Pushing the envelope of retinal ganglion cell genesis: Context dependent
function of Math5 (Atoh7)
Lev Prasov, Tom Glasern
Departments of Human Genetics and Internal Medicine, University of Michigan, 2047 BSRB, Box 2200, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA
a r t i c l e i n f o
Received 16 March 2012
Received in revised form
7 May 2012
Accepted 7 May 2012
Available online 15 May 2012
Retinal ganglion cell (RGC)
Basic helix-loop-helix (bHLH)
Cell fate specification
a b s t r a c t
The basic-helix-loop helix factor Math5 (Atoh7) is required for retinal ganglion cell (RGC) development.
However, only 10% of Math5-expressing cells adopt the RGC fate, and most become photoreceptors. In
principle, Math5 may actively bias progenitors towards RGC fate or passively confer competence to
respond to instructive factors. To distinguish these mechanisms, we misexpressed Math5 in a wide
population of precursors using a Crx BAC or 2.4 kb promoter, and followed cell fates with Cre
recombinase. In mice, the Crx cone–rod homeobox gene and Math5 are expressed shortly after cell
cycle exit, in temporally distinct, but overlapping populations of neurogenic cells that give rise to 85%
and 3% of the adult retina, respectively. The Crx4Math5 transgenes did not stimulate RGC fate or alter
the timing of RGC births. Likewise, retroviral Math5 overexpression in retinal explants did not bias
progenitors towards the RGC fate or induce cell cycle exit. The Crx4Math5 transgene did reduce the
abundance of early-born (E15.5) photoreceptors two-fold, suggesting a limited cell fate shift. None-
theless, retinal histology was grossly normal, despite widespread persistent Math5 expression. In an
RGC-deficient (Math5 knockout) environment, Crx4Math5 partially rescued RGC and optic nerve
development, but the temporal envelope of RGC births was not extended. The number of early-born
RGCs (before E13) remained very low, and this was correlated with axon pathfinding defects and cell
death. Together, these results suggest that Math5 is not sufficient to stimulate RGC fate. Our findings
highlight the robust homeostatic mechanisms, and role of pioneering neurons in RGC development.
& 2012 Elsevier Inc. All rights reserved.
The vertebrate retina is a highly ordered structure composed of
six major types of neurons and one type of glia. These originate from
a common progenitor pool (Holt et al., 1988; Turner and Cepko,
1987; Turner et al., 1990) and include retinal ganglion cells (RGCs),
rod and cone photoreceptors, amacrine, horizontal and bipolar
interneurons, and M¨ uller glia. At the onset of retinal neurogenesis,
embryonic day 11 (E11) in mice, multipotent retinal progenitor cells
(RPCs) begin to exit the cell cycle and differentiate.
Birthdating studies, in which nucleoside analogs are used to
identify progenitors exiting the cell cycle, have defined a fixed,
but overlapping, order for the generation of these major cell
classes in vertebrates (Rapaport et al., 2004; Sidman, 1961;
Young, 1985a). RGCs are the first to exit the cell cycle, at E11 in
mice, with peak birthdates at E14 and termination by P0 (Drager,
1985). This early temporal profile overlaps significantly with
those of cone, horizontal, and amacrine neurons. Rods, M¨ uller
glia and bipolar cells have characteristically later birthdates. In
mice, the distribution of rod births peaks in the neonatal period,
but the tails of the distribution extend across most of the
histogenic period, from E12.5 to P10 (Carter-Dawson and LaVail,
1979; Swaroop et al., 2010), because rods compose ?80% of the
mature retina (Jeon et al., 1998).
Heterochronic co-culture and transplantation experiments, in
which early embryonic and late RPCs were cultured in unequal
ratios, have suggested that fate determination is largely a cell
intrinsic process (Belliveau and Cepko, 1999; Rapaport et al.,
2001; Reh, 1992; Watanabe and Raff, 1990). Indeed, progenitors
cultured at low density can develop into each of the major cell
classes, with similar diversity and proportions as the intact retina,
in the absence of environmental feedback signals (Adler and
Hatlee, 1989; Cayouette et al., 2003; Reh and Kljavin, 1989).
However, extrinsic signals can influence progenitor cell cycle
dynamics and override fate decisions in vivo (Cepko, 1999;
Ezzeddine et al., 1997; Kim et al., 2005; Yang, 2004). Collectively,
these observations are consistent with a temporal, or serial,
competence model for retinal development (Cepko et al., 1996;
Livesey and Cepko, 2001; Reh and Cagan, 1994; Wong and
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nCorresponding author. Current address: Department of Cell Biology and
Human Anatomy, University of California Davis School of Medicine, One Shields
Avenue, Davis CA 95616, USA. Fax: þ1 734 763 2162.
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Developmental Biology 368 (2012) 214–230
Rapaport, 2009). According to this model, RPCs pass through
discrete competence states over time, in which they can adopt a
limited number of cell fates. Within each state, the decision to
exit the cell cycle and the final histotypic choice are influenced by
Two prototypical intrinsic factors important for development
of mouse RPCs into specific types of neurons are the cone-rod
homeodomain (HD) factor Crx and the basic helix-loop-helix
(bHLH) factor Math5 (atonal homolog Atoh7). Crx, and closely
related factor Otx2, are expressed in during or shortly after the
terminal cell cycle in tripotential precursors that give rise to
Muranishi et al., 2011). Because of high degree of spatiotemporal
overlap with Otx2, the precise role of Crx remains unclear. In
mice, Crx expression initiates at E12.5 and is necessary for proper
development of photoreceptors, and may be partially redundant
with Otx2 in conferring competence for photoreceptor specifica-
tion (Chen et al., 1997; Furukawa et al., 1999; Nishida et al., 2003;
Sato et al., 2007). Crx works in concert with other transcription
factor to regulate photoreceptor gene expression (reviewed in
Hennig et al., 2008; Swaroop et al., 2010), and Crx is abundant in
adult rods, cones and bipolar cells. The 50regulatory DNA for Crx
has been extensively characterized, and a critical 2.4 kb promoter
region is thought to faithfully recapitulate the endogenous Crx
pattern. This segment has been used to drive Cre, lacZ, and
regulators of rod photoreceptor specification in transgenic mice
(Cheng et al., 2006; Furukawa et al., 2002; Koike et al., 2005;
Nishida et al., 2003; Oh et al., 2007).
Like Crx, the role of Math5 in fate specification has not been
fully elucidated. Math5 (Atoh7) is a single-exon gene that is
transcribed by retinal progenitors in a spatiotemporal pattern
that mirrors RGC births (Brown et al., 1998; Brzezinski et al.,
2012; Prasov et al., Under revision). This factor is transiently
expressed by RPCs during or after their terminal cell cycle
(Brzezinski et al., 2012; Feng et al., 2010; Kiyama et al., 2011;
Skowronska-Krawczyk et al., 2009) and is required for RGC
development. Math5 mutant mice have very few RGCs (o5%)
and lack optic nerves (Brown et al., 2001; Wang et al., 2001).
Apart from the deficiency of RGCs, all other retinal cell classes are
preserved (Brown et al., 2001; Brzezinski et al., 2005). The mRNA
profiles of Math5 mutant retinas are altered, with downregulation
of genes associated with RGC differentiation (Mu et al., 2005).
Lineage tracing experiments have established that Math5-expres-
sing cells contribute to 3% of the adult retina, and every major cell
class (Brzezinski et al., 2012; Feng et al., 2010; Yang et al., 2003).
Together, these data suggest Math5 acts as an essential compe-
tence factor for RGC development. Orthologous genes in zebrafish,
chicken, and frog have similar functions, lineage properties, and
expression patterns (Kanekar et al., 1997; Kay et al., 2001; Liu
et al., 2001; Matter-Sadzinski et al., 2001; Poggi et al., 2005), and
mutations in human ATOH7 have been linked to optic nerve
aplasia and retinal vascular disease (Ghiasvand et al., 2011;
Khan et al., 2011; Prasov et al., Under revision).
Despite these expression and phenotypic analyses, the precise
role of Math5 in RGC development remains unclear. Gain-of-
function studies in frog and chick, and mouse embryonic stem
cells, have yielded mixed results. In these systems, Math5 biases
proliferating progenitors towards RGC fates when over-expressed
during early developmental stages (Brown et al., 1998; Kanekar
et al., 1997; Liu et al., 2001; Moore et al., 2002; Yao et al., 2007),
but promotes other cell fates when expressed during late
development, or in a cross-species context (Brown et al., 1998;
Moore et al., 2002). In general, the interpretation of these
experiments is confounded by the tendency of proneural bHLH
factors to drive cell cycle exit when overexpressed (Farah et al.,
(Furukawa etal., 1997;
To circumvent these limitations and critically assess the role of
Math5 in biasing RGC development, we generated transgenic
mice that ectopically express Math5 in a large number of retinal
progenitors and newly post-mitotic neurons, under control of a
mouse Crx promoter fragment (Crx4Math5 Tg) or bacterial
artificial chromosome (Crx4Math5 BAC), with a bicistronic Cre
lineage tracer. Although endogenous Crx and Math5 genes mark
overlapping populations, and appear to be co-expressed in some
cells, profound overexpression of transgenic Math5 did not
stimulate RGC production or alter the profile of RGC births.
Instead, the number of early-born photoreceptors was reduced.
Despite sustained high-level expression of Math5 in photorecep-
tors and bipolar cells, retinal histology and cell type distribution
were grossly normal. Likewise, no substantial RGC bias was
observed in retinal explants infected with a Math5-expressing
retrovirus. In mutant mice, the Crx4Math5 transgenes rescued
RGC development. However, because endogenous Crx expression
initiates somewhat later that Math5, early-born RGCs were scarce,
and some rescued ganglion cells exhibited pathfinding defects or
apoptosis during development. These results suggest that Math5
action is context dependent. Our findings also warrant a re-
examination of previous results obtained using conventional Crx
Materials and methods
Conventional and bacterial artifical chromosome (BAC) transgenes
To ectopically express Math5 in a wide population of retinal
cells, we generated a conventional Crx4Math5-IRES-Cre bicis-
tronic transgene. We assembled mouse Math5 cDNA and Cre
recombinase coding sequences, separated by an internal riboso-
mal entry site (IRES2) and followed by a SV40 polyA signal. The
mouse Crx promoter and proximal regulatory region were ampli-
fied by PCR and inserted upstream as a 2.4 kb XhoI-SalI fragment
(Furukawa et al., 2002; Oh et al., 2007). A matched Crx4Cre
transgene was then generated from the Crx4Math5-IRES-Cre
plasmid by precise deletion of Math5 and IRES sequences using
the single-strand oligonucleotide
method (Thomason et al., 2007), with a 70 nt antisense oligo
(Suppl. Table 1) and AscI selection.
To faithfully express Math5 in the endogenous Crx pattern, we
generated BAC transgenes by lRED recombineering (Lee et al.,
2001). The targeting construct was assembled, with short
(400 bp) 50and 30homology arms (H) flanking a Math5-IRES-
Cre-FRT-amp-FRT cassette. This was equivalent to the conven-
tional transgene, but included an FRT-amp-FRT selection cassette
(Gene Bridges, Heidelberg) downstream of the SV40 polyA signal.
The 50homology arm extends from Crx intron 1 to the exon
2 initiation (ATG) codon, while the 30homology arm contains
sequence from Crx intron 2. A matched control (Cre-FRT-amp-
FRT) was then generated by ss oligo recombineering, with the
70 nt antisense oligo (Suppl Table 1) and AscI selection.
Linearized targeting plasmids were used in parallel to target
mouse BAC clone RP23-81H17 by lRED-mediated homologous
recombination in strain SW105 (Warming et al., 2005) after heat
induction. This 219 kb BAC contains 134 kb 50and 69 kb 30DNA
flanking the Crx gene. Targeted BAC clones were selected on
ampicillin and chloramphenicol plates at 30 1C, and verified by
junctional PCR and DNA sequencing. The amp seletion cassette
was then deleted by arabinose induction of Flpe recombinase,
leaving a solitary FRT site (Andrews et al., 1985). Homogeneity
and integrity of the resulting clones was verified by ampicillin
sensitivity, junctional PCRs, restriction mapping, and pulsed-field
L. Prasov, T. Glaser / Developmental Biology 368 (2012) 214–230
Purified circular DNA from BAC transgene constructs or line-
arized plasmid DNA from conventional constructs was injected
into fertilized (C57BL/6J?SJL/2) F2 or R26floxGFP (JAX stock
004077 reporter strain, Mao et al., 2001)?B6SJLF1/J oocytes by
the UM Transgenic Animal Core. Founders were identified by
transgene-specific PCR genotyping (Suppl. Table 1), and lines
were maintained by crossing to C57BL/6J or R26floxGFP reporter
strains. We analyzed 2 founders and 2 lines for each conventional
transgene (Crx4Cre Tg and Crx4Math5 Tg), and Z3 lines for
each BAC transgene. The most extensively characterized trans-
genes in this report were Crx4Math5 Tg 251, Crx4Cre Tg 352
control, Crx4Math5 BAC 60, and Crx4Cre BAC 764 control.
Duplex and competitive triplex RT-PCRs were performed as
described (Prasov et al., 2010) to compare the levels of transgene-
derived and endogenous mRNAs. Total RNA was extracted from
embryonic eyes (E14.5) or adult (P21) tissues of transgenic or wild-
type animals using Trizol reagent (Invitrogen, Carlsbad, CA). First-
strand cDNA was generated by high-fidelity reverse transcription
(RT, TranscriptorTM, Roche) at 50 1C and used as template for PCR,
with primers and conditions in Suppl. Table 1. Triplex competitive
RT-PCRs used a common 6-carboxyfluorescein (FAM)-labeled for-
ward primer in Crx exon 1, and two reverse primers (Suppl.
Table 1). Dual products were closely matched for size and GþC
content, and analyzed using a 3730XL capillary electrophoresis
unit (Applied Biosystems, Carlsbad, CA) and Gene Marker software
(SoftGenetics, State College, PA). Expression copy-number levels
for Crx4Math5 BAC transgenes were determined relative to
endogenous Crx values by direct analysis of peak areas (k).
Quantitative RT-PCRs were performed using custom Taqman
probes and Universal Taqman Mastermix (Applied Biosystems),
and were analyzed on the ABI 7600 Real Time PCR System. Critical
cycle threshold levels were normalized to Gapdh internal controls,
using two-fluorophore (VIC and FAM) detection. Fold activity was
calculated using the ddCt method (Livak and Schmittgen, 2001)
and reported relative to the Crx4Math5 BAC expression level.
Expression copy-number levels for conventional transgenes were
calculated by normalizing to endogenous Crx values (k?a/b),
using the mean ratio determined in triplex competitive RT-PCRs
(k) and the relative ratios of Math5 (a) and Crx (b) transcripts
determined by qPCR. Measurements were obtained using inde-
pendent RNA pools from 2 to 5 mice of each genotype.
For section immunostaining, eyes or embryonic heads were
fixed in 2–4% paraformaldehyde (PFA) 0.1 M NaPO4pH 7.3 for 30–
60 min at 22 1C, processed through a 10–30% graded sucrose
series, embedded in OCT (Tissue-Tek, Torrence, CA) and cryosec-
tioned at 10 mm. For flatmount preparations, eyes of P1 or adult
mice were removed and fixed in 4% PFA for 5 min. The optic
nerves were then transected, and the retinas were teased apart
from other ocular tissues, fixed in 4% PFA for 25 min. After
immunostaining, retinas were incised with 6–8 radial cuts and
flattened with the ganglion cell layer (GCL) facing upward.
For immunodetection, slides or whole retinas were blocked in
a solution of 10% normal donkey serum (NDS), 1% bovine serum
albumin (BSA) in PBTx (0.1 M NaPO4pH 7.3 0.5% Triton X-100) for
1–4 h. To reduce mouse-on-mouse background associated with
mouse monoclonal primary antibodies, donkey anti-mouse IgG
Fab fragments were added at 0.8 mg/mL to some blocking reac-
tions. Primary antibodies were applied overnight at 4 1C and
diluted in 3% NDS 1% BSA in PBTx. Sections or retinas were then
washed in PBS, incubated for 2 h at 22 1C with Dylight-conjugated
secondary antibodies and 40,6-diamidino-2-phenylindole (DAPI),
and mounted in Prolong Gold Antifade (Invitrogen, Grand Island,
NY). Slides were imaged using the Zeiss LSM510 Meta confocal
system or an Olympus BX-51 epifluorescence microscope.
The primary antibodies were mouse anti-AP2a (1:1000, DSHB,
Iowa City, IA); rabbit anti-bgal (1:5000, ICN Cappel, Aurora, OH);
rat anti-b-galactosidase (1:500, (Saul et al., 2008)); rat anti-BrdU
(BU1/75, 1:100, Harlan Seralab, Indianapolis, IN); mouse anti-
calbindin (CB-955, 1:500, Sigma, St. Louis, MO); rabbit anti-
cleaved-caspase3 (1:100, Cell Signaling, Beverly, MA); sheep
anti-Chx10 (1:250, Exalpha, Shirley, MA); mouse anti-Cre (clone
7.23, 1:300, Covance, Princeton, NJ); rabbit anti-Crx (1:1000, (Zhu
and Craft, 2000)); chicken anti-GFP (1:2000, Abcam, Cambridge,
MA); mouse anti-hPLAP (monoclonal 8B6, 1:250, Sigma); mouse
anti-PKC (MC5, 1:100, Sigma); rabbit anti-mCar (1:500, Millipore,
Billerica, MA); mouse anti-syntaxin (HPC-1, 1:1000, Sigma);
rabbit anti-M-opsin (1:1000, Millipore); rabbit anti-S-opsin
(1:5000, (Applebury et al., 2000)); rabbit anti-phosphohistone
H3 (1:400, Upstate, Lake Placid, NY); rabbit anti-rhodamine
(1:500, Invitrogen); rabbit anti-Sox9 (1:250, Millipore); rabbit
anti-TuJ1 (MRB-435P, 1:2000, Covance). The Crx antibody appears
to cross-react weakly with Otx2 antigen (Brzezinski et al., 2010),
most likely through a shared LDYKDQ sequence in the Crx 14-
residue peptide immunogen (Zhu and Craft, 2000).
For detection of BrdU (5-bromo-2-deoxyuridine) and other
antigens, cryosections were fully stained with primary and sec-
ondary antibodies to the other markers. Sections were then treated
with 2.4 N HCl in PBTx for 1 h at 22 1C, and immunostained for
BrdU. Likewise, EdU (5-ethynyl-2-deoxyuridine) was detected after
immunostaining, using an azide-alkyne cycloaddition reaction
(Buck et al., 2008) and with Click-iT-647 reagents (Invitrogen).
For fine histology, mice were perfused transcardially with 2%
PFA and 1.25% glutaraldehyde. The eyes were removed, post-fixed
overnight at 22 1C, dehydrated, embedded in glycol methacrylate
plastic resin (JB-4, Polysciences, Warrington, PA), sectioned at
4 mm with a Leitz 1512 rotary microtome, and stained with basic
fuchsin and methylene blue. Paraffin or cryosections (5–10 mm) of
eyes or optic nerves were stained with hematoxylin and eosin as
described (Brown et al., 2001).
Retrograde axon labeling of RGCs
RGCs were definitively marked by retrograde axon labeling
with rhodamine dextran (Brzezinski et al., 2012; Rachel et al.,
2002). Eyes from adult or P1 mice were removed and immersed in
Hank’s balanced salt solution containing calcium, magnesium and
1 mM glucose (HBSSG). Optic nerves were transected within
1 mm of the sclera, and lysine-fixable tetramethyl rhodamine
dextran 3000 MW powder (Molecular Probes, Eugene, OR) was
applied directly to the cut site. The eyes were positioned with
severed optic nerves facing downward against cubes of surgifoam
(Ethicon, Somerville, NJ) saturated with 3% L-a-lysophosphatidyl
choline (LPC, Sigma) and rhodamine dextran. These were sealed
with 1% agarose, and incubated en bloc in aerated HBSSG for 1 h at
22 1C. The surgifoam was then removed, and the eyes were
incubated overnight in HBSSG under the same conditions. Rho-
damine-labeled eyes were fixed in 4% PFA for 4 h at 22 1C and
processed for sectioning or stained as whole retina preparations.
In some experiments, the signal was enhanced by indirect
immunofluorescence staining with anti-rhodamine antibody.
Cre lineage and dual reporter concordance analysis
To trace the descendants of cells expressing Cre recombinase,
transgenic mice were crossed to R26floxGFP or Z/AP (JAX
stock 003919, Lobe et al., 1999) reporter strains, which activate
L. Prasov, T. Glaser / Developmental Biology 368 (2012) 214–230
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