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Thyroid hormone signaling specifies cone subtypes in human retinal organoids

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Thyroid hormone in color vision development Cone photoreceptors in the eye enable color vision, responding to different wavelengths of light according to what opsin pigments they express. Eldred et al. studied organoids that recapitulate the development of the human retina and found that differentiation of cone cells into their tuned subtypes was regulated by thyroid hormone. Cones expressing short-wavelength (S) opsin developed first, and cones expressing long- and medium-wavelength (L/M) opsin developed later. The switch toward development of L/M cones depended on thyroid hormone signaling through the nuclear thyroid hormone receptor. Science , this issue p. eaau6348
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Thyroid hormone signaling specifies cone subtypes in human
retinal organoids
Kiara C. Eldred1, Sarah E. Hadyniak1, Katarzyna A. Hussey1, Boris Brenerman1, Ping-Wu
Zhang2, Xitiz Chamling2, Valentin M. Sluch2, Derek S. Welsbie3, Samer Hattar4, James
Taylor1,5, Karl Wahlin3, Donald J. Zack2,6,7,8, and Robert J. Johnston Jr.1,*
1Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218,
USA.
2Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
3Shiley Eye Institute, University of California, San Diego, La Jolla, CA 92093, USA.
4National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.
5Department of Computer Science, Johns Hopkins University, 3400 N. Charles Street, Baltimore,
MD 21218, USA.
6Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine,
Baltimore, MD 21287, USA.
7Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD
21287, USA.
8Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD
21287, USA.
Abstract
INTRODUCTION: Cone photoreceptors in the human retina enable daytime, color, and high-
acuity vision. The three subtypes of human cones are defined by the visual pigment that they
express: blue-opsin (short wavelength; S), green-opsin (medium wavelength; M), or red-opsin
(long wavelength; L). Mutations that affect opsin expression or function cause various forms of
color blindness and retinal degeneration.
exclusive licensee American Association for the Advancement of Science. http://creativecommons.org/licenses/by/4.0/
*Corresponding author. robertjohnston@jhu.edu.
Author Contributions: K.C.E.: Conception, data acquisition, new reagent contribution, data analysis, and data interpretation; drafted
and revised manuscript. S.E.H.: Data acquisition and data interpretation. K.A.H.: Data acquisition, data analysis, and data
interpretation. B.B.: Data analysis and data interpretation. P.-W.Z.: New reagent contribution. X.C.: New reagent contribution. V.M.S.:
New reagent contribution. D.S.W.: New reagent contribution. S.H.: Data interpretation. J.T.: Data analysis and data interpretation.
K.W.: Data acquisition and new reagent contribution. D.J.Z.: Data acquisition and new reagent contribution. R.J.J.: Conception and
data interpretation; drafted and revised manuscript.
Competing interests: None.
Data and materials availability: RNA-seq data are available on Gene Expression Omnibus, accession no. GSE119320. All other data
and methods are in the supplementary materials. H7 stem cells are available from WiCell under a materials transfer agreement with
WiCell.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/362/6411/eaau6348/suppl/DC1
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Published in final edited form as:
Science
. 2018 October 12; 362(6411): . doi:10.1126/science.aau6348.
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RATIONALE: Our current understanding of the vertebrate eye has been derived primarily from
the study of model organisms. We studied the human retina to understand the developmental
mechanisms that generate the mosaic of mutually exclusive cone subtypes. Specification of human
cones occurs in a two-step process. First, a decision occurs between S versus L/M cone fates. If
the L/M fate is chosen, a subsequent choice is made between expression of L- or M-opsin. To
determine the mechanism that controls the first decision between S and L/M cone fates, we studied
human retinal organoids derived from stem cells.
RESULTS: We found that human organoids and retinas have similar distributions, gene
expression profiles, and morphologies of cone subtypes. During development, S cones are
specified first, followed by L/M cones. This temporal switch from specification of S cones to
generation of L/M cones is controlled by thyroid hormone (TH) signaling. In retinal organoids that
lacked thyroid hormone receptor β (
Thrβ
), all cones developed into the S subtype.
Thrβ
binds
with high affinity to triiodothyronine (T3), the more active form of TH, to regulate gene
expression. We observed that addition of T3 early during development induced L/M fate in nearly
all cones. Thus, TH signaling through
Thrβ
is necessary and sufficient to induce L/M cone fate
and suppress S fate. TH exists largely in two states: thyroxine (T4), the most abundant circulating
form of TH, and T3, which binds TH receptors with high affinity. We hypothesized that the retina
itself could modulate TH levels to control subtype fates. We found that deiodinase 3 (
DIO3
), an
enzyme that degrades both T3 and T4, was expressed early in organoid and retina development.
Conversely, deiodinase 2 (
DIO2
), an enzyme that converts T4 to active T3, as well as TH carriers
and transporters, were expressed later in development. Temporally dynamic expression of TH-
degrading and -activating proteins supports a model in which the retina itself controls TH levels,
ensuring low TH signaling early to specify S cones and high TH signaling later in development to
produce L/M cones.
CONCLUSION: Studies of model organisms and human epidemiology often generate hypotheses
about human biology that cannot be studied in humans. Organoids provide a system to determine
the mechanisms of human development, enabling direct testing of hypotheses in developing
human tissue. Our studies identify temporal regulation of TH signaling as a mechanism that
controls cone subtype specification in humans. Consistent with our findings, preterm human
infants with low T3 and T4 have an increased incidence of color vision defects. Moreover, our
identification of a mechanism that generates one cone subtype while suppressing the other,
coupled with successful transplantation and incorporation of stem cell-derived photoreceptors in
mice, suggests that the promise of therapies to treat human diseases such as color blindness,
retinitis pigmentosa, and macular degeneration will be achieved in the near future.
Graphical Abstract
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Temporally regulated TH signaling specifies cone subtypes. (A) Embryonic stem cell-derived
human retinal organoids [wild type (WT)] generate S and L/M cones. Blue, S-opsin; green, L/M-
opsin. (B) Organoids that lack thyroid hormone receptor β (
Thrβ
KO) generate all S cones. (C)
Early activation of TH signaling (WT + T3) specifies nearly all L/M cones. (D) TH-degrading
enzymes (such as DIO3) expressed early in development lower TH and promote S fate, whereas
TH-activating regulators (such as DIO2) expressed later promote L/M fate.
Summary
The mechanisms underlying specification of neuronal subtypes within the human nervous system
are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity
daytime and color vision. To determine the mechanism that controls S versus L/M fates, we
studied the differentiation of human retinal organoids. Organoids and retinas have similar
distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first,
followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic
expression of thyroid hormone–degrading and –activating proteins within the retina ensures low
signaling early to specify S cones and high signaling late to produce L/M cones. This work
establishes organoids as a model for determining mechanisms of human development with
promising utility for therapeutics and vision repair.
Cone photoreceptors in the human retina enable daytime, color, and high-acuity vision (1).
The three subtypes of human cones are defined by the visual pigment that they express:
blue-opsin (short wavelength; S), green-opsin (medium wavelength; M), or red-opsin (long
wavelength; L) (2). Specification of human cones occurs in a two-step process. First, a
decision occurs between S versus L/M cone fates (Fig. 1A). If the L/M fate is chosen, a
subsequent choice is made between expression of L- or M-opsins (3–6). Mutations that
affect opsin expression or function cause various forms of color blindness and retinal
degeneration (7–9). Great progress has been made in our understanding of the vertebrate eye
through the study of model organisms. However, little is known about the developmental
mechanisms that generate the mosaic of mutually exclusive cone subtypes in the human
retina. We studied the specification of human cone subtypes using human retinal organoids
differentiated from stem cells (Fig. 1, D to K).
Human retinal organoids generate photoreceptors that respond to light (10–14). We found
that human organoids recapitulate the specification of cone subtypes observed in the human
retina, including the temporal generation of S cones followed by L and M cones. Moreover,
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we found that this regulation is controlled by thyroid hormone signaling, which is necessary
and sufficient to control cone subtype fates through the nuclear hormone receptor thyroid
hormone receptor β (Thr
β
). Expression of thyroid hormone-regulating genes suggests that
retina-intrinsic temporal control of thyroid hormone levels and activity governs cone subtype
specification. Whereas retinal organoids have largely been studied for their promise of
therapeutic applications (15), our work demonstrates that human organoids can also be used
to reveal fundamental mechanisms of human development.
Specification of cone cells in organoids recapitulates development in the
human retina
We compared features of cone subtypes in human organoids with those of adult retinal
tissue. Adult human retinas and organoids at day 200 of differentiation displayed similar
ratios of S to L/M cones as indicated by expression of S- or L/M-opsins (adult, S = 13%,
L/M = 87%; organoid, S = 29%, L/M = 71%) (Fig. 1, B and C, and fig. S1A). The difference
in the ratios is likely due to the immaturity of the organoid at ~6 months compared with the
terminally differentiated adult retina. We examined L/M cones with an antibody that
recognizes both L- and M-opsin proteins because of their extremely high similarity. Both S
and L/M cones expressed the cone-rod-homeobox transcription factor (CRX), a critical
transcription factor for photoreceptor differentiation (Fig. 2, A and E) (16–18), indicating
proper fate specification in organoids. Additionally, cones in organoids and retinas displayed
similar morphologies, with L/M cones that had longer outer segments and wider inner
segments than those of S cones (Fig. 2, B to D and F to H) (19). The outer segments of
cones were shorter in organoids than in adult retinas, which is consistent with postnatal
maturation (Fig. 2, D and H) (20). Thus, cone subtypes in human retinal organoids displayed
distributions, gene expression patterns, and morphologies similar to those of cones of the
human retina.
We next examined the developmental dynamics of cone subtype specification in organoids.
In the human retina, S cones are generated during fetal weeks 11 to 34 (days 77 to 238),
whereas L/M cones are specified later, during fetal weeks 14 to 37 (days 98 to 259) (21, 22).
We tracked the ratios and densities of S and L/M cones in organoids by means of antibody
staining over 360 days of differentiation. Cones expressing S-opsin were first observed at
day 150 (Fig. 2, I, L, and M). The density of S cones leveled off at day 170 (Fig. 2M), at the
time point when cones expressing L/M-opsin began to be observed (Fig. 2, J to M). The
population of L/M cones increased dramatically until day 300 (Fig. 2, K to M), when they
reached a steady-state density. The 20-day difference between S- and L/M-opsin expression
onset in retinal organoids is similar to the 20-day difference observed in the appearance of S
and L/M cones in the fetal retina (21). These observations show a temporal switch from S
cone specification to L/M cone specification during retinal development.
We next conducted RNA sequencing (RNA-seq) through 250 days of induced pluripotent
stem cell (iPSC)-derived organoid development. We found that
S-opsin
RNA was expressed
first at day 111 and leveled off at day 160, whereas
L/M-opsin
RNA was expressed at day
160 and remained steady after day 180, which is consistent with the timeline of
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photoreceptor maturation in organoids and fetal retinas (Fig. 2N and fig. S1B). Moreover,
CRX
RNA and CRX protein were expressed before opsins in organoids, which is similar to
human development (Fig. 2N and fig. S1, B to G) (23). Thus, human organoids recapitulate
many aspects of the developmental timeline of cone subtype specification observed in
human retinas, providing a model system with which to uncover the mechanisms of these
developmental changes.
Thyroid hormone signaling and the temporal switch between S and L/M fate
specification
Seminal work in mice identified Thr
β
2 as a critical regulator of cone subtype specification:
Thrβ2
mutants display a complete loss of M-opsin expression and a complete gain of S-
opsin expression in cone photoreceptors (
24–26
). Similar roles for
Thrβ2
have been
characterized in other organisms with highly divergent cone patterning (27–29).
Additionally, rare human mutations in
Thrβ2
are reported to alter color perception, which is
indicative of a change in the S-to-L/M cone ratio (30). To directly test the role of
Thrβ2
in
human cone subtype specification, we used CRISPR/Cas9 in human embryonic stem cells
(ESCs) to generate a homozygous mutation that resulted in early translational termination in
the first exon of
Thrβ2
(fig. S2A). Surprisingly, organoids derived from these mutant stem
cells displayed no differences in cone subtype ratio from genotypically wild-type organoids
[wild type, S = 62%, L/M = 38%;
Thrβ2
knockout (KO), S = 59%, L/M = 41%;
P
= 0.83].
The S-to-L/M ratio is high for both wild-type controls and
Thrβ2
KO organoids, likely
owing to variability in organoid differentiation. Thus, unlike previous suggestions based on
other species,
Thrβ2
is dispensable for cone subtype specification in humans (Fig. 3, A to
C).
Because
Thrβ2
alone is not required for human cone subtype specification, we reexamined
data from Weiss
et. al
(30) and found that mis-sense mutations in exons 9 and 10 affected
both
Thrβ2
and another isoform of the human
Thrβ
gene,
Thrβ1
(fig. S2A). Thus, we asked
whether
Thrβ1
and
Thrβ2
together are required for cone subtype specification in humans. To
completely ablate Thrβ function (Thr
β
1 and Thr
β
2), we used CRISPR/Cas9 in human ESCs
to delete a shared exon that codes for part of the DNA binding domain of
Thrβ
(fig. S2A).
Thrβ
null mutant retinal organoids displayed a complete conversion of all cones to the S
subtype (wild type, S = 27%, L/M = 73%;
Thrβ
KO, S = 100%, L/M = 0%;
P
< 0.0001)
(Fig. 3, D to E and H). In these mutants, all cones expressed S-opsin and had the S cone
morphology (Fig. 3, I and J). Thus,
Thrβ
is required to activate L/M and to repress S cone
fates in the human retina.
Thrβ binds with high affinity to triiodothyronine (T3), the more active form of thyroid
hormone, to regulate gene expression (31). Depletion or addition of T3 alters the ratios of S
to M cones in rodents (25, 32, 33). Because L/M cones differentiate after S cones, we
hypothesized that T3 acts through Thrβ late in retinal development to induce L/M cone fate
and repress S cone fate. One prediction of this hypothesis is that addition of T3 early in
development will induce L/M fate and repress S fate. To test this model, we added 20 nM T3
to ESC- and iPSC-derived organoids starting from days 20 to 50 and continued until day 200
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of differentiation. We observed a dramatic conversion of cone cells to L/M fate (wild type, S
= 27%, L/M = 73%; wild type + T3, S = 4%, L/M = 96%;
P
< 0.01) (Fig. 3, F and H, and
fig. S2B). Thus, early addition of T3 is sufficient to induce L/M fate and suppress S fate.
To test whether T3 acts specifically through Thr
β
to control cone subtype specification, we
differentiated
Thrβ
mutant organoids with early T3 addition.
Thrβ
mutation completely
suppressed the effects of T3, generating organoids with only S cones (wild type + T3, S =
4%, L/M = 96%;
Thrβ
KO + T3, S = 100%, L/M = 0%;
P
< 0.0001) (Fig. 3, F to H). We
conclude that T3 acts though Thr
β
to promote L/M cone fate and suppress S cone fate.
We confirmed the regulation of L/M-opsin expression through thyroid hormone signaling in
a retinoblastoma cell line, which expresses L/M-opsin when treated with T3 (fig. S2, C and
D) (34). T3-induced activation of
L/M-opsin
expression was suppressed upon RNA
interference knockdown of
Thrβ
(fig. S2, E and F), which is similar to the suppression
observed in human organoids.
In organoids, early T3 addition not only converted cone cells to L/M fate but also
dramatically increased cone density (Fig. 3, F and K). Moreover, T3 acts specifically
through Thr
β
to control cone density (Fig. 3, G and K). Early T3 addition may increase cone
density by advancing and extending the temporal window of L/M cone generation.
Together, these results demonstrate that T3 signals though Thrβ to promote L/M cone fate
and repress S cone fate in developing human retinal tissue.
Dynamic expression of thyroid hormone–regulating genes during
development
Our data suggest that temporal control of thyroid hormone signaling determines the S-
versus-L/M cone fate decision, in which low signaling early induces S fate and high
signaling late induces L/M fate. Thyroid hormone exists largely in two states: thyroxine
(T4), the most abundant circulating form of thyroid hormone, and T3, which binds thyroid
hormone receptors with high affinity (31, 35). Because the culture medium contains low
amounts of T3 and T4, we hypothesized that the retina itself could modulate and/or generate
thyroid hormone to control subtype fates.
Conversion of T4 to T3 occurs locally in target tissues to induce gene expression responses
(36, 37). Deiodinases—enzymes that modulate the levels of T3 and T4—are expressed in the
retinas of mice, fish, and chickens (29, 38–42). Therefore, we predicted that T3- and T4-
degrading enzymes would be expressed during early human eye development to reduce
thyroid hormone signaling and specify S cones, whereas T3-producing enzymes, carriers,
and transporters would be expressed later in human eye development to increase signaling
and generate L/M cones.
To test these predictions, we examined gene expression across 250 days of organoid
development. The expression patterns of thyroid hormone–regulating genes were grouped
into three classes: changing expression (Fig. 4A), consistent expression (Fig. 4B), or no
expression (Fig. 4C). Deiodinase 3 (
DIO3
), an enzyme that degrades T3 and T4 (36), was
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expressed at high levels early in organoid development but at low levels later (Fig. 4A).
Conversely, deiodinase 2 (
DIO2
), an enzyme that converts T4 to active T3 (36), was
expressed at low levels early but then dramatically increased over time (Fig. 4A). We
examined RNA-seq data from Hoshino
et. al
(23) and found that developing human retinas
display similar temporal changes in expression of
DIO3
and
DIO2
(fig. S3A). Deiodinase 1
(
DIO1
), which regulates T3 and T4 predominantly in the liver and kidney (43), was not
expressed in organoids or retinas (Fig. 4C and fig. S3C). Thus, the dynamic expression of
Dio3
and
Dio2
supports low thyroid hormone signaling early in development to generate S
cones and high thyroid hormone signaling late to produce L/M cones.
Consistent with a role for high thyroid hormone signaling in the generation of L/M cones
later in development, expression of transthyretin (
TTR
), a thyroid hormone carrier protein,
increased during organoid and retinal development (Fig. 4A and fig. S3A) (23). By contrast,
albumin (
ALB
) and thyroxine-binding globulin (
SERPINA7
), other carrier proteins of T3
and T4, were not expressed in organoids or retinas (Fig. 4C and fig. S3C) (23).
T3 and T4 are transported into cells via membrane transport proteins (44). The T3/T4
transporters
SLC7A5
and
SLC7A8
increased in expression during organoid differentiation
(Fig. 4A). Additionally, two T3/T4 transporters,
SLC3A2
and
SLC16A2,
were expressed at
high and consistent levels throughout organoid development (Fig. 4B). Other T3/T4
transporters (
SLC16A10, SLCO1C1,
and
SLC5A5
) were not expressed in organoids (Fig.
4C), suggesting tissue-specific regulation of T3/T4 uptake. We observed similar expression
patterns of T3/T4 transporters in human retinas (fig. S3, A to C) (23).
We next examined expression of transcriptional activators and repressors that mediate the
response to thyroid hormone. Consistent with
Thrβ
expression in human cones (45),
expression of
Thrβ
in organoids increased with time as cone cells were specified (Fig. 4A).
Expression of thyroid hormone receptor α (
Thrα
) similarly increased with time (Fig. 4A).
Thyroid hormone receptor cofactors, corepressor
NCoR2
and coactivator
MED1,
were
expressed at steady levels during organoid differentiation (Fig. 4B). Similar temporal
expression patterns were observed in human retinas (fig. S3, A and B) (23). Thus, our data
suggest that expression of Thrβ and other transcriptional regulators enables gene regulatory
responses to differential thyroid hormone levels.
A complex pathway controls production of thyroid hormone. Thyrotropin-releasing hormone
(TRH) is produced by the hypothalamus and other neural tissue. TRH stimulates release of
thyroid-stimulating hormone α (CGA) and thyroid-stimulating hormone β (TSHβ) from the
pituitary gland. CGA and TSHβ bind the thyroid-stimulating hormone receptor (TSHR) in
the thyroid gland. T3 and T4 production requires thyroglobulin (TG), the substrate for T3/T4
synthesis, and thyroid peroxidase (TPO), an enzyme that iodinates tyrosine residues in TG
(46).
TRH
was expressed in organoids and retinas, but the other players were not (Fig. 4, A
to C, and fig. S3, A to C) (23, 47, 48), suggesting that the retina itself does not generate
thyroid hormone; rather, it modulates the relative levels of T3 and T4 and expresses TRH to
signal for thyroid hormone production in other tissues.
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Therefore, the temporal expression of thyroid hormone signaling regulators supports our
model that the retina intrinsically controls T3 and T4 levels, ensuring low thyroid hormone
signaling early to promote S fate and high thyroid hormone signaling late to specify L/M
fate (Fig. 4D).
Organoids provide a powerful system with which to determine the mechanisms of human
development. Model organism and epidemiological studies generate important hypotheses
about human biology that are often experimentally intractable. This work shows that
organoids enable direct testing of hypotheses in developing human tissue.
Our studies identify temporal regulation of thyroid hormone signaling as a mechanism that
controls cone subtype specification in humans. Consistent with our findings, preterm human
infants with low T3/T4 have an increased incidence of color vision defects (49–52).
Moreover, our identification of a mechanism that generates one cone subtype while
suppressing the other, coupled with successful transplantation and incorporation of stem
cell-derived photoreceptors in mice (53–56), suggests that the promise of therapies to treat
human diseases such as color blindness, retinitis pigmentosa, and macular degeneration will
be achieved in the near future.
Materials and methods summary
Cell lines
H7 ESC (WA07, WiCell) and episomal-derived EP1.1 iPSC lines were used for
differentiation. WERI-Rb1 retinoblastoma cells were obtained from ATCC. Cell
maintenance and organoid differentiation protocols are described in the supplementary
materials.
CRISPR mutations
All mutations were generated in H7 ESCs. Cells were modified to express an inducible Cas9
element. Plasmids for guide RNA (gRNA) transfection were generated by using the
pSpCas9(BB)-P2A-Puro plasmid modified from the pX459_V2.0 plasmid (62988, Addgene)
by replacing T2A with a P2A sequence. Mutations were confirmed with polymerase chain
reaction sequencing. Gene diagrams of deletions are displayed in fig. S2A. Detailed
transfection procedures, gRNA sequences, and homology arm sequences are included in the
supplementary materials.
Immunohistochemistry
Primary antibodies were used at the following dilutions: goat anti-SW-opsin (1:200 for
organoids, 1:500 for human retinas) (Santa Cruz Biotechnology), rabbit anti-LW/MW-opsins
(1:200 for organoids, 1:500 for human retinas) (Millipore), mouse anti-CRX (1:500)
(Abnova), and mouse anti-Rhodopsin (1:500) (GeneTex). All secondary antibodies were
Alexa Fluor–conjugated (1:400) and made in donkey (Molecular Probes). Detailed methods
for fixation, microscopy, and image processing of organoids, retinas, and WERI-Rb1 cells
are included in the supplementary materials.
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Organoid age
Opsin expression time course—EP1 iPSC-derived organoids for time course
experiments were binned into 10-day increments for analysis. Organoids were binned into
day 130 [actual day 129 (
n
= 3 organoids)], day 150 [actual day 152 (
n
= 4 organoids)], day
170 [actual day 173 (
n
= 2 organoids)], day 200 [actual days 194 to 199 (
n
= 7 organoids)],
day 290 [actual day 291 (
n
= 3 organoids)], and day 360 [actual day 361 (
n
= 3 organoids)].
Quantifications of outer-segment lengths and inner-segment widths were measured in day
361 organoids (
n
= 3 organoids).
Opsin expression in different conditions—iCas9 H7 ESC–derived organoids for
Thrb2
KOs and controls were analyzed at day 200. Organoids for
Thrb
KO, control, and
wild-type + T3 were analyzed at two time points: two organoids were taken at day 199 for
each group, and one was taken at day 277 for each group. T3-treated organoids were taken at
time points between day 195 and day 200 for different differentiations. For each treatment
group and genotype, organoids were compared with control organoids grown in parallel.
RNA-seq time course—EP1 iPSC-derived organoids were analyzed at time points
ranging from day 10 to day 250 of differentiation. We took samples at day 10 (
n
= 3
organoids), day 20 (
n
= 2 organoids), day 35 (
n
= 3 organoids), day 69 (
n
= 3 organoids),
day 111 (
n
= 3 organoids), day 128 (
n
= 3 organoids), day 158 (
n
= 2 organoids), day 173 (
n
= 3 organoids), day 181 (
n
= 3 organoids), day 200 (
n
= 3 organoids), and day 250 (
n
= 3
organoids). RNA from individual organoids was extracted by using the Zymo Direct-zol
RNA Microprep Kit (Zymo Research) according to manufacturer’s instructions. Libraries
were prepared by using the Illumina TruSeq stranded mRNA kit and sequenced on an
Illumina NextSeq 500 with single 200-base pair reads.
RNA-seq time course analysis
Expression levels were quantified by using Kallisto (version 0.34.1) with the following
parameters: “−b 100 −1 200 −s 10 −t 20–single”. The Gencode release 28 comprehensive
annotation was used as the reference transcriptome (57). Transcripts per million (TPM)
values (table S1) were then used to generate graphs in Prism and heatmaps in R by using
ggplot2. The distributions of transcripts were plotted so as to identify the best low TPM
cutoff (fig. S5A). The threshold was determined to be 0.7 log(TPM + 1)—5 TPM—and this
value was used as an inflection point for the heatmaps. Heatmaps for fig. S3, A to C, were
made similarly, by using CPM values from Hoshino
et. al
(fig. S5B) (23).
Measurements and quantification
Measurements of retinal area and cell morphology were done by using ImageJ software.
Quantifications and statistics (except for RNA-seq data) were done in GraphPad Prism, with
a significance cutoff of 0.01. Statistical tests are listed in figure legends. All error bars
represent the SEM.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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ACKNOWLEDGMENTS
We thank A. Kolodkin, J. Nathans, and members of the Johnston laboratory for helpful comments on the
manuscript.
Funding: K.C.E. was a Howard Hughes Medical Institute Gilliam Fellow and was supported by the National
Science Foundation Graduate Research Fellowship Program under grant 1746891. R.J.J. was supported by the Pew
Scholar Award 00027373.
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Fig. 1. S and L/M cone generation in human retinal organoids.
(A) Decision between S and L/M cone subtype fate. (B and C) S-opsin (blue) and L/M-
opsin (green). (B) Human adult retina age 53. (C)iPSC-derived organoid, day 200 of
differentiation. (D to K) Bright-field images of organoids derived from iPSCs. (D)
Undifferentiated iPSCs. (E) Day 1, aggregation. (F) Day 4, formation of neuronal vesicles.
(G) Day 8, differentiation of retinal vesicles. (H) Day 12, manual isolation of retinal
organoid. (I) Day 43, arrow indicates developing retinal tissue, and arrowhead indicates
developing retinal pigment epithelium. (J) Day 199, arrow indicates outer segments. (K) Day
330, arrow indicates outer segments.
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Fig. 2. Human cone subtype specification is recapitulated in organoids.
(A to K) S-opsin (blue) and L/M-opsin (green) were examined in human iPSC-derived
organoids [(A), (C) to (E), and (G) to (M)] and human retinas [(B), (D), (F), and (H)]. [(A)
to (C) and (E) to (G)] Arrows indicate outer segments, solid arrowheads indicate inner
segments, and open arrowheads indicate nuclei. [(A) and (E)] CRX (a general marker of
photoreceptors) is expressed in S cones and L/M cones. [(B) to (D)] S cones display short
outer segments and thin inner segments in both human retinas and organoids. [(F) to (H)]
L/M cones display long outer segments and wide inner segments in both human retinas and
organoids. [(D) and (H)] Quantification of outer segment lengths and inner segment widths
(adult retina, L/M,
n
= 13 cones, S,
n
= 10 cones; organoid, L/M,
n
= 35 cones, S,
n
= 42
cones). [(I) to (N)] S cones are generated before L/M cones in organoids. (L) Ratio of S:L/M
cones during organoid development. (M) Density of S and L/M cones during organoid
development. (N)
S-opsin
expression precedes
L/M-opsin
expression in human iPSC-
derived organoids.
CRX
expression starts before opsin expression. TPM, transcripts per
kilobase million.
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Fig. 3. Thyroid hormone signaling is necessary and sufficient for the temporal switch between S
and L/M fate specification.
(A to K) S-opsin (blue) and L/M-opsin (green) were examined in human ESC-derived
organoids. (A) Wild-type (WT). (B)
Thrβ2
early termination mutant (
Thrβ2
KO). (C)
Quantification of (A) and (B) (WT,
n
= 3 organoids;
Thrβ2
KO,
n
= 3 organoids). (D) WT.
(E)
Thrβ
KO. (F) WT treated with 20 nM T3 (WT + T3). (G)
Thrβ
KO treated with 20 nM
T3 (
Thrβ
KO + T3). (H) Quantification of (D) to (G) (WT,
n
= 9 organoids;
Thrβ
KO,
n
= 3
organoids; WT + T3,
n
= 6 organoids;
Thrβ
KO + T3,
n
= 3 organoids. Tukey’s multiple
comparisons test: WT versus
Thrβ
KO,
P
< 0.0001; WT versus WT + T3,
P
< 0.01; WT +
T3 versus
Thrβ
KO + T3,
P
< 0.0001). (I) Length of outer segments. WT, L/M
n
= 66 cells;
WT, S
n
= 66 cells;
Thrβ
KO,
n
= 50 cells (Tukey’s multiple comparisons test, WT L/M
versus WT S,
P
< 0.0001; WT L/M versus
Thrβ
KO,
P
< 0.0001; WT S versus
Thrβ
KO, not
significantly different). (J) Width of inner segments. WT, L/M
n
= 78 cells; WT, S
n
= 78
cells;
Thrβ
KO,
n
= 118 cells (Tukey’s multiple comparisons test, WT L/M versus WT S,
P
< 0.0001; WT L/M versus
Thrβ
KO,
P
< 0.0001; WT S versus
Thrβ
KO, not significantly
different). (K) T3 acts through
Thrβ
to increase total cone number. Quantification of density
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of S and L/M cones; WT,
n
= 6 organoids;
Thrβ
KO,
n
= 3 organoids; WT + T3,
n
= 3
organoids;
Thrβ
KO + T3,
n
= 3 organoids (Tukey’s multiple comparisons test between total
cone numbers, WT versus
Thrβ
KO, not significantly different; WT versus WT + T3,
P
<
0.01; WT + T3 versus
Thrβ
KO + T3,
P
< 0.0001).
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Fig. 4. Dynamic expression of thyroid hormone signaling regulators during development.
(A to C) Heat maps of log(TPM + 1) values for genes with (A) changing expression, (B)
consistent expression, and (C) no expression. Numbers at the bottom of heat maps indicate
organoid age in days. (D) Model of the temporal mechanism of cone subtype specification in
humans. For simplicity, only the roles of DIO3 and DIO2 are illustrated. In step 1,
expression of DIO3 degrades T3 and T4, leading to S cone specification. In step 2,
expression of DIO2 converts T4 to T3 to signal Thrβ to repress S and induce L/M cone fate.
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... De plus, dans des organoïdes rétiniens dérivés de cellules ES murines, les enzymes impliquées dans le métabolisme de l'AR (Raldh1, Raldh3 et Cyp26a1) sont apparues drastiquement régulées suivant une temporalité très précise, au début de la phase de maturation des précurseurs des cônes, mais se révèlent favorables ultérieurement pour la spécification des cônes en cônes bleus (Kruczec et al., 2017). Malgré différents protocoles de maturation, la répartition des sous-types de cônes au sein des organoïdes rétiniens humains s'est révélée majoritairement similaire à celle observée dans la rétine humaine in vivo, avec moins de cônes bleus et davantage de cônes rouges/verts (Zhong et al., 2014 ;Reichman et al., 2017 ;Li et al., 2018 ;Capowski et al., 2019 ;Eldred et al., 2018). Le contrôle de la spécification du sous-type des cônes est régi par la signalisation de l'hormone thyroïdienne (Brzezinski et Reh, 2015 ;Wang et Cepko, 2016 ;Eldred et al., 2018). ...
... Malgré différents protocoles de maturation, la répartition des sous-types de cônes au sein des organoïdes rétiniens humains s'est révélée majoritairement similaire à celle observée dans la rétine humaine in vivo, avec moins de cônes bleus et davantage de cônes rouges/verts (Zhong et al., 2014 ;Reichman et al., 2017 ;Li et al., 2018 ;Capowski et al., 2019 ;Eldred et al., 2018). Le contrôle de la spécification du sous-type des cônes est régi par la signalisation de l'hormone thyroïdienne (Brzezinski et Reh, 2015 ;Wang et Cepko, 2016 ;Eldred et al., 2018). Ainsi, il a été montré dans des organoïdes rétiniens dérivés de cellules iPS humaines, qu'en absence de récepteur de l'hormone thyroïdienne (Thrβ), tous les cônes se spécifient en sous-type S. En revanche, l'ajout de triiodothyronine (T3), la forme la plus active de l'hormone thyroïdienne, qui se lie avec une grande affinité à Thrβ induit le devenir de la majorité des cônes en sous-type L/M (Eldred et al., 2018). ...
... Le contrôle de la spécification du sous-type des cônes est régi par la signalisation de l'hormone thyroïdienne (Brzezinski et Reh, 2015 ;Wang et Cepko, 2016 ;Eldred et al., 2018). Ainsi, il a été montré dans des organoïdes rétiniens dérivés de cellules iPS humaines, qu'en absence de récepteur de l'hormone thyroïdienne (Thrβ), tous les cônes se spécifient en sous-type S. En revanche, l'ajout de triiodothyronine (T3), la forme la plus active de l'hormone thyroïdienne, qui se lie avec une grande affinité à Thrβ induit le devenir de la majorité des cônes en sous-type L/M (Eldred et al., 2018). Dans notre protocole, le contrôle de la spécification du sous-type de cône dans les organoïdes rétiniens est probablement assuré par la présence de T3 dans le supplément B27 En optimisant le protocole de différenciation robuste préalablement défini dans notre équipe (Reichman et al., 2014 ;Reichman et al., 2017), nous sommes parvenus à générer des organoïdes rétiniens présentant tous les types cellulaires de la rétine neurale suivant une cinétique d'apparition similaire à celle observée au cours du développement de la rétine in vivo. ...
Thesis
Notre projet consiste à modéliser une forme spécifique de rétinite pigmentaire (RP) en utilisant des cellules iPS de patients. Nous avons d'abord optimisé un protocole de différenciation pour obtenir à partir de cellules iPS des organoïdes rétiniens avec une organisation structurelle plus proche de la rétine in vivo, permettant une maturation avancée des photorécepteurs. Cet outil nous a permis de récapituler entièrement le phénotype RP (dégénérescence des bâtonnets et des cônes), observé chez les patients présentant une mutation du gène RHODOPSINE, codant pour le pigment visuel. Nous avons ensuite utilisé la même approche pour comprendre la pathogénicité des RP liées à des mutations du gène PRPF31, codant pour un facteur d'épissage. Les organoïdes rétiniens ont résumé la dégénérescence des bâtonnets et la perte secondaire des cônes, observées chez les patients. Les cellules de l'épithélium pigmenté de la rétine présentaient également des défauts organisationnels et fonctionnels. Ces phénotypes dégénératifs rétiniens sont corrélés à un niveau d'expression plus faible de la protéine PRPF31, liant la pathogénicité à un mécanisme d'haploinsuffisance. Nous avons donc développé une stratégie d'augmentation du gène, en apportant une copie fonctionnelle de PRPF31 par CRISPR/Cas9 ou en utilisant un vecteur AAV, qui ont permis le sauvetage de la dégénérescence des cellules rétiniennes.
... Contrary to murine models that only contain S-and M− cones, the zebrafish retina contains cones that resemble the human visual system. In fact, the development of L-cones is controlled by TRβ2 in humans and its functional homolog (l-thrb + ) in zebrafish (Suzuki et al., 2013;Eldred et al., 2018;McNerney and Johnston, 2021), whereas in mice, this isoform controls the differentiation of M− cones (Ng et al., 2001). Therefore, this model becomes more interesting in terms of deciphering the TH-mediated molecular mechanisms that regulate the development of L-cones. ...
Article
The zebrafish is an optimal experimental model to study thyroid hormone (TH) involvement in vertebrate development. The use of state-of-the-art zebrafish genetic tools available for the study of the effect of gene silencing, cell fate decisions and cell lineage differentiation have contributed to a more insightful comprehension of molecular, cellular, and tissue-specific TH actions. In contrast to intrauterine development, extrauterine embryogenesis observed in zebrafish has facilitated a more detailed study of the development of the hypothalamic-pituitary-thyroid axis. This model has also enabled a more insightful analysis of TH molecular actions upon the organization and function of the brain, the retina, the heart, and the immune system. Consequently, zebrafish has become a trendy model to address paradigms of TH-related functional and biomedical importance. We here compilate the available knowledge regarding zebrafish developmental events for which specific components of TH signaling are essential.
... The human visual system (HVS) can quickly and accurately capture adequate information in complex visual perception signals, owing to its powerful information processing abilities, such as segmentation, classification, recognition, and discrimination. [1][2][3] The formation of vision involves three processes. [4] First, the optical signals are converted to electrical signals and stored temporarily in the retina. ...
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The human visual attention mechanism enables them to rapidly perceive important information and objects in a complex external scene; this effectively solves the problems of data redundancy, low‐resolution images, and substantial computing resources. The process by which the attention system reconstructs the visual information can be considered as integrating internal attention signals with external visual details in the postsynaptic neuron. However, electronic devices that simulate visual attention modulation by incorporating device characteristics into neuromorphic vision systems (NVSs) to achieve visual attention behavior are rarely reported. Herein, a synapse device that integrates optical and electrical stimulation is designed using ReS2/hBN/monolayer graphene heterojunction to mimic attention regulation and integrate multiple neuron signals successfully. The synapse array can imitate perceptual learning of the human visual system (HVS) to realize visual preprocessing, such as image contrast improvement and weak signal enhancement at the sensory terminal, and overcome data redundancy. Moreover, by applying gate voltage pulses, electric‐tunable synaptic plasticity is successfully observed, attributed to the carrier trapping and de‐trapping mechanism in the floating layer. Attention stabilization, fluctuation, distraction, and reinforcement are exhibited, simulating the attention behaviors of the HVS. Thus, an NVS with attention mechanism is established depending on the optoelectronic hybrid synaptic plasticity of the device, which successfully mimics the HVS to perform a multi‐target recognition task. Furthermore, the effect of device defects on the NVS is rarely evaluated, in which a method is provided to analyze the application results of the NVS when considering uniformity and fault rate. This work may provide new inspiration for developing neuromorphic vision systems for autonomous driving and brainwave control in the future.
... Although there has been an improvement in the cone-to-rod ratio of photoreceptors [27] compared to the mature retina, the response to light stimulation is relatively low. Mostly, studies of retinal organoids have concentrated on photoreceptor development and diseases that severely affect these cells [49,224,225]. In contrast, studies of RGCs within retinal organoids have drawn far less attention. ...
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Retinal organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that mimic the retina's spatial and temporal differentiation, making them useful as in vitro retinal development models. Retinal organoids can be assembled with brain organoids, the 3D self-assembled aggregates derived from hPSCs containing different cell types and cytoarchi-tectures that resemble the human embryonic brain. Recent studies have shown the development of optic cups in brain organoids. The cellular components of a developing optic vesicle-containing or-ganoids include primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. The importance of retinal organoids in ocular diseases such as age-related macular degeneration, Stargardt disease, retinitis pigmentosa, and diabetic retinopathy are described in this review. This review highlights current developments in retinal organoid techniques, and their applications in ocular conditions such as disease modeling, gene therapy, drug screening and development. In addition, recent advancements in utilizing extracellular vesicles secreted by retinal organoids for ocular disease treatments are summarized.
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Background Recurrence of retinoblastoma (RB) following chemoreduction is common and is often managed with local (intra-arterial/intravitreal) chemotherapy. However, some tumors are resistant to even local administration of maximum feasible drug dosages, or effective tumor control and globe preservation may be achieved at the cost of vision loss due to drug-induced retinal toxicity. The aim of this study was to identify drugs with improved antitumor activity and more favorable retinal toxicity profiles via screening of potentially repurposable FDA-approved drugs in patient-derived tumor organoids. Methods Genomic profiling of five RB organoids and the corresponding parental tissues was performed. RB organoids were screened with 133 FDA-approved drugs, and candidate drugs were selected based on cytotoxicity and potency. RNA sequencing was conducted to generate a drug signature from RB organoids, and the effects of drugs on cell cycle progression and proliferative tumor cone restriction were examined. Drug toxicity was assessed with human embryonic stem cell-derived normal retinal organoids. The efficacy/toxicity profiles of candidate drugs were compared with those of drugs in clinical use. Results RB organoids maintained the genomic features of the parental tumors. Sunitinib was identified as highly cytotoxic against both classical RB1-deficient and novel MYCN-amplified RB organoids and inhibited proliferation while inducing differentiation in RB. Sunitinib was a more effective suppressor of proliferative tumor cones in RB organoids and had lower toxicity in normal retinal organoids than either melphalan or topotecan. Conclusion The efficacy and retinal toxicity profiles of sunitinib suggest that it could potentially be repurposed for local chemotherapy of RB.
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The function of a hormone receptor requires mechanisms to control precisely where, when and at what level the receptor gene is expressed. An intriguing case concerns the selective induction of thyroid hormone receptor β2 (TRβ2), encoded by Thrb, in the pituitary and in cone photoreceptors in which it critically regulates expression of the opsin photopigments that mediate color vision. Here, we investigate the physiological significance of a candidate enhancer for induction of TRβ2 by mutagenesis of a conserved intron region in its natural context in the endogenous Thrb gene in mice. Mutation of e-box sites for bHLH (basic-helix-loop-helix) transcription factors preferentially impairs TRβ2 expression in cones whereas mutation of nearby sequences preferentially impairs expression in pituitary. A deletion encompassing all sites impairs expression in both tissues, indicating bifunctional activity. In cones, the e-box mutations disrupt chromatin acetylation, blunt the developmental induction of TRβ2 and ultimately, impair cone opsin expression and sensitivity to longer wavelengths of light. These results demonstrate the necessity of studying an enhancer in its natural chromosomal context for defining biological relevance and reveal surprisingly critical nuances of level and timing of enhancer function. Our findings illustrate the influence of non-coding sequences over thyroid hormone functions.
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Our previous study has shown that individuals with untreated hypothyroidism display significantly higher partial error scores (√PES) along the blue-yellow (B-Y) axis compared to the red-green (R-G) axis than normal individuals, using the Farnsworth-Munsell 100 hue test (FM-100 test) [J. Opt. Soc. Am. A, 37, A18 - A25 (2020)]. We wished to determine how color discrimination may change when hypothyroidism has been treated to the point of euthyroidism. Color discrimination was reassessed for 17 female individuals who had undergone treatment for hypothyroidism, and the results were compared with 22 female individuals without thyroid dysfunction. No statistically significant difference was found in the total error score (√TES) for the first and second measurement for both groups (p > 0.45). The √PES for the hypothyroid group improved significantly in the previously impaired color regions after the treatment. Color discrimination defects found in untreated hypothyroidism can be negated with treatment of the condition over an appropriate time period.
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Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.
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Purpose: Blue cone monochromacy (BCM) is a rare inherited cone disorder in which both long- (L-) and middle- (M-) wavelength sensitive cone classes are either impaired or nonfunctional. Assessing genotype-phenotype relationships in BCM can improve our understanding of retinal development in the absence of functional L- and M-cones. Here we examined foveal cone structure in patients with genetically-confirmed BCM, using adaptive optics scanning light ophthalmoscopy (AOSLO). Methods: Twenty-three male patients (aged 6-75 years) with genetically-confirmed BCM were recruited for high-resolution imaging. Eight patients had a deletion of the locus control region (LCR), and 15 had a missense mutation-Cys203Arg-affecting the first two genes in the opsin gene array. Foveal cone structure was assessed using confocal and non-confocal split-detection AOSLO across a 300 × 300 µm area, centered on the location of peak cell density. Results: Only one of eight patients with LCR deletions and 10 of 15 patients with Cys203Arg mutations had analyzable images. Mean total cone density for Cys203Arg patients was 16,664 ± 11,513 cones/mm2 (n = 10), which is, on average, around 40% of normal. Waveguiding cone density was 2073 ± 963 cones/mm2 (n = 9), which was consistent with published histological estimates of S-cone density in the normal eye. The one patient with an LCR deletion had a total cone density of 10,246 cones/mm2 and waveguiding density of 1535 cones/mm2. Conclusions: Our results show that BCM patients with LCR deletions and Cys203Arg mutations have a population of non-waveguiding photoreceptors, although the spectral identity and level of function remain unknown.
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The eyes of aquatic organisms may be damaged by exposure to pollutants. Zebrafish is a common laboratory model to study ocular toxicity, combining both fish and vertebrate characteristics. The toxic effects on zebrafish eyes caused by pollutants include morphological changes and damage to the retina at the molecular, cellular, and tissue levels; and abnormalities in the visual phototransduction and electrical signal transmission processes. Such damage induces functional disorders of vision-related behaviors. The underlying mechanisms include thyroid hormone signaling, retinoic acid signaling, and retinal glucose metabolism. Here, we review the ocular toxicity phenotypes and related signaling pathways induced by contaminants. We present detection methods and emerging tools for studying ocular toxicity. We also propose a model to predict the potential ocular toxicity of contaminants.
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Purpose: To compare visual dysfunction between very preterm born (VPB) children with no-retinopathy of prematurity (no-ROP) at 6-10 years of age and an age- and sexmatched full-term born controls. Methods: This is an observational, prospective study; included 30 children, 6-10 years of age, born ≤ 32 weeks of gestation, with no-ROP and 30 age- and sex-matched fullterm born controls, conducted from January 2015 until August 2015. All children underwent complete ophthalmic evaluation. Main outcome measures include visual functions (best corrected visual acuity (BCVA), color vision and stereo-acuity), ocular alignment, refractive errors, and the presence of amblyopia and nystagmus. Results: Mean BCVA of the right eyes was 0.04 ± 0.08 logMAR, for VPB children and 0.02 ± 0.05 logMAR for the full-term children (P =0.075). Mean BCVA for the left eyes was 0.07 ± 0.09 logMAR for VPB children and 0.02 ± 0.05 logMAR for the full-term children (P=0.014). Refractive errors were slightly higher though not statistically significant in VPB children compared to full-term children (P=0.125). The incidence of myopia and hypermetropia were 16.7% and 40% respectively in VPB children, and 10% and 23.3% respectively in full-term children. Anisometropia found only in VPB children with an incidence of 16.7%. Amblyopia found in 10% of VPB children compared to 3.3% in full-term children. Strabismus was found equally in 10% of each group. Conclusion: VPB children with no-ROP are at an increased risk of developing decreased BCVA at least in one eye and anisometropia compared to an age matched full-term controls.
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Cell type-specific investigations commonly employ gene reporters or single-cell (sc) analytical techniques. However, reporter line development is arduous and generally limited to a single gene of interest, while scRNA-seq frequently yields equivocal results that preclude definitive cell identification. To examine gene expression profiles of multiple retinal cell types derived from human pluripotent stem cells (hPSCs), we performed scRNA-seq on optic vesicle-like structures (OVs) cultured under cGMP-compatible conditions. However, efforts to apply traditional scRNA-seq analytical methods based on unbiased algorithms were unrevealing. Therefore, we developed a simple, versatile, and universally applicable approach that generates gene expression data akin to those obtained from reporter lines. This method ranks single cells by expression level of a bait gene and searches the transcriptome for genes whose cell-to-cell rank order expression most closely matches that of the bait. Moreover, multiple bait genes can be combined to refine datasets. Using this approach, we provide further evidence for the authenticity of hPSC-derived retinal cell types. This article is protected by copyright. All rights reserved.
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Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017.
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Extrapituitary roles for hypothalamic neurohormones have recently become apparent and clinically relevant, based on the use of synthetic peptide analogs for the treatment of multiple conditions including cancers, pulmonary edema and myocardial infarction. In the eye, it has been suggested that some of these hormones and their receptors may be present in the ciliary body, iris, trabecular meshwork and retina, but their physiological role has yet to be elucidated. Our study intends to comprehensively demonstrate the expression of some hypothalamic neuroendocrine hormones and their receptors within different retinal and extraretinal structures of the human eye. Immunofluorescence, Western blot analysis, and RT-PCR were used to evaluate the qualitative and quantitative expression of Luteinizing Hormone Releasing Hormone (LHRH), Growth Hormone Releasing Hormone (GHRH), Thyrotropin Releasing Hormone (TRH), Gastrin Releasing Peptide (GRP) and Somatostatin as well as their respective receptors (LHRH-R, GHRH-R, TRH-R, GRP-R, SST-R1) in cadaveric human eye tissue and in paraffinized human eye tissue sections. The hypothalamic hormones LHRH, GHRH, TRH, GRP and Somatostatin and their respective receptors (LHRH-R, GHRH-R, TRH-R, GRPR/BB2 and SST-R1), were expressed in the conjunctiva, cornea, trabecular meshwork, ciliary body, lens, retina, and optic nerve.
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The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.
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Purpose: Photoreceptors in the mouse retina express much of the molecular machinery necessary for phototransduction and glutamatergic transmission prior to eye opening at postnatal day 13 (P13). Light responses have been observed collectively from rod and cone photoreceptors via electroretinogram recordings as early as P13 in mouse, and the responses are known to become more robust with maturation, reaching a mature state by P30. Photocurrents from single rod outer segments have been recorded at P12, but no earlier, and similar studies on cone photoreceptors have been done, but only in the adult mouse retina. In this study, we wanted to document the earliest time point in which outer retinal photoreceptors in the mouse retina begin to respond to mid-wavelength light. Methods: Ex-vivo electroretinogram recordings were made from isolated mouse retinae at P7, P8, P9, P10, and P30 at seven different flash energies (561 nm). The a-wave was pharmacologically isolated and measured at each developmental time point across all flash energies. Results: Outer-retinal photoreceptors generated a detectable response to mid-wavelength light as early as P8, but only at photopic flash energies. a-wave intensity response curves and kinetic response properties are similar to the mature retina as early as P10. Conclusion: These data represent the earliest recorded outer retinal light responses in the rodent. Photoreceptors are electrically functional and photoresponsive prior to eye opening, and much earlier than previously thought. Prior to eye opening, critical developmental processes occur that have been thought to be independent of outer retinal photic modulation. However, these data suggest light acting through outer-retinal photoreceptors has the potential to shape these critical developmental processes.
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Hereditary retinal dystrophies, specifically retinitis pigmentosa (RP) are clinically and genetically heterogeneous diseases affecting primarily retinal cells and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Understanding the pathogenicity behind these diseases has been largely precluded by the unavailability of affected tissue from patients, large genetic heterogeneity and animal models that do not faithfully represent some human diseases. A landmark discovery of human induced pluripotent stem cells (hiPSC) permitted the derivation of patient-specific cells. These cells have unlimited self-renewing capacity and the ability to differentiate into RP-affected cell types, allowing the studies of disease mechanism, drug discovery and cell replacement therapies, both as individual cell types and organoid cultures. Together with precise genome editing, the patient specific hiPSC technology offers novel strategies for targeting the pathogenic mutations and design therapies toward retinal dystrophies. We summarize current hiPSC-based RP models and highlight key achievements and challenges of these cellular models, as well as questions that still remain unanswered. This article is protected by copyright. All rights reserved.
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Clinical and genetic heterogeneity associated with retinal diseases makes stem-cell-based therapies an attractive strategy for personalized medicine. However, we have limited understanding of the timing of key events in the developing human retina, and in particular the factors critical for generating the unique architecture of the fovea and surrounding macula. Here we define three key epochs in the transcriptome dynamics of human retina from fetal day (D) 52 to 136. Coincident histological analyses confirmed the cellular basis of transcriptional changes and highlighted the dramatic acceleration of development in the fovea compared with peripheral retina. Human and mouse retinal transcriptomes show remarkable similarity in developmental stages, although morphogenesis was greatly expanded in humans. Integration of DNA accessibility data allowed us to reconstruct transcriptional networks controlling photoreceptor differentiation. Our studies provide insights into human retinal development and serve as a resource for molecular staging of human stem-cell-derived retinal organoids.
Article
Purpose: Mutations in the coding sequence of the L and M opsin genes are often associated with X-linked cone dysfunction (such as Bornholm Eye Disease, BED), though the exact color vision phenotype associated with these disorders is variable. We examined individuals with L/M opsin gene mutations to clarify the link between color vision deficiency and cone dysfunction. Methods: We recruited 17 males for imaging. The thickness and integrity of the photoreceptor layers were evaluated using spectral-domain optical coherence tomography. Cone density was measured using high-resolution images of the cone mosaic obtained with adaptive optics scanning light ophthalmoscopy. The L/M opsin gene array was characterized in 16 subjects, including at least one subject from each family. Results: There were six subjects with the LVAVA haplotype encoded by exon 3, seven with LIAVA, two with the Cys203Arg mutation encoded by exon 4, and two with a novel insertion in exon 2. Foveal cone structure and retinal thickness was disrupted to a variable degree, even among related individuals with the same L/M array. Conclusions: Our findings provide a direct link between disruption of the cone mosaic and L/M opsin variants. We hypothesize that, in addition to large phenotypic differences between different L/M opsin variants, the ratio of expression of first versus downstream genes in the L/M array contributes to phenotypic diversity. While the L/M opsin mutations underlie the cone dysfunction in all of the subjects tested, the color vision defect can be caused either by the same mutation or a gene rearrangement at the same locus.