Note to the Reader
This is a preprint of a paper to be published in The Journal of
Cell Production and Cell Death in the Generation of Variation in Neuron
Richelle C. Strom and Robert W. Williams
Center for Neuroscience, Department of Anatomy and Neurobiology,
University of Tennessee, 855 Monroe Avenue, Memphis, Tennessee 38163
Materials and Methods
Estimating ganglion cell number
Necrotic axons and growth cones
Specificity of strain
Generation and death of retinal
Mechanism generating differences
in ganglion cell production
Variation in retinal ganglion
Nnc1 controls cell
Retinal ganglion cell numbers in adult mice vary from 40,000 to 80,000.
Much of this variation, and the prominent bimodality of strain averages,
is generated by allelic variants at the Nnc1 locus on chromosome
11. The Nnc1 locus may modulate either ganglion cell production or
the severity of ganglion cell death. Here we have determined what the
relative contributions of these two processes are to variation in adult
cell number by estimating total ganglion cell production in 10 strains of
mice (A/J, BALB/cJ, BXD32, C57BL/6J, CAST/Ei, CARL/ChGo, CE/J, C3H/HeSnJ,
DBA/2J, and LP/J). These strains have adult populations that range from
45,000 to 76,000 (data available at
../main/databases.html). We estimated cell production by counting
ganglion cell axons after ganglion cell neurogenesis, but before the onset
of significant cell death.
Total cell production ranges from 131,000 to 224,000, and most of the
variation in adult ganglion cell number is explained by this significant
variation in cell production. In contrast, the percentage of cell death is
relatively uniform in most strains (~69% cell loss). The exceptions are
BXD32, a strain that has an extremely high adult cell population, and
Mus caroli (CARL/ChGo), a wild southeast Asian species that is
distantly related to laboratory strains. In BXD32 and M. caroli
~62% of the population die. Our analysis indicates that substitutions of
single alleles at the Nnc1 locus are responsible for production
differences of ~8,000 ganglion cells.
Numbers of retinal ganglion cells range from fifty thousand in
nocturnal rodents to several million in diurnal birds and primates (Rager
and Rager, 1978; Rakic and Riley, 1983; Finlay and Pallas, 1989; Williams
et al., 1996). Variation is also marked within species: numbers range from
0.7 to 1.5 million in humans (Curcio and Allen, 1990), and from 40,000 to
80,000 in mice. In mice, the distribution of strain averages is bimodal,
with distinct modes centered at 55,000 and 64,000. We have demonstrated
that this variation is primarily genetic, with a heritability of ~0.8
(Williams et al., 1996). We subsequently discovered that most of the
bimodality in strain averages is controlled by a major quantitative trait
locus, neuron number control 1 (Nnc1). This quantitative trait
locus (QTL) is located on chromosome (Chr) 11 and is closely linked to
three genes known to influence retinal development—the retinoic acid
receptor alpha, neuregulin, and the thyroid hormone receptor alpha
(Williams et al., 1998a). Nnc1 may influence ganglion cell number
by modulating either cell production or the severity of natural cell
Our principal objectives in this study were to determine the
developmental mechanism by which allelic variants at Nnc1 control
neuron number and to assess the relative contributions of cell production
and cell death to the consistent and pronounced differences in population
size among strains of mice. We examined ganglion cell production in ten
strains—four selected from the low mode, four from the high mode, and one
each from the high and low extreme (Fig. 1). We specifically included
strains C57BL/6J and DBA/2J—the parental strains of the set of recombinant
inbred mice used to map Nnc1. Mechanisms generating neuron number
differences between these two strains can be more confidently assigned to
Nnc1. Total ganglion cell production was estimated by counting
retinal ganglion cell axons at birth, a time at which ganglion cell
generation in mice is complete (Dräger, 1985), but before significant
ganglion cell death has begun (Linden and Pinto, 1985).
Materials and Methods
Animals. As illustrated in figure 1, strains of mice were chosen
primarily to represent the two major modes in ganglion cell number
(Williams et al., 1996). Three standard inbred strains were selected from
the low mode (C57BL/6J, A/J, and LP/J) and four standard inbred strains
were selected from the high mode (BALB/cJ, C3H/HeSnJ, CE/J, and DBA/2J).
All of these strains were obtained from the Jackson Laboratory, (Bar
Harbor, ME). In addition, we selected two strains—CAST/Ei and BXD32—that
have exceptionally low and high ganglion cell numbers, respectively. BXD32
was obtained from Dr. Benjamin Taylor at the Jackson Laboratory. CAST/Ei
is an inbred strain derived from M. musculus castaneus that we
obtained from Dr. Eva Eicher at the Jackson Laboratory. Finally, we
studied an outbred sample of Mus caroli that we refer to as CARL/ChGo,
a strain that falls into the low ganglion cell mode. CARL/ChGo is a
partially inbred strain of M. caroli given to us by Dr. Dan
Goldowitz at the University of Tennessee. Both CARL/ChGo and CAST/Ei are
representatives of wild species endemic to Southeast Asia. All mice were
mated in our colony to produce neonates. The day of birth was designated
postnatal day zero (P0).
Figure 1. Bimodal distribution of adult ganglion cell averages
for 60 inbred strains. The strains include 38 recombinant inbred strains
and 17 standard inbred strains listed by Williams et al. (1996), and 5
additional strains, 3 of which are included in this study. A Gaussian
probability distribution was computed for each strain and summed to obtain
a probability density plot (see Williams et al., 1998, for methods). The
figure shows that most strains fall into two main modes. The Gaussian
function drawn in the background has a mean of 60.6 ± 6.3 (x1000), the
average and SD of the 60 strains. The arrows designate the strain averages
for the ten strains examined in this study.
Tissue preparation. We anesthetized neonates by placing them on
ice for several minutes. Neonates were then perfused transcardially with
0.1 M phosphate buffered saline (0.9%), followed by fixative (2.5%
glutaraldehyde and 2.0% paraformaldehyde in 0.1 M phosphate buffer).
Midorbital segments of optic nerves were dissected from the neonates,
osmicated, and embedded in Spurr’s resin. Nerves were thin-sectioned,
placed on formvar-coated grids, and stained with lead citrate and uranyl
Estimating ganglion cell number. We estimated ganglion cell
numbers by counting axons in optic nerve cross-sections (Williams et al.,
1996). Previous studies have demonstrated that axon counts are reliable
estimates of ganglion cell number: bifurcating axons, retinopetal and
retinoretinal axons, and axon loss resulting from a ganglion cell
transforming into an amacrine cell, are comparatively rare in mammals even
during development (Perry et al., 1983; Chalupa et al., 1984; Lia et al.,
1986; Williams et al., 1986; Rice et al., 1995, see table 2). Nerves were
photographed in a grid pattern at ~x30,000
using a JEOL EX2000II electron microscope. High and low magnifications
were calibrated for each case by photographing a grid replica (2160
lines/mm, EMS, Fort Washington, PA). Unmyelinated axons were easily
identified (Fig. 2). Axons were counted directly on negatives within a 63
x 86 mm
counting frame. Total axon estimates were calculated by multiplying the
mean axon density by the total area of the optic nerve.
Figure 2. Electron micrograph from a cross-section of a neonatal
optic nerve (C3H/HeSnJ). The scale bar in the bottom left corner
represents 1 µm. Axons at this stage have relatively uniform diameter,
with a mean fiber diameter of ~0.4 µm. Axons can be recognized
unambiguously in well fixed tissue. The two structures marked by arrows
are astrocyte processes and were not counted.
We counted necrotic axons in neonatal optic nerves from two strains
belonging to the high mode and two strains belonging to the low mode. We
did this by systematically scanning the entire optic nerve cross-section
for necrotic axons at x15,000.
The criteria for distinguishing necrotic axons are those described in
Williams et al., (1986). We also searched for growth cones in the sample
of photographs used for counting axons and by scanning several optic
nerves at high magnification (>x40,000).
The retinal ganglion cell population at birth ranged from 131,000 to
224,000 (Table 1). The mean for all 46 cases is 182,500 ± 4,400 SE. This
value is almost three times higher than the average for an equally diverse
sample of adult mice (Williams et al., 1996). On average we counted five
neonates per strain. The coefficient of variation within strains averaged
8.2%—only slightly higher than the 7.2% value obtained for adult mice. The
small increase is probably due to the technical difficulty of counting
axons before they are myelinated. Given the anticipated variation in the
stage of maturation of sets of neonatal mice sacrificed at birth, this CV
is low and suggests that the ganglion cell population within a strain is
comparatively stable at this stage of development. The average coefficient
of error (the standard error divided by the sample mean) averaged 4.5% in
neonates and 2.5% in adults. These values provide an assessment of the
reliability of adult and neonatal ganglion cell counts.
Table 1. Strain Variation in Cell Production and Cell Death
If strain differences in adult ganglion cell numbers result from
differences in the number of neurons that are generated, then at birth
each strain should have a population that is approximately three–fold
higher than its adult mean. The slope of the regression should be close to
1:3 and the correlation should be high. This is what we found. The slope
of a free regression for the ten strains is 0.26 ± 0.07 (Fig. 3). Forcing
the regression line through the origin produces the expected slope of 1:3
with an excellent fit (inset to Fig. 3). The positive y–intercept
(11,600 adult cells) in the free regression may result from sampling
error, non–linearity of cell death, or may indicate a basal level of cell
production. The correlation coefficient of the free regression in Fig. 3
is 0.81, and the corresponding coefficient of determination (r2)
is 0.66. Thus, two–thirds of the variance in adult cell number can be
readily explained by strain differences in cell genesis.
Figure 3. Regression of neonatal (P0) and adult ganglion cell
number averages for ten strains. Error bars represent one standard error
of the mean. The thin regression line includes all strains and the
coefficient of determination for these data is 0.66. The thick
regression line excludes strains CAST/Ei and BXD32 and the coefficient
of determination is 0.77. The inset (bottom right) plots the same data but
with the regression line forced through the origin. [If there is no cell
production, the adult cell number must be zero.]
We were particularly interested in understanding the process that
produces the bimodality of adult strain averages and for this reason we
also restricted the analysis to the eight strains belonging to high and
low modes (Fig. 3, bold line). The coefficient of determination for this
subset of points is 0.77, indicating that the bimodality is generated
primarily by differences in ganglion cell production. The remaining
“unexplained” variance must result from strain differences in the severity
of cell death, developmental noise, and technical error.
Figure 4. Regression of numbers of cells that are lost (number at
P0 minus the number at maturity) and adult ganglion cell number from our
data (4A), and two alternative Monte Carlo simulations (4B
and 4C). The first model (4B) assumes that all differences
in ganglion cell number are caused by cell production differences, whereas
the second model (4C) assumes that all differences are caused by
variation in the severity of cell death. Monte Carlo datasets consisted of
200 numbers randomly selected from normal distributions. In both models,
high and low adult ganglion cell groups, (n = 100 each), were selected
from two normal distributions with seed parameters (mean and standard
deviation) from the 5 high (66,800 ± 5,400) and 5 low (50,920 ± 3,800)
strains that we studied. In the production model (4B), means were
obtained from two normal distributions with seed parameters (mean and
standard deviation) from the 5 high (202,680 ± 15,500) and 5 low (158,100
± 21,200) strains. In the case of the cell death model (4C), where
no production differences are assumed, the neonatal means were obtained
from a single distribution, with a mean and standard deviation of all 10
strains combined (180,390 ± 18,400). The slope obtained with our real data
is +1.5 (4A), while the cell production (4B) and cell death
(4C) models are +1.2 and -1.1, respectively. The positive slope
from our data is close to that of the simulated cell production model,
demonstrating that differences in adult ganglion cell number are
predominantly due to differences in cell production. In this analyses we
used Model I linear least-square regression because the measurement error
term is without bias. Adult ganglion cell number was subtracted from P0
ganglion cell number to make the y-axis formally independent of adult
ganglion cell number plotted on the x-axis.
Our statistical analysis is complicated by two factors: first the
parameters plotted in Fig. 3 are not formally independent—total cell
production cannot be less than the adult population. Second, the
distribution of adult values is far from normal (Fig. 1). Conventional
statistical estimates are therefore difficult to interpret. To address
these problems we carried out Monte Carlo simulations to test cell
production and cell death models using seed parameters taken from the
adult distribution. We also subtracted the adult population from the
neonatal population to insure independence between the parameters (Fig.
4A). Figures 4B and 4C show the outcomes of two typical Monte Carlo
simulations in which we plot adult cell number against the number of lost
cells. The first model (Fig. 4B) assumes that all differences in adult
cell number are caused by matched differences in cell production and that
cell death is strictly proportional to cell production. The second model
(Fig. 4C) assumes that all differences among adult strains are caused by
variation in the severity of cell death and that at birth all strains have
roughly the same cell population (~180,400 ± 18,400 cells). In the cell
production simulation (4B) the regression slope is +1.2, whereas in the
cell death simulation (4C) the slope is -1.1. Our actual dataset (Fig. 4A)
with its slope of +1.5 strongly supports a cell production model.
The ten inbred strains were divided into high (BALB/cJ, C3H/HeSnJ,
CE/J, BXD32, and DBA/2J) and low groups (C57BL/6J, A/J, CAST/Ei, CARL/ChGo,
and LP/J). Mean adult ganglion cell numbers for these groups are 66,800 ±
2,700 and 50,900 ± 1,900, respectively. There are highly significant
differences in ganglion cell production between these groups, with means
of 202,700 ± 7,800 and 158,100 ± 10,600, respectively (t test, p
< 0.001). In contrast, there is no significant difference in the
percentage of ganglion cell loss between high and low groups, with mean
percentage cell loss relative to neonatal values of 66.9% and 67.5%,
respectively (p = 0.42).
Nnc1 was mapped using recombinant inbred strains generated
from the parental strains, DBA/2J and C57BL/6J. For this reason a
comparison between these two strains is especially germane in discovering
how Nnc1 modulates ganglion cell number. The severity of percentage
cell death was closely matched between DBA/2J and C57BL/6J—69% and 70%,
respectively. In contrast, DBA/2J produces ~16,400 more cells than
C57BL/6J. This result, together with our previous finding of additive gene
action (Williams et al., 1998a), indicates that the substitution of a
single allele at Nnc1 is associated with a production difference of
approximately 8,000 cells.
With the exception of strains BXD32, CARL/ChGo, and BALB/cJ, the
average percentage of cell death among strains is relatively uniform—69% ±
1.2% (Table 1). While the percentage of cell death is relatively uniform,
the absolute magnitude of ganglion cell death is variable among strains
and is highly correlated with production values (Table 1).
There are some notable exceptions to this generality. The percent
cell death in BXD32 and CARL/ChGo is significantly lower than in other
strains (t test, p < 0.05, with Bonferonni correction).
Estimates of ganglion cell production are similar in BALB/cJ and C57BL/6J,
yet these strains have adult populations that differ by about 8,000 cells
(Table 1). A slight reduction in the severity of cell death in BALB/cJ
(65% loss) appears to account for this strain’s relatively high cell
number at maturity. In this instance, the marked strain difference in
adult population size results primarily from variation in the severity of
cell death. Differences in cell death can also compensate for differences
in cell production. For example, CARL/ChGo produces an average of 131,000
ganglion cells—20,000 to 40,000 fewer cells than A/J and LP/J,
respectively—yet all three strains have very closely matched adult
populations (Table 1).
Necrotic Axons and Growth Cones
The validity of our quantitative analysis depends on the assurance with
which we can estimate total ganglion cell production in mice. If much cell
loss occurs before birth or much cell addition occurs after birth, then
production estimates based on axon counts in the optic nerve at P0 will be
too low. To rule out the possibility that significant cell death occurs
prenatally, we counted necrotic axons in neonatal optic nerves from
strains belonging to the high and low modes using criteria described by
Williams and colleagues (1986). Necrotic axons are relatively easy to see,
and it was practical to count all sites of necrosis in a single optic
nerve cross-section. Necrotic axons at P0 make up 0.02% and 0.05% of the
fiber population in cases selected from the low strains, A/J and C57BL/6J,
respectively, whereas they make up 0.07% and 0.09% of the population in
cases selected from the high strains, BXD32 and C3H/HeSnJ, respectively.
The fact that a somewhat higher incidence of necrosis was noted in nerves
taken from the high strains makes it very unlikely that variation in early
axon loss accounts for differences between adult values. Growth cones were
exceedingly rare in all material and fewer than 5 profiles among all cases
met even a relatively lax criteria for these structures (Williams et al.,
1986; Williams et al., 1991; Colello and Guillery, 1992).
Specificity of Strain Differences
Do strain differences in retinal ganglion cell number correspond to
differences in total brain weight or are differences among strains
specific to the ganglion cell population? The correlation of ganglion cell
number and brain weight across individual mice is 0.37, but when strain
averages are used the correlation rises to 0.75. This suggests that about
half the variance in neonatal ganglion cell number can be explained
directly or indirectly by differences in brain weight. As assessed by
quantitative DNA analysis, brain weight differences among neonatal mice
are primarily due to differences in total cell number (Zamenhof and
Marthens, 1976). Thus, mechanisms modulating ganglion cell number may have
common effects on cell number in the other parts of the CNS. The
correlation between strain averages of adult brain weight and ganglion
cell number for the same strains is only 0.51. Given the wide confidence
intervals of correlations computed with low numbers of cases, the
difference between the adult and neonatal correlations (0.51 and 0.75,
respectively) may be due to sampling error. But it is also conceivable
that strain variation in cell death decreases an initially high
correlation between brain weight and retinal ganglion cell number. In any
case, the cellular specificity of the strain differences is likely to be
low, and we expect differences in numerous other neuronal cell populations
to be closely matched with the differences we find in ganglion cell
Synopsis. Our analysis demonstrates that most of the variation
in adult ganglion cell number among strains of mice can be traced to
differences in cell production. Allelic variants at the Nnc1 locus
on Chr 11 (Williams et al., 1996; Williams et al., 1998a) generate the
pronounced bimodality in ganglion cell population size by modulating
ganglion cell production.
Generation and Death of Retinal Ganglion Cells
Generation of retinal ganglion cells in mice begins on embryonic day 11
(E11) and lasts until just before birth (Dräger, 1985). There is a short
delay between neurogenesis and the time at which ganglion cell axons
extend into the optic nerve (Colello et al., 1992). This delay could
deflate estimates of total cell production. However, very few ganglion
cells are produced after E18 (Dräger, 1985) and as anticipated from the
work of Colello and Guillery (1992), we did not observe growth cones in
neonatal optic nerves. It is therefore unlikely that our estimates of
total production are biased downward by late ganglion cell generation.
In contrast, ganglion cell death begins at, or just before,
birth, peaks between postnatal days 4–6, and is essentially complete by
P12 (Linden et al., 1985). At the peak of cell loss, between 5,000 and
10,000 ganglion cells are eliminated per day (Williams et al., 1990).
However, fewer than 2,000 cells are lost on the day of birth in mice,
consistent with our observation of very few necrotic axons—fewer than 300
per nerve. At this rate it is improbable that more than a total of 10,000
ganglion cells are lost prenatally. In chickens there is as much as a
three day delay between the onset of ganglion cell degeneration in the
retina and the elimination of axons in the optic nerve (Rager et al.,
1978). If there is a similar delay in mice then axon counts should
preserve our production estimates. Nonetheless, our estimates of total
production maybe biased downward slightly by the early loss of ganglion
cell axons. However, the magnitude of this error is sufficiently small
(~10,000 cells) that we did not think this loss warranted correction.
Mechanism GeneratingDifferences in Ganglion Cell Production
We recently mapped a gene, Nnc1, that is responsible for more
than half of the genetic variance in ganglion cell number in mice, and
generates the pronounced bimodality that we discovered among strain
averages (Williams et al., 1998a). Nnc1 is the first locus known to
control normal variation in cell number in the vertebrate CNS. The thyroid
hormone receptor alpha gene (Thra) is a superb candidate gene.
Thra maps within 1–2 cM of Nnc1 on chromosome 11 (Montgomery et
al., 1997), and is expressed within the developing chick retina (Sjöberg
et al., 1992). The ligand of THRA, triiodothyronine, is known to influence
retinal ganglion cell fate determination (Hoskins, 1985), retinal
maturation rate (Macaione et al., 1984), and hypothyroidism during retinal
development results in decreased cell density in the ganglion cell layer
(Hoskins, 1985; Navagantes et al., 1996).
Nnc1, possibly Thra, could influence ganglion cell
production by affecting, 1. numbers of retinal progenitor cells, 2.
pathways of cell determination, or 3. kinetics of progenitor cell
proliferation. Genetically determined differences in numbers of
multipotent retinal progenitors would have consistent effects on the
number of many retinal cell types. However, a comparison of horizontal
cell and ganglion cell numbers for six strains demonstrates that ratios in
these early generated cell types are not always matched (Williams et al.,
1998b). We have also examined other retinal cell populations and in
preliminary work have found a weak negative correlation between ganglion
cells and photoreceptors (Williams et al., 1998a). This suggests that
there may be a reciprocal relationship between the generation of early-
and late-generated retinal cell types. An example of this type of
reciprocal relationship is found when the Notch signaling pathway is
perturbed. This results in a shift in the ratio of early to late–generated
retinal cell types (Dorsky et al., 1997). The idea of temporally regulated
competence is supported by a confluence of work (Watanabe and Raff, 1990;
Anchan et al., 1991; Williams and Goldowitz, 1992; Guillemot and Joyner,
1993; Cepko et al., 1996; Alexiades and Cepko, 1997).
The progressive slowing of the cell cycle and its eventual cessation
results in part from decreased availability of key exogenous factors
(Jacobson, 1991; Alexiades and Cepko, 1996). Studies have identified
multiple mitogenic factors for retinal progenitors— FGF, TGFa (Lillien and
Cepko, 1992); TGFb, EGF (Anchan et al., 1991), and IGF–1 (Hernández-Sánchez
et al., 1995; Frade et al., 1996). Interestingly, the addition of the THRA
ligand, triiodothyronine, to cultured fetal rat hypothalamus cells
stimulates the release IGF–1 into the culture medium (Binoux et al.,
1985). Finally, genetic variants of Nnc1 could alter the
proliferation kinetics of progenitors that give rise to ganglion cells.
Rates of mitosis may be influenced through inhibitory molecules and
pathways. One extremely interesting example, is dopa-a tyrosine metabolite
that normally has inhibitory effects on cell genesis in retina (Ilia and
Jeffery, 1996). The absence of dopa in albino rats leads to an anomalous
upregulation of ganglion cell production followed by an increase in the
severity of cell death (Ilia and Jeffery, 1998). Nnc1 could have
effects within any of these mitotic regulatory pathways.
Variation in Retinal Ganglion Cell Death
The severity of cell death is close to 68–70% in most strains of mice.
However, there are three exceptional strains with less severe loss. Three
to nine percent fewer cells are lost in BXD32, CARL/ChGo, and BALB/cJ.
BXD32 is particularly interesting because it has the highest adult
population (75,800 ± 1,900) among the 60 strains we have now examined. Yet
at birth BXD32 has an unexceptional number—199,500—that is lower than
three other strains. Clearly, one or more genes controlling rates of
ganglion cell death are responsible for the high adult cell number in this
strain. It would be feasible to map a cell death gene by crossing BXD32 to
a strain with similar ganglion cell production but higher cell death.
Variation in the severity of cell death may result from differences
in titers of neurotrophic factors. The neurotrophins—BDNF and neurotrophin–3/4—have
been found to increase survival of retinal ganglion cells in chicken and
rat (Rosa et al., 1993; Ma et al., 1998). Neuregulin, found on the cell
surface and as a secreted protein, can also increase survival of neonatal
rat retinal ganglion cells in culture (Bermingham-McDonogh et al., 1996).
Differences in the concentration or expression time of these neurotrophic
factors, their receptors, or components within their signaling pathways
could produce variation in the severity of naturally occurring ganglion
Nnc1 Controls Cell ProductionÅ
We have shown that as much as 77% of the variation among adult strains
results from differences in the production of ganglion cells. The
percentage of cell death in high and low groups does not differ
significantly (66.9% and 67.5%, respectively). We conclude that variation
in adult ganglion cell number among inbred mouse strains results
predominantly from differences in cell production. Comparison of our data
with the Monte Carlo simulations (Fig. 4) corroborates this conclusion.
A major motivation for undertaking the present study was to determine
how and when allelic variants at Nnc1 influence the size of the
ganglion cell population. Collectively, our results strongly indicate that
Nnc1 modulates ganglion cell number by influencing cell production,
and because ganglion cell production occurs before birth, our results
indicate a timeframe for the action of Nnc1.
We thank K. Troughton for technical help and D. Goldowitz for providing
us with M. caroli pups. This research was supported by grants from
the National Institutes of Health to R.W. (NINDS R01 NS35485 and the NEI
EY08868). R.C.S. was supported in part by U.S. Public Health Service
Training Grant RNS-07323.
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Materials and Methods
Estimating ganglion cell number
Necrotic axons and growth cones
Specificity of strain
Generation and death of retinal
Mechanism generating differences
in ganglion cell production
Variation in retinal ganglion
Nnc1 controls cell
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