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Developmental Biology 276 (2004) 416–430 www.elsevier.com/locate/ydbio

Integrin a4h1 function is required for cell survival in developing retina

Sergiu T. Leua, Susan A.L. Jacquesa, Kevin L. Wingerda, Sherry T. Hikitaa, Erin C. Tolhursta, Jan L. Pringa, Derek Wiswella, Lisa Kinneya, Nichol L. Goodmana,1, David Y. Jacksonb, Dennis O. Clegga,*

aNeuroscience Research Institute and Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, United States bDepartment of Bioorganic Chemistry, Genentech Inc., South San Francisco, CA 94080, United States

Received for publication 10 April 2004, revised 31 August 2004, accepted 1 September 2004 Available online 1 October 2004

Abstract

In the retina, in the h1 family have been shown to be important in many phases of neuronal development, particularly neuroblast migration and axon outgrowth. However, the functions of specific heterodimers are not well defined. In this study, we investigated the functions of h1 integrins in developing chicken retina by expression of a dominant-negative h1A construct using a replication-competent retrovirus. Inhibition of integrins using this approach resulted in alteration of cell morphology and increased apoptosis, but did not preclude migration and axon elongation. In an attempt to identify which specific h1 heterodimer was important, expression and function of the a4h1 heterodimer were also investigated. At early developmental stages, a4 and mRNA were detected in undifferentiated neuroblasts throughout the retina. At later stages, expression was confined to retinal ganglion cells (RGCs) and amacrine cells. A small molecule antagonist of a4 integrins was shown to inhibit neurite outgrowth on recombinant soluble vascular molecule-1 (VCAM-1), a known ligand of a4h1. Introduction of a4 antagonist in vivo gave rise to increased apoptosis and led to a thinning of the retina and reduced numbers of retinal ganglion cells (RGCs). We conclude that the integrin a4h1 is important for survival of developing retinal neurons, including RGCs. D 2004 Elsevier Inc. All rights reserved.

Keywords: Integrin a4h1; Retina; Chicken; VCAM-1; Cell adhesion; Axon outgrowth; Apoptosis; RCAS; Retinal ganglion cells

Introduction developing retina, perturbation experiments have shown the importance of h1 family of integrins. For example, in During development of the nervous system, adhesive Xenopus, expression of dominant-negative h1 integrins interactions between cells are crucial at every step, from the inhibited extension of retinal ganglion cell (RGC) dendrites proliferation and migration of neuroblasts to the generation and axons (Lilienbaum et al., 1995). Immunological block- and modulation of synapses. Integrins, a family of hetero- ade of h1 integrins in combination with N- resulted dimeric receptors that mediate cell–cell and cell–extracel- in axon pathfinding errors by RGCs (Stone and Sakaguchi, lular matrix (ECM) interactions, are abundantly expressed 1996). Introduction of function blocking anti-h1 antibodies by neurons and have been implicated in many phases of into the embryonic chicken retina resulted in dramatic neural development (Clegg et al., 2000, 2003). In the disruption of retinal lamination and growth (Svennevik and Linser, 1993), which may be due in part to disruption of h1- dependent RGC migration (Cann et al., 1996). * Corresponding author. Fax: +1 805 893 2005. Integrins are also important in regulating apoptosis in E-mail address: [email protected] (D.O. Clegg). 1 Current address: Program in Biomedical Science, School of both nonneuronal and neuronal cells. Many cells cultured in Medicine, University of California, San Diego, La Jolla, CA 92093, United the absence of an ECM integrin ligand undergo apoptosis or States. banoikisQ (Frisch and Francis, 1994). Occupied integrins

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.09.003 S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 417 signal to increase the synthesis and activity of anti-apoptotic a4 die at embryonic day 11 (E11) due to defective formation BCL family and increase the phosphorylation and of the heart and placenta (Hynes, 1996; Yang et al., 1995). inactivation of the pro-apoptotic BAD proteins via multiple A neuronal role for a4h1 integrins is emerging from pathways involving focal adhesion kinase and AKT recent studies of the nervous system. a4 integrins are (Stupack and Cheresh, 2002). Similar signaling is thought abundantly expressed on neural crest cells, where they have to go on in neuronal cells (Banks and Noakes, 2002; been implicated in migration and survival (Haack and Bonfoco et al., 2000; Marchionini et al., 2003). Inhibition of Hynes, 2001; Kil et al., 1998; Stepp et al., 1994). Recently, h1 integrins in the chicken tectum via retroviral expression it was shown that a4 integrins are expressed on dorsal root of antisense RNA was shown to inhibit not only migration, ganglion growth cones in regenerating sciatic nerve but also caused loss of cells, possibly due to apoptosis (Vogelezang et al., 2001) and by sympathetic neurons (Galileo et al., 1992). Additional experiments in the tectum innervating the heart (Wingerd et al., 2002). Inhibition of showed that inhibition of a6 expression reduced migration a4h1 in vivo decreased outgrowth of sympathetic fibers in and inhibition of a8 reduced survival. In the retina, the developing postnatal rat heart (Wingerd et al., 2002). expression of antisense h1 RNA during retinogenesis Here we show that expression of a dominant-negative h1 reduced the resulting clone size derived from each precursor integrin construct in the developing chicken retina alters cell (Skeith et al., 1999). However, the specific h1 heterodimers morphology and increases apoptosis. We also characterize involved in retinal neuron survival are not known. the expression of the a4 protein and mRNA, and show that The h1 integrin subunit is known to pair with at least 11 a4 integrins are important for cell survival in the developing different alpha subunits, and six of these have been detected retina. in retina (a1, a2, a3, a4, a6, a8, and av) (Bosco et al., 1994; Bossy et al., 1991; Bradshaw et al., 1995; Cann et al., 1996; Cohen et al., 1987; de Curtis et al., 1991; Duband et Materials and methods al., 1992; Hall et al., 1987; Sheppard et al., 1994). The a2h1 heterodimer has been shown to mediate neurite outgrowth Construction of dominant-negative integrin b1 expression and adhesion of retinal cells to collagens, and the a6h1 vectors heterodimer is the primary laminin-1 (Ln-1) receptor in the retina (Bradshaw et al., 1995; de Curtis and Reichardt, The dominant-negative h1A construct (Lukashev et al., 1993). Aberrant retinal lamination was observed in retinas 1994) was introduced into the replication-competent of mice lacking the a6 , suggesting defective neuroblast chicken retroviral vector RCAS (http://home.ncifcrf.gov/ migration (Georges-Labouesse et al., 1998). avh3 and avh5 hivdrp/RCAS/plasmid.html). The CH-1 (dominant negative) integrins, which act as vitronectin receptors in other cells, and CH-2 (control) coding regions were excised from have also been detected in developing retina (Gervin et al., pBSKS-CH1 and pBSKS-CH2 (from Matvey Lukashev; 1996). Lukashev et al., 1994) using XbaI and XhoI, and inserts Integrin a4 immunoreactivity was observed on undiffer- were ligated between the XbaI and SalI sites of the vector entiated neuroblasts and on RGCs in mouse (Hikita et al., pCla12 (enzymes from Promega, Madison, WI). This step 2003; Sheppard et al., 1994). In chicken retina, integrin a4 was carried out to convert these inserts into ClaI fragments subunit mRNA was detected throughout retinal development that could be conveniently cloned into the RCAS vectors. (Cann et al., 1996), but cells expressing it have not been The CH-1 and CH-2 inserts were then excised with ClaI and determined. The a4h1 heterodimer (also known as VLA4) inserted into the pRCASBPA and pRCASBPB, and is best characterized as a key T-cell receptor for fibronectin orientation was confirmed by digestion with XbaI and ClaI (primarily recognizing the LDV motif within the alterna- using standard molecular cloning methods. tively spliced CS-1 region) and vascular cell adhesion Virus production was carried out in uninfected chick molecule-1 (VCAM-1) that functions during leukocyte embryo fibroblast (CEF-O) cells as described by Morgan extravasation during inflammation (Lobb and Hemler, and Fekete (1996) from viral-free SPF white leghorn 1994). However, it is known to recognize at least nine chicken eggs from Charles River SPAFAS (Preston, CT) to additional ligands: a4h1 itself, osteopontin (OPN), throm- obtain concentrated viral stocks of 106 to 108 per ml. Virus bospondin-1, intercellular adhesion molecule-4 (ICAM-4), was tittered by staining for the viral p19 protein using the junctional -2 (JAM-2), invasin, AMV-3C2 monoclonal antibody from the Developmental ADAM-28, the precursor to von Willebrand factor, and Studies Hybridoma Bank (U. Iowa) as described in transglutaminase C (Altevogt et al., 1995; Bayless and Morgan and Fekete (1996). In some experiments, titers Davis, 2001; Cunningham et al., 2002; Elices et al., 1990; were also determined by staining for CD4 (1:40 rat anti- Guan and Hynes, 1990; Isobe et al., 1997, 1999; Mould et mouse CD4 (BabCo/Covance, Richmond, CA) followed al., 1990; Qian et al., 1994; Spring et al., 2001; Wayner et by 1:800 donkey anti-rat Cy-3 (Jackson Labs, West Grove, al., 1989; Yabkowitz et al., 1993). The a4 subunit can also PA)). pair with h7 to form a receptor for MadCAM, which is The dominant-negative h1 integrin construct was intro- important in lymphocyte homing. Mouse mutants lacking duced into a plasmid expression vector (pGL, Invitrogen, 418 S.T. Leu et al. / Developmental Biology 276 (2004) 416–430

Carlsbad, CA) that also expressed green fluorescent protein NaPO4 pH7 for 4 h at 48C, then cryoprotected by incubation (GFP). The CH1 and CH2 inserts were excised from pCla12 in 20% sucrose in 0.1 M NaPO4 pH7 at 48C overnight. with ClaI (Promega), isolated via gel extraction, and Tissue was embedded in 15% sucrose, 7.5% gelatin in PBS, inserted into the ClaI site in pGL. Plasmid DNA from 10 and 16-Am cryosections were cut using a Leica cryostat and colonies per construct was purified via MiniPrep (Qiagen, mounted on Superfrost Plus slides (Fisher Scientific, Santa Clarita, CA) and cut with EcoRI (Promega) to Pittsburgh, PA). The chicken a4 integrin C-terminal peptide determine forward or reverse orientation of insert. (MNRKESWSFTSGNKDD) was synthesized by the Cal- ifornia Institute of Technology Biopolymer Synthesis and Infection and transfection Analysis Facility and coupled to hemacyanin. Rabbits were immunized to produce antibodies in the UCSB Animal Fertilized White Leghorn chicken eggs (AA Laborato- Resource Center. Affinity purified antibody (4 Ag/ml) was ries, Westminster, CA) were windowed and injected as applied to sections and incubated at 48C overnight. described in Morgan and Fekete (1996). Sterile India ink Sections were also stained with antibodies to the RGC was injected under the embryos and fast green (0.025%) marker islet-1 (mouse anti-chicken 39.4D5; 1:100; Devel- was added to viral stocks to allow visualization. High titer opmental Studies Hybridoma Bank) and the RGC axonal viral stocks (approximately 100 nl) were delivered onto the marker neurofilament (rabbit anti-neurofilament H; 1:1000; anterior neural tube at 36–48 h of incubation (HH stages 11– Chemicon Inc., Temecula, CA; and mouse anti-pig neuro- 12) using pulled glass capillary needles attached to a filament 200; 1:200; Sigma), or with DAPI (1 AM). picospritzer and controlled by a micromanipulator. Poly- Secondary antibodies were donkey anti-rat Cy-3 (1:200; brene (80 Ag/ml) was added to RCAS-B virus to improve Jackson Labs), goat anti-rabbit Cy-3(1:800; Jackson Labs), infection. Survival rate varied from experiment to experi- goat anti-mouse Alexa-488 (1:200; Molecular Probes, ment and depended on the quality of the eggs, but was Eugene, OR). At the ages used in this study, islet-1 has always greater than 50%. been shown to be a specific RGC marker (Austin et al., To quantify cell death, infected retinas were stained with 1995; Pimentel et al., 2000). anti-CD4 and DAPI. Infected patches of retina were identified by CD4 staining and the length of the patch Immunohistochemistry along the inner retinal surface was recorded. DAPI fluorescence was observed to determine the number of Tissue was placed overnight in 10% neutral-buffered pyknotic nuclei within each patch. For each determination, formalin (Fisher Scientific). The fixed tissues were then at least 25 infected patches covering 5–10 mm of the inner placed in an automated tissue processor (Ventana Medical retinal surface were measured. Systems, Tucson, AZ) for further fixation, dehydration, and For transfection of cultured retinal cells, embryonic day 6 paraffin infiltration. A microtome (American Optical Corp., (E6) retinas were dissected as described in Cann et al. Buffalo, NY) was used to cut 4-Am tissue sections. The (1996). Dissociated cells were incubated overnight in F12 sections were placed onto charged, 75-Am cap-gap slides media supplemented with insulin, selenium, and transferrin (Fisher) and incubated overnight at 658C. (ITSE, Invitrogen) and penicillin, streptomycin, and fungi- Prior to staining, sections were hydrated using a series zone (PSF, Invitrogen) in 5% CO2 at 378C on 8-well of xylene, ethanol, and water rinses. Once hydrated, each chambered plastic slides (NalgeNunc Inc, Rochester, NY) section was paired with a blank gap slide (when paired coated with 10 Ag/ml human placental laminin (LN-2) with a blank gap slide, reagents move up the slide by (Sigma, St. Louis, MO), 10 Ag/ml human plasma fibronectin capillary action) and immersed in citrate buffer (0.1 M (Collaborative Biomedical Products, Bedford, MA), 10 Ag/ Citrate pH 5.5). To unmask or retrieve epitopes that were ml purified chicken osteopontin (gift of Dr. Samy Ashkar, altered by fixation, slides were heated by steam for 20 Harvard University), or 10 Ag/ml recombinant soluble min. Once cooled, the slide pairs were loaded onto an human VCAM-1 (gift of Dr. Roy Lobb, Biogen Corp., auto-stainer (TechMate 500, Biotek Solutions, Goleta, CA) Cambridge, MA). Plasmid DNA (0.25 Ag) was mixed with and blocked with blocking buffer (0.01 M phosphate, 1 Al Lipofectamine 2000 (Invitrogen) according to the 0.151 M NaCl, 1% bovine serum albumin (BSA), 4.8% manufacturer’s instructions, added to cells in a volume of normal goat serum, 0.2% Tween 20, and 0.1% Proclin 200 Al, incubated for 5 h at 378C, and then replaced with 300). The C-terminal anti-a4 antibody was applied to the media. After incubation for 2 days, GFP-expressing cells tissue and incubated at 4 Ag/ml with an overnight were viewed in an Olympus IMT2 microscope equipped for incubation (E6 and E12) or for 1 h at 8 Ag/ml epifluorescence and photographed. concentration (P1 and Adult). After several buffer rinses, the tissue was then incubated with a biotinylated goat anti- Immunofluorescence rabbit antibody at a 1:250 dilution (Vector Laboratories, Burlingame, CA). To visualize positive staining, an Embryos (E3–E9) or eyes (E12–postnatal day 1 (P1)) avidin–biotin complex with a horseradish peroxidase were dissected and fixed in 4% paraformaldehyde in 0.1 M enzyme conjugate (Vector Laboratories) was used in S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 419 conjunction with a DAB chromogen (Moss Laboratories, then STE (10 mM Tris–HCL, 0.1 M Tris–HCL, 0.1 M Pasadena, MD) that ultimately produced a brown precip- NaCl, 1 mM EDTA 0.1% Tween 20, pH 8.0). Finally, slides itate. Hematoxylin (Anatech, Battle Creek, MI) was used were treated with RNAse (1:200, 5 min, Roche Laborato- as a nuclear counter stain. Slides were removed from the ries) to digest any single-stranded RNA. instrument, dehydrated, and cover slipped with a perma- After being rinsed in STE, the sections were next nent mounting media. Primary antibody was replaced with immersed in a blocking buffer without goat serum. Once rabbit IgG (Jackson Immunoresearch, West Grove, PA) as drained, they were then blocked using the same blocking a negative control. Specificity of the antibody was buffer but containing 2% goat serum. To visualize the confirmed by blocking with the peptide antigen. A bound probe, sections were immersed in a blocking microscope (Olympus BX-60) with an attached digital buffer (100 mM Tris–HCl, 150 mM NaCl, 5 N NaOH, camera system (Olympus Oly-750) was used to visualize pH 7.5; 2% goat serum) and then incubated with a 1:500 and capture images of stained sections. dilution of sheep anti-DIG conjugated to alkaline phosphatase (Roche Laboratories), followed by BCIP/ In situ hybridization NBT chromogen (Moss Laboratories). The sections were then removed from the instrument and placed in a stop Sections were prepared as described above, rinsed with buffer (1 mM EDTA, 10 mM Tris–HCL, 1 ml Tween 20, 1Â PBS (0.1 M Phosphate, 1.51 M NaCl, 0.1% Proclin 300, 5 N NaOH), rinsed with water, and dried. As a positive pH 6.7), drained, and then digested with proteinase K at a control, sections were stained with a DIG-labeled ribo- 1:40 dilution (Ready to Use Solution-Dakocytomation, probe transcribed from beta-actin (Roche Laboratories). Carpinteria, CA) for 3 min at 378C. Digested slide pairs Images were captured on an Olympus BX-60 as were loaded onto the auto-stainer and washed with a series described above. of buffers including; 1Â PBS, DEPC-treated water, and 2Â SSC (Invitrogen). To help reduce nonspecific labeling, Western blotting slides placed in triethanolamine buffer (0.1 M, pH 8.0) acetylated with 0.25% acetic anhydride for 10 min. Slides The chicken macrophage cell line HD11 was main- pairs were then placed in a final rinse of 2Â SSC. tained in RPMI media supplemented with 10% fetal calf Probes were prepared from 220- and 3-kb fragments of serum (FCS) and 1Â PSF (Invitrogen) at 378Cin5% the chicken a4 cDNA described in Cann et al. (1996) and CO2. Chicken embryonic fibroblasts (CEF) were isolated Kil et al. (1998), respectively. The 220-bp cDNA was from trypsinized (0.25% trypsin for 10 min) E10 chicken subcloned into the EcoRI site of the pBluescript KS+ vector embryos. CEF cells were maintained in F12 media and the 3-kb fragment was inserted into the SmaI site of the supplemented with 10% FCS at 378Cin5%CO2 pBluescript KS-vector (Stratagene, La Jolla, CA). Prior to (Morgan and Fekete, 1996). Retinas from E6 chicken transcription, the plasmid containing the 220-bp fragment embryos were dissected, quick-frozen on dry ice, and was linearized with XbaI and HindIII to generate anti-sense crude membranes were isolated (Bono et al., 1983). and sense templates, respectively. SalI and XbaI were used Proteins from each sample were separated by SDS-PAGE to generate anti-sense and sense templates for the 3-kb and transferred to nitrocellulose for Western blotting. The cDNA. Each transcription was carried out with 1 Agof chicken a4 integrin C-terminal antibody described above linearized template using a (Roche Laboratories, Indian- (1:100) was used to detect the presence of a4 integrins apolis, IN) to incorporate digoxygenin (DIG)-labeled UTP. and a goat-anti-rabbit-HRP secondary was used to visual- RNA probes were precipitated with 0.1 M LiCl and ethanol, ize bands using ECL detection (Amersham Pharmacia characterized by gel electrophoresis, and quantified by a Biotech, Piscataway, NJ). direct detection assay according to the manufacturer’s instructions. In order to achieve optimal probe penetration, Neurite outgrowth the 3-kb probe was hydrolyzed to 200–1000 bp in carbonate buffer (60 mM Na2CO3, 40 mM NaHCO3, pH 10.2) at 608C To assay for a4 integrin-dependent neurite outgrowth, E6 for 40 min. An equal volume of neutralization buffer (200 chicken retinal cells were cultured as described above on mM sodium acetate, 1% acetic acid, pH 6.0) was added to substrates of 5 Ag/ml purified recombinant soluble VCAM-1 stop the reaction. diluted in a 50 mM carbonate buffer, pH 9.5 (Lobb et al., Probes were diluted to 0.2 ng/Al in prehybridization 1994). As controls, cells were incubated on LN-2, plasma buffer (200 Ag/ml salmon sperm DNA, 10 SSPE, and 10Â fibronectin (FN; Sigma), and 3% bovine serum albumin Denhardt’s solution; Sigma) containing 50% formamide. (BSA) in PBS. Live cells were stained with the vital dye Probes were heated to 908C for 5 min, then added to calcein AM (Molecular Probes) and photographed using an sections and placed in a 428C oven for overnight hybrid- inverted Olympus IMT2 microscope equipped with epi- ization. To remove unbound probe, sections were rinsed fluorescence optics (Culley et al., 2001). At least 40 cells with the following buffer series: 2Â SSC, 1Â SSC, 4Â SSC were analyzed for each point. All neurites, regardless of containing 50% formamide, 0.5Â SSC, 0.02Â SSC, and length, were measured. 420 S.T. Leu et al. / Developmental Biology 276 (2004) 416–430

Perturbation of a4 integrin in vivo

The small molecule a4 integrin antagonist was dissolved in 50% PEG and 100 Al was dripped onto or injected into the neural tube opening of 36–40 h chicken embryos in windowed eggs, as described above for viral particles. This molecule is a phenylalanine derivative (similar to the Genentech example in Table 12 of Jackson, 2002) based on the peptide within VCAM-1 that is recognized by a4h1. The antagonist has been shown to be specific for a4 integrins in binding assays using purified integrins and integrin ligands. Specificity is similar to lead peptides and similar compounds that have been reported in earlier studies (Castanedo et al., 2002; Dubree et al., 2002; Jackson, 2002; Jackson et al., 1997). As a control, the same volume of 50% PEG carrier was introduced. The average egg volume was 50 ml, so 100 Al of 40 mg/ml antagonist should yield a total egg concentration of 80 AM, twice that necessary to completely block neurite outgrowth on rsVCAM-1. The small molecule readily diffuses through tissues, so locally high concentrations rapidly dissipate. Over the course of these studies, a total of 120 embryos were treated (50 control, 70 experimental). Any embryos showing signs of necrosis due to dehydration were not analyzed further. Staining for islet-1 was as described above. TUNEL analysis was carried out as described in Sakaietal. (2003), and the number of TUNEL-positive cells for the entire section was determined. At least 36 horizontal sections, from the same depth of section (using the lens as a reference point), for each data point were analyzed.

Results

Perturbation of b1 integrins alters cell shape and increases apoptosis

To investigate the functions of integrins in the develop- ing chicken retina, we expressed a dominant-negative version of the human h1A gene (Lukashev et al., 1994). Fig. 1. Expression of dominant-negative h1 integrin in E6 chicken retinal The CH-1 construct fuses the extracellular domain of an cells reduces neurite outgrowth on laminin, fibronectin, osteopontin, and VCAM-1. Embryonic retinal cells were transfected with the plasmid irrelevant protein CD4 with the transmembrane and h h encoding the CH-1 dominant-negative integrin 1 or CH-2 or pGL control cytoplasmic domain (66 aa) of human 1A. Expression of plasmids (A) and cultured on laminin-2 for 2 days. The percent of GFP- this protein, which does not dimerize with a subunits, expressing cells with neurites (B) and the average neurite length (C) were disrupts integrin signaling by diverting signaling compo- quantified. In panel (D), cells transfected with CH-1 (white bars) or CH-2 nents away from intact heterodimers (Faraldo et al., 1998; (black bars) were cultured 2 days on fibronectin (FN), osteopontin (OPN), N N Lukashev et al., 1994; Oguey et al., 2000). This chimeric or VCAM-1. *P 90% different from control; **P 99%. CMV promoter, h h h cytomegalovirus intermediate early promoter; TM, transmembrane; Cyto, protein inhibits 1 integrins as well as 3 and 5 integrins cytoplasmic; SV40pA, simian virus-40 poly-A addition site. in human and murine cells. A control construct CH-2 lacked the cytoplasmic h1 tail and does not affect integrin function (Fig. 1A). To test for inhibition of integrin function in and C). A similar reduction was seen on OPN, Fn, and chicken retinal cells, the construct was cloned into a GFP VCAM-1 substrates where outgrowth is mediated partially expression plasmid (pGL) and transfected into E6 chicken or in whole by integrin a4h1(Fig. 1D). Neurite outgrowth retinal cells that were cultured on laminin-2. Expression of was not completely eliminated, and adhesion of cells to CH-1 reduced both the percent of cells with neurites and the laminin could still occurs, indicating that this is a bknock- neurite length on laminin-2 by greater than 50% (Figs. 1B downQ of h1 integrins rather than a complete ablation of S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 421 function. Transfection of CH-2 did not affect neurite nearby replicating cells. Infected patches of retina, which outgrowth compared to control cells transfected with the ranged from a few cells to most of the retina, were GFP expression vector pGL. identified by staining for the CD4 portion of the chimeric The CH-1 and CH-2 constructs were inserted into the CH-1 and CH-2 proteins in anatomically analogous replication-competent RCAS retroviral vector (Hughes et sections of retina. Expression was first visible 2 days al., 1987) and expressed in developing embryos. Virus postinfection. Examples of cells infected with RCAS are was introduced at HH Stage 10–11 (36–48 h), just as the shown in Fig. 2. Many of the retinal cells expressing CH- neural tube was closing. Windowed eggs were returned to 1 and CH-2 were similar in appearance, especially after the incubator, and developing retinas were analyzed at short incubations. CH-1-expressing cells contributed to the various times. The RCAS virus will infect replicating cells optic fiber layer (OFL) and to the optic nerve, indicating and viral particles produced can spread the infection to that RGC migration, differentiation, and axon outgrowth

Fig. 2. Expression of dominant-negative h1 integrin in embryonic retina alters cell morphology. Embryos were infected with RCAS-CH-1 or CH-2 (control) viral particles at 36 h and embryos were harvested and analyzed at the times indicated. Cryostat sections were stained with anti-CD4 antibodies to detect infected cells expressing CH-1 and CH-2. Embryonic age and viral constructs used are indicated on the left. Peripheral-most sections are shown on the left and central-most sections are shown on the right, and all sections are oriented with the retinal pigment epithelium (RPE) at the top and vitreous (Vit) and optic fiber layer (OFL) at the bottom. Panels A, R, and S were infected with RCAS subtype B; all others were RCAS subtype A. Autofluorescent cells behind the RPE layer are indicated with asterisks. See text for detailed description. Scale bar = 75 AminA;50Am in B–X. 422 S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 were not completely inhibited. However, irregular mor- notably shorter projections than CH-2-infected cells, which phologies were noted in some CH-1-expressing cells, displayed long processes terminating in endfeet (Figs. 2M especially after longer incubations. and N). At this age, the more central areas have a thick OFL, At E5.5, individual retinal neuroblasts infected with and again, both CH-1 and CH-2 proteins were detected in either CH-1 or CH-2 were observed in the peripheral retina RGC axons within the OFL, although there was more CH-2 with endfeet extended and in contact with both the vitreal immunofluorescence visible (Figs. 2N, O, and P). and retinal pigment epithelium (RPE) retinal borders (Figs. At E8, CH-1-positive cells (Figs. 2Q, R, and S) were 2A, B, E, and F). On occasion, rounded cells (probably harder to find, and areas of infection were generally smaller dividing) were noted at the RPE border, sometimes in pairs than observed with CH-2 (Figs. 2U, V, and W). However, (curved arrows). In more central portions of the retina, there were some exceptions where a larger patch of CH-1 rounded clusters of CH-1-infected cells without extensive infection was observed (Fig. 2T). Again, CH-1 cells had less endfeet were noted (arrowheads, Figs. 2C and D), whereas prominent projections, but CH-1 immunofluorescence was CH-2-expressing cells had more extensive endfeet (Figs. 2G observed in the OFL. In Fig. 2X, a central region of a CH-2- and H). Developing RGCs at the vitreal border of central infected retina showing a thick layer of CH-2-positive RGC retina were noted expressing both CH-1 and CH-2 (arrows, axons in the OFL. Figs. 2D and G). Furthermore, expression of CH-1 and CH- We attempted to follow the fate of CH-1-expressing cells 2 was detected in RGC axons in the OFL. by examining retinas after longer periods of infection. At E6.5, in peripheral retina, CH-1-infected patches were However, as the embryos got older, fewer and fewer CH-1- smaller and CH-1-expressing cells (Figs. 2I and J) had positive cells were observed. At E16, no CH-1 cells could

Fig. 3. Expression of dominant-negative h1 integrin in embryonic retina increases apoptosis. Embryos were infected at 36 h, harvested at E6.5, and sections were stained with anti-CD4 (green, B and E) and DAPI (blue, A and D). Sections from the central retina of CH-1-infected (A–C) and CH-2-infected retinas (D–F) are shown. C and F are merged images of A and B, and D and E, respectively. Pyknotic nuclei per linear millimeter of inner retinal (vitreal) surface, from infected (CD4+) and uninfected (CD4À) regions, is quantified in G. RGC, retinal ganglion cells. Scale bar = 50 Am. *P N 90% compared to CH-2. S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 423 be identified. No abnormalities in retinal histogenesis could be detected in E16 retinas of CH-1-infected embryos. This suggested that cells expressing CH-1 were eliminated during development, or expression was lost. One possible explanation for the loss of CH-1-expressing cells over time was that inhibition of integrins caused cells to lose attachment to ECM and undergo apoptosis. To detect apoptotic cells, embryos were infected, and E6.5 retinal sections were stained for CD4 to detect infected patches and DAPI to detect nuclei. Pyknotic nuclei, indicative of apoptotic cells, were detected more frequently within CH- 1-infected areas (Figs. 3A–C) compared to CH-2-infected areas (Figs. 3D–F). Pyknotic nuclei were common in both the undifferentiated neuroblast layer and in the RGC layer of CH-1-infected patches. Quantification of pyknotic nuclei per linear millimeter of retinal inner surface showed that CH-1-expressing regions contained 5-fold more pyknotic nuclei than CH-2-infected regions. We also carried out experiments where infected retinas were labeled with CD4 to detect infected patches and stained using the TUNEL method to detect apoptotic cells. In CH-1-infected patches, we detected 23.9 F 1.8 TUNEL-positive cells per linear millimeter of inner retinal surface, which is in good agreement with the number of pyknotic nuclei detected (22.9 F 4.7). These data show that expression of CH-1 increased apoptosis of developing retinal cells.

Expression of the integrin a4 subunit in developing retina Fig. 4. a4 integrin is proteolytically processed in E6 retina. A Western blot of a nonreducing gel of membrane extracts from E6 retina, HD11, and CEF cells, probed with the anti-a4-C-terminal antibody, reveals bands at 150 and The specific integrin heterodimers of the beta-1 family 70 kDa. The band at 70 kDa in the retinal extract is likely to be due to that are important in retinal cell survival and morphology proteolysis after R558 (B), which has been characterized in human a4. are not known. One integrin heterodimer that might be Positions of MW markers are shown on the left. involved is integrin a4h1. a4 subunit mRNA has been detected throughout retinal development in the chicken reactivity was observed throughout the developing retina (Cann et al., 1996), but cells expressing a4 have not been (Fig. 5A). Staining is most abundant at this age. At E6–E9, identified. To determine which cells express a4, we newly differentiated RGCs as well as undifferentiated generated antibodies to three different peptides from the neuroblasts express a4 protein (Figs. 5B and C). As chicken a4 sequence. Antibodies were prepared to development proceeds, immunoreactivity is most prominent predicted surface peptides R2 and R3 (Irie et al., 1997) in the RGC layer, where expression persists in the mature P1 and to a C-terminal peptide. All three peptide antibodies retina (Figs. 5D–F). Control sections blocked with the stained the surface of chicken embryo fibroblasts (CEF) peptide antigen (Fig. 5G) or stained with nonimmune rabbit and recognized a 150-kDa band in Western blots. Fig. 4 IgG (Fig. 4H) or with primary antibody omitted (Fig. 5I) shows a Western blot stained with the anti-C-terminal were negative. peptide antibody. The antibody also recognizes a 150-kDa Expression on RGCs was investigated further. To band in membrane extracts of chicken HD11 cells. determine if a4 was expressed on migrating RGCs, However, in E6 chicken retinal extracts, a faint 150-kDa peripheral E6 sections were labeled for the RGC marker band and a stronger 70-kDa band were detected, indicating islet-1 and for a4. Figs. 6A–D show that migrating RGCs that the a4 subunit is proteolytically processed in the with processes spanning the retina express both islet-1 chicken retina, similar to what has been described in and a4. The a4 immunoreactivity was observed on cell human cell lines and tissues (Fig. 4B; Texido et al., 1992). bodies and processes of migrating RGC. Furthermore, a4 Previous studies have shown that the cleaved fragments are can be detected on E6 RGC axons in the optic nerve, held together noncovalently and that proteolysis does not which were identified by double labeling with antibodies affect a4h1 adhesive activity (Bergeron et al., 2003; to neurofilament. Texido et al., 1992). The a4 subunit was also localized in paraffin sections, The C-terminal antibody was used to stain cryosections which yielded better morphology (Fig. 7). Immunoreactivity of developing retina (Fig. 5). At E3, abundant immuno- is observed on neuroblasts and RGCs at E6 and on RGCs at 424 S.T. Leu et al. / Developmental Biology 276 (2004) 416–430

Fig. 5. a4 integrin expression in developing retina. Horizontal cryostat sections from medial retina from E3 (A), E6 (B), E9 (C), E12 (D), E16 (E), and P1 (F) stained with the anti-a4 C-terminal antibody are shown. Sections were taken from either temporal or nasal retina, approximately halfway between the peripheral boundary with the ciliary margin and the central retina. Controls show an E6 section stained in the presence of peptide antigen (G); an E12 section stained with rabbit IgG instead of primary antibody (H), and a P1 section with primary antibody omitted (I). Scale bar = 50 Am.

E12 (Figs. 7A and B) In addition, a4 was detected in the Perturbation of a4 integrins increases apoptosis and thins developing inner plexiform layer at E12. At P1, immunor- the RGC fiber layer eactivity was observed on RGCs and also on cells in the innermost portion of the inner nuclear layer (Fig. 7C). Based To determine what functions a4 integrins might fulfill in on their location and size, these were identified as amacrine the developing retina, we utilized a small molecule anta- cells. Faint immunoreactivity in the OPL was also observed gonist of human a4h1 from Genentech (Jackson, 2002). on occasion (Fig. 7C). However, some OPL immunoreac- First, we tested the antagonist for its ability to block chicken tivity was also seen in peptide block and nonimmune IgG a4h1. Since a4h1 is the only known retinal receptor for controls, and was attributed to nonspecific staining (data not VCAM-1 (Hikita et al., 2003), neurite outgrowth on shown). The RGC fiber layer was only faintly stained in recombinant soluble VCAM-1 was used as an assay of paraffin sections. a4h1 function in chicken retinal cells (Fig. 9). Dissociated To confirm protein expression patterns, a4 mRNA was E6 cells were plated on VCAM-1 in the presence of also localized in developing retina using in situ hybrid- increasing amounts of antagonist. As a control, cells were ization (Fig. 8). At E6, the a4 antisense probe hybridized to plated on LN-2. The antagonist inhibited outgrowth on cells throughout the retina, consistent with protein staining VCAM-1 in a dose-dependent fashion, but did not affect in cryostat and paraffin sections described above (Fig. 8A). outgrowth on LN-2 (Figs. 9I and J). On a substrate of plasma At E12, staining is present in neuroblasts as well as RGCs FN, the antagonist did not significantly affect the percent of (Fig. 8B). The most prominent staining was present in cells with neurites on FN, but did cause a slight reduction in presumptive amacrine cells (arrows, Figs. 8B and C) and in the average neurite length. This result indicates that retinal RGCs (arrowheads, Figs. 8A–C). Both the 220- and 3-kb neurons use other integrin (or non-integrin) receptors for probes gave similar results. outgrowth on FN. S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 425

Fig. 6. a4 integrin is expressed on migrating RGCs and RGC axons. Cryostat sections from peripheral (A–D) and central (E–H) E6 retina were labeled with anti- a4 C-terminal antibody (red, A and E), antibodies to RGC markers islet-1 (green; B) and neurofilament (green; G), and DAPI (blue; C and F). Merged images are shown in D and H. The arrow in A–D indicates a migrating RGC. Panels E–H include the optic nerve head. Scale bar = 25 Am in A–D; 50 Am in E–H.

The antagonist was then introduced into developing that blocked outgrowth in vitro. At E6, embryos were chicken embryos in windowed eggs from E2 to E5. Enough harvested and TUNEL labeled or stained for the RGC antagonist was introduced to achieve final concentrations marker islet-1 or the RGC axonal marker neurofilament

Fig. 7. a4 integrin expression in developing retina. Horizontal paraffin sections from E6 (A and D), E12 (B and E), and P1 (inner portion of retina) (C and F) were labeled with anti-a4 C-terminal antibody (brown; A–C). Sections were counterstained with hematoxylin (blue; A–F). Corresponding peptide block controls are shown in D–F. Examples of a4-positive RGCs are shown indicated by arrowheads, and a putative a4-positive amacrine cell is indicated by an arrow. Outer plexiform layer, OPL; inner nuclear layer, INL; inner plexiform layer, IPL. Scale bar = 100 Am. 426 S.T. Leu et al. / Developmental Biology 276 (2004) 416–430

Fig. 8. a4 integrin mRNA expression in developing retina. Paraffin sections from E6 (A and D), E12 (B and E), and P1 (C and F) were hybridized to the 3KB antisense (A–C) and sense control (D–F) a4 probes. As in Fig. 7, arrows indicate amacrine cells and arrowheads indicate RGCs. Outer nuclear layer, ONL; Scale bar = 100 Am.

(Fig. 10). As a control, embryos were treated with the carrier differences were noted in general appearance or size of the 50% PEG solution. In general, the eyes of treated embryos embryos. However, consistent defects were noted in the had the same gross morphology as untreated eyes, and no mesencephalon, which appeared appreciably smaller in

Fig. 9. An a4 antagonist inhibits retinal neurite outgrowth on VCAM-1. E6 retinal cells were cultured overnight on LN-2 (A and E), rsVCAM-1 (B and F), FN (C and G), or BSA (D and H) in the presence of increasing concentrations of the a4 antagonist and live cells were visualized by calcein-AM staining. Shown are cells incubated in the absence of antagonist (A–D) and in the presence of 40 Ag/ml antagonist (E–H). Note the decrease in outgrowth on VCAM-1 (B vs. D). Percent of cells with neurites and neurite lengths are indicated in I and J. *P N 90% compared to no antagonist; **P N 95%. S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 427

Fig. 10. Inhibition of a4 integrins increases apoptosis. Embryos were treated from E2 to E5 with a4 antagonist (30 mg/ml; B, D, and F) or carrier (50% PEG; A, C, and E) as a control. Embryos were harvested at E6 and horizontal cryostat sections were labeled using the TUNEL method (red; A and B) to identify apoptotic cells and with anti-islet-1 (green; A and B) to identify RGCs. The same regions of peripheral retina from control and experimental retinas are shown, approximately 500 Am from the ciliary margin. Sections from peripheral (C and D) and central (E and F) regions of treated and control retinas were stained with anti-neurofilament to show the optic fiber layer. TUNEL-positive cells per section are quantified in G. Scale bar = 50 Am. embryos treated with antagonist. In the retina, an increased one integrin heterodimer that is important in preventing number of TUNEL-positive cells was observed in retinas apoptosis. In the first part of this study, a dominant-negative that had been treated with antagonist (Figs. 10A and B). human h1 construct was used to perturb integrin function. These apoptotic cells were apparent in both the neuroblast This effect is brought about by expression of a chimeric layer and in the RGC layer. In embryos treated with high protein that combines the extracellular portion of the CD4 concentrations of antagonist, the retinas appeared thinner. protein with the transmembrane and cytoplasmic domain of Staining with antibodies to islet-1 revealed a decreased the human h1 protein (Lukashev et al., 1994). A number of number of RGCs (Fig. 10B), with approximately 50% fewer previous studies have shown that expression of the islet-1-positive cells. Staining with antibodies to neurofila- cytoplasmic domain diverts signaling proteins from normal ment showed a thinning of the RGC fiber layer (Figs. 10D integrins and inhibits their activities, resulting in detachment and F) compared to anatomically equal positions of control and cell death of cultured cells (LaFlamme et al., 1994; retinas. Quantification of TUNEL-positive cells per section Lukashev et al., 1994; Oguey et al., 2000; Smilenov et al., is shown in Fig. 10G. A 3-fold increase in apoptotic cells 1994). Expression of the h1 cytoplasmic domain also was brought about by treatment with 30 mg/ml antagonist. inhibits other h integrins through a transdominant effect (Oguey et al., 2000). In the chicken retina, it is expected that at least eight integrins would be inhibited (see Introduction). Discussion Expression of the dominant-negative chimera in cultured chicken retinal neurons inhibited neurite outgrowth on The results presented here demonstrate an important role laminin by about 50% (Fig. 1). Treatment of cultured for integrins in maintaining cell survival in the developing neurons with blocking h1 antibodies will completely block retina. The a4h1 heterodimer, which is expressed by neurite outgrowth (Cann et al., 1996; Hall et al., 1987), neuroblasts, retinal ganglion cells, and amacrine cells, is suggesting that the dominant negative perturbs but does not 428 S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 completely ablate integrin function. This may be due to possibility that integrins and integrin ligands could also decreased expression of the CH-1 protein by the RCAS regulate the naturally occurring apoptosis. vector compared to other high copy vectors used in previous Which specific integrin heterodimers are important in studies (Lukashev et al., 1994). Nevertheless, this level of preventing apoptosis? One candidate is a4h1. This integrin is perturbation had significant effects on retinal cells in vivo. known to fulfill important functions in the immune system, Infected cells that expressed the dominant-negative CH-1 but its activities in neural cells are just becoming apparent. a4 protein showed morphologies that were different than mRNAwas detected in chicken retina some time ago (Cann et controls. This was especially noticeable after longer periods al., 1996). However, in earlier attempts, we were unable to of expression. Some, but not all, CH-1-expressing retinal detect a4 using an antibody to the C-terminus of human a4. neuroblasts lacked the long processes that normally extend Sequence analysis of the chicken a4 homologue showed that to the retinal borders. Such cells were not observed with any the chicken C-terminus is only 50% similar to human (Kil et frequency in CH-2-expressing areas, where more orderly al., 1998). We generated specific antisera to synthetic chicken columnar arrangements of neuroblasts were detected. Such a a4 peptides and showed that this integrin subunit is difference could not result from sectioning artifacts, since expressed by neuroblasts, RGCs, and amacrine cells. results were reproducible in multiple animals at multiple Interestingly, immunoreactivity was detected on migrating ages. Care was taken to process CH-1- and CH-2-infected RGCs that had not yet reached their final destination at the embryos in the same way and to analyze sections from vitreal border. RGCs are known to express several specific anatomically equivalent retinal areas. Although CH-1 markers of differentiation before reaching their final desti- expression altered morphology, cells expressing the CH-1 nation (McLoon and Barnes, 1989). a4 was also detected on protein were able to extend some processes and RGCs could RGC axons in the developing optic fiber layer and in the optic migrate and extend axons. Although fewer RGC axons were nerve. The expression pattern, confirmed by in situ hybrid- positive for CH-1 immunofluorescence compared to CH-2, ization experiments, is similar to that observed in developing this could have been due to the decreased number of mouse retina (Hikita et al., 2003). infected cells. The lack of obvious defects in some CH-1- The expression pattern of a4 suggests that a4 integrins expressing cells may be due to low levels of expression could potentially be involved in early stages of neuro- brought about by the viral vector. genesis, such as neuroblast proliferation, migration, and The second significant effect of CH-1 expression was determination of cell fate. They could also play a role in increased apoptosis. CH-1-infected areas contained more axon and dendrite formation by RGCs. To address a4 pyknotic nuclei throughout the width of the retina. function, we took advantage of the accessibility of chicken Apoptosis induced by loss of integrin function in cultured embryos and introduced a potent small molecule antagonist cells is well characterized (Stupack et al., 2002). This may into windowed eggs. The most striking effect was increased explain why we could not detect CH-1-infected cells later in apoptosis, detected by TUNEL labeling. We did not observe development. These cells may have simply failed to survive any general effects on the vasculature, and since the chick and were eliminated. Our results are similar in some ways to retina is avascular, effects of the antagonist are unlikely to other knock down studies of h1 integrins. For example, in be due to indirect effects on blood supply. The simplest the developing chicken tectum, Galileo et al. (1992) explanation of the data is that inhibition of retinal a4h1 observed a similar disappearance of clones expressing h1 increases apoptosis. Since the TUNEL method only detects antisense from a replication-incompetent virus. Skeith et al. a short window of apoptosis, the actual effects may be (1999) observed decreased clonal size in retinal cells greater. In some of the most affected embryos, as much as a infected with the same retrovirus. 50% reduction in RGCs was observed. Our results indicate Apoptosis is an important regulatory device in the that a4h1 integrins are important in maintaining survival of developing retina as it is thought to sculpt the size and some, but not all, developing retinal cells. placement of neurons (Bahr, 2000; de la Rosa et al., Many questions remain. First, the ligands recognized by 2000). Two periods of cell death have been observed, one a4h1 in retina are not completely characterized. Hikita et al. early during neurogenesis (E5–7 in chicken) and another (2003) recently showed that osteopontin, expressed by that coincides with tectal synapse formation (E15–18). RGCs, has a novel neurite outgrowth-promoting activity The latter has been traced in part to neurotrophin that is mediated in part by a4h1. However, there are likely dependence and elimination of RGCs that have failed to to be additional ligands. Second, the exact mechanism of make proper connections (Bovolenta et al., 1996; de la cell death caused by integrin-perturbing reagents is not Rosa et al., 1994). The early phase of apoptosis is less understood. This could be a result of aberrant integrin- well understood. One physiological reason for some early dependent migration or adhesion, or improper matrix/ cell death is to make way for RGCs turning into the growth factor localization. Third, the surviving RGCs in retina to form the optic nerve head (Cuadros and Rios, embryos treated with the a4 antagonist had normal looking 1988; Martin-Partido et al., 1988). This early cell death axons, within the retina, but the more central projections of appears to be regulated in part by insulin-like growth these axons have not yet been analyzed. Finally, a4 may be factor (Diaz et al., 1999, 2000). Our results raise the important in other parts of the CNS. We observed that S.T. Leu et al. / Developmental Biology 276 (2004) 416–430 429 embryos treated with the a4 antagonist had consistently Bradshaw, A.D., McNagny, K.M., Gervin, D.B., Cann, G.M., Graf, T., smaller mesencephalons. Further studies will be needed to Clegg, D.O., 1995. beta 1 mediates interactions between developing embryonic retinal cells and collagen. Development address a4 functions in the brain. 121, 3593–3602. Cann, G.M., Bradshaw, A.D., Gervin, D.B., Hunter, A.W., Clegg, D.O., 1996. Widespread expression of beta1 integrins in the developing Acknowledgments chicken retina: evidence for a role in migration of retinal ganglion cells. Dev. Biol. 180, 82–96. Castanedo, G.M., Sailes, F.C., Dubree, N., Nicholas, J., Caris, L., Kevin, We thank Marianne Bronner-Fraser, in whose lab a C.K., Susan, K.S., Chiu, H., Fong, S., Marsters, J., Sutherlin, D.P., portion of this work was carried out while DOC was on Jackson, D.Y., 2002. 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