A role for bHLH transcription factors in retinal degeneration and dysfunction.
ABSTRACT The basic helix loop helix (bHLH) transcription factors collectively mediate cellular differentiation in almost every type
of tissue including the retina (Murre et al. 1989; Jan and Jan 1993; Cepko 1999). Class A factors are ubiquitously expressed throughout mammalian tissue, while the expression
of class B factors are cell type specific. These factors have both a DNA binding domain and helix loop helix domain (HLH)
protein dimerization domain. Class B factors usually heterodimerize with the ubiquitously expressed, bHLH factors, such as
E12/E47. Because of their importance during photoreceptor development, bHLH factors are candidate genes for photoreceptor
degeneration. We have examined the roles of two bHLH factors, both which are expressed during retinal development, but also
share the property of continued expression in the adult retina.
- SourceAvailable from: Casonya Johnson[Show abstract] [Hide abstract]
ABSTRACT: In Caenorhabditis elegans, the dopamine transporter DAT-1 regulates synaptic dopamine (DA) signaling by controlling extracellular DA levels. In dat-1(ok157) animals, DA is not taken back up presynaptically but instead reaches extrasynpatic sites where it activates the dopamine receptor DOP-3 on choligeneric motor neurons and causes animals to become paralyzed in water. This phenotype is called swimming induced paralysis (SWIP) and is dependent on dat-1 and dop-3. Upstream regulators of dat-1 and dop-3 have yet to be described in C. elegans. In our previous studies, we defined a role for HLH-17 during dopamine response through its regulation of the dopamine receptors. Here we continue our characterization of the effects of HLH-17 on dopamine signaling. Our results suggest that HLH-17 acts downstream of dopamine synthesis to regulate the expression of dop-3 and dat-1. First, we show that hlh-17 animals display a SWIP phenotype that is consistent with its regulation of dop-3 and dat-1. Second, we show that this behavior is enhanced by treatment with the dopamine reuptake inhibitor, bupropion, in both hlh-17 and dat-1 animals, a result suggesting that SWIP behavior is regulated via a mechanism that is both dependent and independent of DAT-1. Third, and finally, we show that although the SWIP phenotype of hlh-17 animals is unresponsive to the dopamine agonist, reserpine, and to the antidepressant, fluoxetine, hlh-17 animals are not defective in acetylcholine signaling. Taken together, our work suggests that HLH-17 is required to maintain normal levels of dopamine in the synaptic cleft through its regulation of dop-3 and dat-1.G3-Genes Genomes Genetics 04/2014; · 2.51 Impact Factor
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ABSTRACT: This article summarizes our recent works on stratum-by-stratum structure-function rules for synaptic contacts between retinal bipolar cells and third-order retinal neurons in the inner plexiform layer. These rules were derived from large-scale voltage clamp recordings of various types of bipolar cells in the tiger salamander retina, and they appear applicable to bipolar cells in the mouse and other mammalian species. This review also gives a brief account of how we used pathway-specific knockout mouse models to dissect rod and cone signaling channels in the mammalian retina. Furthermore, studies on cellular and genetic mechanisms underlying several neurodegenerative retinal disorders are described.Vision research 11/2008; 49(9):992-5. · 2.29 Impact Factor
A ROLE FOR BHLH TRANSCRIPTION FACTORS IN
RETINAL DEGENERATION AND DYSFUNCTION
Mark E. Pennesi, Debra E. Bramblett, Jang-Hyeon Cho, Ming-Jer Tsai,
Samuel M. Wu*
The basic helix loop helix (bHLH) transcription factors collectively mediate
cellular differentiation in almost every type of tissue including the retina (Murre et al.
1989; Jan and Jan 1993; Cepko 1999). Class A factors are ubiquitously expressed
throughout mammalian tissue, while the expression of class B factors are cell type
specific. These factors have both a DNA binding domain and helix loop helix domain
(HLH) protein dimerization domain. Class B factors usually heterodimerize with the
ubiquitously expressed, bHLH factors, such as E12/E47. Because of their importance
during photoreceptor development, bHLH factors are candidate genes for photoreceptor
degeneration. We have examined the roles of two bHLH factors, both which are
expressed during retinal development, but also share the property of continued expression
in the adult retina.
Beta2, a bHLH transcription factor, was cloned as a regulator for insulin gene
expression (Naya et al. 1995). It was also isolated from embryos and referred to as
NeuroD because it could convert epidermal cell fate into neuronal (Lee et al. 1995).
Beta2/NeuroD is widely expressed throughout the nervous system and thought to act as a
neuronal differentiator (Lee 1997; Cho and Tsai 2004). Mice homozygous null for this
gene have decreased insulin production and defects in the limbic, vestibular, and auditory
systems (Naya et al. 1997; Liu et al. 2000a; Liu et al. 2000b; Kim et al. 2001). Studies in
rodent retinal explants demonstrated multiple roles for this gene in retinal development
and predicted its importance in photoreceptor survival (Morrow et al. 1999). Expression
in the mouse retina begins at E10.5 in the outer neuroblastic layer and encompasses all
three retinal layers by E18.5 (Brown et al. 1998; Morrow et al. 1999). Additionally,
* Mark E. Pennesi, Samuel M. Wu, Department of Ophthalmology, Debra E. Bramblett. Jang Cho, Ming-Jer
Tsai, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, 77030.
Current Address for Debra Bramblett: Department of Biology, University of St. Thomas, Houston TX 77006
2 M.E. PENNESI ET AL.
Figure 1. Schematic showing how bHLH proteins combine to form transcription factors. Family tree showing
the relationship of Beta2/NeuroD and Bhlhb4 (Bramblett et al. 2002).
robust expression of Beta2/NeuroD in the outer nuclear layer continues in the adult
mouse, suggesting a possible role for this gene in not only the development of these cells,
but also in their survival (Pennesi et al. 2003).
Bhlhb4 is a previously uncharacterized bHLH transcription factor related to
Beta2/NeuroD which is also highly expressed in the retina (Bramblett et al. 2002).
Figure 1 shows the relationship of Beta2/NeuroD and Bhlhb4 within the family of class B
of bHLH genes. Both the temporal and spatial expression patterns of Bhlhb4 differ from
that of Beta2/NeuroD. Its expression was first detected at P5 in a restricted population
of cells in the INL, which morphologically and immunohistochemically resemble rod
bipolar cells. Expression of this gene transiently drops between P9 and P12, but returns
at P14 and is maintained in the adult retina. Thus, it appeared that Bhlhb4 may have both
a developmental role and as well as a functional role in the adult retina.
To explore the roles of Beta2/NeuroD and Bhlhb4 in retinal development and to
help elucidate their role in the adult retina, we developed mice lacking these genes and
studied their retinal histology and function.
Detailed methods are described elsewhere (Pennesi et al. 2003; Bramblett et al.
2004). Beta2/NeuroD null mice were generated on the 129/SvJ background as previously
described (Liu et al. 2000b). Bhlhb4 null mice were generated on the 129/SvEv
background and backcrossed to C57Bl6 mice (Bramblett et al. 2004). For histology, eyes
were placed in 4% paraformaldehyde containing PBS. Eyecups were dehydrated,
orientated, and embedded in JB-4 for cutting. Sections were stained with
bHLH proteins combine to form
dimers which act as transcription
BHLH TRANSCRIPTION FACTORS IN RETINAL DEGENERATION 3
Figure 2. On the left are retinal slices from two month old Beta2/NeuroD +/+ mice, two month old -/- mice,
and, 18 month old -/- mice. On the right are retinal slices from two month old Bhlhb4 +/+ and -/- mice.
hematoxylin and eosin. For ERGs, mice were anesthetized under dim red light with a
solution of ketamine and xylazine. Methylcellulose gel and a platinum electrode were
applied to the cornea. Flashes for scotopic and a-wave measurements were generated by
a Grass PS-33+ photostimulator and a 1500-watt Novatron xenon flash, respectively.
3. RETINAL HISTOLOGY FROM KNOCKOUT MICE
Figure 2 shows light micrographs of retinas taken from adult BETA2/NeuroD
and Bhlhb4 mice. In BETA2/NeuroD null mice the cell loss was most prominent in the
outer nuclear layer (ONL). The ONL, which contains the photoreceptors, normally
measures 10-12 cells thick (Carter-Dawson and LaVail 1979). In BETA2/NeuroD null
mice at 2 months of age, the thickness of the ONL was reduced to 5-6 cells. In addition,
there also appeared to be a thinning of the outer plexiform layer (OPL) and a shortening
of the outer and inner segments when analyzed by electron microscopy (Pennesi et al.
2003). There was no change in the thickness or appearance of the INL, inner plexiform
layer (IPL), or ganglion cell layer (GCL) in null mice. Light microscopy from 18-month-
old -/- mice revealed that the ONL was completely devoid of photoreceptors.
Bhlhb4 null mice displayed a markedly different phenotype. Unlike
Beta2/NeuroD null retinas, which exhibit a degeneration of photoreceptors, the ONL of
Bhlhb4 null retinas were normal in thickness and cell count. However, there was a
notable difference in thickness of the INL. Detailed cell counts revealed that the average
ratio of the number of INL cells to the number of ONL cells in the Bhlhb4 per section
was, 21 percent less than wild type. Immunohistochemical studies showed that the
identity of the missing cells was the rod bipolar cell, and that the death of these cells
occurred during the postnatal development of the retina (Bramblett et al. 2004).
4. ELECTRORETINOGRAMS FROM KNOCKOUT MICE
Under scotopic conditions, the electroretinogram (ERG) detects responses from
rod-driven circuitry, while under photopic conditions it detects responses from cone-
-/- 18 mo.
4 M.E. PENNESI ET AL.
Figure 3. The top row shows scotopic ERGs recorded from two month old Beta2/NeuroD mice, while the second row
shows the response to a saturating flash. The bottom two rows show the responses to similar stimuli in Bhlhb4 mice.
driven circuitry. Figure 3 shows scotopic ERG recordings from the BETA2/NeuroD and
Bhlhb4 lines of mice. In BETA2/NeuroD wild-type mice, the maximum scotopic b-wave
(bmax) measured 650 µV. Heterozygous mice were indistinguishable from control mice
for this and other tests of visual function. Homozygous null mice had a severe decrease
in the scotopic b-wave with bmax measuring 300 µV. To establish if the decrease in the
ERG worsened with age, we tested several mice older than 9 months. Neither rod-driven
nor cone-driven responses were detectable from null mice at these ages. ERGs from
corresponding control mice were only slightly decreased, consistent with aging (data not
shown). In Bhlhb4 wild-type mice, bmax measured 640 µV. Heterozygous mice
demonstrated a small, but significant decrease in the scotopic b-waves with bmax
averaging 485 µV. Scotopic b-wave recordings from Bhlhb4 null mice were profoundly
decreased with bmax measuring only 165 µV. To directly characterize rod photoreceptor
function, we used intense flashes to measure the scotopic a-wave. In Beta2/NeuroD
wild-type mice this stimulus elicited a saturated a-wave, or amax, measuring 600 µV . In
null mice, the saturated a-wave was reduced by almost half to 300 µV. In Bhlhb4 wild-
type mice, amax measured about 825 µV. While, in heterozygous and null mice amax,
BHLH TRANSCRIPTION FACTORS IN RETINAL DEGENERATION 5
measured 700 µV and 825 µV, respectively. In contrast to the Beta2/NeuroD mice, there
was no significant difference between wild-type, heterozygous, or Bhlhb4 null mice.
While both Beta2/NeuroD and Bhlhb4 null mice showed decreased scotopic b-
waves, the origin of the deficit in each was different. The scotopic b-wave is the
extracellular field potential that primarily arises from rod bipolar cells in response to dim
flashes of light (Pugh et al. 1998). The maximum amplitude of the scotopic b-wave is
dependent on the number and activity of bipolar cells, the integrity of the photoreceptor
bipolar synapse, and the number and activity of rod photoreceptor cells. In
Beta2/NeuroD null mice, there was a 50% reduction in both the scotopic b-wave and rod
a-wave. There was no change in the number of rod bipolar cells and although we cannot
rule out synaptic defects, the most likely cause of the diminished b-wave is decreased
input to the bipolar cell due to the loss of photoreceptors and functional impairment of
remaining photoreceptors due to shortened outer segments. In Bhlhb4 null mice, ERG
recordings displayed a dramatic reduction in the scotopic b-wave with a preserved a-
wave. With normal rod morphology, the disruption of b-wave in these mice is certainly
due to the death of the rod bipolar cells. The residual rod b-wave at observed higher
intensities likely represents contribution from cone bipolar cells, since these stimuli are
above the cone threshold.
The cause of death of photoreceptors in Beta2/NeuroD null mice and bipolar
cells in Bhlhb4 null mice is not clear. The loss of Beta2/NeuroD has been linked to the
down regulation of developmental markers and defects in differentiation in the pancreas,
dentate gyrus, and auditory system (Naya et al. 1997; Mutoh et al. 1997; Liu et al. 2000b;
Kim et al. 2001). For example, Beta2/NeuroD is necessary for the expression of insulin
in β-cells of the pancreas and proper formation of the islets. Beta2/NeuroD may play
similar role in the retina by inducing and maintaining the expression of photoreceptor
specific genes. Bhlhb4 likely plays a similar role in the terminal differentiation and
survival of rod bipolar cells, although no specific gene targets have been elucidated.
While the importance of bHLH genes in retinal development has been known for
some time, the idea that continued expression of these genes may be necessary for
survival of retinal cells is new and implies that mutations in these genes may result in
degenerative diseases of the retina. No visual disease in humans has been linked to either
the Beta2/NeuroD or Bhlhb4 loci. Heterozygous mutations in BETA2/NeuroD are
associated with the development of both type 1 and type 2 diabetes mellitus in humans,
but have not been implicated in retinal degeneration (Malecki et al. 1999; Iwata et al.
1999). The loss of Bhlhb4 leads to a ERG phenotype that is commonly referred to as
“negative ERG”. In humans, “negative ERG” is often associated with congenital
stationary night blindness (CSNB) (Dryja 2000). Bhlhb4 is excluded as the determinate
of X-linked CSNB because this has been mapped to the distal end of human chromosome
20 (Bramblett et al. 2002). However, variations of CSNB exist, including autosomal
dominant and recessive forms (Dryja 2000; Fitzgerald et al. 2001) and perhaps Bhlhb4
plays a role in these disorders. Our results indicate that the loss of bHLH factors could
play a role in retinal disease and should be screened in the future for mutations.
6 M.E. PENNESI ET AL.
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