RESEARCH REPORT
805
Development 135, 805-811 (2008) doi:10.1242/dev.012088
Evidence for an evolutionary conserved role of
homothorax/Meis1/2 during vertebrate retina development
Peer Heine, Eva Dohle, Keely Bumsted-O’Brien*, Dieter Engelkamp and Dorothea Schulte†
During eye development in D. melanogaster, the TALE-homeodomain protein Homothorax (Hth) is expressed by progenitor cells
ahead of the neurogenic wave front, promotes rapid proliferation of these cells and is downregulated before cells exit the cell cycle
and differentiate. Here, we present evidence that hth function is partially conserved in vertebrates. Retinal progenitor cells (RPCs) in
chicks and mice express two Hth-related proteins, Meis1 and Meis2 (Mrg1), in species-specific temporal sequences. Meis1 marks
RPCs throughout the period of neurogenesis in the retina, whereas Meis2 is specific for RPCs prior to the onset of retinal
differentiation. Transfection of Meis-inactivating constructs impaired RPC proliferation and led to microphthalmia. RNAinterference-mediated knock-down of expression indicated that progenitor cells expressing Meis1 together with Meis2 proliferate
more rapidly than cells expressing Meis1 alone. Transfection of Meis-inactivating constructs reduced the expression of cyclin D1
(Ccnd1) in the eye primordium and co-transfection of cyclin D1 partially rescued RPC proliferation. Collectively, these results suggest
that (1) Meis1 and Meis2, similar to hth, maintain retinal progenitor cells in a rapidly proliferating state; (2) they control the
expression of some ocular-determination genes and components of the cell cycle machinery; and (3) together with the speciesspecific differences in Meis1/Meis2 expression, combinatorial expression of Meis family proteins might be a candidate mechanism
for the differential regulation of eye growth among vertebrate species.
KEY WORDS: Proliferation, Retina, Meis, Cyclin D1, Pax6, Mouse, Chick
Department of Neuroanatomy, Max-Planck-Institute for Brain Research,
Deutschordenstr. 46, 60528 Frankfurt, Germany.
*Present address: Department of Optometry and Vision Science, University of
Auckland, Auckland, New Zealand
†
Author for correspondence (e-mail: [email protected])
Accepted 27 December 2007
Pax6 homolog Eyeless (Ey) and the zinc-finger protein Teashirt
(Tsh) promotes rapid, asynchronous proliferation of retinal
progenitor cells and prevents their premature differentiation (Bessa
et al., 2002). The targets of hth in the eye imaginal disc and its
mechanism of action are not yet known.
Vertebrate homologs of Hth are the Meis family proteins
Meis1, Meis2 (Mrg1) and Meis3 (Mrg2). Meis proteins were first
identified as co-factors of other homeodomain-containing
proteins and play multiple roles in development and disease
(Berkes et al., 2004; Burglin, 1997; Mercader et al., 1999; Moens
and Selleri, 2006; Nakamura et al., 1996). Meis1 and Meis2 are
expressed in the eye, but only Meis1-deficient embryos have been
generated to date, and these display defects in angiogenesis and
eye development (Azcoitia et al., 2005; Hisa et al., 2004). The
precise function of Meis1 and Meis2 in the retina is still largely
unknown. Here, we provide evidence based on gain-of-function
and knock-down experiments, and on the expression of functionblocking constructs in chick embryos that both proteins play an
evolutionary conserved role in maintaining the rapidly
proliferating state of early retinal progenitor cells.
MATERIALS AND METHODS
In situ hybridization and immunochemical detection
The following probes were used to analyze gene expression in mice and
chicks: chick Meis1 [nucleotides (NT) 1-711], chick Meis2 (NT 1-818),
mouse Meis1 (NT 247-1122), mouse Meis2 (NT 558-1056) and mouse Pax6
(Grindley et al., 1995). All other probes were gifts of C. Cepko (Harvard
Medical School, Boston, MA), C. Tabin (Harvard Medical School, Boston,
MA), F. Pituello (University P. Sabatier, Toulouse, France) and J.-M. Matter
(University of Geneva, Geneva, Switzerland) or were cloned with genespecific primers (primer sequences are available upon request). The dilutions
of primary antibodies were: polyclonal anti-Meis2 1:2000 (A. Buchberg);
purified monoclonal anti-Pax6 Fab-fragments 1:5000 (Developmental
Studies Hybridoma Bank), polyclonal anti-RCAS (p27) 1:10000 (Charles
River Laboratories, CT), polyclonal anti-GFP 1:1000 (Molecular Probes,
OR), polyclonal anti-phosphorylated histone H3 (pH3) 1:1000 (Upstate
DEVELOPMENT
INTRODUCTION
Despite the different structure and developmental origin of the
vertebrate lens-eye and the invertebrate compound eye, some
components of the regulatory network that directs eye development
are conserved across the animal kingdom. The transcription factor
eyeless/Pax6, for instance, is essential for eye development in
vertebrates and invertebrates, and atonal and its vertebrate homolog
Ath5 (also known as Atoh7) are important regulators of retinal
neurogenesis in both systems (Kozmik, 2005; Vetter and Brown,
2001). However, examples of non-conservation are also known,
which include dachshund (dac) and its vertebrate homologs Dach1
or Dach2, or apterous and the closely related Lhx2 (Davis et al.,
2001; Davis et al., 2006; Porter et al., 1997). The molecular network
that controls ocular differentiation is, thus, only partially conserved.
During development of the vertebrate neural retina, six classes of
neurons and one class of glia are generated from multipotent
progenitor cells over a long developmental period and in
overlapping neurogenic waves (Livesey and Cepko, 2001). Retinal
ganglion cells (RGCs) are always born first (beginning by E10.5 in
the mouse or late E2 in the chick), followed by the other retinal cell
classes (Prada et al., 1991; Young, 1985). Postmitotic neurons in the
compound eye of Drosophila melanogaster, by contrast, are
generated in the wake of a single neurogenic wave, the
morphogenetic furrow (MF), which sweeps across the eye imaginal
disc during the third larval instar (Heberlein and Treisman, 2000).
Proliferating progenitor cells ahead of the MF express the TALEhomeodomain protein Homothorax (Hth), which together with the
RESEARCH REPORT
Biotechnology, NY), polyclonal anti-Myc tag 1:300 (Upstate
Biotechnology). In situ hybridization and immunohistochemical analyses
were performed as described previously (Engelkamp et al., 1999; Schulte et
al., 1999; Bumsted-O’Brien et al., 2007).
Construction of gene-delivery vectors
The coding sequence of Meis2a was cloned by RT-PCR from total RNA
extracted from HH10-14 chick embryonic heads using the primers
5⬘-CCATGGCGCAAAGGTAC and 5⬘-AGTGTAACTTGGCTGTG.
Meis2HA is full-length chick Meis2a fused to a triple HA-tag. For
Meis2EnR, Meis2a was fused to the repressor domain of D. melanogaster
engrailed (EnR), which contained a single Myc-tag at its C-terminus for
immunohistochemical detection. Meis1EnR is a fusion of the chick Meis1a
partial coding sequence (NCBI AF202933) extended by the first four amino
acids of mouse Meis1 (NCBI NM_010789). Controls included SOHo1, GH6
or Nkx5.1 (Hmx3) fused to EnR. The expression vector was either pMIWIII
or pMES, which contains an IRES-GFP cassette (Suemori et al., 1990;
Swartz et al., 2001). Unless stated otherwise, 1 ␮g/␮l of the expression
constructs were electroporated into the right optic vesicle as described
(Schulte et al., 1999). In experiments performed with pMIWIII, 0.7 ␮g/␮l
pMIWIII-GFP was co-electroporated for visualization. RIS(B) is a
replication-incompetent derivative of RCASBP(B), in which the env-gene
has been interrupted by introducing a premature stop codon. Infectious
RIS(B) viral particles were produced in cell culture by supplying the avian
(A)-envelope in trans. For RNA-interference (RNAi)-mediated knock-down
of expression, the psiSTRIKE-U6 Hairpin Cloning System was used
(Promega, Mannheim, Germany; see Fig. S1 in the supplementary material)
and 0.5-1 ␮g/␮l of the RNAi targeting constructs was electroporated into
the right optic vesicle.
Analysis of cell proliferation and cell death
pMES or pMES-MeisEnR were electroporated into the right optic vesicle at
HH11. Forty-eight hours later, at HH22, BrdU labeling was performed with
the BrdU Labeling and Detection Kit I (Roche, Mannheim, Germany);
labeling duration was 2 hours. Apoptotic cells were determined with the In
Situ Cell Death Detection Kit (Roche). As a control for the labeling
procedure, sections were treated with 200 U/ml DNAse I for 10 minutes at
room temperature.
Development 135 (5)
RESULTS AND DISCUSSION
Spatial-temporal expression of Meis family
members in mouse and chicken
Meis2 is expressed in the anterior neural plate of HamburgerHamilton stage 7 (HH7) chick embryos (Fig. 1A). Later, Meis2
was strongly expressed in the optic vesicle and optic cup, where
it co-localized with Pax6 (Fig. 1B,F-F⬙). Strikingly, expression
of Meis2 was downregulated in the retina after late E2 in a
central-to-peripheral wave, which paralleled the appearance of
RGC-competent, Ath5-expressing cells (Fig. 1C,D). By HH25,
Meis2 was absent from RPCs with the exception of the nasal
retinal periphery, which reflects the slight delay in maturation of
the nasal compared with the temporal retina in chicken (Fig. 1E,
arrowhead) (Prada et al., 1991). Meis2 expression in the chick is,
therefore, specific for progenitor cells ahead of the RGC
differentiation wave. This expression pattern is unique amongst
known RPC markers (e.g. Pax6, Six3 and Optx2), which
continue to be expressed when postmitotic neurons and glia are
generated. The related protein Meis1 was also expressed by
RPCs, but onset of expression lagged behind that of Meis2 and
expression continued throughout the period of proliferation (Fig.
1G-J). Meis2-specific transcripts were also detected in the mouse
eye anlage, but expression was lost prior to optic vesicle
invagination (Fig. 1K,L). Meis1 was strongly expressed in the
mouse optic vesicle and expression continued into postnatal
development (Fig. 1M,N and data not shown). Meis3 was not
detected in the eyes of either species at the embryonic stages
analyzed (data not shown). RPCs in chicks and mice, thus,
express different combinations of Meis1 and Meis2 over time:
self-renewing RPCs in the early eye anlage of both species
express Meis1 together with Meis2. Meis2 expression is
terminated before the onset of retinal differentiation, at the late
optic vesicle stage in mice, but just ahead of the RGC production
front in chicken.
Fig. 1. Spatial and temporal expression of
Meis1 and Meis2. (A-C,E) Expression of
Meis2 in chick at (A) HH7 (arrow, anterior
neural plate), (B) HH15 (E2; arrow, eye cup),
(C) HH19 (early E3) and (E) HH25 (E4.5).
Meis2 expression is lost in RPCs after late E2.
Arrows in C indicate the central domain,
where Meis2 is downregulated. (D) Expression
of Ath5 in chick at HH19. Arrows indicate
where Ath5+ cells appear at HH19.
(F-F⬙) Double immunostaining for Meis2 and
Pax6 at HH11. (G-J) Expression of Meis1 in
chick at (G) HH11, (H) HH15, (I) HH19 and (J)
HH25. Arrow in J indicates Meis1
downregulation in the RGC layer.
(K) Expression of Meis2 in mouse eye anlage
at E9.5. (L) Meis2 is not expressed in the
E12.5 mouse retina. (M,N) Meis1 expression
in the mouse optic vesicle (M) and neural
retina (N). Expression is downregulated in the
RGC layer (arrow). Boxed areas in E and J are
shown at high magnification in the insets. Le,
lens; lp, lens placode; mes, mesencephalon;
nR, neural retina; ov, optic vesicle; PE,
pigmented epithelium; SE, surface ectoderm.
Scale bars: 200 ␮m in C,D,I; 500 ␮m in
B,E,H,J; 50 ␮m in K,M; 100 ␮m in L,N.
DEVELOPMENT
806
Meis1 and 2 are required for rapid progenitor cell
proliferation
The presence of Meis1 and Meis2 in proliferating and
undifferentiated cells of the early retina suggests that both proteins
promote self-renewal of these cells. Moreover, rapid asynchronous
cell proliferation in the D. melanogaster eye imaginal disc is
restricted to cells expressing the Meis-related protein Hth, and lossof-function and gain-of-function studies have demonstrated that hth
is both necessary and sufficient for progenitor cell proliferation
(Bessa et al., 2002; Dominguez and Casares, 2005; Heberlein and
Treisman, 2000). We therefore hypothesized that the normal
proliferative capacity of early vertebrate RPCs might, at least in part,
depend on the presence of functional Meis proteins in these cells.
To test this idea, we first blocked Meis function by ectopically
expressing the coding region of Meis1a or Meis2a fused to the
engrailed repressor domain (Meis1EnR or Meis2EnR, collectively
termed MeisEnR) from D. melanogaster. These fusion proteins
compete with the endogenous Meis proteins for DNA binding and
can act as true antimorphs (Dibner et al., 2001; Inbal et al., 2001).
Twenty-four hours following introduction of MeisEnR and/or GFP
into the right optic vesicle (HH15-16), the right transfected and left
control eyes were similar in size (Fig. 4 and data not shown). From
HH21-22 onwards, however, the transfected eyes were severely
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807
microphthalmic (80%, n=12/15) compared with non-electroporated,
GFP- or Meis2HA-transfected eyes (wild type, n=13 embryos; GFP,
n=12; Meis2HA, n=9; Fig. 2A-B⬘ and data not shown) and often
exhibited an apparent partial transformation of the pigmented
epithelium to retina (Fig. 2C). MeisEnR-induced microphthalmia was
associated with a reduction in the total number of cells per retina.
Upon dissociation of transfected HH25 retinae, we counted an
average of 9.06⫻105 cells/GFP-transfected retina (s.e.m.=0.3001;
n=3) compared with 4.41⫻105 cells/MeisEnR-transfected retina
(s.e.m.=0.6899; n=3). Retinae transfected with RNAi-targeting
constructs against Meis1 and Meis2 contained an average of
5.15⫻105 (s.e.m.=0.856; n=3) cells per retina. These figures correlate
well with those determined by others (Dutting et al., 1983) and
demonstrate that transfection of Meis-inactivating constructs reduced
retinal cell counts. To determine whether this resulted from reduced
progenitor cell proliferation or enhanced cell death, we labeled
retinae 24 hours after transfection, a time before morphological
changes of the eye became apparent, with the thymidine analog
BrdU. Two hours after BrdU injection, 61.7% (±1.07 s.e.m.; n=13
embryos) of the GFP-expressing cells, but only 27.8% (±2.63 s.e.m.;
n=17) of the MeisEnR/GFP-expressing cells had incorporated the
label (Fig. 2D-F). To control for possible non-specific effects, several
other homeodomain-EnR fusion proteins were misexpressed in the
Fig. 2. Forced expression of a dominant-negative Meis
interferes with RPC proliferation. (A-C) MeisEnRtransfected chick eyes are microphthalmic. (D-E⬘) BrdU
incorporation following ectopic expression of GFP (D,D⬘) or
MeisEnR together with GFP (E,E⬘). (F) Quantification of the
results from D-E⬘. Error bars indicate s.d. (G,H) TUNEL
labeling of HH16 retinae transfected with nuclear red
fluorescent protein (nRFP; G) or MeisEnR together with nRFP
(H). (I) TUNEL labeling on a HH16 chick retina treated with
DNase I to visualize the effectiveness of the labeling strategy.
(J-L) Clonal analysis. Representative clones infected with
RIS(B)-GFP (J), RIS(B)-Meis2 (K) and RIS(B)-MeisEnR (L)
visualized with the virus-specific antibody p27 (green).
RIS(B)-MeisEnR-infected cells express the Myc-tag Cterminally fused to EnR (red, L). (M) Number of cells per
clone and per section plotted as a function of the frequency
at which clones of the respective sizes were observed.
DEVELOPMENT
Meis and vertebrate retina development
808
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Development 135 (5)
eye and were found to not affect progenitor cell proliferation (data
not shown). TUNEL labeling indicated that MeisEnR transfection
does not compromise cell survival (Fig. 2G-I).
To substantiate these results, we performed clonal analysis with a
replication-incompetent avian retrovirus [RIS(B)]. We infected
chick embryos at the optic vesicle stage with low-titer RIS(B)
viruses that contained either GFP, Meis2HA or the dominantnegative MeisEnR, and analyzed the number of progeny that
infected cells had given rise to 48 hours later (HH20, Fig. 2J-L).
Following infection with RIS(B)-GFP, the majority of clones
contained 5-7 cells/section with a second peak of 13
cells/clone/section, which most likely reflects a cohort of cells that
had undergone an additional round of cell division (n=74 clones;
Fig. 2J). Clone sizes did not change upon retroviral misexpression
of Meis2, suggesting that RPCs do not overproliferate in the
presence of elevated Meis2 levels (n=80 clones; Fig. 2K and data not
shown). This contrasts with published data on homothorax, as clones
overexpressing hth in the D. melanogaster eye imaginal disc can
grow very large (Bessa et al., 2002). Upon retroviral misexpression
of MeisEnR, the vast majority of clones contained only 2-3
cells/section with a second peak of 5 cells/clone/section (n=85
clones; Fig. 2L). Although retinal progenitor cells can divide in the
presence of MeisEnR, Meis function nevertheless appears to be
required for the rapid proliferation characteristic of early RPCs (Li
et al., 2000).
To determine the relative contribution of Meis1 and Meis2 to RPC
proliferation we performed RNAi-mediated knock-down of each
gene alone and in combination (Fig. 3A-E and see Fig. S1 in the
supplementary material). Following knock-down of Meis1, the
mitotic index of RPCs, visualized by detecting the mitosis-specific
phosphorylated form of histone H3 (pH3), decreased by 26.8%
(±1.4 s.e.m.; n=6 embryos) compared with RPCs transfected with
GFP or a randomized targeting sequence. Following Meis2 knockdown, the mitotic index decreased by 30.2% (±1.5 s.e.m.; n=6)
compared with the control, whereas simultaneous knock-down of
Meis1 and Meis2 or forced expression of Meis2EnR lead to a
59.53% (±0.19 s.e.m.; n=5) and 56.03% (±1.5 s.e.m.; n=3) reduction
of the percentage of mitotic cells, respectively. Thus, the mitotic
index of RPCs was more profoundly reduced when expression of
both Meis genes was knocked-down than in each of the single
knock-downs. These results led us to conclude that Meis1 and Meis2
do not function redundantly in the early chick retina, but instead act
together to control RPC proliferation. If this were the case, one
would expect that RPC proliferation would drop during normal
DEVELOPMENT
Fig. 3. Expression of Meis-inactivating constructs reduces RPC proliferation and cyclin D1 expression. (A-E) HH15 chick eyes transfected
with the listed expression constructs together with GFP (green) and stained for phosphorylated histone H3 (pH3, red). (A) Control siRNA construct
containing a randomized targeting sequence. pH3+/GFP+ indicates percentage of transfected cells in mitosis. (F-J) HH15 chick eyes stained for cyclin
D1 or cyclin D2 following transfection with the constructs indicated. (F,H) cyclin D1, (G,J) cyclin D2. (K-M) Retinae transfected with MeisEnR+GFP
(K), GFP (L) or MeisEnR+GFP together with cyclin D1 (M), stained for GFP (green) and pH3 (red); cell nuclei are counterstained with DAPI (blue). The
position of pH3-labeled cell nuclei is indicated in the right-hand and middle panels of K. (N) Percentage of cells in mitosis (y-axis) for each
transfected construct (x-axis). (O-R) Expression of Ath5 (O,Q) following transfection with the constructs indicated. The boxed areas in O,Q are
shown at high magnification in O⬘,Q⬘, respectively. F,G, H-J, O,P and Q,R show neighboring sections. (I,P,R) Transfection controls.
Meis and vertebrate retina development
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Fig. 4. Decrease in Pax6, Six3 and Chx10
expression following MeisEnR transfection.
(A-H) Expression of Pax6 (A-D), Chx10 (E,F) or
Six3 (G,H) in HH15 chick eyes transfected with
control-EnR (A,C,E,G) or MeisEnR (B,D,F,H).
(A,B,E,F) Wholemounts. (C,D,G,H) Vibratome
sections. (I) Quantification of the gene expression
changes observed. (J) RT-PCR of two independent
experiments (Exp_a, Exp_b) for Pax6 and ␤-actin
of chick HH15 eyes transfected with GFP (control)
or M1si_I and M2si_I together (Msi). PCR
conditions were: 30 seconds 94°C, 30 seconds
60°C, 1 minute 72°C for 27 cycles. (K) Relative
pixel intensity of the ethidium bromide-stained
bands from J (Pax6/␤-actin). Simultaneous knockdown of Meis1 and Meis2 expression decreases
Pax6 transcript levels in the eye. (L,M) Pax6 fails
to rescue cyclin D1 expression in MeisEnRtransfected chick retinae. Arrows indicate
domains of MeisEnR/Pax6 misexpression and
reduced cyclin D1 expression. (M) cyclin D1
expression following transfection of MeisEnR
together with Pax6. (N-P) Expression of cyclin D1
(N) or cyclin D2 (P) following Pax6EnR
misexpression. (O,L) Transfection control. L,M and
N-P are neighboring sections. (Q) Quantification
of the observed gene expression changes from
Fig. 3F-J and data not shown.
retina past HH24, and those that were present were clumped
together at the vitreal surface and did not show features of
differentiated neurons (see Fig. S3 in the supplementary material).
This was surprising, as retinal differentiation is normal in cyclin
D1 mutant mice despite the insufficient cell number in their
retinae. We therefore investigated whether the expression of other
genes that are essential for eye development also depends on Meis
function.
Meis proteins are components of the regulatory
network that controls eye development in
vertebrates
The paired-type transcription factor Pax6 is crucial for vertebrate eye
development and Meis proteins have been shown to directly regulate
expression of Pax6 in the developing lens and pancreas (Zhang et al.,
2002; Zhang et al., 2006). Pax6-specific transcripts were decreased in
eyes transfected with MeisEnR (Fig. 4A-D and see Fig. S3 in the
supplementary material). This suggests that Meis1 and Meis2
contribute to the regulation of retinal Pax6 expression. This view is
supported by a series of co-transfection experiments in which
transfection of Meis1 or Meis2 together with Pax6 stimulated the
activity of an ␣-enhancer-driven ␤-galactosidase reporter construct
about 1.6-fold over the previously reported autostimulation by Pax6
alone, whereas co-transfection of Pax6 with unrelated homeodomain
proteins did not elevate enhancer activity (see Fig. S3 in the
supplementary material) (Kammandel et al., 1999). Transfection of
MeisEnR effectively repressed expression of Six3 and Chx10 (Vsx2),
but not that of other ocular-determination genes (Fig. 4E-I). RNAimediated knock-down of Meis1 together with Meis2 also reduced
retinal Pax6 transcript levels, albeit to a lesser degree than did
MeisEnR transfection (Fig. 4J,K). Together, these results place
DEVELOPMENT
chick development when cells downregulate Meis2 expression (but
retain Meis1) after late E2. Indeed, the BrdU index of RPCs was
seen to decline between HH18 and HH25 following a central-toperipheral gradient (see Fig. S2 in the supplementary material).
To pinpoint the molecular basis of this effect, we transfected
retinal progenitor cells at HH10-11 with MeisEnR (to
simultaneously block Meis1 and Meis2 function) and assessed the
expression of cell cycle components 16-20 hours later. D-type
cyclins are important regulators of the progression through the G1
phase of the cell cycle and the retinae of cyclin D1-deficient animals
are hypoplastic, with each retinal layer containing significantly
fewer cells than those of wild-type littermates (Fantl et al., 1995;
Sicinski et al., 1995). Following MeisEnR expression, cyclin D1 was
markedly reduced (in 86% of the embryos, n=6/7; Fig. 3F,H,I),
whereas expression of the related cyclin D2 was normal (Fig. 3G,J).
Transfection of cyclin D1 together with MeisEnR partially rescued
the RPC proliferation defects we had observed after transfection of
MeisEnR alone (Fig. 3K-N; n=6 embryos for each condition). Meis2
overexpression failed to induce cyclin D1 expression (data not
shown; n=12). Our finding that Meis function is important for RPC
proliferation and cyclin D1 expression, together with earlier studies
that have associated cyclin D1 levels with the modulation of cell
cycle kinetics in the retina, suggest that Meis proteins play a role in
the regulation of retinal progenitor cell number through influencing
the expression of cell cycle components (Green et al., 2003; Locker
et al., 2006; Skapek et al., 2001; Wang et al., 2005; Yamaguchi et al.,
2005). Similar observations have been made independently by Bessa
et al. (Bessa et al., 2008).
MeisEnR-transfected cells also failed to express the proneural
genes NeuroM and Ath5 (Fig. 3O-R and data not shown; n=5/5).
In addition, few MeisEnR-expressing cells could be detected in the
RESEARCH REPORT
Meis1/Meis2 upstream of Pax6 in retinal development and indicate
that Meis proteins are part of the genetic cascade that drives
neuroepithelial cells towards ocular differentiation.
Progenitor cell proliferation in the developing retina and cortex is
sensitive to changes in the level of Pax6 expression (Berger et al.,
2007; Grindley et al., 1995; Manuel et al., 2007; Schedl et al., 1996).
This raises the possibility that the observed reduction in cyclin D1
expression by MeisEnR might be a secondary effect of the
concomitant decrease in Pax6 expression in the eye. We therefore
tested whether cyclin D1 expression could be rescued by cotransfection with Pax6. This was, however, not observed (Fig.
4L,M). Overexpression of Pax6 also failed to elevate cyclin D1 or
cyclin D2 expression in the retina, and cyclin D1 and cyclin D2
expression levels were normal following expression of a Pax6-EnR
fusion protein, which had previously been shown to effectively
block Pax6 function in the developing central nervous system (Fig.
4N-P) (Yamasaki et al., 2001). Therefore, Meis1/Meis2 control over
cyclin D1 expression appears to be independent of their ability to
regulate Pax6 expression.
Collectively, our data integrate Meis proteins into the genetic
network that regulates eye development in vertebrates, place them
upstream of cyclin D1 and Pax6 in RPCs and suggest that they,
similar to their invertebrate homolog Hth, help to maintain retinal
progenitor cells in a rapidly self-renewing state. Homeodomain
transcription factors have been implicated in the tissue-specific
regulation of progenitor cell proliferation in the nervous system and
several of the homeodomain proteins that are expressed in the
vertebrate eye anlage, such as Chx10 and Optx2, are directly or
indirectly required for retinal progenitor proliferation or enhance eye
growth when overexpressed (Burmeister et al., 1996; Green et al.,
2003; Zuber et al., 1999). Unlike these proteins, the duration of Meis
expression is highly species-specific and correlates well with the
different eye sizes in birds and mammals. For instance, chicken,
which express Meis2 together with Meis1 over a longer
developmental period than mice, contain approximately 50-fold
more RGCs than do mice (Rager and Rager, 1976; Strom and
Williams, 1998). Although other genes clearly contribute to eye
growth too, the species-specific regulation of Meis family proteins
together with their influence on retinal progenitor cell proliferation
present a likely mechanism as to how species-specific differences in
retinal size could have evolved.
We are grateful to E. Laufer for suggesting the strategy to construct RIS(B) and
to A. Buchberg, C. Krull, J.-M. Matter, F. Pituello, M. Kengaku and C. Cepko
for reagents. We also thank C. Ziegler for excellent technical assistance, H.
Martin for help with the RNAi experiments, G. Pflugfelder for helpful
discussions and F. Casares for sharing data prior to publication.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/5/805/DC1
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DEVELOPMENT
Meis and vertebrate retina development