1 Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center,
University of Utah, Salt Lake City, UT 84132, USA
2 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT
84132, USA
* Author for correspondence (e-mail: elevine{at}hmbg.utah.edu)
Accepted 31 October 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Proliferation, Cell cycle, Cyclin-dependent kinase inhibitor, Ocular retardation, Microphthalmia, Homeobox, Retina, Chx10, p27Kip1, Cyclin D1
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Surprisingly little is known about the molecular mechanisms that regulate
region-specific proliferation in the CNS. One reason for this is that cell
number variation between different CNS tissues is also dependent on processes
that are distinct from proliferation. For example, although regulation of
proliferation is an essential aspect of both the development and evolution of
the mammalian cerebral cortex (Kornack,
2000; Rakic and Caviness,
1995
), identifying the mechanism of proliferation control in the
cortex is complicated by intricate patterning
(Monuki and Walsh, 2001
;
Redies and Puelles, 2001
;
Sur and Leamey, 2001
), cell
migration (Gleeson and Walsh,
2000
, Hatten,
1999
; Maricich et al.,
2001
; Ross and Walsh,
2001
) and cell death (Blaschke
et al., 1996
; de la Rosa and
de Pablo, 2000
; Voyvodic,
1996
).
We have focused on the mechanism of proliferation control in the mammalian
neural retina because cell proliferation is a primary determinant of retinal
size and cell number. In contrast to other regions of the CNS, cell migration
(Stone and Dreher, 1987;
Watanabe and Raff, 1988
) and
cell death (Beazley et al.,
1987
; Voyvodic et al.,
1995
; Young, 1984
)
do not contribute significantly to the size of the total cell population
during retinal development. Both processes occur late and involve relatively
few cells. Like other areas of the CNS, however, the mammalian neural retina
undergoes a massive expansion in cell number by proliferation. From embryonic
day 14 (E14) until postnatal day 8 (P8; 16 days), total retinal cell number
increases
400 fold, from 60,000 cells to 25 million cells in the rat
(Alexiades and Cepko, 1996
).
Furthermore, this expansion in cell number is region specific. The neural
retina and retinal pigmented epithelium (RPE) are adjacent tissues, and are
both patterned from the optic vesicle. However, the total cell number of the
neural retina is much larger than that of the RPE, due primarily to
differential regulation of proliferation. Evidence of this kind suggests that
tissue-specific regulators of proliferation must exist in different regions of
the CNS.
Within the eye, the homeodomain-containing transcription factor Chx10 and
its orthologs in fish (Vsx2, Alx1), chicken (Chx10-1), cow
and humans are exclusively expressed in the neural retina and adjacent ciliary
margin. Within the retina, Chx10 is expressed in retinal progenitor cells
(RPCs) throughout the period of proliferation
(Barabino et al., 1997;
Belecky-Adams et al., 1997
;
Chen and Cepko, 2000
;
Levine et al., 1994
;
Levine et al., 1997a
;
Liu et al., 1994
;
Passini et al., 1997
). As RPCs
become postmitotic and differentiate, Chx10 expression is terminated in all
cell types, except bipolar interneurons, which are the last neuronal class to
be generated during retinal histogenesis.
Null mutations in Chx10 cause congenital microphthalmia in humans
(Ferda Percin et al., 2000)
and mice (Burmeister et al.,
1996
) (previously referred to as ocular retardation or
orJ), and antisense RNA injections into zebrafish embryos
cause a failure of retinal development
(Barabino et al., 1997
). Gross
abnormalities shared between Chx10-null humans and mice include small
eyes, cataracts, iris coloboma and blindness
(Ferda Percin et al., 2000
;
Robb et al., 1978
). Although
the whole eye is affected by loss of Chx10 function, the primary genetic
defect is specific to the retina and is characterized by two major
developmental defects: a dramatic reduction in retinal cell number and an
absence of bipolar interneurons
(Bone-Larson et al., 2000
;
Burmeister et al., 1996
;
Konyukhov and Sazhina, 1971
).
Although it has been suggested that Chx10 acts in combination with the
neurogenic bHLH gene Mash1 (Ascl1 Mouse Genome
Informatics) to promote the bipolar cell fate
(Hatakeyama et al., 2001
), the
function of Chx10 in regulating cell number is unknown.
Recent studies have demonstrated the importance of cyclin-dependent kinase
inhibitor (CDKI) proteins as negative regulators of proliferation in the
developing CNS (Cunningham and Roussel,
2001). Two families of mammalian CDKI proteins are known: the Ink4
family, comprising p15Ink4a, p16Ink4b,
p18Ink4c and p19Ink4d; and the Cip/Kip family,
comprising p21Cip1, p27Kip1 (hereafter referred to as
Kip1; Cdkn1b Mouse Genome Informatics) and p57Kip2. Ink4
and Cip/Kip proteins are functionally distinct in that Ink4 proteins inhibit
Cdk4 and Cdk6, and Cip/Kip proteins inhibit Cdk2, but high levels of
expression of any of these proteins is sufficient to block progression through
the cell cycle (Nakayama,
1998
; Sherr and Roberts,
1999
; Vidal and Koff,
2000
). Although all of the CDKI genes are expressed in the CNS,
only p19Ink4d, Kip1 and p57Kip2 have been identified as
regulators of proliferation in the retina
(Cunningham et al., 2002
;
Dyer and Cepko, 2000
;
Levine et al., 2000
). Of these
three genes, Kip1 appears to be the most important with respect to retinal
cell number regulation. Kip1 protein is expressed in most, if not all, retinal
cells as they exit the cell cycle during differentiation
(Dyer and Cepko, 2001
;
Levine et al., 2000
), and the
Kip1 knockout retinal phenotype is the most severe, as demonstrated
by a high level of ectopic RPC proliferation and focal dysplasia
(Cunningham et al., 2002
;
Dyer and Cepko, 2001
;
Levine et al., 2000
;
Nakayama et al., 1996
).
Although Kip1 regulates proliferation in a large number of tissues
throughout the developing embryo (Fero et
al., 1996; Kiyokawa et al.,
1996
; Nakayama et al.,
1996
), its activity may be differentially regulated by
tissue-specific factors, in order to control the increase in cell number
during the proliferative expansion of developing tissues. To investigate this
possibility, we sought to determine whether a genetic interaction exists
between Chx10 and Kip1 in the developing mouse retina. In this study, we show
that Kip1 protein is abnormally present in retinal progenitor cells of
Chx10-null mice, and that the genetic elimination of Kip1
alleviates the cell number deficit in the Chx10-null retina.
Interestingly, lamination is restored in the Chx10, Kip1 double null
retina, but bipolar cells are still absent. We further show that Chx10 is not
likely to be a repressor of Kip1 gene transcription, and that cyclin
D1 (CycD1; CycD1 Mouse Genome Informatics) may mediate the ability of
Chx10 to prevent Kip1 protein accumulation in progenitors.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry
Retinal tissue was obtained by dissecting the surrounding ocular tissues
away from the retina in Hanks buffered saline solution (HBSS). For
immunohistochemistry, retinas were fixed in 4% paraformaldehyde (PFA) in
phosphate-buffered saline (PBS) for 1 hour. The tissue was then cryoprotected
in 20% sucrose in PBS, embedded in OCT, and stored at -80°C until
sectioning. Sections (12 µm) were used for immunohistochemistry.
The following antibodies were used in this study: rabbit anti-neuronal
class III ß-tubulin (Covance, Richmond, CA); sheep anti Chx10 (Exalpha
Biologicals, Boston, MA); rabbit anti-cellular retinaldehyde binding protein
(CRALBP; Dr J. Saari, University of Washington, Seattle); mouse monoclonal
anti-CycD1 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-CycD1/bcl-1
(Lab Vision, Fremont, CA); mouse anti-Kip1/p27 (Transduction Laboratories,
Lexington, KY); mouse anti-nestin (Developmental Studies Hybridoma Bank, Iowa
City, IA); rabbit anti-phospho-Histone H3 (Upstate Biotechnology, Lake Placid,
NY); mouse monoclonal anti-proliferation cell nuclear antigen (PCNA Clone
PC10; Dako, Denmark); rabbit anti-protein kinase C (PKC
; Sigma,
St. Louis, MO); rabbit anti-recoverin (Dr J. Hurley, University of Washington,
Seattle, WA); mouse monoclonal anti-Rhodopsin (Rho 4D2; Dr R. Molday,
University of British Columbia); and rabbit anti-calbindin (Chemicon
International, Temecula, CA). All antibodies were used at appropriate
dilutions in 2% normal goat or donkey serum, 0.15% Triton X-100 and 0.01%
sodium azide in PBS. Primary antibodies were followed with species-specific
secondary antibodies conjugated to Fluorescein (FITC; Jackson Immunoresearch,
West Grove, PA), rhodamine (TRITC, Jackson Immunoresearch), Alexa Fluor 488
(Molecular Probes, Eugene, OR) or Alexa Fluor 568 (Molecular Probes). Nuclei
were stained with 4,6-diamidino-2-phenylindole (DAPI; Fluka).
Total cell counts/quantification of markers
To obtain total cell counts for all genotypes at P0, retinas were dissected
as above, then trypsinized and subsequently triturated and resuspended in
media. Cells were counted on a hemocytometer and the total number of cells per
retina calculated. Cells were plated on poly-d-lysine coverslips and allowed
to settle for 1 hour at 37°C and 5% CO2. Cells were then fixed
with 4% PFA in PBS and stored at 4°C. Coverslips were stained with
antibodies as above.
Quantification of the percentage of cells expressing immunohistochemical markers was done by random field analysis on each coverslip, counting the number of cells positive for that marker and the total number of cells in that field (stained with DAPI). A minimum of 500 cells was counted per coverslip, and the percentage of cells expressing the marker for that coverslip was determined. At least three different animals were analyzed per condition and an average percentage was determined, with each animal counted as n=1. Statistical significance was determined by unpaired t-tests using StatView (Abacus Concepts, Cary, NC).
In situ hybridization
P0 wild-type tissue was obtained as above for immunohistochemistry. Retinas
were fixed on ice for 2 hours in 4% formaldehyde in PBS/2mM EGTA, followed by
cryoprotection and storage as above. Sections were cut as above and stored at
-80°C until use. In situ hybridization was performed on sections as
previously described (Schaeren-Wiemers and
Gerfin-Moser, 1993).
Double in situ labeling was performed as for single in situ reactions, with
the following modifications: One RNA probe was labeled with dig-UTP and one
probe was labeled with Fluorescein-12-uridine-5'-triphosphate (F-UTP,
Roche). Hybridization of both probes was carried out at the same time, with
each probe at a concentration of 400 ng/ml. After hybridization, sections were
incubated with anti-fluorescein-AP (Roche). Probes were visualized by
incubating sections with Fast Red tablets (Roche) dissolved in 0.1 M Tris (pH
8.2). After sufficient staining, the reaction was stopped by rinsing in TE (pH
8) and then washing for 10 minutes in 0.1 M Glycine (pH 2). The tissue
was then incubated with anti-dig-AP as above and visualized using B3 with
BCIP/NBT.
Northern blot hybridization
Total RNA (1.8 µg) from P0 wild-type and Chx10-null retinas
(Trizol, Invitrogen, Carlsbad, CA) was electophoresed in agarose under
denaturing conditions and transferred to Nytran SuperCharge membrane
(Schleicher and Schuell, Keene, NH). [32P]-dCTP labeled random
primed DNA probes (Ladderman, Takara Biochemical, Berkeley, CA) for Kip1 and
CycD1 (see below) were hybridized overnight at 42°C and washed at a final
stringency of 0.1xSSC at 72°C (for Kip1) and 90°C (for CycD1).
Filters were exposed to Biomax-MS film (Kodak, Rochester, NY) overnight at
-80°C with an intensifying screen. Kip1 probe was synthesized from a 590
bp cDNA fragment corresponding to the complete open reading frame (ORF), and
CycD1 probe was synthesized from a 250 bp PCR clone that spans 120 nucleotides
(nt) of the 5'-untranslated region and 105 nt of the ORF containing the
N-terminal domain. Specific details for electrophoresis, transfer and
hybridization have been described previously
(Chow et al., 1998;
Levine and Schechter,
1993
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To determine if the presence of Kip1 protein contributes to the reduced cell number in the Chx10-null retina, we generated Chx10, Kip1 double null mice. We found that many of the qualitative phenotypic defects of the Chx10-null mouse retina are rescued in the Chx10, Kip1 double null mouse. Differences between a wild-type retina, a Chx10-null retina, a Chx10, Kip1 double null retina, and a Kip1-null retina at P19 can be seen at the macroscopic level (Fig. 2). Much of the size deficit of the Chx10-null is rescued in the Chx10, Kip1 double null retina. Radial cross-sections of these retinas reveal that the defective lamination of Chx10-null mice is also rescued in the double null retina. In the double null, the outer nuclear layer, inner nuclear layer, ganglion cell layer and the intervening plexiform layers are all easily distinguishable. These results support the hypothesis that Chx10 is necessary to antagonize Kip1 function during retinal development.
|
To determine if the rescue includes all of the major retinal cell types,
retinal sections from P19 mice of each genotype were analyzed with cell
type-specific markers. At P19, retinal histogenesis is complete
(Sidman, 1961;
Young, 1985b
). Photoreceptors
were visible with recoverin labeling (Fig.
3A-D), and opsin labeling (not shown). Calbindin was used to
identify horizontal cells and amacrine cells in the INL, and displaced
amacrine and ganglion cells in the GCL
(Fig. 3E-H), and Müller
glia were seen with CRALBP labeling (Fig.
3I-L). AII and starburst amacrine cells were visible with
calretinin labeling (Fig. 3M-P)
and ChAT labeling (Fig. 3Q-T),
respectively. Although all of these major cell types are present in
Chx10-null retinas, they do not have normal morphology and are not
organized into layers (Fig.
3B,F,J.N,R). By contrast, cells comprising each of these cell
types have grossly normal structure and location in the Chx10, Kip1
double null retina (Fig.
3C,G,K,O,S).
|
|
Changes in proliferation account for the cell number rescue in
Chx10, Kip1 double null mice
The adult Chx10, Kip1 double null retina is intermediate in size
between retinas from Chx10-null and wild-type mice. Part of the
difference between wild-type and double null retinas probably arises from the
lack of bipolar cells in the double null, as these cells normally constitute
10% of the total cell number (Young,
1985a
). We therefore analyzed cell number at P0, a time before the
birthday of bipolar cells (Sidman,
1961
; Young,
1985a
). Interestingly, DAPI stained sections show that the
Chx10, Kip1 double null retina has an intermediate number of nuclei
at P0 relative to Chx10-null and wild-type retinas
(Fig. 5A-C). Calculation of
total cell number from dissociated P0 retinas revealed that
Chx10-null retinas have 19-fold fewer cells than wild-type retinas,
and that Chx10, Kip1 double null retinas are reduced fourfold
compared with wild type (Fig.
5D). The proportion of the Chx10-null cell number defect
that is independent of bipolar cell genesis is therefore significantly, but
not completely, rescued by deletion of Kip1 (see Discussion).
|
The greatly reduced cell number in Chx10-null retinas could be due to defects in proliferation, increased cell death, premature differentiation or to some combination thereof. Similarly, the rescue of cell number in the Chx10, Kip1 double null retina could be due to compensatory changes in any or all of these processes. To begin to dissect the relative contributions of these factors to cell number, we measured the extent of apoptosis, differentiation and proliferation in Chx10-null, Chx10, Kip1 double null and wild-type retinas.
TUNEL labeling was performed at P0 (Fig. 5E-G), P10 and P19 (not shown) to measure apoptosis. We found that there were no significant differences in the tissue distribution of apoptotic cells (Fig. 5E-G), or in the percentage of TUNEL-positive cells between wild-type, Chx10-null and Chx10, Kip1 double null retinas (Fig. 5H). This suggests that the cell number rescue in Chx10, Kip1 double null retinas cannot be accounted for by changes in cell death.
If Chx10 and Kip1 regulate cell number by influencing how and when RPCs
choose to exit the cell cycle, then the ratios of progenitor cells and
differentiated cells should change between genotypes. For example, if the loss
of Chx10 function causes premature differentiation similar to that observed
due to inactivation of Hes1 (Tomita et
al., 1996), then the percentage of progenitor cells in the
Chx10-null retina should decrease, and the percentage of
differentiated cells should increase in comparison with wild type.
Furthermore, if the loss of Kip1 compensates for the loss of Chx10 function in
the double null retina by delaying differentiation, then the percentage of
progenitors should increase at the expense of the differentiated cell
population. To address this, we calculated the percentages of retinal cells in
mice expressing markers for progenitors (PCNA) and neurons (Class III
ß-tubulin, recoverin and neurofilament) at P0. Interestingly, the
percentages of progenitors and neurons do not change in the
Chx10-null and Chx10, Kip1 double null retinas when compared
with wild type or to each other (Fig.
5I). At this age, progenitors constitute about 50% of cells in all
genotypes, despite the large differences in total cell number between
genotypes. To verify this, we labeled sections through central retina from
each genotype with antibodies for PCNA
(Fig. 6A-C). Additionally, we
performed in situ hybridization for Chx10 mRNA in sections from each
genotype (not shown). Using both methods, we found that the progenitor
population size was proportionally maintained across genotypes. The
Chx10-null hypocellular phenotype, and the rescue of cell number in
the double null, are therefore not attributable to changes in the timing of
cell cycle exit, or in the onset of cellular differentiation.
|
These observations lead to several important conclusions. First, as we see no evidence of changes in the ratios of progenitors, differentiated cells or apoptotic cells across genotypes, the rescued cell number in Chx10, Kip1 double null retinas must be primarily due to an increase in proliferation. Specifically, it is probably due to an increase in the rate of proliferation (i.e. the number of cell cycles over time), as opposed to changes in timing of exit from the cell cycle. If the latter were true, we should have observed an increase in the fraction of differentiated cells, and a decrease in the fraction of progenitors. Second, the rescued proliferation in the double null must be occurring specifically among progenitor cells, as they proportionally maintain their population size in all genotypes. Third, because the proportion of cells that are progenitors in Chx10-null retinas is the same as in wild-type retinas (50%, Fig. 5I), the increase in Kip1 in Chx10-null cells (to 80%, Fig. 1) must be occurring in progenitor cells. Therefore, Chx10 must have some role in preventing Kip1 protein accumulation in retinal progenitors, and the removal of Kip1 from Chx10-null RPCs must be responsible for their increased proliferation in the double null retinas.
Chx10 regulates the balance of G1 phase regulatory proteins
Chx10 and Kip1 protein, but not mRNA, are present in mutually
exclusive cell populations
Although it has previously been shown that Chx10 is expressed in RPCs
(Liu et al., 1994), and that
Kip1 is expressed primarily in postmitotic differentiating cells
(Levine et al., 2000
), these
protein patterns have not been directly compared. Double label
immunofluorescence with antibodies to Chx10 and Kip1 on the same P0 wild-type
mouse section show that these proteins are predominantly expressed in mutually
exclusive cell populations (Fig.
7A-C). This result is consistent with the hypothesis that Chx10
negatively regulates expression of Kip1.
|
|
CycD1 is important for the suppression of Kip1 protein levels in
RPCs
Previous studies have demonstrated that CycD1 has a function in promoting
RPC proliferation (Fantl et al.,
1995; Ma et al.,
1998
; Sicinski et al.,
1995
). Interestingly, deletion of the Kip1 gene abrogates
this requirement for CycD1 (Geng et al.,
2001
; Tong and Pollard,
2001
). It has also been shown that in addition to their canonical
role as activators of Cdk4, D-type cyclins can sequester Kip1 away from the
CycE:Cdk2 complex in human keratinocytes and mink lung epithelial cells,
thereby contributing to progression of cells through G1 phase
(Polyak et al., 1994
;
Reynisdottir et al., 1995
;
Toyoshima and Hunter, 1994
).
We therefore hypothesized that in RPCs, CycD1 may mediate negative regulation
of Kip1 by Chx10. We first examined the localization of CycD1 protein relative
to Chx10 and Kip1 on cross-sections from P0 wild-type retinas. As expected, we
found that CycD1 and Chx10 are expressed in the same cells
(Fig. 8A-C), while CycD1 and
Kip1 are expressed in mutually exclusive cells
(Fig. 8D-F). Next, we examined
the localization patterns of CycD1 and Kip1 in retinas that were null for
Chx10 (Fig. 8G-I). We
found that CycD1 for the most part retained its complementary expression with
Kip1. Finally, we examined the localization patterns of Chx10 and Kip1 in the
retinas of P0 CycD1-null mice
(Fig. 8J-L,N). In these mice,
Chx10 and Kip1 had significant co-expression, and cells that co-expressed the
two proteins were in the neuroblast layer. This result contrasts starkly with
the mutual exclusion of Chx10 and Kip1 proteins in wild-type retinas, and it
supports the hypothesis that CycD1 mediates the antagonism between Chx10 and
Kip1 (Fig. 8M).
|
To quantify these observations, we performed cell counts on dissociated cells that were double labeled with combinations of Kip1, Chx10 and CycD1 antibodies. Kip1 was co-localized with Chx10 or CycD1 in only about 1% of cells in wild-type retinas (Fig. 9A,B). In CycD1-null retinas, however, 24% of cells were positive for both Chx10 and Kip1 (Fig. 9A). In Chx10-null retinas, 8% of cells co-expressed CycD1 and Kip1 (Fig. 9B). These results show that the negative regulation of Kip1 protein levels in RPCs is critically dependent on the presence of both Chx10 and CycD1.
Chx10 is required for normal CycD1 expression
Fig. 9B also shows that the
percentage of cells expressing CycD1 is significantly reduced in
Chx10-null retinas, compared with wild-type (29% versus 48%,
P<0.0003). In contrast to Kip1, the change in CycD1 protein
expression is correlated with a reduction in steady state levels of the
CycD1 mRNA in the Chx10-null retina
(Fig. 9C). Interestingly, even
though cell number is significantly restored in the Chx10, Kip1
double null retina, the percentage of cells expressing CycD1 (30%) is not
restored to normal (Fig. 10).
Therefore Chx10 appears to be required to maintain the correct expression of
CycD1. Together with the evidence that CycD1 is involved in the negative
regulation of Kip1, this result establishes a link between Chx10 and both
positive and negative regulators of the G1 phase of the cell cycle. These
results suggest that the decreased cell number observed in the
Chx10-null retina is due, in large part, to a change in the balance
of CycD1 and Kip1 in retinal progenitors.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results and earlier studies of Chx10
(Barabino et al., 1997;
Belecky-Adams et al., 1997
;
Burmeister et al., 1996
;
Levine et al., 1997a
;
Passini et al., 1997
) provide
important support for the notion that homeobox genes in general might play
tissue-specific roles in directing proliferation, especially in the nervous
system. The activity of these genes may link tissue-specific regulation of
cell number to the internal mechanics and timing of the cell cycle. Other
examples of homeobox genes with tissue-specific roles in proliferation include
Emx2 and Otx1 in development of cerebral cortex, and Optx2 in Xenopus
eyes (Cecchi et al., 2000
;
Tole et al., 2000
;
Zuber et al., 1999
). If
different homeobox genes independently regulate proliferation in different
tissues by their influence on cell cycle dynamics, then these genes may have
been excellent targets for evolutionary changes of tissue size between
vertebrate species.
This study is among the first to demonstrate a genetic interaction between a homeobox gene and regulatory proteins of the cell cycle, and the first to show rescue in an animal model of microphthalmia through deletion of a cell cycle regulator. A study by Bone-Larson et al. demonstrated rescue of the Chx10-null phenotype by breeding a Chx10-null allele in 129/Sv mice into a Mus musculus castaneus background. Some mice of this background that were homozygous null for Chx10 had improved retinal size, bipolar cells and measurable ERGs. Although the genetic modifiers that produced this effect are unknown, we believe that a malfunctioning Kip1 protein in this background could not be the sole modifier, as Chx10, Kip1 double null mice do not have bipolar cells. The rescue we observe in Chx10, Kip1 double null mice is specific to the proliferation defect, and we believe it reflects the antagonistic actions of these two proteins during development. Unknown genetic modifiers from Kip1-null mice can not account for the rescue we observed, as the phenotype was tightly linked to the Kip1 allele. Our study also shows that Chx10 is still required for bipolar cell genesis or maintenance even when proliferation is restored. Additionally, it shows that Chx10 is not required for the formation of normal retinal lamination and architecture, except to the extent that it is needed to generate the building blocks of the retinal cytoarchitecture through regulation of cell number.
The phenotypes of null mouse strains reflect normal and abnormal
regulation of Kip1
Kip1 is a tumor suppressor that can inhibit the S phase-promoting kinase
Cdk2 (Polyak et al., 1994;
Toyoshima and Hunter, 1994
),
and it has been shown to be a key regulator of cell cycle exit in the
developing rodent retina (Cunningham et
al., 2002
; Dyer and Cepko,
2001
; Levine et al.,
2000
). Kip1 has been suggested to accumulate in late G2 and early
G1 phases of the RPC cell cycle (Dyer and
Cepko, 2001
), possibly only in the last cell cycle of a cell that
will go on to differentiate. We find that in the wild-type retina, Kip1
protein is almost never seen in cells that express Chx10
(Fig. 7A-C). Furthermore, Kip1
protein levels are deregulated in the Chx10-null retina
(Fig. 1;
Fig. 8H-I). The ectopic
expression of Kip1 in RPCs does not appear to cause premature cell cycle exit
in the Chx10-null retina, as the ratio of progenitors to
differentiated cells is normal at P0, a relatively advanced stage of retinal
development (Fig. 5I). However,
it is clear that Kip1 contributes to the decreased retinal cell number of
these mice. The rescued phenotype of the Chx10, Kip1 double null
mouse retina demonstrates that in fact a significant amount of the severity of
the Chx10-null retina can be attributed to the presence of Kip1. We
found that at P0, the Chx10-null retina is 19 times smaller than wild
type, while the double null retina is four times smaller than wild type.
An important question is whether the rescue we see in the Chx10,
Kip1 double null retina reflects a specific interaction between these two
genes among progenitor cells or if, instead, it reflects a nonspecific
increase in cell number in another cell population, caused by the removal of
the cell cycle inhibitor Kip1. Our findings that Kip1 expression is increased
specifically in progenitor cells in the Chx10-null retina, and that
progenitor cells contribute proportionally to the increased cell number in the
Chx10, Kip1 double null retina, demonstrate that the rescue reflects
an interaction that is specific to progenitors. Interestingly, in a study
examining the relationship between Myc, a positive cell cycle regulatory
protein, and Kip1, generation of a double null mouse did not rescue the severe
cell number defects of the Myc single null
(Trumpp et al., 2001). Their
study demonstrates that genetic elimination of Kip1 does not always compensate
for the lack of positive cell cycle regulators.
As Kip1 contributes significantly to the phenotype of the
Chx10-null retina, and because it does not do so by promoting
premature cell cycle exit, we propose that its abnormal presence in RPCs is in
large part responsible for the reduced proliferation observed in
Chx10-null mice. An interesting possibility is that increased Kip1
expression lengthens the G1 phase in RPCs, leading to an increase in the total
cell cycle time. This suggestion is supported by two independent observations:
that transgenic Kip1 mis-expression in cortical progenitors lengthens their G1
phase, and as a consequence, total cell cycle time
(Mitsuhashi et al., 2001); and
that the cell cycle time in Chx10-null RPCs is longer than in their
wild-type counterparts, possibly because of a specific increase in G1
(Konyukhov and Sazhina, 1971
).
More studies are needed to determine whether Kip1 is in fact responsible for
longer cell cycle times in Chx10-null retinas. It is somewhat
surprising that the abnormal presence of Kip1 in RPCs of Chx10-null
mice does not cause premature cell cycle exit, as Kip1 is a component of the
cell cycle exit mechanism at the onset of RPC differentiation
(Dyer and Cepko, 2001
;
Levine et al., 2000
). It is
possible that Kip1 normally works in combination with other intracellular and
extracellular factors (Fuhrmann et al.,
2000
) to promote cell cycle exit, and that its abnormal presence
in Chx10-null RPCs has only the effect of slowing the cell cycle.
Mechanisms of Kip1 regulation
In the developing retina, Kip1 is regulated
post-transcriptionally
In other systems, Kip1 has been shown to be regulated transcriptionally,
translationally and by at least two post-translational mechanisms
(Cheng et al., 1998;
Hengst and Reed, 1996
;
Kolluri et al., 1999
;
Pagano et al., 1995
;
Servant et al., 2000
).
Homeodomain-containing proteins such as Chx10 have long been recognized for
their importance in transcriptional control of developmental events, but no
transcriptional targets have been identified for the paired-like:CVC
homeobox family to which Chx10 belongs. Our data demonstrate that the
inhibition of Kip1 by Chx10 cannot be accounted for by a direct
transcriptional repression mechanism. First, we find that Kip1 mRNA
is present in precisely the same population of cells as both Chx10
mRNA and Chx10 protein. Second, Kip1 mRNA levels do not change in the
Chx10-null compared with wild type. Chx10 must therefore have some
post-transcriptional inhibitory effect on Kip1. Furthermore, our results
demonstrate that the expression pattern of Kip1 in the wild-type retina is
somewhat unusual, with the mRNA and protein seemingly present in complementary
cell populations. We suspect that these cell populations are distinct in
timing only, with Kip1 mRNA expressed in proliferating RPCs, and Kip1
protein upregulated in the progenitors that exit the cell cycle. This suggests
that there must be a significant change in the translational efficiency of
Kip1 mRNA or in the stability of the Kip1 protein that occurs around
the time of cell cycle exit.
CycD1 may mediate the interaction between Chx10 and Kip1
At present, we do not understand the molecular interaction between Chx10
and Kip1. We believe, however, that CycD1 is involved. In addition to
activating Cdk4/6, CycD1 can sequester Kip1 away from Cyclin E:Cdk2 complexes,
and this may facilitate the targeting of Kip1 for degradation
(Hengst and Reed, 1996;
Malek et al., 2001
;
Pagano et al., 1995
). The
expression patterns we observed suggest that Chx10 may promote CycD1
expression, and consequently prevent Kip1 protein accumulation. We find that
the percentage of CycD1 expressing cells is reduced in the Chx10-null
mouse retina, and is not restored in the Chx10, Kip1 double null
retina, even though cell number is partially restored. This shows that Chx10
is necessary for proper regulation of CycD1 expression. Second, in contrast to
wild type retinas, we find that CycD1-null retinas have many cells
that co-express Chx10 and Kip1. Over 50% of Chx10-positive cells in the
CycD1-null retina were also Kip1 positive (compared with 3% in
wild-type retina). This shows that CycD1 is necessary to keep Kip1 protein at
low levels or absent in Chx10-positive progenitors. Finally, Kip1 is present
in an abnormally high percentage of cells in both CycD1- and
Chx10-null retinas, suggesting that the activity of both proteins is
needed to prevent Kip1 protein from accumulating in cells. Note that it is
unlikely that other D-type cyclins are targets of Chx10 function, or
inhibitors of Kip1, as neither cyclin D2 nor cyclin D3 is expressed at
significant levels in the developing retina
(Geng et al., 1999
).
Kip1 and CycD1 are known to influence the activity of G1-phase CDKs, and the ectopic expression of Kip1 in Chx10-null retinas is seen in few, if any, cells expressing markers for the S, G2 and M phases of the cell cycle (data not shown). Normal cell cycle progression in retinal progenitors therefore appears to depend heavily on the ability of Chx10, which acts at least in part through CycD1, to prevent Kip1 protein accumulation during G1 phase.
Although our data suggest that Chx10 could be a transcriptional activator
of CycD1, it is also possible that its effects on CycD1 may be more indirect.
We did not observe Chx10 DNA binding sites in the CycD1 promoter, and CycD1
protein expression does not appear reduced in the cells that express it in
Chx10-null mice. It may be that Chx10 promotes the expression of
other genes required for G1 progression, or that it mediates the transduction
of mitogenic signals from the extracellular environment
(Anchan and Reh, 1995;
Anchan et al., 1991
;
Chow et al., 1998
;
Das et al., 2000
;
Ilia and Jeffery, 1999
;
Jensen and Wallace, 1997
;
Levine et al., 1997b
;
Lillien and Cepko, 1992
;
McConnell and Kaznowski,
1991
).
Additional roles for Chx10 in regulating RPC proliferation
Our data demonstrate that a significant part of the microphthalmic
condition of Chx10-null mice (and therefore possibly of humans with
mutations in CHX10) is due to the unchecked activity of Kip1 protein
in RPCs. However, the intermediate total cell number in the Chx10,
Kip1 double null retina compared with wild-type and Chx10-null
retinas indicates that the rescue of proliferation is not complete.
Furthermore, the hypoproliferative defect of CycD1-null mice at P0 is
significantly less severe than that of Chx10-null mice. This evidence
demonstrates that Chx10 must act to some extent through mechanisms that are
independent of CycD1 and Kip1 to promote proliferation. A complete
understanding of the function of Chx10 will require analysis of its potential
regulation of other CDKIs, and of other possible roles for Chx10 within the
cell cycle.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexiades, M. R. and Cepko, C. (1996). Quantitative analysis of proliferation and cell cycle length during development of the rat retina. Dev. Dyn. 205,293 -307.[CrossRef][Medline]
Anchan, R. M. and Reh, T. A. (1995). Transforming growth factor-beta-3 is mitogenic for rat retinal progenitor cells in vitro. J. Neurobiol. 28,133 -145.[Medline]
Anchan, R. M., Reh, T. A., Angello, J., Balliet, A. and Walker, M. (1991). EGF and TGF-alpha stimulate retinal neuroepithelial cell proliferation in vitro. Neuron 6, 923-936.[Medline]
Barabino, S. M., Spada, F., Cotelli, F. and Boncinelli, E. (1997). Inactivation of the zebrafish homologue of Chx10 by antisense oligonucleotides causes eye malformations similar to the ocular retardation phenotype. Mech. Dev. 63,133 -143.[CrossRef][Medline]
Beazley, L. D., Perry, V. H., Baker, B. and Darby, J. E. (1987). An investigation into the role of ganglion cells in the regulation of division and death of other retinal cells. Brain Res. 430,169 -184.[Medline]
Belecky-Adams, T., Tomarev, S., Li, H. S., Ploder, L., McInnes, R. R., Sundin, O. and Adler, R. (1997). Pax-6, Prox 1, and Chx10 homeobox gene expression correlates with phenotypic fate of retinal precursor cells. Invest. Ophthalmol. Vis. Sci. 38,1293 -1303.[Abstract]
Blaschke, A. J., Staley, K. and Chun, J.
(1996). Widespread programmed cell death in proliferative and
postmitotic regions of the fetal cerebral cortex.
Development 122,1165
-1174.
Bone-Larson, C., Basu, S., Radel, J. D., Liang, M., Perozek, T., Kapousta-Bruneau, N., Green, D. G., Burmeister, M. and Hankin, M. H. (2000). Partial rescue of the ocular retardation phenotype by genetic modifiers. J. Neurobiol. 42,232 -247.[CrossRef][Medline]
Burmeister, M., Novak, J., Liang, M. Y., Basu, S., Ploder, L., Hawes, N. L., Vidgen, D., Hoover, F., Goldman, D., Kalnins, V. I. et al. (1996). Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. Nat. Genet. 12,376 -384.[Medline]
Cecchi, C., Mallamaci, A. and Boncinelli, E. (2000). Otx and Emx homeobox genes in brain development. Int. J. Dev. Biol. 44,663 -668.[Medline]
Chen, C. M. and Cepko, C. L. (2000). Expression of Chx10 and Chx10-1 in the developing chicken retina. Mech. Dev. 90,293 -297.[CrossRef][Medline]
Chen, P. and Segil, N. (1999). p27(Kip1) links
cell proliferation to morphogenesis in the developing organ of Corti.
Development 126,1581
-1590.
Cheng, M., Sexl, V., Sherr, C. J. and Roussel, M. F.
(1998). Assembly of cyclin D-dependent kinase and titration of
p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1).
Proc. Natl. Acad. Sci. USA
95,1091
-1096.
Chow, L., Levine, E. M. and Reh, T. A. (1998). The nuclear receptor transcription factor, retinoid-related orphan receptor beta, regulates retinal progenitor proliferation. Mech. Dev. 77,149 -164.[CrossRef][Medline]
Cunningham, J. J. and Roussel, M. F. (2001).
Cyclin-dependent kinase inhibitors in the development of the central nervous
system. Cell Growth Differ.
12,387
-396.
Cunningham, J. J., Levine, E. M., Zindy, F., Goloubeva, O., Roussel, M. F. and Smeyne, R. J. (2002). The cyclin-dependent kinase inhibitors p19(Ink4d) and p27(Kip1) are coexpressed in select retinal cells and act cooperatively to control cell cycle exit. Mol. Cell Neurosci. 19,359 -374.[CrossRef][Medline]
Das, I., Sparrow, J. R., Lin, M. I., Shih, E., Mikawa, T. and
Hempstead, B. L. (2000). Trk C signaling is required for
retinal progenitor cell proliferation. J. Neurosci.
20,2887
-2895.
de la Rosa, E. J. and de Pablo, F. (2000). Cell death in early neural development: beyond the neurotrophic theory. Trends Neurosci. 23,454 -458.[CrossRef][Medline]
Dyer, M. A. and Cepko, C. L. (2000). p57(Kip2)
regulates progenitor cell proliferation and amacrine interneuron development
in the mouse retina. Development
127,3593
-3605.
Dyer, M. A. and Cepko, C. L. (2001). p27Kip1
and p57Kip2 regulate proliferation in distinct retinal progenitor cell
populations. J. Neurosci.
21,4259
-4271.
Fantl, V., Stamp, G., Andrews, A., Rosewell, I. and Dickson, C. (1995). Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. Genes Dev. 9,2364 -2372.[Abstract]
Ferda Percin, E., Ploder, L. A., Yu, J. J., Arici, K., Horsford, D. J., Rutherford, A., Bapat, B., Cox, D. W., Duncan, A. M., Kalnins, V. I. et al. (2000). Human microphthalmia associated with mutations in the retinal homeobox gene CHX10. Nat. Genet. 25,397 -401.[CrossRef][Medline]
Fero, M. L., Rivkin, M., Tasch, M., Porter, P., Carow, C. E., Firpo, E., Polyak, K., Tsai, L. H., Broudy, V., Perlmutter, R. M. et al. (1996). A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85,733 -744.[Medline]
Fisher, R. P. (1997). CDKs and cyclins in transition(s). Curr. Opin. Genet. Dev. 7, 32-38.[CrossRef][Medline]
Follette, P. J. and O'Farrell, P. H. (1997). Cdks and the Drosophila cell cycle. Curr. Opin. Genet. Dev. 7,17 -22.[CrossRef][Medline]
Fuhrmann, S., Chow, L. and Reh, T. A. (2000). Molecular control of cell diversification in the vertebrate retina. In Vertebrate Eye Development, Vol.31 (ed. E. Fini), pp. 69-84. New York: Springer Verlag.
Geng, Y., Whoriskey, W., Park, M. Y., Bronson, R. T., Medema, R. H., Li, T., Weinberg, R. A. and Sicinski, P. (1999). Rescue of cyclin D1 deficiency by knockin cyclin E. Cell 97,767 -777.[Medline]
Geng, Y., Yu, Q., Sicinska, E., Das, M., Bronson, R. T. and
Sicinski, P. (2001). Deletion of the p27Kip1 gene restores
normal development in cyclin D1-deficient mice. Proc. Natl. Acad.
Sci. USA 98,194
-199.
Gleeson, J. G. and Walsh, C. A. (2000). Neuronal migration disorders: from genetic diseases to developmental mechanisms. Trends Neurosci. 23,352 -359.[CrossRef][Medline]
Hatakeyama, J., Tomita, K., Inoue, T. and Kageyama, R.
(2001). Roles of homeobox and bHLH genes in specification of a
retinal cell type. Development
128,1313
-1322.
Hatten, M. E. (1999). Central nervous system neuronal migration. Annu. Rev. Neurosci. 22,511 -539.[CrossRef][Medline]
Hengst, L. and Reed, S. I. (1996). Translational control of p27Kip1 accumulation during the cell cycle. Science 271,1861 -1864.[Abstract]
Ilia, M. and Jeffery, G. (1999). Retinal mitosis is regulated by dopa, a melanin precursor that may influence the time at which cells exit the cell cycle: analysis of patterns of cell production in pigmented and albino retinae. J. Comp. Neurol. 405,394 -405.[CrossRef][Medline]
Jensen, A. M. and Wallace, V. A. (1997).
Expression of Sonic hedgehog and its putative role as a precursor cell mitogen
in the developing mouse retina. Development
124,363
-371.
Kiyokawa, H., Kineman, R. D., Manova-Todorova, K. O., Soares, V. C., Hoffman, E. S., Ono, M., Khanam, D., Hayday, A. C., Frohman, L. A. and Koff, A. (1996). Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell 85,721 -732.[Medline]
Kolluri, S. K., Weiss, C., Koff, A. and Gottlicher, M.
(1999). p27(Kip1) induction and inhibition of proliferation by
the intracellular Ah receptor in developing thymus and hepatoma cells.
Genes Dev. 13,1742
-1753.
Konyukhov, B. V. and Sazhina, M. V. (1971). Genetic control over the duration of G 1 phase. Experientia 27,970 -971.[Medline]
Kornack, D. R. (2000). Neurogenesis and the evolution of cortical diversity: mode, tempo, and partitioning during development and persistence in adulthood. Brain Behav. Evol. 55,336 -344.[CrossRef][Medline]
Levine, E. M. and Schechter, N. (1993). Homeobox genes are expressed in the retina and brain of adult goldfish. Proc. Natl. Acad. Sci. USA 90,2729 -2733.[Abstract]
Levine, E. M., Hitchcock, P. F., Glasgow, E. and Schechter, N. (1994). Restricted expression of a new paired-class homeobox gene in normal and regenerating adult goldfish retina. J. Comp. Neurol. 348,596 -606.[Medline]
Levine, E. M., Passini, M., Hitchcock, P. F., Glasgow, E. and Schechter, N. (1997a). Vsx-1 and Vsx-2: two Chx10-like homeobox genes expressed in overlapping domains in the adult goldfish retina. J. Comp. Neurol. 387,439 -448.[CrossRef][Medline]
Levine, E. M., Roelink, H., Turner, J. and Reh, T. A.
(1997b). Sonic hedgehog promotes rod photoreceptor
differentiation in mammalian retinal cells in vitro. J.
Neurosci. 17,6277
-6288.
Levine, E. M., Close, J., Fero, M., Ostrovsky, A. and Reh, T. A. (2000). p27(Kip1) regulates cell cycle withdrawal of late multipotent progenitor cells in the mammalian retina. Dev. Biol. 219,299 -314.[CrossRef][Medline]
Lillien, L. and Cepko, C. (1992). Control of proliferation in the retina: temporal changes in responsiveness to FGF and TGF alpha. Development 115,253 -266.[Abstract]
Liu, I. S., Chen, J. D., Ploder, L., Vidgen, D., van der Kooy, D., Kalnins, V. I. and McInnes, R. R. (1994). Developmental expression of a novel murine homeobox gene (Chx10): evidence for roles in determination of the neuroretina and inner nuclear layer. Neuron 13,377 -393.[Medline]
Lowenheim, H., Furness, D. N., Kil, J., Zinn, C., Gultig, K.,
Fero, M. L., Frost, D., Gummer, A. W., Roberts, J. M., Rubel, E. W. et al.
(1999). Gene disruption of p27(Kip1) allows cell proliferation in
the postnatal and adult organ of corti. Proc. Natl. Acad. Sci.
USA 96,4084
-4088.
Ma, C., Papermaster, D. and Cepko, C. L.
(1998). A unique pattern of photoreceptor degeneration in cyclin
D1 mutant mice. Proc. Natl. Acad. Sci. USA
95,9938
-9943.
Malek, N. P., Sundberg, H., McGrew, S., Nakayama, K., Kyriakidis, T. R. and Roberts, J. M. (2001). A mouse knock-in model exposes sequential proteolytic pathways that regulate p27Kip1 in G1 and S phase. Nature 413,323 -327.[CrossRef][Medline]
Maricich, S. M., Gilmore, E. C. and Herrup, K. (2001). The role of tangential migration in the establishment of mammalian cortex. Neuron 31,175 -178.[Medline]
McConnell, S. K. and Kaznowski, C. E. (1991). Cell cycle dependence of laminar determination in developing neocortex. Science 254,282 -285.[Medline]
Mitsuhashi, T., Aoki, Y., Eksioglu, Y. Z., Takahashi, T., Bhide,
P. G., Reeves, S. A. and Caviness, V. S., Jr (2001).
Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets
inducible gene expression to central nervous system progenitor cells.
Proc. Natl. Acad. Sci. USA
98,6435
-6440.
Monuki, E. S. and Walsh, C. A. (2001). Mechanisms of cerebral cortical patterning in mice and humans. Nat. Neurosci. 4 Suppl.,1199 -1206.
Nakayama, K. (1998). Cip/Kip cyclin-dependent kinase inhibitors: brakes of the cell cycle engine during development. BioEssays 20,1020 -1029.[CrossRef][Medline]
Nakayama, K., Ishida, N., Shirane, M., Inomata, A., Inoue, T., Shishido, N., Horii, I. and Loh, D. Y. (1996). Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85,707 -720.[Medline]
Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., del Sal, G., Chau, V., Yew, P. R., Draetta, G. F. and Rolfe, M. (1995). Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269,682 -685.[Medline]
Passini, M. A., Levine, E. M., Canger, A. K., Raymond, P. A. and Schechter, N. (1997). Vsx-1 and Vsx-2: differential expression of two paired-like homeobox genes during zebrafish and goldfish retinogenesis. J. Comp. Neurol. 388,495 -505.[CrossRef][Medline]
Polyak, K., Kato, J. Y., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J. M. and Koff, A. (1994). p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 8, 9-22.[Abstract]
Rakic, P. and Caviness, V. S., Jr (1995). Cortical development: view from neurological mutants two decades later. Neuron 14,1101 -1104.[Medline]
Redies, C. and Puelles, L. (2001). Modularity in vertebrate brain development and evolution. BioEssays 23,1100 -1111.[CrossRef][Medline]
Reynisdottir, I., Polyak, K., Iavarone, A. and Massague, J. (1995). Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta. Genes Dev. 9,1831 -1845.[Abstract]
Robb, R. M., Silver, J. and Sullivan, R. T. (1978). Ocular retardation (or) in the mouse. Invest. Ophthalmol. Vis. Sci. 17,468 -473.[Abstract]
Ross, M. E. and Walsh, C. A. (2001). Human brain malformations and their lessons for neuronal migration. Annu. Rev. Neurosci. 24,1041 -1070.[CrossRef][Medline]
Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100,431 -440.[Medline]
Servant, M. J., Coulombe, P., Turgeon, B. and Meloche, S.
(2000). Differential regulation of p27(Kip1) expression by
mitogenic and hypertrophic factors: Involvement of transcriptional and
posttranscriptional mechanisms. J. Cell Biol.
148,543
-556.
Sherr, C. J. and Roberts, J. M. (1999). CDK
inhibitors: positive and negative regulators of G1-phase progression.
Genes Dev. 13,1501
-1512.
Sicinski, P., Donaher, J. L., Parker, S. B., Li, T., Fazeli, A., Gardner, H., Haslam, S. Z., Bronson, R. T., Elledge, S. J. and Weinberg, R. A. (1995). Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82,621 -630.[Medline]
Sidman, R. L. (1961). Histogenesis of mouse retina studies with [3H]thymidine. In The Structure of the Eye (ed. G. Smelser), pp. 487-506. New York: Academic Press.
Stone, J. and Dreher, Z. (1987). Relationship between astrocytes, ganglion cells and vasculature of the retina. J. Comp. Neurol. 255,35 -49.[Medline]
Sur, M. and Leamey, C. A. (2001). Development and plasticity of cortical areas and networks. Nat. Rev. Neurosci. 2,251 -262.[CrossRef][Medline]
Tole, S., Goudreau, G., Assimacopoulos, S. and Grove, E. A.
(2000). Emx2 is required for growth of the hippocampus but not
for hippocampal field specification. J. Neurosci.
20,2618
-2625.
Tomita, K., Ishibashi, M., Nakahara, K., Ang, S. L., Nakanishi, S., Guillemot, F. and Kageyama, R. (1996). Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis. Neuron 16,723 -734.[Medline]
Tong, W. and Pollard, J. W. (2001). Genetic
evidence for the interactions of cyclin D1 and p27(Kip1) in mice.
Mol. Cell. Biol. 21,1319
-1328.
Toyoshima, H. and Hunter, T. (1994). p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78,67 -74.[Medline]
Trumpp, A., Refaeli, Y., Oskarsson, T., Gasser, S., Murphy, M., Martin, G. R. and Bishop, J. M. (2001). c-Myc regulates mammalian body size by controlling cell number but not cell size. Nature 414,768 -773.[CrossRef][Medline]
Vidal, A. and Koff, A. (2000). Cell-cycle inhibitors: three families united by a common cause. Gene 247,1 -15.[CrossRef][Medline]
Voyvodic, J. T. (1996). Cell death in cortical development: How much? Why? So what? Neuron 16,693 -696.[Medline]
Voyvodic, J. T., Burne, J. F. and Raff, M. C. (1995). Quantification of normal cell death in the rat retina: implications for clone composition in cell lineage analysis. Eur. J. Neurosci. 7,2469 -2478.[Medline]
Walsh, C. A. (1999). Genetic malformations of the human cerebral cortex. Neuron 23, 19-29.[Medline]
Watanabe, T. and Raff, M. C. (1988). Retinal astrocytes are immigrants from the optic nerve. Nature 332,834 -837.[CrossRef][Medline]
Young, R. W. (1984). Cell death during differentiation of the retina in the mouse. J. Comp. Neurol. 229,362 -373.[Medline]
Young, R. W. (1985a). Cell differentiation in the retina of the mouse. Anat. Rec. 212,199 -205.[Medline]
Young, R. W. (1985b). Cell proliferation during postnatal development of the retina in the mouse. Brain Res. 353,229 -239.[Medline]
Zuber, M. E., Perron, M., Philpott, A., Bang, A. and Harris, W. A. (1999). Giant eyes in Xenopus laevis by overexpression of XOptx2. Cell 98,341 -352.[Medline]