Pax6 is a member of the mammalian Pax transcription factor family. Many of the Pax genes display semi-dominant loss-of-function heterozygous phenotypes, yet the underlying cause for this dosage requirement is not known. Mice heterozygous for Pax6 mutations exhibit small eyes (Sey) and in embryos the most obvious defect is a small lens. We have studied lens development in Pax6(Sey)(−1Neu)/+ embryos to understand the basis of the haploinsufficiency. The formation of the lens pre-placode appears to be unaffected in heterozygotes, as deduced from the number of cells, the mitotic index, the amount of apoptosis and the expression of SOX2 and Pax6 in the pre-placode. However, the formation of the lens placode is delayed. The cells at the edge of the lens cup fail to express N-cadherin and undergo apoptosis and the lens fails to detach completely from the surface ectoderm. After formation, the lens, which has 50% of the cells found in wild-type embryos, grows at a rate that is indistinguishable from wild type. We rule out the possibility that monoallelic expression of Pax6 at the time of lens placode formation accounts for the 50% reduction in cell number by showing that expression of Pax6 is biallelic in the lens placode and optic vesicle. We propose instead that a critical threshold of PAX6 protein is required for lens placode formation and that the time in development at which this level is reached is delayed in heterozygotes.

REFERENCES

REFERENCES
Baldwin
C. T.
,
Hoth
C. F.
,
Amos
J. A.
,
da-Silva
E. O.
,
Milunsky
A.
(
1992
)
An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome.
Nature
355
,
637
638
Collinson
J. M.
,
Hill
R. E.
,
West
J. D.
(
2000
)
Different roles for Pax6 in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye.
Development
127
,
945
956
Cvekl
A.
,
Piatigorsky
J.
(
1996
)
Lens development and crystallin gene expression: many roles for Pax-6.
BioEssays
18
,
621
630
Dressler
G. R.
,
Wilkinson
J. E.
,
Rothenpieler
U. W.
,
Patterson
L. T.
,
Williams-Simons
L.
,
Westphal
H.
(
1993
)
Deregulation of Pax-2 expression in transgenic mice generates severe kidney abnormalities.
Nature
362
,
65
67
Engelkamp
D.
,
van Heyningen
V.
(
1996
)
Transcription factors in disease.
Curr. Opin. Genet. Dev
6
,
334
342
Epstein
D. J.
,
Vekemans
M.
,
Gros
P.
(
1991
)
Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3.
Cell
67
,
767
774
Fisher
E.
,
Scambler
P.
(
1994
)
Human haploinsufficiency—one for sorrow, two for joy.
Nat. Genet
7
,
5
7
Fujiwara
M.
,
Uchida
T.
,
Osumi-Yamashita
N.
,
Eto
K.
(
1994
)
Uchida rat (rSey): a new mutant rat with craniofacial abnormalities resembling those of the mouse Sey mutant.
Differentiation
57
,
31
38
Furuta
Y.
,
Hogan
B. L. M.
(
1998
)
BMP4 is essential for lens induction in the mouse embryo.
Genes. Dev
12
,
3764
3775
Gage
P. J.
,
Suh
H.
,
Camper
S. A.
(
1999
)
Dosage requirement of Pitx2 for development of multiple organs.
Development
126
,
4643
4651
Glaser
T.
,
Lane
J.
,
Housman
D.
(
1990
)
A mouse model of the aniridia-Wilms tumor deletion syndrome.
Science
250
,
823
827
Grainger
R. M.
,
Mannion
J. E.
,
Cook
T. L.
Jr.
,
Zygar
C. A.
(
1997
)
Defining intermediate stages in cell determination: acquisition of a lens-forming bias in head ectoderm during lens determination.
Dev. Genet
20
,
246
257
Grindley
J. C.
,
Davidson
D. R.
,
Hill
R. E.
(
1995
)
The role of Pax-6 in eye and nasal development.
Development
121
,
1433
1442
Hanson
I. M.
,
Fletcher
J. M.
,
Jordan
T.
,
Brown
A.
,
Taylor
D.
,
Adams
R. J.
,
Punnett
H. H.
,
van Heyningen
V.
(
1994
)
Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly.
Nat. Genet
6
,
168
173
Hatta
K.
,
Takeichi
M.
(
1986
)
Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development.
Nature
320
,
447
449
Hendzel
M. J.
,
Wei
Y.
,
Mancini
M. A.
,
Van Hooser
A.
,
Ranalli
T.
,
Brinkley
B. R.
,
Bazett-Jones
D. P.
,
Allis
C. D.
(
1997
)
Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.
Chromosoma
106
,
348
360
Herrmann
B. G.
,
Labeit
S.
,
Poustka
A.
,
King
T. R.
,
Lehrach
H.
(
1990
)
Cloning of the T gene required in mesoderm formation in the mouse.
Nature
343
,
617
622
Hill
R. E.
,
Favor
J.
,
Hogan
B. L.
,
Ton
C. C.
,
Saunders
G. F.
,
Hanson
I. M.
,
Prosser
J.
,
Jordan
T.
,
Hastie
N. D.
,
van Heyningen
V.
(
1991
)
Mouse small eye results from mutations in a paired-like homeobox-containing gene.
Nature
354
,
522
525
Hogan
B. L.
,
Horsburgh
G.
,
Cohen
J.
,
Hetherington
C. M.
,
Fisher
G.
,
Lyon
M. F.
(
1986
)
Small eyes (Sey): a homozygous lethal mutation on chromosome 2 which affects the differentiation of both lens and nasal placodes in the mouse.
J. Embryol. Exp. Morphol
97
,
95
110
Johnson
C. V.
,
Singer
R. H.
,
Lawrence
J. B.
(
1991
)
Fluorescent detection of nuclear RNA and DNA: implications for genome organization.
Methods Cell Biol
35
,
73
99
Jordan
T.
,
Hanson
I.
,
Zaletayev
D.
,
Hodgson
S.
,
Prosser
J.
,
Seawright
A.
,
Hastie
N.
,
van Heyningen
V.
(
1992
)
The human PAX6 gene is mutated in two patients with aniridia.
Nat. Genet
1
,
328
332
Kamachi
Y.
,
Uchikawa
M.
,
Collignon
J.
,
Lovell-Badge
R.
,
Kondoh
H.
(
1998
)
Involvement of Sox1, 2 and 3 in the early and subsequent molecular events of lens induction.
Development
125
,
2521
2532
Keller
S. A.
,
Jones
J. M.
,
Boyle
A.
,
Barrow
L. L.
,
Killen
P. D.
,
Green
D. G.
,
Kapousta
N. V.
,
Hitchcock
P. F.
,
Swank
R. T.
,
Meisler
M. H.
(
1994
)
Kidney and retinal defects (Krd), a transgene-induced mutation with a deletion of mouse chromosome 19 that includes the Pax2 locus.
Genomics
23
,
309
320
Macchia
P. E.
,
Lapi
P.
,
Krude
H.
,
Pirro
M. T.
,
Missero
C.
,
Chiovato
L.
,
Souabni
A.
,
Baserga
M.
,
Tassi
V.
,
Pinchera
A.
,
Fenzi
G.
,
Gruters
A.
,
Busslinger
M.
,
Di Lauro
R.
(
1998
)
PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis.
Nat. Genet
19
,
83
86
Makarenkova
H. P.
,
Ito
M.
,
Govindarajan
V.
,
Faber
S. C.
,
Sun
L.
,
McMahon
G.
,
Overbeek
P. A.
,
Lang
R. A.
(
2000
)
FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.
Development
127
,
2563
2572
Mannervik
M.
,
Nibu
Y.
,
Zhang
H.
,
Levine
M.
(
1999
)
Transcriptional coregulators in development.
Science
284
,
606
609
Mansouri
A.
,
Goudreau
G.
,
Gruss
P.
(
1999
)
Pax genes and their role in organogenesis.
Cancer Res
59
,
1707
1710
Nishiguchi
S.
,
Wood
H.
,
Kondoh
H.
,
Lovell-Badge
R.
,
Episkopou
V.
(
1998
)
Sox1 directly regulates the gamma-crystallin genes and is essential for lens development in mice.
Genes Dev
12
,
776
781
Nutt
S. L.
,
Busslinger
M.
(
1999
)
Monoallelic expression of Pax5: a paradigm for the haploinsufficiency of mammalian Pax genes?.
Biol. Chem
380
,
601
611
Nutt
S. L.
,
Vambrie
S.
,
Steinlein
P.
,
Kozmik
Z.
,
Rolink
A.
,
Weith
A.
,
Busslinger
M.
(
1999
)
Independent regulation of the two Pax5 alleles during B-cell development.
Nat. Genet
21
,
390
395
Ostrom
L.
,
Tang
M. J.
,
Gruss
P.
,
Dressler
G. R.
(
2000
)
Reduced Pax2 Gene Dosage Increases Apoptosis and Slows the Progression of Renal Cystic Disease.
Dev. Biol
219
,
250
258
Piatigorsky
J.
(
1981
)
Lens differentiation in vertebrates. A review of cellular and molecular features.
Differentiation
19
,
134
153
Quinn
J. C.
,
West
J. D.
,
Hill
R. E.
(
1996
)
Multiple functions for Pax6 in mouse eye and nasal development.
Genes Dev
10
,
435
446
Rhoades
K. L.
,
Singh
N.
,
Simon
I.
,
Glidden
B.
,
Cedar
H.
,
Chess
A.
(
2000
)
Allele-specific expression patterns of interleukin-2 and Pax-5 revealed by a sensitive single-cell RT-PCR analysis.
Curr. Biol
10
,
789
792
Sanyanusin
P.
,
Schimmenti
L. A.
,
McNoe
L. A.
,
Ward
T. A.
,
Pierpont
M. E.
,
Sullivan
M. J.
,
Dobyns
W. B.
,
Eccles
M. R.
(
1995
)
Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux.
Nat. Genet
9
,
358
364
Schedl
A.
,
Ross
A.
,
Lee
M.
,
Engelkamp
D.
,
Rashbass
P.
,
van Heyningen
V.
,
Hastie
N. D.
(
1996
)
Influence of PAX6 gene dosage ondevelopment: overexpression causes severe eye abnormalities.
Cell
86
,
71
82
Silver
J.
,
Hughes
A. F. W.
(
1973
)
The role of cell death during morphogenesis of the mammalian eye.
J. Morph
140
,
159
170
Stockton
D. W.
,
Das
P.
,
Goldenberg
M.
,
D'Souza
R. N.
,
Patel
P. I.
(
2000
)
Mutation of PAX9 is associated with oligodontia.
Nat. Genet
24
,
18
19
Stoykova
A.
,
Gotz
M.
,
Gruss
P.
,
Prince
J.
(
1997
)
Pax6-dependent regulation of adhesive patterning, R-cadherin expression and boundary formation in developing forebrain.
Development
124
,
3765
3777
Stuart
E. T.
,
Haffner
R.
,
Oren
M.
,
Gruss
P.
(
1995
)
Loss of p53 function through PAX-mediated transcriptional repression.
EMBO J
14
,
5638
45
Takeichi
M.
(
1988
)
The cadherins: cell-cell adhesion molecules controlling animal morphogenesis.
Development
102
,
639
655
Theiler
K.
,
Varnum
D. S.
,
Stevens
L. C.
(
1978
)
Development of Dickie's small eye, a mutation in the house mouse.
Anat. Embryol. (Berl.)
155
,
81
86
Ton
C. C.
,
Hirvonen
H.
,
Miwa
H.
,
Weil
M. M.
,
Monaghan
P.
,
Jordan
T.
,
van Heyningen
V.
,
Hastie
N. D.
,
Meijers-Heijboer
H.
,
Drechsler
M.
, et al. 
(
1991
)
Positional cloning and characterization of a paired box-and homeobox-containing gene from the aniridia region.
Cell
67
,
1059
1074
Walther
C.
,
Gruss
P.
(
1991
)
Pax-6, a murine paired box gene, is expressed in the developing CNS.
Development
113
,
1435
1449
Wawersik
S.
,
Purcell
P.
,
Rauchman
M.
,
Dudley
A. T.
,
Robertson
E. J.
,
Maas
R.
(
1999
)
BMP7 acts in murine lens placode development.
Dev. Biol
207
,
176
188
Weil
M.
,
Jacobson
M. D.
,
Raff
M. C.
(
1997
)
Is programmed cell death required for neural tube closure?.
Curr. Biol
7
,
281
284
Wilkie
A. O.
(
1994
)
The molecular basis of genetic dominance.
J. Med. Genet
31
,
89
98
Wilkinson
D. G.
,
Nieto
M. A.
(
1993
)
Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.
Methods Enzymol
225
,
361
373
Wilm
B.
,
Dahl
E.
,
Peters
H.
,
Balling
R.
,
Imai
K.
(
1998
)
Targeted disruption of Pax1 defines its null phenotype and proves haploinsufficiency.
Proc. Natl. Acad. Sci. USA
95
,
8692
8697
This content is only available via PDF.