First published online December 8, 2005
doi: 10.1242/10.1242/dev.02181
Development 133, 89-98 (2006)
Published by The Company of Biologists 2006
A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo
Nilima Prakash1,*,
Claude Brodski2,*,
Thorsten Naserke1,*,
Eduardo Puelles3,*,
Robindra Gogoi3,
Anita Hall4,
Markus Panhuysen1,
Diego Echevarria5,
Lori Sussel6,
Daniela M. Vogt Weisenhorn1,
Salvador Martinez5,
Ernest Arenas4,
Antonio Simeone3,7,8,
and
Wolfgang Wurst1,
1 GSF-National Research Center for Environment and Health, Technical University
Munich, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1,
85764 Munich/Neuherberg, Germany, and Max-Planck-Institute of Psychiatry,
Kraepelinstrasse 2, 80804 Munich, Germany.
2 Ben-Gurion University of the Negev, Faculty of Health Sciences, Zlotowski
Center for Neuroscience, Department of Morphology, Be'er Sheva 84105,
Israel.
3 MRC Centre for Developmental Neurobiology, 4th floor, New Hunt's House, King's
College London, Guy's Campus, London Bridge, London SE1 UL, UK.
4 Laboratory of Molecular Neurobiology, MBB, Karolinska Institute, 17177
Stockholm, Sweden.
5 Instituto de Neurociencias, Universidad Miguel Hernandez, San Juan, 03550
Alicante, Spain.
6 Barbara Davis Center, University of Colorado Health Science Center, 4200 E.
9th Avenue, Denver, CO 8020, USA.
7 CEINGE Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples,
Italy.
8 Institute of Genetics and Biophysics `ABT', Via Guglielmo Marconi 12, 80125
Naples, Italy.
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Fig. 1. Ectopic expression of Wnt1 in the rostral hindbrain leads to
induction of Otx2 and concomitant generation of ectopic mDA neurons
only within the hindbrain FP. (A) Midsagittal sections at the level
of the FP of wild-type and En1+/Wnt1 (n=6)
embryos at E10.5 hybridized with probes for Otx2, Aldh1a1, Fgf8 and
Wnt1. Top row shows bright-field images; red rectangles depict the
region of the dark-field images from consecutive sections shown below, except
for those hybridized with Wnt1, which correspond to embryos of a
distinct litter. Black arrowheads indicate the normal (wild-type) position of
the MHB. White arrows indicate the ectopic expression domains. (B)
Midsagittal sections of wild-type and En1+/Wnt1
(n=4) embryos at E12.5 hybridized with probes for Wnt1, Nr4a2,
Th and Pitx3 (white arrows indicate ectopic expression).
(C) Pseudocolored overlays of the corresponding dark-field images from
coronal sections of E10.5 wild-type and En1+/Wnt1
(n=4) embryos hybridized with probes for Gbx2 (green),
Aldh1a1 (red) and Wnt5a (green). Overlapping domains appear
yellow. (D) Immunodetection of BrdU in sagittal sections of E11.5
wild-type and En1+/Wnt1 (n=2) embryos after 6
hours of cumulative labeling did not reveal in mutant embryos obvious
abnormalities in proliferating activity along the FP region of the mid- and
hindbrain. (E) Schematic drawing of a cross-section through the rostral
hindbrain floor plate and basal plate of En1+/Wnt1 mutants
at two different time points of development, summarizing the events occurring
in this region. The ectopic expression of Wnt1 throughout the entire
rostral hindbrain (rhombomere 1) of the En1+/Wnt1 mutants
has been omitted for clarity.
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Fig. 2. Ectopic Nkx2-2 is sufficient to repress the mDA neuronal fate and to
induce rostral 5HT neurons in the ventral midbrain. (A) Fluorescent
immunostaining for Otx2 (red), Nkx2-2 (green), Shh (red), Th (green) and 5HT
(green), or in situ hybridization with Wnt1 probe on coronal sections
of wild-type, conditional En1+/Cre;
Otx2flox/flox, Nkx2-2-/- single and compound
En1+/Cre; Otx2flox/flox;
Nkx2-2-/- triple mutant mouse embryos at E12.5
(n=3 for each genotype). Wnt1 expression in the dorsal
midline (RP) of the midbrain remained unaffected in all mutants (white
arrows). (B) Schematic drawing of a cross-section through the ventral
midbrain of E12.5 wild-type mouse embryos summarizing our results.
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Fig. 3. The ectopic mDA progenitor domain in En1+/Wnt1
mutants is established through partial repression of Nkx2-2 in the
ventral hindbrain by Otx2. (A,B) Coronal sections of E12.5
wild-type and En1+/Wnt1 (n=4) embryos at the
height of rhombomere 1-2 (black bars in
Fig. 1B) hybridized with probes
for Wnt1, Shh, Otx2 (green), Th (red) and the serotonin
transporter (Slc6a4, green), or immunostained for Otx2, Nkx2-2 and
Th. Top row in A are bright-field pictures corresponding to the dark-field
images. Pictures in A are partly pseudocolored overlays of the corresponding
dark-field images. Brackets in B indicate the region where the most ventral
part of the Nkx2-2 domain in the hindbrain was repressed by the ectopically
induced Otx2. (C) Sketch of a cross-section through the
En1+/Wnt1 mutant rostral hindbrain at E12.5 summarizing
these results. 5HT neurons were probably generated from the dorsolateral
Nkx2-2-positive domain within the mutant BP in this region.
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Fig. 4. Wnt1 is necessary for terminal differentiation of Th-expressing mDA
precursors. (A) Consecutive sagittal sections of wild-type and
Wnt1-/- embryos at E9.5 (n=8) hybridized with
probes for En1 and Aldh1a1. (B) Sagittal sections of
wild-type and Wnt1-/- embryos at E11.5 (n=8)
hybridized with En1 or immunostained for RC2 (a radial glia marker,
red). (C) Fluorescent immunodetection of Th (green) and Pitx3 (red) on
sagittal sections of wild-type and Wnt1-/- mutant embryos
at E11.5 (n=3). The white square in B corresponds to the region of
the sections in C. Graph shows that the number of Th-positive cells was
drastically reduced in the Wnt1-/- mutant at this stage
(mean±s.d./s.e.m. from three sibling pairs: wild type,
1958±129/92 cells; Wnt1-/-,14±11/8 cells).
Triple asterisks indicate numbers that differ at P<0.0024, paired
t-test. (D) Schematic drawing of a cross-section through the
E11.5 ventral midbrain of wild-type mouse embryos, suggesting a possible role
of Wnt1 in the generation of Pitx3-expressing mDA neurons.
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Fig. 5. Wnt1 is required for ectopic induction of mDA neurons by Fgf8
and Shh. (A) In situ hybridization with Th probe (dark
blue) on E8.0-8.5 wild-type (Wnt1+/+ and
+/-; n=20/28) and Wnt1-/-
(n=8/9) mouse anterior neural plate explants 6 days after
implantation of Fgf8b-coated beads (arrowheads) close to the ventral midline
of the presumptive forebrain. No induction of Th-positive cells was
seen after implantation of BSA-coated (control) beads (data not shown;
n=8/9 for wild-type and n=4/5 for mutant explants).
(B) In situ hybridization of wild-type E8.0-8.5 mouse anterior neural
plate explants 24 hours after implantation of Fgf8b-coated beads with
Foxg1b (light blue) and Shh (red) to confirm the forebrain
and ventral identity of the tissue where the bead was implanted (arrowhead).
No Wnt1 expression (dark blue) was detected in the tissue surrounding
the bead located outside the endogenous Wnt1 domain (arrowhead).
However, expression of Wnt1 was maintained at a distance from the
bead when compared with the contralateral control side of the explant (broken
line coincides with the ventral midline). Repression of endogenous
Wnt1 around another Fgf8b-coated bead located in the presumptive
dorsal midbrain is apparent (arrow). (C) RT-PCR for Th, Pitx3,
Nr4a2, Aldh1a1 and Wnt1 on total RNA from pooled wild-type and
Wnt1-/- explants cultured under the same conditions.
Detection of ubiquitously expressed glyceraldehyde-3-phosphate dehydrogenase
(Gapd) was used as control. Expression of Nr4a2 and
Aldh1a1 is not restricted to mDA neurons and was therefore detected
in wild-type as well as in Wnt1-/- explants.
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Fig. 6. Wnt1 may control two different steps in the generation of mDA neurons in
vivo. (A) Schematic drawing of a cross section through the mouse
ventral midbrain close to the MHB at E9.5-10.5 showing the overlapping
expression of Shh (black) and Wnt1 (green) in the midbrain FP and BP. Wnt1 is
spared from the ventral midline (medial FP), where only Shh is expressed. Otx2
(yellow) is expressed throughout the midbrain neuroepithelium at this stage,
overlapping with a narrow stripe corresponding to the Nkx2-2 expression domain
(red). Both Wnt1 and Otx2 are engaged in a positive feedback loop controlling
their expression within the ventral midbrain such that secreted Wnt1 protein
induces and/or maintains Otx2 expression in the FP and BP of the
midbrain and vice versa. Otx2 protein is in turn required for repression of
Nkx2-2 within this territory of the neural tube. The mDA progenitor
domain is thus established by this Wnt1-controlled regulatory network during
early neural development. (B) Schematic drawing of a cross-section
through the mouse ventral midbrain close to the MHB at E12.5. The Shh (black)
and Wnt1 (green) expression domains have refined to a narrow area
corresponding to the ventricular and subventricular zone and partly
overlapping with the Otx2-positive (yellow) proliferative neuroepithelium of
the midbrain FP and BP. The Nkx2-2 (red) domain is now restricted to a
wedge-shaped transversal stripe at the alar-basal boundary corresponding to
the region of lowest Otx2 protein levels. At this stage, proliferating mDA
progenitors within the midbrain FP/BP have already generated Th-expressing mDA
precursors. Wnt1 may be required for terminal differentiation of these cells
by inducing expression of Pitx3 (blue). The as yet hypothetical nature of a
direct role of Wnt1 in the generation of mDA neurons is indicated by broken
lines.
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