Lior I. Schenk1, Praseeda Venugopalan1,2, Xiong Zhang1, and Jeffrey L. Goldberg1,3 1 University of California, San Diego, La Jolla, CA; 2 University of Miami, Miami, FL; 3 UCSD Shiley Eye Center, La Jolla, CA

1. Characterization of RNA binding protein RBPMS
as a novel marker for retinal ganglion cells.
ABSTRACT
The recently discovered RNA binding protein
RBPMS appears to be a strong candidate as an
RGC marker. To test its specificity and reliability, we
performed immunostaining for RBPMS and Brn3a
(the latter which labels a large percentage of RGCs),
and compared them to retrogradely labeled RGCs.
Previous studies demonstrate that retrograde
labeling with Fluorogold yields over 99% labeling of
RGCs. Based on FG labeling, results indicate that
RBPMS was expressed specifically in RGCs, and
closely matched Fluorogold's labeling efficiency. This
indicates that RBPMS is expressed in over 99% of
RGCs, unlike Brn3a--which is expressed in only
about 80% of RGCs. Thus, our studies provide
strong evidence that RBPMS is a more reliable and
specific marker for RGCs, compared to other
commonly used markers.
Lior I. Schenk1, Praseeda Venugopalan1,2, Xiong Zhang1, and Jeffrey L. Goldberg1,3
1 University of California, San Diego, La Jolla, CA; 2 University of Miami, Miami, FL; 3 UCSD Shiley Eye Center, La Jolla, CA
FG labeling colocalizes with immunostaining against
RBPMS in over 99% of cells
RBPMS Brn3a
CONCLUSION
Abridged References
Acknowledgements
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Major funding for this work was provided by the National Eye Institute (grant 7R01EY020913-04 ). Thanks
also go to the UCSD Division of Biological Sciences and the Undergraduate Research Scholarship
program for additional funding—and to Ms. W. Kwok (UCSD) for her generous support through the Eureka!
Scholarship to LIS. Original poster layout was provided by Ms. E.K. Nguyen (UCLA). I also thank PV and
XZ for their guidance, Mr. A. Kreymerman for help with editing, Dr. T. Stiles for help with printing, and the
other members of the Goldberg lab for their continual support and good will.
Immunostaining against RBPMS labels RGCs in a
more consistent manner than against current markers
Brn3a RBPMS
Brn3a
DAPI
RBPMS
RBPMS labels more RGCs than other markers do.
Immunostaining against RBPMS yields higher levels
of RGC labeling than staining against other markers
BACKGROUND
How can we identify the cellular impact of retinal
disease? One challenge lies in accurately
quantifying the loss of cells such as retinal ganglion
cells (RGCs). This requires reliable markers that
identify all the different subtypes of RGCs. Several
studies investigating different RGC markers have
been performed--but have yielded varying levels of
success, due to inconsistent expression and
specificity. As such, there is a need in retinal
research for a more efficient RGC marker.
 RBPMS yields labeling of RGCs with over 99%
specificity,
 in a consistent reliable manner,
 and with higher output than that of current
antibodies.
By utilizing this powerful new marker, we can even better visualize each
disease’s pathway of action--putting us one step further in the fight for sight.
METHODS
Retrograde Labeling of RGCs
 Fluorogold dye is first applied to the
surface of the superior colliculus.
 FG taken up by RGC axon terminals
is transported retrogradely up the
optic nerve to somata in the retina.
 Complete FG transport results in
labeling of about 98% of RGCs.
Immunohistochemistry
1. Kwong JM, Caprioli J, Piri N (2010). RNA binding protein with multiple splicing: a new marker for retinal ganglion
cells. Invest Ophthalmol Vis Sci.
2. Nadal-Nicolás FM, Jiménez-López M, Sobrado-Calvo P, Nieto-López L, Cánovas-Martínez I, Salinas-Navarro M,
Vidal-Sanz M, Agudo M (2009). Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time
course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci.
3. Chiu K, Lau WM, Yeung SC, Chang RC, So KF (2008). Retrograde labeling of retinal ganglion cells by application of
fluoro-gold on the surface of superior colliculus. J Vis Exp.
All animals were housed, maintained, and examined with
accordance to the IACUC and UCSD’s policies and
procedures as set forth by the Animal Research Committee.
Antibodies:
 RBPMS: uniquely expressed in retinal
ganglion cells, for reasons unknown. RNA
binding protein; stains cytoplasm of RGCs.
 BRN-3: a family of proteins , each with
various roles in RGC development.
Transcription factors; stain RGC variably
depending on developmental stage (as well
as blood vessels due to peroxidase activity).
DAPI: used as a nuclear and chromosome
counterstain. Binds strongly to A,T regions of
DNA resulting in strong labeling of nuclei.
RESULTS
FG
<1
0
66
5
<1
A B C
D (A,B) More RGCs are present in the
RBPMS stain than in the Brn3a stain.
(B) Brn3a additionally stains blood
vessels, demonstrating a lack of
specificity for RGCs alone. (C) A merged
image with DAPI staining depicts
additional populations of non-RGCs in
the amacrine cell layer. (D) A merged
image compares RPMS and Brn3a IHC.
Arrowheads: Multiple cells were
positive for RBPMS but not for Brn3a.
RBPMS
Brn3a
DAPI
RBPMS RGC labeling (relative to RBPMS)
Brn3a
100%
90%
FG
80%
70%
60%
50%
40%
30%
20%
10%
0%
RBPMS Brn3a
• Over 99% of cells labeled by FG
were also positive for RBPMS.
• RBPMS staining colocalizes
with FG labeling at a higher
rate than Brn3a and FG.
• Though not all cells quantified
were positive for FG or for
Brn3a, over 99% were positive
for RBPMS.
• n=1; more experiments
necessary for conclusive data.
(A) RBPMS immunofluorescence overlayed onto Brn3a
immunofluorescence and FG retrograde labeling. Arrows: Multiple
RGCs (identify confirmed by FG) were positive for RBPMS and FG, but
negative for Brn3a—indicating that RBPMS may be more effective
than Brn3a as an RGC marker. (B) Quantification performed in 4
retinae (stained for RBPMS, Brn3a, and DAPI) demonstrated higher
RBPMS staining overall. On average, and in all zones of the retina,
RBPMS achieved higher RGC labeling than Brn3a.
• A portion of the retina was not labeled
by FG. This could be due to afferents
traveling to areas other than the SC,
such as the LGN and the SCN.
• Though Brn3a staining covered the
entirety of the retina, relevant areas of
examination resulted in higher RBPMS
staining overall.
RBPMS labels RGCs in a highly consistent and reliable manner.
RBPMS
Brn3a
FG
A B
Counterstaining against FG confirms RGC specificity.