doi: 10.1242/10.1242/dev.00421
Loss of Emx2 function leads to ectopic expression of Wnt1 in the developing telencephalon and cortical dysplasia
Keith L. Ligon1,2,3,
Yann Echelard4,
Stavroula Assimacopoulos5,
Paul S. Danielian6,
Sovann Kaing1,
Elizabeth A. Grove5,
Andrew P. McMahon6 and
David H. Rowitch1,7,*
1 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard
Medical School, 44 Binney Street, Boston, MA 0215, USA
2 Department of Pathology, Division of Neuropathology, Brigham and Women's
Hospital, 75 Francis Street, Boston, MA 02115, USA
3 Division of Neuropathology, Children's Hospital Boston, 300 Longwood Avenue,
Boston, MA 02115, USA
4 GTC Biotherapeutics, 5 Mountain Rd, Framingham, MA 01701-9322, USA
5 Department of Neurobiology, Pharmacology and Physiology, University of
Chicago, Chicago, IL 60637, USA
6 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity
Avenue, Cambridge, MA 02138, USA
7 Division of Newborn Medicine, Children's Hospital Boston, 300 Longwood Avenue,
Boston, MA 02115, USA
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Fig. 1. Marginal zone heterotopias in Emx2–/– mice
are similar to human heterotopias in individuals with cortical dysplasia.
Histological analysis of Hematoxylin and Eosin stained sections from
Emx2–/– mouse brains demonstrated diffuse
changes in the marginal zone with increased cellularity and poor distinction
of the marginal zone from the underlying cortical plate proper (D-F). Nodular
protrusions of cells into the marginal zone and overlying leptomeninges
(arrowheads) were also detected and were frequently associated with focal
disorganization of the subplate and full thickness abnormalities of the
cortical plate in the same regions (broken line). No such abnormalities were
detected in Emx2+/– littermates (A-C). Hematoxylin
and Eosin stained human leptomeningeal glioneuronal heterotopia from the
cortex of a 36-week-old human infant had a similar morphology (G).
Immunohistochemical analysis of human and
Emx2–/– lesions demonstrated the presence of
NeuN-positive neurons within both lesions (H,J arrows). Immunohistochemistry
in the human (I) and in situ hybridization in the mouse (K) demonstrated
GFAP-positive astrocytes within the lesions (arrowheads). mz, marginal zone;
cp, cortical plate; sp,subplate; iz, intermediate zone. Scale bar: in G, 200
µm for G; in C, 50 µm for C,F,J,K.
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Fig. 2. Ectopic Wnt1 expression in the cortical hem and cortical
ventricular zone of Emx2-null mutant mice. Whole-mount in situ
hybridization analysis of Wnt1 expression in 12.5 dpc wild-type
embryos showed normal expression of Wnt1 within the roof plate of the
neural tube and extending to the rostral diencephalon (A). In addition to the
wild-type pattern, Emx2–/– embryos (B)
exhibited strong ectopic expression of Wnt1 within the dorsomedial
telencephalon, cortical hem organizer region and in the diencephalon (arrows).
A dissected coronal view (C) of same embryo at the level of the broken line in
B showed ectopic Wnt1 within the cortical hem, hippocampal primordium
and cortical ventricular zone (arrow). tel, telencephalon; di, diencephalon;
hem, cortical hem; hp, hippocampal primordium; ncx, neocortex.
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Fig. 4. Deletion of the HBS1 site in the 1.1 kb 3' Wnt1 enhancer element
results in ectopic expression of transgenes within the telencephalon. (A-C)
Whole-mount histochemical analysis of ß-galactosidase expression in
Wnt1-lacZ- HBS1 transgenic mice at 8.75 (A), 10.5 (B)
and 16.5 dpc (C). Ectopic expression was highest in the dorsomedial
telencephalon (A-C, red arrowheads) with a gradient of expression diminishing
ventrally (C). Ectopic expression was also noted in the rostral diencephalon
(B,C). Expression of the transgene was relatively weak in the endogenous
Wnt1 expression domain, including the roof plate and mid-hindbrain
junction compared with previous analyses of the full-length Wnt1 1.1
kb enhancer (Iler et al.,
1995 ; Rowitch et al.,
1998) (B, red arrow). (D) Summary of the wild-type endogenous
Wnt1 expression pattern (blue) and ectopic telencephalic expression
(red), as observed in Wnt1-Tg and
Emx2–/– transgenic mice. (E-H) Representative
results of in situ hybridization analysis of coronal sections from 12.5 dpc
control (n=4) (E,F) and Wnt1-Tg (n=4) (G,H)
telencephalon demonstrated a striking gradient of ectopic Wnt1
expression in the cortical hem and cortical ventricular zone of the transgenic
(G,H) mice in a pattern recapitulating that of endogenous Emx2 (E).
mes, mesencephalon; di, diencephalon; tel, telencephalon; rp, roof plate of
neural tube; r1, rhombomere 1; hem, cortical hem; cpp, choroid plexus
primordium; ncx, neocortical ventricular zone.
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Fig. 5. Absence of cortical hem patterning defects in Wnt1-Tg mice.
Hematoxylin and Eosin staining of control littermates (A,B) and
Wnt1-Tg mice (E,F) at 12.5 dpc showed only mild disorganization of
the cortical ventricular zone and cortical hem. Poor definition of the choroid
plexus primordium from the surrounding thalamic eminence was noted. Expression
of the cortical hem marker Wnt2b (D,H) and the roofplate organizer
marker Bmp4 (C,G) were normal in Wnt1-Tg mice by in situ
hybridization at 12.5 dpc. cpp, choroid plexus primordium; hem, cortical hem;
cp, cortical ventricular zone; di, diencephalon. The bracket delineates the
cortical hem region. All sections are coronal.
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Fig. 6. Emx2–/– and Wnt1-Tg mice exhibit
diffuse abnormalities of the marginal zone and subplate at 18.5 dpc. (A-C) In
situ hybridization analysis shows a reduction in the number of reelin-positive
Cajal-Retzius (CR) cells in the anterior regions of the marginal zone and
subplate of Emx2–/– (B) and a patchy reduction
in Wnt1-Tg (C) mice when compared with control wild-type littermates
(A) at 18.5 dpc. Note, that scattered reelin-positive cells (arrowheads) with
small cell morphology persisted at 18.5 dpc but few cells with clear CR type
morphology were observed in anterior dorsolateral cortex. (D-F)
Immunohistochemistry demonstrating a marked reduction in the number and
complexity of calretinin-positive cells and CR cells (arrowheads), and their
processes within the marginal zone and subplate of
Emx2–/– and Wnt1-Tg mice. cp,
cortical plate proper (layers II-VI); mz, marginal zone (layer I); sp,
subplate. Scale bar: 50 µm.
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Fig. 7. Ectopic Wnt1 expression in the telencephalon produced heterotopias
identical to those seen in Emx2–/– mice. (A-C)
Representative histological analysis (Hematoxylin and Eosin staining) of the
18.5 dpc cortex of Emx2–/– (n=6) (B)
and Wnt1-Tg (n=2) (C) mice demonstrated heterotopias that
were identical in morphological appearance. A further Wnt1-Tg founder
so analyzed at P1 also showed heterotopias. Lesions in the Wnt1-Tg
mice contained NeuN-positive neurons (D, arrowheads) as well as
calretinin-positive neuronal cell bodies and processes (F, arrowheads) similar
to those seen in Emx2–/– mice (E, arrowheads).
Immunohistochemistry for Nestin demonstrated focal defects in the subpial glia
limitans in direct association with heterotopias of
Emx2–/– (H) and Wnt1-Tg (I) mice,
which were not seen in wild-type littermates (G). Staining was normal away
from the lesions (red arrowheads). Radial glial processes (white arrowheads)
were seen to extend directly into the lesions from the underlying cortical
plate but did not form a glia limitans (H,I). mz, marginal zone; cp, cortical
plate.
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Fig. 8. Early heterotopias and defects of preplate derivatives in Wnt1-Tg
mice. (A, part a) Immunohistochemical staining in wild-type and
Wnt1-Tg mice at 13.5 dpc showed a marked reduction in the number of
Calretinin-positive cells and processes (arrowheads) within the marginal zone
and subplate of Wnt1-Tg transgenic mice in anterior and lateral
neocortex. (A, part b) In situ hybridization for reelin mRNA transcripts
demonstrated a similar reduction of Cajal-Retzius cells in the same
distribution (arrowheads). Note that the cortex of Wnt1-Tg mice also
exhibited a marked increase in size. (All sections cut in the horizontal
plane; boxed regions correspond to sections photographed in B, parts a-l). (B,
parts a-f) Analysis of anterior regions of the neocortex of Wnt1
transgenic animals showed a loss of calretinin-positive processes and cells
within the marginal zone and subplate as well as loss of reelin-positive
Cajal-Retzius cells within the marginal zone at 13.5 dpc. (B, parts g-l)
Reductions in cell number and processes were not as evident in more posterior
regions of neocortex. No significant abnormalities of cortical plate
architecture were detected in Wnt1-Tg animals by Hematoxylin and
Eosin morphological evaluation (a,b,g,h). (C, parts a,b) Heterotopias were
detected within the marginal zone and leptomeninges at 13.5 dpc in
Wnt1-Tg mice (b) by Hematoxylin and Eosin staining (arrowheads,
broken lines) and were associated with defects in the subplate morphology. (C,
part c) Marginal zone heterotopias were not consistently associated with an
absence of calretinin-positive cells as some positive cells were detected in
close proximity to an overlying heterotopia (arrowheads). (C, part d) Defects
in the laminin-positive pial basement membrane were not detected in
association with or distant from Wnt1-Tg heterotopias (white
arrowheads), or at any other location by immunohistochemistry at 13.5 dpc. mz,
marginal zone; cp, cortical plate; sp,subplate.
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© The Company of Biologists Ltd 2003
