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Figure 1. The basic structure of the mammalian retina:
Left: A vertical section of mouse retina showing labelling of the major neuronal cell types.
Immunostaining for cone photoreceptors (anti-cone arrestin, blue), horizontal cells (anticalbindin, pink), bipolar cell terminals (anti-synaptotagmin2 and anti-PKC, red),
amacrine cells (anti-calretinin, purple), and ganglion cells (SMI-32, white).Right: A
schematic showing the layers of the mouse retina corresponding to the left side image.
PKC = Protein Kinase C, SMI-32 = anti hypo-phosphorylated neurofilament heavy chain,
ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer, OPL =
outer plexiform layer, IPL = inner plexiform layer, HC = horizontal cell, BC = bipolar
cell, AC = amacrine cell, RGC = retinal ganglion cell. (adapted from reference 2)
37
Figure 2. Development timeline of the retina:
Top: a depiction of the progressively changing competence of progenitor cells as they
divide to form daughter progenitor cells, neurons or asymmetrically forming both
progenitor and neuronal cells. Bottom: The overlapping time points at which different
cell types of the retina are generated (adapted from reference 6).
38
Figure 3. Basic helix-loop-helix and homeodomain core transcription factors
regulate generation of retinal cell types:
Vsx2 is expressed in the common progenitor pool preventing differentiation by
repressing Atoh7, Ptf1a and Vsx1. As development progresses Vsx2 expression in most
RPCs ceases allowing formation of the different cell types. The remainder of the Vsx2expressing RPCs generate a subset of BCs and MG . RPC=Retinal progenitor cell, BC =
Bipolar cell, GC = Ganglion cell, PR = Photoreceptor cell, HC = Horizontal cell, AC =
amacrine cell (adapted from reference 10)
39
Figure 4. Histone modification crosstalk
Histone modifications on adjacent or distant sites interact in order to fine tune their effects
on chromatin. Arrow ends indicate positive interactions while flat ends indicate negative
interactions (adapted from reference 19).
40
Figure 5. Histone methylation patterns at mammalian genes
H3K4me3 commonly occupies the promoters of active genes whilst H3K36me3 occupies
their gene bodies. At inactive genes H3K27me3 is the major player occupying their gene
bodies and flanking regions. H3K9me3 and H4K20me3 also occupy inactive genes but
to a lesser extent (adapted from reference 22).
41
42
Figure 6. Utx expression pattern in the mouse retina
A: In prenatal stages of development (E14-E16) Utx is expressed in RPCs (Ki67 positive)
of the NBL and in the GCL. Perinatally Utx is highly expressed in postmitotic cells in the
INBL and GCL (E18-P3). B: In mature retina (P14) Utx is expressed in the INL and GCL
and co-stains with markers of all cells in these layers. C: Utx mRNA expression in the
retina is stable from developmental to mature stage on RT-qPCR. D: The expression of
Jmjd3 in the mature retina (P14) is very low both in the INL and GCL; there is no
expression in the ONL. E: RNA sequencing also showed that Utx is stably expressed
while Jmjd3 expression peaks at mid postnatal stages and is low before and after that
(accession number GSE71462). (NBL= neuroblastic layer, ONBL/INBL= outer/inner
neuroblastic layer respectively, GCL= ganglion cell layer). FPKM = Fragments Per
Kilobase of transcript per Million mapped reads. Scale bar =25 µm (A&B), 30 µm.
43
Figure 7. Utx expression was efficiently knocked down using two different shRNAs
Utx expression decreased after knockdown with two different shRNA (D-I) in
comparison to control (A-C). shRNAs or pU6 empty vector were transfected together
with an EGFP-expressing plasmid in P1 retinae which were then cultured for 3days as
explants. Utx expression was analysed by immunohistochemistry. EGFP and Utx double
positive cells were counted over 440µm long sections. The average number of cells from
three independent experiments is shown. ** p-value <0 .01, p-value > 0.05 = not
significant (ns) by ANOVA followed by post-hoc Tukey’s HSD test. Scale bar 25µm.
44
Figure 8. Rod bipolar cell number is reduced by knockdown of Utx.
Knockdown of Utx using the two shRNAs reduced PKC-expressing rod bipolar cells
(PKC). A control vector (A-C) or shRNA(D-I) was transfected together with an EGFPexpressing plasmid at P1 then retinae were cultured for 12 days as explants. The
percentage of EGFP+PKC+ cells in the INL was obtained from at least three independent
experiments, ** p-value <0 .01 by ANOVA followed by post-hoc Tukey’s HSD test.
ONL = outer nuclear layer, INL= inner nuclear layer, GCL= ganglion cell layer, PKCα
(Protein kinase C alpha). Scale bar = 25µm
45
Figure 9. Overall bipolar cell number slightly decreased after knockdown of Utx
Overall bipolar cell number decreased slightly after transfection with shUtx . A control
vector (A-C) or shRNA(D-I) was transfected together with an EGFP-expressing plasmid
at P1 then, explants were cultured for 12 days. The percentage of EGFP+Chx10+ cells in
the INL was obtained from at least three independent experiments. p-value > 0.05 by
ANOVA followed by post-hoc Tukey’s HSD test. ONL = outer nuclear layer, INL= inner
nuclear layer, GCL= ganglion cell layer, Chx10 (pan bipolar cell marker). Scale bar =
25µm.
46
47
Figure 10. Other bipolar cell subtypes were not affected by knocking down Utx:
A: Cone OFF bipolar subtype number was not significantly affected by knock down of
Utx . The U6 promoter (control) or shRNA targeting Utx was transfected together with
an EGFP-expressing plasmid at P2 then, explants were cultured for 11 days. B: Cone ON
bipolar cells represented by weak Isl1 signals (arrow heads) were not significantly
changed by knockdown of Utx while rod ON bipolar cells represented by strong Isl1
signals (full arrows) were significantly reduced as expected. C & D : Counting results
showed no significant difference in cone OFF bipolar and cone ON bipolar cell subtypes.
Counting was done over 438µm long images; cells from two images were counted for
each marker and the percentage of EGFP+marker+ cells in the INL was obtained from
three independent experiments. *p-value < 0.05, **< 0.01 by ANOVA followed by posthoc Tukey’s HSD test. ns = not significant ONL = outer nuclear layer, INL= inner nuclear
layer, GCL= ganglion cell layer, Recoverin = cone OFF bipolar subtype marker, Isl1 =
cone ON and rod ON bipolar subtype marker. Scale bar = 25µm
48
49
Figure 11. Knockdown of Utx does not affect the differentiation of other retinal
cell types
Immunohistochemical analysis of other cell types revealed no difference in cell number
after knockdown of Utx. P1 retina were electroporated with shRNA targeting Utx along
with an EGFP expressing plasmid then cultured for 12 days. A: amacrine cells stained by
transcription factor AP-2; alpha (Tfap2a), horizontal cells stained by calbindin, Müller
glia stained by glutamine synthetase (GS) and rod photoreceptors stained by PNR also
known as nuclear receptor subfamily 2, group E, member 3 (Nr2e3). B: Counting results
over 438µm long images; cells from two images were counted for each marker and the
average number from three independent experiments, calculated. The thickness of the
outer nuclear layer reflecting photoreceptor number was measured in the left, mid and
right thirds of the outer nuclear layer and the lengths, averaged. Two images were
measured in Utx knockdown and control retina. p-value> 0.05 = not significant(ns) by
Student t test. Scale bar = 12µm
50
51
52
Figure 12. Utx overexpression rescues rod bipolar cells in shUtx transfected retina:
A: Utx knockdown at P0 also leads to loss of PKC+ cells. B: Utx over expression on its
own did not affect rod bipolar cell structure or significantly alter the PKC+ cell number.
C: Overexpression of Utx after its knockdown rescued PKC+ cell number. D: The
percentage of EGFP+PKC+ cells and the average number of PKC+ cells in the INL were
obtained from three independent experiments. E: In the same explants, Utx expression in
the INL was reduced by shUtx. F: Utx was successfully overexpressed in Utx-transfected
retina with stronger signals in the ONL than in the INL; only some EGFP+ cells in the
INL overexpressed Utx. P0 or P2 (Overexpression of Utx alone) retinae were transfected
with shUtx or the U6 promoter as control(A), a Utx over expression vector or U6
promoter as control (B), shUtx or shUtx and Utx overexpression vector (C) along with
the EGFP plasmid before they were cultured as explants for 12 days. Scale bar = 18µm
(A,C,E,F), 25µm(B). **p< 0.01,*p< 0.05, ns= not significant. ONL = outer nuclear layer,
INL= inner nuclear layer, GCL= ganglion cell layer, PKC α (Protein kinase C alpha).
53
54
Figure 13. Generation of Utx conditional knockout mice
A: Top- deletion of exon 11&12 that encodes Utx including the region of the JmjC
domain. Heterozygous Utx-floxed and Dkk3-cre expressing male mice (a gift from Prof.
Honda) were crossed with homozygous Utx floxed female mice to give Utx conditional
knockout (Utx cKO) female mice. The exons containing the TRP domain are shown in
yellow, and the exons containing JmjC domain are shown in red. Bottom- schematic of
the Utx (Kdm6a) gene showing the tetratricopeptide repeats (TPR) and the catalytic
Jumonji C (JmjC) domain (adopted from reference 34) B: Utx transcript level was
depleted in Utx cKO retinae. Total RNA extracted from cKO and WT retinae was
quantified by RT-qPCR and cq values used to calculate relative expression levels. C: Leftimmuno-staining for H3K27me3 showed increase of H3K27me3 in the INL and GCL
after knocking out Utx (In-figure A & B). Right- H3K27me3 grey value was measured
over 200µm (GCL), 100µm (INL), 50µm (ONL) of WT and cKO images. The ratio of
the Utx cKO average grey to that of WT is shown. ONL = outer nuclear layer, INL=
inner nuclear layer, GCL= ganglion cell layer, CKO = conditional knockout, WT = wild
type. Scale bar = 25µm
55
56
Figure 14. Rod bipolar cells decrease when Utx is knocked out
A: Rod bipolar cells were decreased by Utx cKO compared to WT in developing and
adult retina at P10, P14, P24 and P40 stages. C: Overall bipolar cell number decreased
with varying significance at different stages. B & D: Counting data corresponding to the
images in A & B; PKCα was consistently decreased by Utx cKO while Chx10 expression
also decreased to different extents. Two 438µm wide images were counted for both
markers in WT and Utx cKO retinae. The average number of cells in three independent
experiments is shown. *p<0.05, **p<0.01 by student t test, ns = not significant. ONL =
outer nuclear layer, INL = inner nuclear layer, INL= inner nuclear layer, GCL= ganglion
cell layer, CKO = conditional knockout, WT = wild type, PKC α = rod bipolar cell marker,
Chx10 pan bipolar cell marker. Scale bar = 25µm
57
58
Figure 15. Other bipolar subtypes were not affected by loss of Utx:
A: Cone ON bipolar cell number was not significantly different between WT and Utx
cKO at P14.The arrow head shows weakly Isl1+Pax6- cells which represent cone ON
bipolar cells and their number was the same after Utx cKO, while the full arrow shows
strongly Isl1+Pax6- cells in the outer most part of the inner nuclear layer that represent
rod ON bipolar cells and were significantly decreased as expected. B: Graphical
representation shows a significant decrease in rod ON bipolar cells (left side) and no
significant change in cone ON bipolar cells after cKO of Utx. C: Cone OFF bipolar cell
number was not affected by knockout of Utx. D: The average number of cone OFF bipolar
cells shown in C was not different between WT and Utx cKO retinae. Counting was done
over 438µm long images; cells from two images were counted for each marker and the
average number from three independent experiments, calculated cells. *p<0.05 by student
t test. Isl1 (cone ON and rod ON bipolar cell marker), Pax6 (pan amacrine cell marker),
Recoverin (cone OFF bipolar cell marker ), Bhlhb5 (cone OFF bipolar cell marker). CKO
= conditional knockout, WT = wild type Scale bar = 25 µm
59
Fig.16 Amacrine cell number was not affected by knockout of Utx:
The number of amacrine cells did not change in Utx cKO compared to WT . Retinae were
harvested at postnatal day 3 (P3),cryo-sectioned and stained with the indicated marker .
NBL= neuroblastic layer ,GCL= Ganglion cell layer, Transcription factor ap2a (Tfap2a)
= pan-amacrines cell marker . Scale bar = 25µm.
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Fig17. Proliferation and apoptosis were not affected by knockout of Utx:
The number of proliferating cells at P3 did not change after knockout of Utx . Apoptosis
also seemed to not be very different between WT and Utx cKO retina. Retinae were
harvested at postnatal day 3 (P3),cryo-sectioned and stained with the indicated markers .
NBL= neuro-blastic layer ,GCL= Ganglion cell layer, Ki67= mitotic cell marker, AC3=
marker of apoptosis . Scale bar = 25µm.
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