient for lens formation. Smad1; Smad5DCKO E10 embryo heads. (OV—optic vesicle, OC—optic cup). Another example of redundant BMP receptor function is seen during 314 R. Rajagopal et al. / Developmental Biology 335 (2009) 305–316 retinal development. Developing retinae lacking Bmpr1a and Bmpr1b double knockout ectoderm had diminished ability to sustain contact (Alk6) exhibit severe eye defects resulting from reduced growth and with the optic vesicle late in placode formation, especially in the failure of retinal , defects not seen in the single receptor ventral region of the placode. Cells in this region were also not as thick knockouts (Murali et al., 2005). In addition to demonstrating their as cells in the dorsal region of the placode. Cells of Bmpr1a; Acvr1DCKO redundancy, the present study provides a unique example of placodes had normal apical-basal polarity and contributed to a dissimilar and shared functions performed by two BMP receptors basement membrane that appeared normal in extent and composi- during the formation of a single tissue. tion. Therefore, it seems less likely that precocious separation was caused by defects in the secretion of components of the extracellular Bmpr1a and Acvr1, regulate diverse functions during lens placode matrix. Contact between the optic vesicle and the ectoderm was formation initially established, but was not maintained as the optic vesicle began to invaginate. The invagination of the ventral portion of the optic Deletion of individual type I BMP receptors showed they have vesicle differs from invagination of the dorsal and lateral regions, due unique functions, which are not required for lens formation. Bmpr1a to the formation of the ventral optic fissure (Morcillo et al., 2006). This promotes the survival of placode cells, whereas Acvr1 promotes their may account for the separation of the optic vesicle from the ectoderm proliferation. Acvr1 signaling is mitogenic for other cell types during in this region. Separation of the surface ectoderm and the optic vesicle their differentiation, specifically, the -derived cells of was also observed in Bmp4 null embryos (Furuta and Hogan, 1998). It Meckel's cartilage (Dudas et al., 2004). As in the lens placode, deletion seems most probable that the inability of the knock out surface of Bmpr1a in the lung epithelium leads to increased apoptosis ectoderm to invaginate synchronously with the optic vesicle caused (Eblaghie et al., 2006). However, unlike its function in the placode, these tissues to separate. loss of Bmpr1a in the lung epithelium also results in decreased Previous studies showed that neural crest cells could inhibit the proliferation (Eblaghie et al., 2006). The distinct functions of Bmpr1a formation of a lens from head ectoderm (Sullivan et al., 2004). and Acvr1 in the lens appears to be mediated by different downstream Mesenchyme cells were present in the space between the ventral pathways. While promotion of cell survival appears to require the R- placode and the optic vesicle, raising the possibility that that these Smads, Smad1 and Smad5, cell proliferation is maintained by one or cells inhibited lens formation. However, it is not clear what more Smad-independent mechanisms. Analyzing Bmp4 and Bmp7 mechanism would have induced the mesenchyme cells to invade null surface ectoderm for defects in cell survival and proliferation may the space between the placode and the optic vesicle, since no determine whether one or both ligands elicit these distinct responses mesenchyme cells were present in this space at earlier stages of from the type I BMP receptors. placode formation. Therefore, it seems unlikely that neural crest cell invasion was responsible for inhibiting lens formation. BMP receptors activate multiple signaling pathways during lens formation Defects in placode thickening, F-actin redistribution and lens morphogenesis in Bmpr1a; Acvr1DCKO embryos Our studies revealed that, among the several facets of lens formation regulated by BMP signaling, some are mediated by the canonical Smads, The underlying cellular mechanisms that lead to failure of lens but most are not. Several instances of Smad-independent signaling formation in Bmp4 or 7 knockouts were unclear (Furuta and Hogan, downstream of BMP receptors have been reported [reviewed in 1998; Jena et al., 1997; Wawersik et al., 1999). In Bmp4 null mice, (Moustakas and Heldin, 2005)]. Most of these involve the activation of mRNA encoding Sox2, a transcription factor that is normally expressed specific MAP kinase modules, particularly the p38 pathway. Type II BMP in the lens placode, fails to accumulate (Furuta and Hogan, 1998). In receptors may also signal through the cytoplasmic kinase, LIMK1 mice lacking BMP7, expression of Pax6, Sox2 and secreted frizzled- (Foletta et al., 2003; Lee-Hoeflich et al., 2004; Wen et al., 2007). Further related protein-2 (sFRP2), an antagonist of the Wnt signaling studies are needed to determine whether Bmpr1a and Acvr1 promote pathway, is reduced in the surface ectoderm (Wawersik et al., lens formation by activating one of these pathways, or an as yet 1999). Conditional deletion of Pax6 in the prospective lens-forming unknown Smad-independent signaling cascade. ectoderm prevents lens formation (Ashery-Padan et al., 2000). Mice BMP signaling promoted placode cell survival and maintained full lacking Sfrp1 and 2 do not appear to have obvious lens defects (Satoh expression of Sox2 protein in a Smad-dependent manner. Lens et al., 2006) and mice lacking Sox2 in the lens forming-ectoderm have placodes lacking Smad1, Smad5, or both R-Smads showed increased not been reported. Thus, it seems possible that decreased expression cell death, with no change in cell proliferation. However, maintaining of Pax6 and Sox2 contributes to the failure of lens formation in Bmp4 cell survival did not require the Co-Smad, Smad4, providing evidence or 7 null mice. However, deletion of Bmpr1a and Acvr1 did not for an R-Smad-dependent, Smad4-independent signaling mechanism. decrease the levels of Pax6 protein. Sox2 levels were lower in the This is reminiscent of the effect of deleting Smad4 from the mouse double knockouts, but were also lower in ectoderm cells lacking epiblast, where only a subset of BMP-dependent responses were Smad4 or the R-Smads, which formed lenses. affected (Chu et al., 2004). However, the requirement for the R-Smads The results of the present study differ in some ways from those in these Smad4-independent responses has not been examined. obtained from germline deletion of Bmp7 (Furuta and Hogan, 1998; Although Smad4 is usually considered to play a central role in Smad- Wawersik et al., 1999), where absence of Bmp7 resulted in a dependent signaling, emerging evidence suggests that R-Smads can gradual loss of Pax6 mRNA and a marked reduction in Sox2 bind to factors other than Smad4, such as TIF-1γ (He et al., 2006). transcripts in the lens-forming ectoderm. This difference may be Although Smad4 is expressed during early stages of lens development because proteins would be expected to persist longer than their and localizes to the nuclei of placode cells, it is possible that surrogates mRNAs. Germline deletion of Bmp7 could also impair the earlier of Smad4, similar to TIF-1γ, mediate some of the effects of Bmpr1a and development of tissues that contribute to Pax6 and Sox2 expression Acvr1 during early lens development. in the placode. It is not clear to what extent thickening of the lens placode is Loss of BMP signaling leads to separation of the lens placode from necessary for the subsequent invagination of the surface ectoderm. the optic vesicle Hendrix and Zwaan (1975) proposed that adhesion between the placode cells and underlying optic vesicle promoted lens invagination Bmpr1aCKO or Acvr1CKO lens placodes thicken normally and by preventing spreading of the surface ectoderm, resulting in placode maintain contact with the underlying optic vesicle. However, the thickening and subsequent invagination. Enhancement of placode R. Rajagopal et al. / Developmental Biology 335 (2009) 305–316 315

Distinct molecular and cellular regulation of lens induction by BMP receptor signaling

Lens induction is defined by two hallmark features. At the molecular level, it is characterized by the expression of an array of transcription factors and lens-specific proteins. The cellular aspects of lens formation involve the generation of a placode, followed by its invagination. Our study demonstrates that BMP receptors mediate both the molecular and cellular aspects of lens formation. BMP receptor signaling influences the expression of three molecular markers, Sox2, FoxE3 and αA-crystallin. Both cellular mechanisms contributing to lens formation, lens placode formation and lens invagination, are impaired when BMP receptors are deleted from the lens forming ectoderm. Defects in cell proliferation and survival and/ or loss of contact with the underlying optic vesicle may reduce the extent of placode formation, but a placode does form. Failure of cytoskeletal rearrangement appears to be the principal cause of failure of lens invagination in the BMP receptor knockouts.

Acknowledgments

We thank Drs. Ruth Ashery-Padan and Judy West-Mays for providing the Le-Cre mice and are grateful for the expert technical assistance of Chenghua Wu and Mary Feldmeier with genotyping and Fig. 7. A schematic comparison of lens invagination to a drawstring mechanism. (A) Belinda McMahan and Jean Jones with histology and immunohisto- Drawing of the string pulls the fabric together along its axis. (B) Apical actin localization chemistry. We are indebted to Drs. Jeffrey Miner and Peter Carlsson and its axial contraction in the wild-type pulls the cells together facilitating lens for the laminin and FoxE3 antibodies, respectively. This work was invagination. (C) Failure of apical actin localization in the Bmpr1a; Acvr1aDCKO precludes an axial contraction and lens invagination. supported by an unrestricted grant from Research to Prevent Blindness and NIH Core Grant EY02687 to the Department of Ophthalmology and Visual Sciences, NIH grant EY04853 to DCB and thickening, either by ethanol treatment or ligature of the ectoderm, 5R01HL074862 and 5R01DE013085 to VK. Drs. Lieve Umans and An CKO resulted in more rapid invagination (Wakely, 1984). Pax6 surface Zwijsen are indebted to Dr. Danny Huylebroeck for his support of their ectoderm does not thicken at all (unpublished data) and Bmpr1a; work. Acvr1DCKO ectoderm forms thinner placodes. In neither case was a lens formed. These observations support the idea that lens placode Appendix A. Supplementary data formation may be required for lens invagination. 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