Palm is expressed in both developing and adult mouse lens and retina
© Castellini et al; licensee BioMed Central Ltd. 2005
Received: 01 March 2005
Accepted: 21 June 2005
Published: 21 June 2005
Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6.
The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR.
In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens.
Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation.
The retina and lens form from the neural tube and head ectoderm respectively. Despite these different origins, the development of the mature eye requires mutually inductive interactions between these two cell layers . Further, in many cases, the lens and retina express the same developmentally important transcription factors [2–6]. In addition, a number of studies have identified the expression of proteins with known roles in neuronal function in the lens [7–12] and proteins important in lens function in the retina [13, 14]. This may partially be due to the need of both retinal neurons and lens fiber cells to develop elaborated plasma membranes for their function [15–17].
Pax6 is a paired and homeodomain containing transcription factor that is required for the formation of the lens placode from the head ectoderm . Specific loss of Pax6 expression from retinal progenitor cells results in the conversion of all retinal cell types to amacrine interneurons  and lens epithelial cells heterozygous for a Pax6 mutation preferentially differentiate into lens fiber cells . Overexpression of the canonical form of Pax6 in lens fiber cells (Pax6 con transgenics) results in cataracts typified by incomplete lens fiber cell elongation and denucleation, instability of the transcription factor c-Maf and a drastic downregulation of βB1-crystallin expression  while overexpression of the Pax6 (5a) splice form also results in cataracts without the changes in cMaf stability . Microarray analysis was previously performed on lenses from both Pax6 (con) transgenics and mice heterozygous for a Pax6 null allele and 13 genes were found to be upregulated in the transgenics and downregulated in the heterozygous knockout mice .
One of these genes, paralemmin (Palm), encodes a protein present at the plasma membrane in axons, dendrites and perikarya of differentiating neuronal cell lines, and at high levels in the processes of the cerebellar molecular layer . Further, this gene is downregulated in lenses overexpressing the Pax6(5a) splice variant  and the protein is detected in lens cells from both mice and chickens [25, 26]. Overexpression of Palm in both neuronal and non-neuronal cell lines initiates the expansion of the plasma membrane and the development of extended processes and microspikes which is dependent on Palm targeting to the cytoplasmic face of the plasma membrane via a palmitoyl group covalently linked near the protein's C-terminus [24, 27].
Here we investigate the distribution of Palm in the developing lens and retina, and compare its mRNA levels with two other members of the paralemmin family, paralemmin-2 (Palm-2) and palmdelphin/paralemmin-like (PalmD) [28, 29].
All experiments using animals were approved by the both the University of Delaware and Albert Einstein College of Medicine Institutional Animal Care Committees and conform to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. C57Bl/6 mice were generated in-house from breeding stock obtained from Harlan Sprague Dawley (Indianapolis, IN). CD-1 mice were obtained directly from Charles River Laboratories (Wilmington, MA). Embryonic mice were staged by designating noon of the day on which a semen plug was observed in the dam as 0.5 days post-coitum (dpc). Postnatal mice were staged by designating the day of birth as 1 day postnatal (DPN). All mice were maintained in a 12-hour light/dark cycle at 21–24°C and were given food and water ad libitum.
Immunofluorescent detection of Palm in tissue sections
Palm was detected by indirect immunofluorescence following the protocol previously described . Briefly, tissue or embryos were excised from C57Bl/6 mice, embedded in tissue freezing media (TFM, Triangle Biomedical Sciences, Durham, NC) and sectioned at 16 μM on a Leica CM 3050 S Cryostat (Leica, Deerfield, IL). Sections were mounted on Colorfrost-plus™ slides (Fisher Scientific; Pittsburgh, PA), fixed in ice-cold acetone:methanol (1:1 vol/vol) for 15 minutes, dried and blocked with 1% BSA in phosphate buffered saline (PBS), pH 7.4. The blocking solution was removed and the sections incubated with a 1:150 dilution of rabbit polyclonal anti-Palm antibody  in 1% BSA-PBS for one hour at room temperature. The bound primary antibody was detected with AlexaFluor 568 goat anti-rabbit IgG (Molecular Probes, Inc. Eugene, OR) and cell nuclei were detected by counter-staining with TO-PRO-3 (1:3000 dilution in 1% BSA-PBS; Molecular Probes, Inc). Negative controls consisted of parallel staining experiments that omitted the primary antibody. Images were captured on a Zeiss LSM 510 Confocal Microscope configured with an Argon/Krypton laser (488 nm and 568 nm excitation lines) and Helium Neon laser (633 nm excitation line)(Carl Zeiss Inc, Göttingen, Germany).
Transfections and reporter assays
Four copies of the Pax6-binding site previously identified in the human PALM promoter  were cloned into E4TATA-pGL3  using a synthetic double stranded oligonucleotide 5'-ctagGGCTACTTTCACTCTGCGATGGCAGAGCAGGGCTACTTTCACTCTGCGATGGCAGAGCA-3'. Nucleotides containing Pax6-binding sites are in bold and nucleotides used for subcloning are indicated by lower case letters. Transient transfection assays were performed in 293T cells, which do not express endogenous Pax6 proteins, as described earlier .
Immunofluorescent detection of Palm in cultured chick retina
Fertile White Leghorn eggs were obtained from the Department of Animal and Food Sciences at the University of Delaware and kept in a humidified, forced-draft incubator until embryonic day (E) 7. Retinas were dissected in calcium and magnesium-free saline solution (CMF). The neural retina was separated from the pigmented epithelium with fine forceps. The neural retina was minced with fine scissors and incubated in 0.25% trypsin in CMF for 20 minutes at 37°C. Retinas were dissociated into single cells by trituration with a Pasteur pipet in a 0.3 mg/ml soybean trypsin inhibitor/ 0.03 mg/ml DNaseI in Medium 199 (Cellgro, Herndon, Virginia). Cells were plated at a density of 5 × 105 cells / 12 mm diameter round glass coverslip in wells of a 24-well plate in one milliliter of Medium 199 (Cellgro) supplemented with 10% fetal bovine serum. Retina cultures were kept in a standard humidified culture incubator with 5% CO2.
Two days or one week after plating, cultures were fixed in 1% paraformaldehyde in PBS pH 7.4 for 30 minutes and then rinsed in PBS. Cells were then incubated for approximately 1 hour in a mixture of 1:200 rabbit polyclonal anti-chicken Palm  and 1:2 mouse monoclonal anti-neurofilament (RT-97) hybridoma supernatant (Developmental Studies Hybridoma Bank, Iowa City, IA; [33, 34]) in PBS supplemented with 5% normal goat serum (NGS) and 0.03% Triton X-100 (TX-100). Cultures were rinsed in PBS and then incubated for approximately 1 hour in a mixture of 1:200 Alexa 488-goat anti-rabbit and 1:200 Alexa 594-goat anti-mouse secondary antibodies (both from Molecular Probes, Inc., Eugene OR) in the PBS/NGS/TX-100 mixture. Cultures were then rinsed in PBS and coverslips were mounted on glass slides in a buffered glycerol mounting medium containing ρ-phenylenediamine to retard photo-bleaching. Cultures were observed and photographed using a Nikon Microphot FX epifluorescence microscope equipped with a Nikon DXM-1200 CCD camera. Red and green channel images were merged using Adobe Photoshop.
Real Time RT-PCR
Tissue microdissected from the lens, cerebellum and telencephalon was stored in RNA later (Qiagen, Valencia, California). Total RNA from the lens, cerebellum and forebrain of newborn CD-1 mice was isolated using the RNeasy Protect Mini Kit (Qiagen). Retinal P0, P4 and P22 RNA was kindly provided by Drs. Mike Dorrel and Kenneth Mitton, respectively. DNaseI digestion was performed during RNA isolation with RNase-Free DNase Set (Qiagen). The RNA was quantified with an Agilent 2100 Bioanalyzer and first strand cDNA was then synthesized using 5 μg of RNA, Oligo(dT)12–18 primer and Superscript II RT (Invitrogen, Carlsbad, California) as per manufacturer's instructions. The cDNA was diluted 1:10 and PCR reactions were conducted using 2 μl of cDNA, 50 nm of forward and reverse primers, and 2X SYBR Green PCR Master Mix (Applied Biosystems, Foster City, California). Amplification of the cDNA was performed using a 7900 HP Applied Biosystems Real Time PCR machine. The cDNA was initially denatured at 94°C for 5 minutes, followed by 45 cycles of 94°C for 10 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 30 seconds. A final extension at 72°C for 5 minutes was then conducted. Each gene was amplified nine times (three times as triplicate experiments). The primers used with Ensembl or NCBI accession numbers follow: Palm (ENSMUSG00000035863) (5' -AGCAGGCAGAGATTGAGAGC-3' and 5' -AGCCAGCGTTCCCTCAGT-3'); Palm2 (NM 172868) (5' -CGCAGGCAGTCTGAAGAAG-3' and 5' -TTTCGAGCGCTTGTATTTCC-3'); PalmD (ENSMUSG00000033377) (5' -AGTAGCTGGAGACGGGACTG-3' and 5' -CACGGCTCTCAGATCACCTT-3'). The housekeeping genes β2-microglobulin, B2M (ENSMUSG00000033376) (5' -TGGTGCTTGTCTCACTGACC-3' and 5' -TATGTTCGGCTTCCCATTCT-3'); Hypoxanthine-guanine phosphoribosyltransferase, HPRT (ENSMUSG00000025630) (5' -GTTGTTGGATATGCCCTTGA-3' and 5' -GGCTTTGTATTTGGCTTTTCC-3'): and succinate dehydrogenase, SDHA (ENSMUSG00000021577) (5'-GAGGAAGCACACCCTCTCATA-3' and 5' -GCACAGTCAGCCTCATTCAA-3') were used for normalization of gene expression levels. Each primer set was designed using Primer3  and specificity verified by NCBI Blast . Standard PCR was then performed to verify amplification of a single PCR product bearing the correct size. The dissociation curve of each PCR amplicon was analyzed using ABI PRISM SDS 2.0 and revealed a single peak, indicating specific PCR amplification .
The mRNA levels were normalized to the internal housekeeping gene, B2M and the change in Ct values for each gene (ΔCt) were determined according to the standard method [38, 39]. The standard deviation calculated for each sample was less than 5% and was therefore not shown in Figure 5. The primers used had similar efficiencies for amplification as determined by serial dilution experiments .
Results and discussion
Previously, we determined that Palm gene expression is downregulated in lenses from mice lacking one copy of the Pax6 gene  and upregulated in lenses overexpressing Pax6 . Since potential Pax6 binding sites were identified upstream of the transcriptional start site of Palm , Palm may be a direct Pax6 target gene. Thus, we undertook a developmental expression study of Palm in the eye to assess the extent that its expression overlaps that of Pax6.
In neuronal cell lines, Palm was previously detected at the cell membrane of the cell body and developing axons as well as in a granular localization intracellularly. In vivo, Palm co-purifies with chick brain synaptic plasma membranes consistent with its palmitoylation . While the staining pattern of Palm in the developing mouse retina is consistent with this membrane localization, we wanted to confirm this in dissociated retinal cultures. The neural retina of the E7 chick is at a period of extensive neurogenesis, migration, and process formation in vivo, especially of ganglion cells [51–53]. This ability to extend neurites is also manifest in cultures made from this age retinal tissue [54, 55]. Chick retinas were dissociated, plated and stained for Palm either 2 days or 7 days after plating. After 2 days in culture (Figure 5A–C), Palm appears expressed by most cells and is evident at the plasma membrane and as intracellular puncta. Fine processes resembling axons (arrows) that sometimes stain with the anti-neurofilament antibody RT-97  are also positive for Palm immunoreactivity. After 7 days in culture (Figure 5D–F), Palm staining appears punctate but more diffuse in the cell body, and does not appear to be localized on the numerous long processes stained for neurofilament. Thus, like in the mouse retina in vivo, Palm is detected in retinal cultures undergoing active process formation while it is less evident in mature cells, which are undergoing less process extension.
Palm is a member of a multigene family consisting of two other family members, paralemmin-2 (Palm-2) and palmdelphin/paralemmin-like (PalmD/PalmL) [28, 29]. Palm2 shares 37% amino acid identity with Palm and like Palm has a C-terminal CaaX motif that could potentially be prenylated. However, the Palm2 gene is alternatively spliced and not all variants contain the prenylation motif. PalmD is 23% identical to Palm but generally lacks a C-terminal prenylation motif although rare splice variants have an alternative C-terminus containing a prenylation motif similar to Palm. Experimentally, the majority of PalmD is cytoplasmic and does not co-purify with plasma membrane fractions [28, 29]. Since Palm is potentially able to modulate plasma membrane growth in the lens, retina and brain, while Palm2 and PalmD are of related sequence, we performed quantitative rt-PCR to compare the relative expression levels of all three paralemmin family members in the lens, retina, cerebellum and forebrain.
The ratio between the housekeeping genes tested, B2M, HPRT and SDHA, in the different tissues analyzed was found to range between 0.98–1.04. Since the ratio of an ideal internal control between various tissues would be 1 and the variability of each of our internal normalizing genes between the various tissues assayed was low, we normalized our data to one housekeeping gene, B2M [39, 56].
The lens and retina express paralemmin during development with its transient upregulation during the formation of optic nerve and formation of both plexiform layers. Further, the putative PALM promoter contains a functional Pax6 binding site and the developmental expression pattern of Palm in the eye generally correlates well with that reported for Pax6, leading credence to the idea that Palm is a Pax6 directly-regulated gene.
days post coitum
phosphate buffered saline
inner nuclear layer
outer nuclear layer
outer plexiform layer
inner plexiform layer
retinal pigmented epithelium.
We thank the staff of the Albert Einstein College of Medicine Biotechnology Center for the qPCR analysis, Dr. Jean Hebert of AECOM for helpful suggestions. Dr. Harry Maisel for the anti-chick PALM antibody, Drs. M. Busslinger and R. Maas respectively for the Pax6 and PAX6(5a) expression vectors and Dr. Kirk Czymmek of the University of Delaware Core Imaging Facility for confocal microscopy support. This work was funded by National Eye Institute grants EY015279 and EY012221 to MKD and EY12200 and EY14237 to AC; National Institute of Neurological Diseases and Stroke grant NS40317 to DSG and INBRE program grant P20 RR16472 supporting the University of Delaware Core Imaging facility.
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