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Originally published In Press as doi:10.1167/iovs.08-2556 on January 24, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:1996-2003.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2556
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Multiple Genes on Chromosome 7 Regulate Dopaminergic Amacrine Cell Number in the Mouse Retina

Irene E. Whitney,1 Mary A. Raven,1 Daniel C. Ciobanu,2 Robert W. Williams,2 and Benjamin E. Reese1

1From the Neuroscience Research Institute and Department of Psychology, University of California, Santa Barbara, California; and the 2Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. The size of neuronal populations is modulated by gene variants that influence cell production and survival, in turn influencing neuronal connectivity, function, and disease risk. The size of the dopaminergic amacrine (DA) cell population is a highly heritable trait exhibiting sixfold variation among inbred strains of mice and is used here to identify genes that modulate the number of DA cells.

METHODS. The entire population was counted in retinal wholemounts from 37 genetically defined lines of mice, including six standard inbred strains, 25 recombinant inbred strains (AXB/BXA), reciprocal F1 hybrids, a chromosome (Chr) 7 consomic line, and three additional genetically modified lines.

RESULTS. Much of this variation was mapped to a broad locus on Chr 7 (Dopaminergic amacrine cell number control, Chr 7 [Dacnc7]). The Dacnc7 locus is flanked by two candidate genes known to modulate the number of other types of retinal neuron—the proapoptotic gene, Bax, and tyrosinase. The Tyr mutation was shown to modulate DA cell number modestly, though in the direction opposite that predicted. In contrast, Bax deficiency increased the population fourfold. Bax expression was significantly greater in the A/J than in the C57BL/6J strain, an effect that may be attributed to an SNP in a p53 consensus binding site known to modulate transcription. Finally, we note a strong candidate situated at the peak of the Dacnc7 locus, Lrrk1, a Parkinson’s disease gene exhibiting missense mutations segregating within the AXB/BXA cross.

CONCLUSIONS. Multiple polymorphic genes on Chr 7 modulate the size of the population of DA cells.

Natural variation in neuronal number between individuals is a product of environmental determinants and genetic variants that modulate the processes of cellular production and survival. Inbred strains of mice provide an excellent resource with which to examine each separately by looking at the phenotypic differences within an isogenic line or by comparing the phenotypic variance between different lines of mice. The latter approach has made it possible to uncover chromosomal loci that contribute to differences in brain structure and neuronal number in a variety of regions within the central nervous system, from which one can identify candidate polymorphic genes existing between such strains that may be directly responsible for this genetic component in the phenotypic variation.1 2 3 4 5

Success in mapping such quantitative traits to genomic loci requires sufficient variability among strains in the absence of conspicuous interindividual variability within a strain. The dopaminergic (DA) amacrine cells constitute less than one-hundredth of a percent of the total population of retinal neurons.6 Remarkably, we show here that the number of DA amacrine cells in the retina is tightly conserved within a strain despite their meager absolute number, yet between strains there is a substantial variance in the size of this population. To identify gene variants that modulate the number of DA amacrine cells, we used recombinant inbred strains derived from the A/J and C57BL/6J (B6/J) laboratory strains. We provide evidence for a broad quantitative trait locus (QTL) on Chr 7 controlling DA cell number and identify three candidate genes, two of which, when disrupted, modulate the size of this population; the third is associated with Parkinson disease.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Six inbred laboratory strains of mice [A/J, C57BL/6NCrl (hereafter B6/NCrl), C57BL/6J (hereafter B6/J), DBA2/J, ALR/LtJ, and ALS/LtJ], 25 recombinant inbred (RI) strains derived from the A/J and B6/J strains (the AXB/BXA strain-set; see Supplementary Table S1, online at http://www.iovs.org/cgi/content/full/50/5/1996/DC1), two F1 crosses (AB6F1/J and B6AF1/J), one chromosome substitution strain (C57BL/6J-Chr 7A/JNaJ; hereafter B6.A<7>), one strain with a point mutation in the Tyr gene (B6(Cg)-Tyrc-2J/J) that is coisogenic with B6/J, one strain with a segment of Chr 7 from B6/J introgressed onto an A/J background, including wild-type Tyr alleles (A.B6-Tyr+/J), and one strain congenic with B6/J containing a targeted deletion of the Bax gene (B6.129 x 1-Baxtm1Sjk/J) were obtained. B6/NCrl stock was ordered from Charles River Laboratories and bred in the Animal Resource Center at the University of California at Santa Barbara (UCSB). All other stock was obtained from The Jackson Laboratory for immediate use or for use after a single generation. All experiments were conducted under authorization by the Institutional Animal Care and Use Committee at UCSB and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Mice were perfused with 2 mL of 0.9% saline followed by 50 mL of 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2 at 20°C). Whole retinas were dissected immediately, rinsed in phosphate buffer, and immunolabeled at 4°C using standard indirect immunofluorescence techniques. All incubation solutions included 1% Triton X-100 in phosphate-buffered saline (PBS). Retinas were incubated in a blocking solution (5% normal donkey serum and 2% bovine serum albumin; Sigma, St. Louis, MO) for 3 hours and then rinsed with PBS. Retinas were then incubated in mouse monoclonal antibody to tyrosine hydroxylase (1:10,000; T1299, Sigma) over three nights, rinsed in PBS, and incubated in donkey anti–mouse secondary antibody conjugated to Cy3 (1:200; 715–165-151; Jackson ImmunoResearch Laboratories, West Grove, PA) overnight. Retinas were rinsed with PBS followed by 0.1 M phosphate buffer.

After rinses, retinas were mounted under a coverslip in phosphate buffer and examined under a fluorescence microscope (BH2; Olympus, Tokyo, Japan) coupled through a Sony video camera to a computer running image analysis software (Bioquant Nova Prime; R&M Biometrics, Nashville, TN). The entire retinal wholemount was quantified (retinal wholemounts missing a small portion were excluded from the analysis), while the position of every large, TH-immunoreactive (dopaminergic) amacrine cell in the inner nuclear and ganglion cell layers (the latter are rare, as previously reported,7 amounting to fewer than four cells per retina) was plotted. A minimum of three mice were sampled in each strain; n was indicated within every bar in each histogram. The mean total number and SE of DA cells in each strain is plotted in all histograms and described in the text, except at those locations in the text where the SD within a population is specifically quoted. Individual fields for illustration were imaged using a laser scanning confocal microscope (Fluoview; Olympus) with a 20x objective in which image stacks were collected at 1-µm intervals.

Quantitative trait locus (QTL) mapping was performed with the WebQTL mapping module of GeneNetwork (www.genenetwork.org). We mapped the loci that modulate DA cell number using a weighted interval mapping method that takes into account the significant differences in errors of strain means (see the function labeled "Use SE or Variance for Weighted Regression" toward the bottom of the Trait Data and Analysis Form), excluding the parental strains in the mapping. After standard interval mapping, we also mapped residual differences in DA cell number using a composite interval procedure that controls for the confirmed presence of a locus on Chr 7. High-resolution consensus sequence maps are available for this RI strain set.8 9 The phenotype data in this article have been entered into the AXB/BXA Phenotypes database in GeneNetwork as record ID 10127. All megabase (Mb) position values in this article refer to the Mouse Genome Assembly of 2006 (mm8).

WebQTL makes use of a permutation test of the RI strain data to determine the probability of achieving likelihood ratio statistic (LRS) scores by chance. Thresholds for suggestive and significant LRS scores are indicated (see Figs. 3a 3b ). WebQTL also performs a bootstrap test of the RI strain data, examining the robustness of the site of the peak LRS. This is indicated in the yellow histogram of Figure 3b . Both procedures are described in detail online (www.genenetwork.org; see Glossary).


Figure 1
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  		FIGURE 1. DA cell number is tightly regulated within a laboratory strain yet shows substantial variation between strains. (a) DA cell distribution in the retina of an A/J mouse and an ALS/LtJ mouse showing the extremes across these laboratory strains. Confocal images of labeled cells and their processes are shown on the right. Scale bar, 150 µm. (b) Means and standard errors for six laboratory strains and the F1 crosses between two of those strains (A/J and C57BL/6J, hereafter B6/J). In this and all subsequent figures, the number of mice analyzed per strain is indicated at the base of each bar in the histogram.

 

Figure 2
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  		FIGURE 2. DA cell number varies across the RI strain-set AXB/BXA (gray bars), suggesting the presence of multiple genes with allelic variants that participate in the control of DA cell number. The parental strains are now (and hereafter) shown in black (B6/J) and white (A/J) for comparison, whereas the F1 crosses are striped.

 

Figure 3
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  		FIGURE 3. QTL mapping for DA cell number across the RI strain-set AXB/BXA. (a) Whole genome map. The red trace indicates genomic loci where B alleles increase trait value, whereas the green trace indicates loci where A alleles increase trait value (additive effect, cells per allele; right y-axis). The blue trace indicates the genomewide likelihood ratio statistic (LRS) associated with the linkage between trait variation and genomic locus. The horizontal lines indicate LRS values associated with suggestive (gray; P < 0.63) and significant (pink; P < 0.05) effects. (b) Chromosome 7 map. The yellow bars show the results of bootstrap testing, revealing the consistent presence of a locus near 67 Mb, where Lrrk1 is positioned. Also shown are the locations of two other prospective candidate genes that flank this locus, Tyr and Bax, as well as the introgressed segment of B alleles on an otherwise genetically A strain background in A.B6-Tyr+/J mice (shaded). Lighter shading indicates regions of uncertain haplotype identity. Other conventions as in (a).

 
We used Bax the mouse DNA sequence available in GenBank (NM_007527) and designed primers to amplify (GoTaq Flexi DNA polymerase; Promega Corporation, Madison, WI) the entire Bax gene of the A/J and B6/J strains. We performed reverse transcription of total RNA by using Moloney murine leukemia virus reverse transcriptase and hexanucleotide priming according to the manufacturer’s protocol (GE Healthcare, Piscataway, NJ). We used RNA extracted from postnatal day (P)10 retinas of A/J and B6/J strains to amplify the 5' cDNA ends using a rapid amplification of cDNA ends protocol kit (FirstChoice RLM-RACE; Ambion, Austin, TX) according to instructions of the manufacturer. We also amplified approximately 1 kb upstream of the transcription start of Bax. We sequenced the PCR products and 5' cDNA ends using cycle sequencing reagents (BigDye Terminator, version 3.1; Applied Biosystems, Foster City, CA) and the genetic analyzer instrument (ABI3130xl; Applied Biosystems). We used sequence assembly software (Sequencher, version 4.8; GeneCodes, Ann Arbor, MI) to assemble and align DNA sequences of A/J and B6/J and to identify polymorphisms.

To examine the expression of Bax and Lrrk1 during development, retinas were collected from multiple P1, P5, and P10 litters of the A/J and B6/J strains using RNase-free reagents and were then stored in tissue storage reagent (RNAlater; Ambion) at –20°C. The retinas were disrupted for extraction using RNase-free plastic pestles and tubes (Kontes, Rochester, NY) and homogenized on spin columns (QIAshredder; Qiagen, Valencia, CA). RNA was then extracted with a kit (RNeasy Plus Mini; Qiagen) according to the manufacturer’s instructions. RNA concentration was measured with a spectrometer (Nanodrop-1000; Thermo Scientific, Wilmington, DE), and RNA integrity was verified (2100 Bioanalyzer; Agilent, Santa Clara, CA). Twenty-seven individual RNA samples were used to generate cDNA; 250 ng of each sample was reverse transcribed using a cDNA kit (170–8891; iScript; Bio-Rad, Hercules, CA). PCR master mix (10 µL; final concentration, 50 mM KCl, 10 mM Tris-HCl [pH 9.0 at 25°C], 2.5 mM MgCl2, 0.1% Triton X-100, 0.4 mM dNTPs [U1515; Promega Corporation], 0.20 U Platinum Taq DNA Polymerase [10966; Invitrogen, Carlsbad, CA], 1x SYBR Green I [S-7563, Invitrogen], and 20 nM fluorescein [170–8780; Bio-Rad]) and cDNA were added to 10 µL of 1 µM sense and anti–sense primers for each reaction. Primers were designed with real-time PCR assay software (Beacon Designer, version 7.01; Premier Biosoft International, Palo Alto, CA) and purchased from Operon (Huntsville, AL). Each individual sample was pipetted in quadruplicate (Biomek 2000 Laboratory Automation Work Station; Beckman Coulter, Fullerton, CA) for the Bax and Lrrk1 primers and four housekeeping genes. A detection system (MyiQ Single Color Real-Time PCR Detection System; Bio-Rad) was used to perform PCR amplifications and to generate CT values. Data were corrected for product size and the temperature at which it was analyzed. PCR efficiencies were corrected using linear regression software version 7.2 (LinReg PCR; Jan M. Ruijter, Department of Anatomy and Embryology, Academic Medical Centre, Amsterdam, The Netherlands; available at http://www.gene-quantification.de/lin_reg_pcr_help.pdf).10 The geometric mean of the housekeeping genes was used to normalize average amounts calculated from quadruplicate reactions.11


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
DA Cell Number Varies between Laboratory Strains
Analysis of six inbred laboratory strains revealed a remarkable fourfold variation in DA cell number (Figs. 1a 1b) . All retinas stained consistently for DA cells and their processes across the full extent of the retina (Fig. 1a) , discounting variation in immunolabeling as the source of this variation in cell number. Retinas of A/J contained the fewest DA cells, with an average of 261 ± 7.7 (SEM), whereas ALS/LtJ contained the most, with an average of 962 ± 11.9 (Fig. 1a) . ALR/LtJ averaged 732 ± 13.3 cells, and DBA2/J averaged 756 ± 7.3 cells. The two C57BL/6 substrains we studied showed significant differences: that from the Jackson Laboratory, C57BL/6J (B6/J), had 617 ± 8.3 cells, whereas that from Charles River Laboratories, C57BL/6NCrl (B6/NCrl), had 470 ± 19.8 cells (Student’s t-test; P < 0.05). The B6/NCrl stock was separated in 1951 from the Jackson Laboratory’s stock at generation F32 and has been maintained separately for almost 200 generations.

A small variance can be observed within any of the strains (Fig. 1b) , the SD averaging approximately 5% of the mean number of DA cells. The heritability estimate of this trait (h2)12 was 0.84 for the laboratory strains; that is, the variable strain accounts for most of the variance within the dataset. Such high heritability makes DA cell number a particularly attractive trait for genetic dissection.

We counted retinas of reciprocal F1 hybrids (AB6F1/J and B6AF1/J) generated by crossing the A/J and B6/J strains, two parental strains that differ approximately 2.5-fold in DA cell number. If gene variants modulating DA cell numbers acted in a purely additive and independent manner, then the F1 hybrids would have a population size close to the midparental value of 439 cells. Values for the two F1 hybrids were 509 ± 7.4 for AB6F1/J and 505 ± 4.8 for B6AF1/J (Fig. 1b) , suggesting a mild dominance deviation toward the value of the B6/J parent. If DA cell number were influenced by a parent-of-origin effect (e.g., imprinting, maternal environment, or a mitochondrial effect), one would expect a difference between the reciprocal hybrids. In fact, no significant difference was detected (Student’s t-test; P > 0.05).

Analysis of Recombinant Inbred Strains Indicates Multiple Polymorphic Genes Contribute to DA Cell Number
The AXB/BXA recombinant inbred (RI) strain set consists of 25 independent strains derived from reciprocal crosses between A/J and B6/J strains.8 13 We determined DA cell number within each RI strain (Fig. 2) . As for the inbred laboratory strains, the individual RI strains also showed low within-strain variance, with an average coefficient of variation of 6%. Strain means for the 25 AXB/BXA RI strains, by contrast, were highly variable. Retinas of AXB12 contained merely 160 ± 20 cells, whereas BXA12 contained 637 ± 4.3 cells. This variation was unrelated to the age of animals (r = 0.017; range, 25–88 days) or to the total retinal area (r = 0.047; range, 14.11–19.31 mm2). The wide range of variation that extended beyond the parental values and the fact that the distribution was not obviously bimodal or multimodal suggests that allelic variants in multiple genes participate in the regulation of DA cell number.

QTL Mapping Reveals a Locus on Chromosome 7
We mapped the variation in DA cell number (see GeneNetwork AXB/BXA Phenotype ID 10127) using interval mapping and detected significant linkage on Chr 7 between 45 and 100 Mb (Fig. 3a) . In this interval, the B allele has the anticipated effect on DA cell number, being an additive effect of approximately 65 DA cells per allele. The correlation between variation in DA cell number (strain means) and SNP genotypes at rs6160140 is 0.7, indicating that this single locus can account for up to 50% of the total genetic variance in DA cell number segregating in the AXB/BXA cross. This is a remarkably strong effect, and the presence of B alleles at this locus accounts for as much as 36% of the difference we detected between the parental strains. The Chr 7 locus, which we have named Dopaminergic amacrine cell number control, Chr 7 (Dacnc7), was detected whether we mapped with conventional Haley-Knott linear regression equations or with a weighted regression procedure implemented in WebQTL that factors in the SEM for each strain.

Consomic B6.A<7> Mice Confirm the Presence of the QTL
Singer et al.14 generated a series of chromosome substitution strains in which entire chromosomes from A/J have been introgressed onto a B6/J background. We phenotyped the Chr 7 line (B6.A<7>), which has a complete A/J Chr 7 on an otherwise B6/J genetic background. This B6.A<7> consomic strain had an average of 509 ± 12.0 DA cells or approximately 108 cells fewer than observed in the B6/J strain (Fig. 4) . These results confirm the presence of the QTL on Chr 7. The size of this effect is approximately 83% that expected from the additive effect detected in the AXB/BXA cross, a reasonable concordance with expectation. This result strongly supports Dacnc7 as a locus on Chr 7 that accounts for about one-third of the phenotypic difference between the parental strains.


Figure 4
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  		FIGURE 4. Consomic mice containing A alleles on chromosome 7 contain fewer DA cells. The chromosome substitution strain B6.A<7> had 108 fewer cells than did the B6/J strain, confirming the presence of a QTL on chromosome 7.

 
In addition to Dacnc7, we detected several secondary loci by composite interval mapping on Chr 3, 4, 9, and 15 (not shown). None of these intervals achieve statistical significance. However, an interval on Chr 9 centered at approximately 65 Mb near Rora (RAR-related orphan receptor alpha) is of some interest. The B allele in this interval is associated with lower numbers of DA cells. Although such an "A-high" interval cannot explain the pronounced difference between parental phenotypes, it can help explain the observation that some of the AXB/BXA strains have numbers that are either lower or higher than the parental strains.

The AXB/BXA RI strain-set is comparatively small, and, as a result, the 2-LOD score confidence interval of the QTL of Chr 7 is large and extends from approximately 45.5 to 100.8 Mb (Fig. 3b) . This locus likely contains multiple polymorphic genes that affect DA cell number. Bootstrap re-samples of the strain phenotypes revealed approximately 60% that had a peak LRS score near 67 Mb, but the remaining 40% were scattered throughout the locus, consistent with this interpretation (Fig. 3b , yellow bars). We proceeded to examine two particularly attractive candidates flanking the center of Dacnc7.

Disrupting Tyrosinase Gene Function Modulates DA Cell Number
The distal region of Dacnc7 contains one strong candidate gene that is already known to modulate cell proliferation in the retina, the tyrosinase (Tyr) gene. The mouse Tyr gene is located at 87.3 Mb (indicated in Fig. 3b ), on the fringe of the Dacnc7 locus, but still a gene worthy of attention. Tyr encodes the enzyme tyrosinase, critical for the synthesis of melanin in the skin, hair, and eyes. Mutations in Tyr produce oculocutaneous albinism. Ocular hypopigmentation has been associated with abnormalities in cell division and in cell-cycle kinetics during retinal development15 16 17 and in the number of certain types of retinal neurons at maturity,18 19 raising the possibility that variants of this gene also modulate the production of DA cells. Wild-type B6/J mice contain a functional Tyr gene, whereas A/J contains a point mutation rendering Tyr nonfunctional, producing the albino phenotype. We consequently examined B6 (Cg)-Tyrc-2J/J (albino) mice. This substrain is coisogenic with B6/J except for the presence of a distinct, spontaneously arising point mutation at the Jackson Laboratory that also inactivates Tyr.20 Rather than being decreased, DA cell number was unexpectedly increased significantly by 63 cells (Fig. 5a ; Student’s t-test; P < 0.01), suggesting that the effect of the mutation in Tyr within A/J is masked by other alleles present within the QTL that lower DA cell number. Effects of the Tyr null mutation in A/J could also have contributed to the observation that the consomic B6.A<7> strains had slightly higher DA neurons (approximately 20) than expected given the effect size of Dacnc7.


Figure 5
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  		FIGURE 5. (a) Disrupting a functional Tyr gene in the B6/J strain increased DA cell number. (b) A.B6-Tyr+/J mice contained more DA cells than did A/J mice, indicating that other alleles present in the distal portion of the QTL outweigh any effect of the functional Tyr alleles in this retina. Shown, for comparison, in each case are the data derived from their comparison laboratory strains, A/J and B6/J, respectively.

 
Congenic A.B6-Tyr+/J Mice Confirm Other Loci in the Distal QTL Modulate DA Cell Number
The wild-type Tyr allele has been backcrossed for more than 10 generations to A/J, to yield a pigmented A.B6 congenic line. This strain was genotyped to determine the extent of the chromosomal interval around the Tyr gene that is derived from B6/J. A 55-Mb segment encompassing Tyr (73.8–122.3Mb) was confirmed to contain B alleles (Fig. 3b , shaded region). When compared with A/J, these congenic mice contained 86 more DA amacrine cells (Fig. 5b) , a statistically significant increase (Student’s t-test; P < 0.01). Because the B6 (Cg)-Tyrc-2J/J mice had more DA cells than did the wild-type (pigmented) B6/J mice, the increase in DA cell number in the A.B6-Tyr+/J mice was unlikely to be due to the effect of the Tyr+/+ alleles, indicating some other polymorphic gene (or genes) in the distal half of the QTL, yet to be identified, contributes to this phenotypic difference between the parental strains.

A Gene in the Proximal Portion of the QTL Modulates DA Cell Number
Another noteworthy candidate within the QTL is the gene for the Bcl2-associated X protein, Bax, positioned proximal to the introgressed segment in the A.B6-Tyr+/J strain, at 45.3 Mb (also indicated in Fig. 3b ). Bax is a proapoptotic gene that antagonizes the action of Bcl2, regulating the release of cytochrome c from the mitochondrion and leading to the induction of apoptosis.21 Bcl2-overexpressing transgenic mice are known to have an excessive number of DA amacrine cells and other retinal neurons,22 whereas Bax–/– mice have reduced levels of apoptosis and thicker ganglion cell and inner nuclear layers.23 24 We consequently examined the effect of knocking out Bax gene function on DA cell number. Bax+/– mice showed a conspicuous increase in their number of DA cells (an average of 2696 ± 140.8 cells), whereas the Bax+/+ and Bax+/– littermate mice had numbers of DA cells comparable to the parental B6/J strain, being less than one quarter of this number (Fig. 6) .


Figure 6
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  		FIGURE 6. Bax knockout produces a greater than fourfold increase in DA cell number. (a) DA cell distribution in the retina of a Bax+/+ mouse and a Bax–/– mouse and associated confocal images. Scale bar, 200 µm. (b) Means and standard errors for Bax+/+, Bax+/–, and Bax–/– mice.

 
To investigate whether polymorphisms in the Bax gene may contribute to this natural variation in DA cell number between the A/J and B6/J strains, we sequenced the entire Bax gene from 45,329,773 to 45,334,934 Kb in both strains. We found three additional SNPs and a polymorphic STR (short tandem repeat). These polymorphisms, their locations, and the alleles for each strain are listed first in Supplementary Table S2, http://www.iovs.org/cgi/content/full/50/5/1996/DC1 and are followed by a previously identified SNP. All these polymorphisms were located in introns. The STR and two of the SNPs are part of low-complexity DNA sequences and are less likely to be part of any potential regulatory element.

We also investigated whether the A/J and B6/J strains use the same start site for Bax transcription and whether there are any structural differences between the Bax transcripts at the 5' end. We were encouraged to look at this potential difference because of an interesting 5' end alternative transcript (AY095934) expressed in the P3 x 63-Ag8.653 cell line (genome.ucsc.edu). Sequencing of the 5' cDNA ends did not reveal any difference between the strains relative to the transcription start site or structural differences at the 5' end.

We did, however, find a difference in Bax gene expression throughout postnatal development using real time RT-PCR. mRNA was extracted from whole dissected A/J and B6/J retinas at postnatal day (P) 1, P5, and P10. Two-way ANOVA confirmed this effect of strain to be significant (P < 0.01), yet revealed no significant effect of age or the presence of any interaction (Fig. 7) . This difference in Bax expression, in the direction predicted, suggests that regulatory variants within the Bax promoter segregate in the AXB RI panel. To examine this possibility, we sequenced the DNA upstream of the transcription start of Bax and uncovered an informative SNP (rs31477291) at the position –475 bp. The SNP variant of B6/J abolishes a perfect p53 binding site that is otherwise present in A/J (Semaan SJ, et al. IOVS 2007;48:ARVO E-Abstract 562).


Figure 7
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  		FIGURE 7. Bax gene expression during postnatal development differs between the A/J and B6/J strains. The number of litters, with each litter consisting of at least three retinas at P1, P5, and P10 for each strain, is indicated at the base of each bar in the histogram.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The present results show tremendous variation in DA cell number across different strains of mice in the absence of substantial variation within any individual strain. There are two significant implications of these results. The first is that there should be polymorphic genes contributing to this natural variation between strains, and we have confirmed that this is the case by identifying a significant QTL on Chr 7, Dacnc7. The second is that the specification of DA cell number within any strain of mice must be precisely controlled. We will consider each of these separately, in reverse order:

The specification of DA cell number shows a remarkable level of precision when one considers that developmental mechanisms produce on the order of 6 million nerve cells in the mouse retina.25 Somehow, a small fraction of 1% of the cells exiting the cell cycle gets assigned to a DA cell fate. Subsequently, some proportion of these cells is eliminated during normal development to yield the final number of DA cells. The manner by which fate-determining events or cell survival decisions might reproducibly yield a periodic patterning of even a sparse number of nerve cells is not difficult to envision,26 27 but the DA cells do not conform to a regular array in the mouse retina. Rather, they are nearly random in their local distribution except for a large exclusion zone reducing the tendency of close-neighbor pairings.7 Such exclusion zones are often sufficient for producing regularity in nerve cell patterning.28 29 In the case of the DA cells, however, their packing is still far below the theoretical packing limit imposed by such an exclusion zone, yielding regularity indexes close to those generated by random simulations.7 Consequently, it remains intriguing how fate-determining events and cell survival processes produce such tightly defined numbers of DA cells in the absence of any precise patterning in their spatial distribution.

The present results show that Chr 7 contains multiple genes that influence DA cell number. Tyr and another nearby gene (or genes) antagonize one another in their effect on DA cell number, while more proximally Bax gene function has been shown to have a profound effect on DA cell number. Tyrosinase-mutant mice are known to have altered cell-cycle kinetics and reductions in some,16 18 19 30 31 if not all, retinal cell types,32 so the fact that DA cell number was increased in the Tyr–/– (B6(Cg)-Tyrc-2J/J) retina was unexpected, indicating that it does not contribute to the phenotypic difference between the two parental strains; rather, its effect is masked by other genes. That at least one of those other genes is also present in the QTL is provided by the fact that A.B6-Tyr+/J mice also show an increase in DA cell number relative to the A/J strain. For these mice, the increase cannot be associated with the functional Tyr alleles because they alone are associated with a reduction in DA cell number. Some other polymorphic gene or genes within a 55-Mb region surrounding Tyr must also modulate DA cell number. In addition, bootstrap analysis revealed the consistent presence of another candidate(s) residing near 67 Mb. Together, their effects should sum to yield (assuming no nonlinear epistatic interactions) the phenotypic difference observed in the congenic B6.A<7> chromosome substitution mouse. Polymorphic genes on other chromosomes should in turn combine to yield the entire phenotypic difference between the parental strains, but these did not segregate reliably with the haplotypes associated with this RI strain-set. Some of those, like Tyr and Bax, may affect multiple types of retinal nerve cell by modulating cellular processes controlling proliferation or survival, whereas others may act more specifically to set the number of DA cells alone.

We demonstrated a conspicuous role for the Bax gene in the control of DA cell number. In conjunction with other studies showing a role for the antiapoptotic gene Bcl2,22 the present results suggest that some of the polymorphic genes modulating DA cell number may do so not by modulating only cell production but also by modulating cell survival, affecting multiple cell types.22 24 No functional polymorphisms were detected within the coding region for the Bax gene itself, but a polymorphism in the Bax promoter was detected within a p53 consensus binding sequence. p53 is a known modulator of the cellular response after stress and is known to induce Bax activation,33 while the polymorphism detected has been shown to affect transcription (Semaan SJ, et al. IOVS 2007;48:ARVO E-Abstract 562). Consistent with this, we have confirmed a significant difference in Bax expression during postnatal development, with the A/J strain showing higher expression levels, as predicted. Consequently, this variant in B6/J may reduce Bax expression levels but is less likely responsible for a dramatic reduction in the number of DA cells becasue Bax+/– mice did not show any evidence for an effect of gene dosage.

The present results indicate that DA cell number is a complex trait controlled by multiple polymorphic genes and that other potential candidates near the Dacnc7 locus are worthy of investigation. Bootstrap analysis indicates a polymorphic gene near 67 Mb. There are three interesting candidates at 78 to 79 Mb, each of which contains missense mutations: a gene that codes for aggrecan (Acan), a proteoglycan; a gene that codes for hyaluronan (Hapln3), a proteoglycan link protein 3; and a gene that codes for milk fat globule epidermal growth factor E8 (Mfge8). Acan and Hapln3 are linked in chromosomal location and function.34 Acan is found in the developing retina35 and is thought to be important for neural patterning,36 while Hapln3 interacts with Acan.34 Mfge8 expression is also found in the retina37 and is thought to be important for retinal cell adhesion,38 as are Hapln3 and Acan.35 The recent report that a mutation in the cell adhesion gene Dscam increases DA cell density within the mouse retina39 suggests that cell adhesion molecules may participate in the regulation of DA cell number, encouraging the future pursuit of these as candidate genes.

Far more promising, however, is Lrrk1, positioned at the very locus identified in the bootstrap analysis (Fig. 3b) . This gene codes for a leucine-rich repeat kinase 1, is expressed in early neural retina, and is identified as a potential growth regulatory factor.40 41 Real time RT-PCR results for this gene showed no significant strain difference in expression levels during postnatal development (not shown), but of the 251 known SNPs discriminating A from B6, six of them are missense mutations (Supplementary Table S3, http://www.iovs.org/cgi/content/full/50/5/1996/DC1). Most important, Lrrk1, a paralog of Lrrk2, is linked to the loss of midbrain dopaminergic neurons resulting from familial Parkinson disease,42 43 making it a particularly promising candidate for the control of dopaminergic amacrine cells. Indeed, of the candidate genes identified, Lrrk1 may turn out to be the most specific, modulating exclusively this population of retinal nerve cell.


   Acknowledgements
 
The authors thank Robin Bishop for assistance with the data collection described in Figure 1 and Lu Lu for genotyping the A.B6-Tyr+/J mice.


   Footnotes
 
Supported by National Institutes of Health Grants R01 EY-011087 and S10 RR-022585.

Submitted for publication July 14, 2008; revised September 19, 2008; accepted March 23, 2009.

Disclosure: I.E. Whitney, None; M.A. Raven, None; D.C. Ciobanu, None; R.W. Williams, None; B.E. Reese, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Benjamin E. Reese, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106-5060; breese{at}psych.ucsb.edu.


   References
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 Abstract
 Methods
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 References
 

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