Screening genes related to development and injury of the mouse optic nerve by cDNA microarrays

The aim of this study was to screen genes related to the development and injury of the mouse optic nerve so as to provide possible target genes for gene-engineering therapy of central nervous system (CNS) injury. Gene expression was profiled by cDNA microarrays in the mouse superior colliculus at 8-time points during the development or following injury of the optic nerve; consequently, 1,095 highly expressed genes (ratio ≥2) were identified. Then, these genes were categorized functionally; there were 561 genes (51.19%) with unidentified functions and 534 genes (48.81%) with identified or partially identified functions. After discounting the overlapping genes, 486 genes with identified or partially identified functions were categorized into 17 functional groups. The 17 functional groups were as follows: I transcription regulation, II signal transduction, III protein synthesis, IV materials transporting, V RNA processing, VI metabolism-related genes, VII cell cycle or apoptosis-related genes, VIII extracellular matrix, IX protein folding and degradation, X cytoskeleton, XI histone metabolism, XII nervous system specific functional genes, XIII tumor related genes, XIV DNA replication and repair, XV axon growth and guidance, XVI immune response, and XVII cell adhesion. These genes may play key roles in the development, injury, and repairment of the optic nerve.

cDNA microarray, Optic nerve, Development, Injury

The relationship between CNS development and injury is spectacular in present research. The proliferation, differentiation, migration, axon growth, and orientation of nerve cells are prominent during the embryonic stage. Tempo-spatially specific expression of many development-related genes is the molecular basis for the normal structure and function of the central nervous system (Jain 2009; Koso et al. 2007; Vilpoux et al. 2006). These development-related genes either have limited expression during adulthood or are not expressed at all during adulthood. It has been found recently that some development-related genes are re-expressed following injury in adulthood. Hence, it is believed that some development-related genes participate in CNS injury and self-repair (Dalton et al. 2009; Minor et al. 2009; Brustle 1999). We postulate that CNS regeneration can be promoted by using gene-engineering technology that introduces the genes which are expressed during optic-nerve development and helps regeneration by suppressing the expression of genes that prevent regeneration. Accordingly, through well-rounded research, revealing the genes that are related to CNS development and injury is imperative to gain better understanding of the genes that are crucial for the development, injury, and regeneration of the CNS. Optic-nerve transaction or eyeball enucleation has long been used as a model for studies on CNS injury and regeneration (Ananthakrishnan et al. 2008; Dunlop et al. 2007). Many studies show that the superior colliculus (SC) undergoes a prominent morphological and molecular reaction after monocular enucleation. Therefore, we supposed that the indirect lesion in the SC caused by monocular enucleation might be used as a model for research on CNS injury. Compared with conventional gene detection methods such as in situ hybridization and RT-PCT, cDNA microarray technology has many advantages such as being simple, automatic, applicable to a number of mRNA sequences, and of high detection efficiency. Some crucial genes involved in the brain development, such as those related to the proliferation, differentiation, axon growth, and guidance of nerve cells, are silenced during adulthood. Thereby, it is difficult to detect these genes by conventional tissue-derived expression profiling chips. Using mouse embryonic tissues, obtained during the peri-implantation period, mesonephrois and genital glands of 12.5-day-old mouse embryos, and neonatal mouse ovary tissues, Tanaka et al. constructed a developmental cDNA library that included 15,264 genes (Tanaka et al. 2000). We prepared developmental cDNA library gene chips on the basis of this library to detect gene expression of the mouse SCs at various developmental stages of the optic nerve and at various time points after the removal of a unilateral eyeball. Our aim was to screen the genes related to neural development and injury and the genes contributing to CNS regeneration. This study may help us better understand CNS regeneration.

Groups of Animals Pure Kunming mice were provided by the Experimental Animal Center of the university. Fifteen-day-old and 18-day-old embryos (E), 0- and 7-day-old postnatal (P) mice, and mice 1, 7, 14, and 21 days after injury (I) were used in the study (n = 20). There were 150 35-day-old adult Kunming mice in the control group (male, 75; female, 75). Specimen Collection The mouse SCs of the developmental groups were harvested from E15 (E0 refers to the time when a vaginal plug was observed), E18, P0, and P7 mice. Of the injury groups, mice’s left eyeballs were removed while the mice were under ether anesthesia. These mice were killed 1, 7, 14, and 21 days after the operation, and then their superior colliculi were also harvested, respectively. Total RNA Extraction The tissue piece was minced and homogenized. Tripure was added (7 ml/gram tissue), and the mixture was allowed to stand for 10 min. Then, trichloromethane was added (0.2 ml/ml Tripure), mixed vigorously for 40 s, and allowed to stand for 15 min. The mixture was centrifuged at 4°C, 12,000g for 15 min. The supernatant was collected and isopropanol was added (0.5 ml/ml Tripure). The mixture was shaken and allowed to stand for 15 min and then was centrifuged at 4°C, 12,000g for 15 min. The pellet was washed three times with 75% ethanol. After the evaporation of ethanol, the pellet was dissolved in 50-μl RNA dissolvent. For each group, 1 μl of the resulting solution was subject to ultraviolet spectrometric quantification and electrophoresis. cDNA Microarray Hybridization and Detection Developmental library cDNA microarrays were prepared by the Department of Biochemistry of City University of Hong Kong, China. Each chip contained 7,680 mouse developmental cDNAs. Three chips were repeatedly used for each sample. The chips were processed as follows. Reverse Transcription Hundred micrograms of total experimental sample RNA and 100 μg of total control RNA were dripped into two centrifuge tubes, respectively, which was followed by the addition of 6-μl oligo dT, 0.75-μl RNAase inhibitor, and DEPC water (final volume 30 μl). The tubes were incubated at 70°C for 10 min then submerged in an ice bath for 10 min, which was followed by the addition of 12 μl 5× buffer, 6 μl 10× lowT dNTP, 6-μl DTT, and 3-μl SS-II reverse transcriptase. Three microliters Cy5-labeled dUTP was added to the experimental sample, and 3-μl Cy3-labeled dUTP was added to the control sample. Reverse transcription and labeling was carried out at 42°C for 2 h. Then 15 μl of 0.5 M NaOH/0.1 M EDTA was added to each sample. Then the samples were incubated at 65°C for 10 min to hydrolyze remnant RNA, and finally, the samples were neutralized with 15 μl of 0.5 M HCl/1 M Tris. Wash The experimental sample and control sample were mixed and applied to a YM-30 centrifuge column. Centrifugation at 12,000g for 4 min followed. Four hundred and fifty microliters of TE buffer was added. Centrifugation at 12,000g for 12 min followed. The procedure was carried out three times. Then, the column was centrifuged at 1,000 rpm for 3 min to collect the labeled cDNA. The resulting solution was subject to vacuum drying, and the final volume was adjusted to 31.5 μl. Hybridization We added 13.5 μl of the blocking solution and 0.45 μl of 10% SDS to the sample mixture. This was, followed by denaturation at 100°C for 2 min and rapid cooling in an ice bath. The mixture was spotted onto the chip, and the chip was mounted and incubated at 65°C for 16 h in a humid box. Following hybridization, the chip was rinsed twice in 2× SSC/0.1% SDS and 0.2× SSC at 37°C for 15 min. After dried, the chip was scanned under a laser confocal scanning microscope. Data Processing and Screening The scanned images were processed using GenePix 4.0 software (http://www.moleculardevices.com/pages/software/gn_genepix_pro.html) to obtain the fluorescence intensity data of the hybridization blots, and the data were then standardized. All genes with a ratio ≥2 were screened as highly expressed genes. Then, highly expressed genes were categorized according to their functions described by GENEBANK (http://www.ncbi.nlm.nih.gov/Genbank/) and GENEKARD (http://www.genecards.org/).

Scannned cDNA Microarrays Results Cy5-labeled cDNA of the experimental groups and Cy3-labeled cDNA of the control group were hybridized to the probes on the chips. If the expression of a gene was higher in the experimental group than in the control group, the Cy5 signal intensity would be higher than the Cy3 signal intensity on the chip, as indicated by a red blot on the chip. Otherwise, the blot on the chip would green. If the Cy5 signal intensity was equivalent to the Cy3 signal intensity, the blot would be yellow. cDNA microarray results of the parts scanned are shown in Fig. 1. cDNA Microarrays Hybridization Data Processing The scanned images under laser confocal scanning microscope were converted by GenePix 4.0 software to data, i.e., the background and optical intensity of CY3 and CY5 fluorescence of each hybridization blot. Three chips were repeatedly used for each of the eight samples, and each chip contained 7,680 hybridization blots. For a single blot, if the fluorescence intensity of both Cy3 and Cy5 was higher than 200, or that of Cy3 or Cy5 or both was higher than 800, the gene labeling was valid. The cy5/cy3 ratios of the same sample on the three chips were averaged. Of the 3,604 blots, valid data were obtained at no less than one-time point. The valid data were automatically standardized by GenePix 4.0 software, and genes with a ratio ≥2 were defined as highly expressed genes. Of the 3,604 valid genes detected, 1,095 genes were highly expressed at no less than one time point. There were 560 genes highly expressed only during optic-nerve development, 406 genes highly expressed only following injury, and 119 genes highly expressed during both development and following injury. Functional Retrieve of the Highly Expressed Genes The 1,095 highly expressed genes (ratio ≥ 2) were categorized functionally according to the gene databases NCBI GenBank and GeneCards. There were 561 genes (51.19%) with unidentified functions and 534 genes (48.81%) with identified or partially identified functions. After discounting the overlapping genes, 486 genes with identified or partially identified functions were categorized into 17 functional groups. These groups are as follows: I transcription regulation, II signal transduction, III protein synthesis, IV materials transporting, V RNA processing, VI metabolism-related genes, VII cell cycle or apoptosis-related genes, VIII extracellular matrix, IX protein folding and degradation, X cytoskeleton, XI histone metabolism, XII nervous system specific functional genes, XIII tumor related genes, XIV DNA replication and repair, XV axon growth and guidance, XVI immune response, and XVII cell adhesion. Then, all functional groups were ranked according to the number of genes. 1. There were 227 genes only highly expressed during optic-nerve development, the functional distribution of which is shown in Fig. 2. The gene names and functional groups they belong to are as follows: I: Pcbp4, Ruvbl2, Hmga2, BG073146, RasGAP, BG072075, Tcfubf, BG071570, BG072261, Zfp238, Fstl, BG075630, Tcfl1, Tcea1, Usf1, E2f6, Idb3, Trim28, Csda, BG069902, BG070520, BG070297, Mkrn1, Nasp, Trp53, BG075341, Nfix, BG073938, BG070980, BG070656, D6Wsu147e, Hmgb1, Bzw1, BG074358, Bcap37. II: Macs, Meis1, BG074554, BG069869, Ptprs, RasGAP, Ulk2, BG074999, BG073268, Basp2, BG074531, Ddah2, BG075453, BG073777, Nr2f2, Cdkn1c, BG073596, BG072686, BG075889, BG072821, BG075428, BG074086, BG076018, BG075693, BG072837, Anxa2, Ran, BG072299, BG074161, Rab11b, BG072068, Avpr1a, Bcap37, BG074050, Apbb2. III: BG070206, BG073247, BG074873, BG072542, BG074285, BG072863, BG074798, BG073074, BG073619, BG074035, BG075670, BG075060, BG072547, BG072993, BG075946, BG073693, BG074574, BG075161, BG070597. IV: BG075876, Ap2m1, BG075546, BG072959, BG074733, LOC227683, BG072859, BG070707, BG075712, BG073876, BG075627, BG073650, BG074367, BG074636, 2510027N19, BG072299, BG074087, BG075690, BG073116. V: BG072173, BG073807, BG074524, BG074108, BG075142, BG075863, BG074984, BG075645, BG072533, BG074779, BG070353, BG074041, BG069999, BG072225, BG073254, BG072534, BG073536. VI: BG070015, BG075362, LOC227683, BG070867, BG073732, BG070116, BG073164, LOC228572, Idh1, 2510027N19, BG075693, BG072884, BG074849, BG075171, BG074463, BG071682. VII: Ccnd2, BG074638, BG072956, Cdkn1c, Cenpa, Cdc42, BG073680, Trp53, Pten, Cdkl2, BG072077, Fyn, BG070529, Erh, Mcmd6. VIII: BG073735, BG073709, BG074718, BG073227, BG072998, BG074668, BG073716, BG074327, BG072504, BG073196, BG073809, BG075779, BG072787. IX: Psma4, Pcdhga1, BG074847, BG075782, BG073680, BG074012, BG073636, BG076036, BG073415, BG073146, BG071935, BG071408. X: BG070612, Tubb5, Tuba1, LOC192176, BG074797, Tuba6, BG071120, BG074513, BG074802, Odf2, BG072752, BG074030. XI: BG070156, BG073485, BG075175, BG074103, BG074072, BG075176, BG072755. XII: Hn1, BG074194, Basp2, Cpd2, LL5 protein, Meis1, BG075643, Basp2, Pafaha/Lis1 exon 3. XIII: BREAST-CANCER SUPPRESSOR CANDIDATE 1, Maged2, BG075630, Trp53, Lag. XIV: BG075289, BG074767, BG072968, BG074189, BG075357. XV: EFNB3, Ptn, Nrp, Dbn1. XVI: BG073051, BG075468. XVII: 1110007F2. 2. There were 202 genes highly expressed only following injury, the functional distribution of which is shown in Fig. 3. The gene names and functional groups they belong to are as follows: I: Nfe2l1, Nfatc4, BG075892, Pfdn5, BG071317, Fosb, BG073276, BG075741, BG075451, Zdhhc7, Sirt7. II: BG072300, BG075384, BG070958, Adcyap1r1, Fkbp3, BG071826, BG075111, Map2k1ip1, Cdk4, BG070721, Rab9, Arhgap6, BG074345, BG075975, Csnk2b, Gng3lg, BG076202, BG074454, BG076011, BG075972, BG074892, BG071544, Dlgh3, BG073989, BG073644, Ly6a. III: BG074155, BG073398, BG071485, BG072526, BG074430, BG073107, BG075667, BG075997, BG072588, BG073685, BG074953, BG074938, BG075285, BG071604, BG074455, BG072780, BG073120, BG072862. IV: BG072639, BG076257, BG074434, BG076101, BG072086, BG071754, BG074688, Vti1b-pending, BG073133, BG073062, Hbb-b1, Hba-a1, 2510022J06, BG071893, BG071793, Atox1, BG070376, BG072808, Atp5j2, BG073617, BG070959, BG072912, BG073868, BG075174, BG075811, BG075878, BG072362, BG072634, BG073610, BG073608, BG075903, BG071869, BG072607, BG074864, BG071282, BG073545, BG075047. V: BG072173, BG073807, BG074108, BG075142, BG075863, BG074984, BG075645, BG072533, BG074779, BG070353, BG074041, BG069999, BG072225, BG073254, BG072534, BG073536. VI: BG073288, BG076244, BG073744, BG071754, BG073133, BG076406, BG074747, BG075616, BG073274, BG071953, BG071826, BG070376, BG071511, BG072457, BG070959, BG072912, BG075174, BG075878, BG072634, BG075903, BG071869, BG071282, BG073545, BG074186, BG075740, BG075767, BG075047, BG072509, Spph1-pending, BG076253, BG071516, BG071472, BG074498, BG076088. VII: Rad2, Cd63, Lgals7, ZFP36L1, Fosb, Gas6, Gas5, BG073181, Cdc42. VIII: BG072321, BG075826, BG074440, BG076176. IX: BG070557, BG076166, Ube4b, BG074419, BG073224, BG072180, BG074692, BG076342, BG074772, BG075549, BG073484, BG075084. X: BG072963, BG075908, BG074479, BG071735, Cnn2, BG072419, BG072239, BG074570, BG070433, BG074772, BG075541, BG074505, BG071723, BG076126, BG072716. XI: BG075488. XII: neuronal protein 15.6, Ehd3, Brp44 l, BG075937, BAI1-associated protein 1, Tm4sf8. XIII: Eplin-pending, BG074692. XIV: BG071892, BG070240. XV: Rtn4. XVI: BG073301, BG072379, BG072793. XVII: Dgcr6, CD151, Lgals7, BG070884, cadherin 2. 3. There were 57 genes highly expressed during both development and following injury, the functional distribution of which is shown in Fig. 4. The gene names and functional groups they belong to are as follows: I: HMG-2, BG075393, Smt3h2, Nr2f1, ZFP36L1, BG072143, Hmgn1. II: Cd24a, Cdk4, BG076069, Ran, Anxa6, Ptdsr. III: BG072653, BG072601, BG072597, Rpl32, BG073765, BG073641, BG072855, BG076248, BG072983, BG071840, BG072480, BG073219, BG072179, BG073850, BG072600, BG072620, BG072476, BG071486, BG072862, BG072732. IV: BG073044, BG073045, BG073042, BG073212, BG073043. V: BG070811, BG074318. VI: BG075782, BG072994, Gpx1, BG076239, BG070912. VII: Rps4x. VIII: BG074512. IX: BG071432. X: none. XI: BG074329, BG073954, BG074908, BG074812, BG072463. XII: none. XIII: Tpt1. XIV: BG073276. XV: Mdk. XVI: BG073161. XVII: none.

In the present study, we detected gene expression in the SC during the development and following injury of the optic nerve (where the lateral geniculate body is located which is the first projection site of the optic nerve). Of the 7,680 genes detected, 1,095 highly expressed genes were screened. The functions of 51.19% of the 1,095 highly expressed genes were unidentified, and the implications of expression of these genes during the development and after injury of the optic nerve are unclear. The other genes with identified or partially identified functions were ranked according to the number of genes in a given functional group. The biggest two groups of genes highly expressed during the optic-nerve’s development were related to transcription regulation (35 genes) and signal transduction (also 35 genes). During the optic-nerve’s development, only through precise signal transduction and transcription regulation by these highly abundant genes can nerve cells differentiate, proliferate and migrate, axons grow, and synapses form. All of these genes are silenced during adulthood and following injury; therefore, it is interesting to observe what will happen if we can introduce the re-expression of these genes by gene-engineering technology after CNS injury. The biggest two groups of genes highly expressed following injury were related to material-transporting (37 genes) and metabolism (34 genes), which is in agreement with the fact that substances and energy are needed in large amounts during inflammation, regeneration, and repair after injury. Of those genes highly expressed both during the development and following injury of the optic nerve, 50% were related to protein synthesis, metabolism, and material-transporting, suggesting that large amounts of new proteins were synthesized and large amounts of energy and active material-transporting were needed both during neural development and injury repair. We also noticed that some genes related to the cell cycle, proliferation, transcription regulation, and signal transduction were highly expressed during the development and following injury of the optic nerve. Examples of these genes are the transcription-regulation genes HMG-2, Smt3h2, Nr2f1, ZFP36L1, Hmgn1, and the signal-transduction-related genes Cd24a, Cdk4, Ran, Anxa6, Ptdsr, and the cell-cycle-regulation-related genes Rps4x, and Mdk. These genes played important roles during neural development and became silenced after neural development but were reactivated to express during injury repair. These genes may play crucial roles in the self-repair of the CNS, hence, more concretely functional research to these genes would facilitate to better understanding of CNS regeneration. An important mechanism of CNS regenerative impairment lies in the fact that regenerated axons cannot penetrate the injured site to reach target sites and form synapses. In the CNS, axon growth, guidance, and blockage involve complex and precise mechanisms, which are not fully understood yet. In the present study, we also noticed a differential expression of some genes related to axon growth, guidance, and/or blockage. For instance, Efnb3, Ptn, Nrp, and Dbn1 were highly expressed only during neural development. Efnb3 is a recently discovered ligand of the EPH receptor family, which is crucial for axon guidance and neuron migration. Efnb3 is highly expressed in the midline of early-stage neural tubes and during development of retinoblastoma (Katoh and Katoh 2006). Ptn, a gene widely expressed at early developmental stages, is highly expressed in tumors and endometriosis tissues. It promotes angiogenesis, cell differentiation, and axon growth (Blondet et al. 2005). Nrp (neuropilin) is a receptor for multiple ligands such as VEGF and SEMAPHORINS and plays a crucial role in angiogenesis and neural development (Chen et al. 2005). Dbn1 is a newly discovered neurofilament-binding protein that is specifically expressed in brain tissues. It regulates cytoskeletal rearrangement and dentritic spine formation (Ivanov et al. 2009). We found that Dbn1 was very highly expressed at various developmental stages; hence, we postulate that it may play a critical role in synaptic development of the optic nerve but it is no longer expressed after the development is completed. These four genes and their pathways might be important for the regulation of axon growth, guidance, and synaptic formation of the optic nerve. However, they did not appear after injury. Re-activation of these pathways may benefit CNS regeneration. We did not find any genes that promote and guide axon growth, the expression of which was increased during injury. However, we did find a gene that prevents axon growth, i.e., Rtn4 (Nogo-A). The expression of the gene was increased 14 days after injury. Rtn4 is a member of the reticuloma protein family, which includes two hydrophobic transmembrane domains and potently suppresses axon regeneration (Jurewicz et al. 2007). Following optic-nerve injury, high expression of Rtn4 might be an important factor preventing axon regeneration of the optic nerve. Mdk (midkine), a 13Kd protein, is widely expressed in the nervous system, kidneys, lungs, and blood vessels at early developmental stages. Mdk promotes cell survival, neuron migration, and axon growth during the development of the nervous system. During ischemia/reperfusion of the kidneys and after ischemic retinal injury, Mdk is highly expressed (Sato et al. 2001). High expression of Mdk after CNS trauma has not been reported yet. We found that Mdk was highly expressed during the development and following injury of the optic nerve. Hence, we postulate that Mdk may guide axon growth and regulate neuron migration during optic-nerve development. Mdk was virtually no longer expressed after the optic-nerve’s development was complete, but it resumed high expression following injury to the optic nerve. Hence, Mdk might promote neuron survival and induce axon regeneration during spontaneous optic-nerve injury. Some tumor-related genes were highly expressed during the development and following injury of the optic nerve. The genes that are highly expressed during optic-nerve development include a putative oral cancer suppressor and breast-cancer suppressor candidate 1, Maged2, BG075630, Trp53 and Lag. The genes highly expressed following injury include BG075908 and BG074692; Tpt1 was highly expressed both during the development and following injury of the optic nerve. Although the functions of these genes remained unclear, according to the literature, most of these genes first discovered in tumors were later demonstrated that which were related to cell cycle, cell proliferation, differentiation, transcription regulation, and signal transduction. These genes are exemplified by the NOV family (genes over-expressed in nephroblastomas) and Rb family (genes over-expressed in retinoblastomas) (Su et al. 2001; Scambia et al. 2006). These genes might play roles in cell cycle modulation, cell proliferation, differentiation, transcription regulation, and signal transduction during optic-nerve development and/or following optic-nerve injury. In addition, some CNS-specific genes were found to be highly expressed in the present study. The genes highly expressed during optic-nerve development include Pafaha/Lis1 exon 3 (a gene related to brain development and neural differentiation), Meis1 (a specific transcription factor during brain development), Basp2 (a gene related to signal transduction), PLM-like protein (a gene closely related to myelinogenesis), and Hn1, Cpd2, and LL5 protein with unidentified functions. The genes highly expressed following optic-nerve injury (mentioned in Fig. 3) include BAI1-associated protein 1 (a cell-adhesion molecule), Tm4sf8 (a gene related to oligodendrocyte migration and myelination) and some genes with unidentified functions such as neuronal protein 15.6, Ehd3, Brp44 l, and BG075937. No CNS-specific gene highly expressed both during the development and after injury of the optic nerve were found in the present study. Most of CNS-specific highly expressed genes have CNS-specific functions. Although the functions of some of these genes remain unclear, we postulate that these genes play specific roles during the development and after injury of the optic nerve. In summary, in the present study, gene expression in the SC was investigated in a comparative manner at various time points during the development and after injury of the optic nerve; the genes highly expressed during the aforementioned parameters were screened and categorized functionally. The present study revealed the expression patterns of genes crucial for optic-nerve development and injury, and might help further investigate the molecular mechanisms of CNS development and injury. Further research is required to demonstrate the in vivo expression patterns and functions of most of these differentially expressed genes.

This work was financially supported by a key project of the People’s Liberation Army of China 10th Five-Year Plan (titled “Research on genes crucial for axon orientation during central nervous system development and their functions”) (No: 01Z068) and also by the Natural Science Foundation of Chongqing, China (No: 2007BB5048).

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