Abstract
purpose. To localize tubby-like protein 1 (TULP1) in developing and adult human retinas.
methods. TULP1 was localized by immunofluorescence microscopy in human retinas,
aged 8.4 fetal weeks to adult. TULP1-positive cells were identified by
double labeling with antibodies specific for cones, rods, and astrocytes.
results. In adult retinas, anti-TULP1 labels cone and rod inner segments,
somata, and synapses; outer segments are TULP1-negative. A few inner
nuclear and ganglion cells are weakly TULP1- positive. In fetal
retinas, cells at the outer retinal border are TULP1-positive at 8.4
weeks. At 11 weeks, the differentiating central cones are strongly
TULP1-reactive and some are positive for blue cone opsin. At 15.4
weeks, all central cones are strongly positive for TULP1 and many are
reactive for red/green cone opsin. At 17.4 weeks, central rods are
weakly TULP-reactive. In peripheral retina at 15.4 weeks to 1 month
after birth, displaced cones in the nerve fiber layer are positive for
TULP1, recoverin, and blue cone opsin. Some ganglion cells are weakly
reactive for TULP1 at 11 weeks and later, but astrocytes and the optic
nerve are TULP1-negative at all ages examined.
conclusions. The finding of TULP1 labeling of cones before they are reactive for
blue or red/green cone opsin suggests an important role for TULP1 in
development. TULP1 expression in both developing and mature cones and
rods is consistent with a primary photoreceptor defect in retinitis
pigmentosa (RP) caused by TULP1 mutations. Weak
TULP1-immunolabeling of some inner retinal neurons in developing and
adult retinas suggests that optic disc changes in patients with RP who
have TULP1 mutations may be primary as well as secondary to
photoreceptor degeneration.
In tubby mice, also known as retinal degeneration 5
(
rd5), a recessive mutation in the
tub gene
causes progressive retinal and cochlear degeneration and adult-onset
obesity with insulin resistance.
1 2 The mutation in the
tub gene that leads to these phenotypic alterations is a
G→T transversion that abolishes a donor splice site, leading to
replacement of the carboxyl-terminal 44 amino acids with a 20–amino
acid sequence not found in the wild-type protein.
3 Tub is a member of a small neuronally expressed gene
family that includes human
TUB (homologous to mouse
tub), tubby-like protein 1 (
TULP1),
TULP2, and
TULP3. The tubby proteins have a
highly conserved carboxyl terminal region but divergent amino
terminals, and related proteins are found in lower animals and plants.
The
TUB gene is expressed in multiple human tissues
including retina, whereas the
TULP1 gene product is found
mainly in retina.
TULP2 is expressed primarily in
testis,
4 and
TULP3 is found in multiple
tissues, including the retina.
5 6
Recent evidence suggests that TULP1 is a transcription factor involved
in control of downstream genes in retinal photoreceptors.
7 Autosomal recessive retinitis pigmentosa (RP) develops in patients
homozygous for mutations in the
TULP1 gene.
8 9 10 11 A recent report of a large family in the
Dominican Republic described the phenotype of patients homozygous for a
splice site mutation (IVS14 + 1, G→A) in the conserved carboxyl
region of
TULP1 gene.
11 In the first decade of
life, the affected persons have nystagmus, absent rod function, and
severely impaired cone function throughout the retina. The early vision
loss is suggested to result from photoreceptor maldevelopment,
dysfunction, or degeneration.
11
The Dominican Republic family members with RP also show early optic
disc abnormalities suggestive of degenerative changes in the ganglion
cells and/or the optic nerve. One study of mouse retina found
tub expression restricted to ganglion cells during
embryogenesis and mainly in photoreceptors in postnatal and adult
animals.
12 A more recent study found
tub expressed in both ganglion cells and photoreceptors in adult mouse
retina.
6 The clinical findings, together with the
tub expression pattern in retina, raises the question of
whether
TULP1 is expressed not only in photoreceptors but
also in ganglion cells and the optic nerve in developing human
retinas.
11 Recent studies
6 13 demonstrate
TULP1-immunolabeling of mouse photoreceptors. However, cones are a
minor population in mouse retina, and we wanted to know whether human
cones also express this protein. To resolve these questions and provide
needed information on the pathophysiology of retinal degeneration
caused by
TULP1 mutations, we used immunocytochemistry to
localize the TULP1 protein in developing and adult human retinas.
Adult human eyes were obtained through the donor programs of the
Foundation Fighting Blindness (Hunt Valley, MD) and the University of
Washington Lions Eye Bank (Seattle). Fetal human eyes were obtained
from the University of Washington Human Embryology Laboratory. The
research followed the tenets of the Declaration of Helsinki, and
informed consent was obtained from all donors. The research was
approved by the institutional human subjects review boards of the
University of Washington and the University of Pennsylvania. Eight
normal adult retinas were evaluated, all fixed at 6 hours or less after
death, along with retinas fixed within 1 hour after death at fetal ages
8.4, 9, 11, 14, 15.4, 17.4, 18, 19, and 21.5 weeks and postnatal ages 3
days and 1 month. The globes were fixed for several weeks to months in
4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3), and stored
thereafter in 2% paraformaldehyde in the same buffer.
Retinal samples were cryosectioned at 12 μm and processed for
immunofluorescence according to published techniques.
14 The secondary antibodies (goat anti-rabbit or anti-mouse IgG) were
labeled with fluorescein isothiocyanate (green), Cy-2 (green), or Cy-3
(red; Jackson ImmunoResearch, West Grove, PA). Nuclei were stained with
DAPI (1 μg/ml; Molecular Probes, Eugene, OR). Control sections were
treated in the same way with omission of primary antibody.
The anti-TULP1 (TULP1-N) was a rat polyclonal antibody (pAb) against
the amino-terminal half of human TULP1,
6 used at a
dilution of 1:750 to 1:1000. Cell-specific antibodies prepared in
rabbits or mice were used for double labeling to analyze the various
retinal cell types labeled with anti-TULP1. Rods were identified with
anti-rhodopsin, a mouse monoclonal antibody (mAb; 4D2; 1:40; from
Robert Molday, University of British Columbia, Vancouver,
Canada). Cones were marked with rabbit pAb anti-blue cone opsin (JH455;
1:5000–1:10,000) and anti-red/green cone opsin (JH492;
1:5000–1:10,000; from Jeremy Nathans, Johns Hopkins University,
Baltimore, MD). Cones were also labeled with rabbit pAb anti-red/green
cone opsin (1:200; from John Saari, University of Washington, Seattle).
The rods, cones, and flat midget bipolar cells were identified with
rabbit pAb anti-recoverin (1:1000; from Alexander Dizhoor, Wayne State
University, Detroit, MI). Astrocytes were labeled with pAb anti-glial
fibrillary acidic protein (GFAP; 1:500; Dako, Carpinteria, CA).
Immunolabeled retinal sections were photographed with an
epifluorescence microscope (DMR; Leica, Deerfield, IL) using Elite
Chrome film (ASA 400; Eastman Kodak, Rochester, NY). Images were
digitized with a flatbed scanner (HiRes; Saphir, Heidelberg, Germany)
with Elite 5.1 software (LinoColor, Heidelberg, Germany) and imported
into a graphics program (Photoshop 5.0; Adobe, San Jose, CA) from which
dye-sublimation prints were generated.
The pattern of TULP1-labeling in the newborn (3 day and 1 month)
retinas was essentially the same as in the adult. Strong
TULP1-reactivity was found in the cone and rod inner segments, somata,
and synapses, and a few inner nuclear layer and ganglion cells were
weakly labeled. In the periphery, TULP1- and recoverin-positive cells
were present in the nerve fiber layer, and a few of these were reactive
for blue cone opsin. The optic nerve head and astrocytes were
TULP1-negative.