First published online January 12, 2006
doi: 10.1242/10.1242/dev.02223
Development 133, 495-505 (2006)
Published by The Company of Biologists 2006
Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons
Julianna Kele1,*,
Nicolas Simplicio2,*,
Anna L. M. Ferri3,
Helena Mira1,
,
François Guillemot2,
Ernest Arenas1,
,
and
Siew-Lan Ang3,,
1 Laboratory of Molecular Neurobiology, MBB, Karolinska Institutet,
Scheelesväg 1, Retzius building A1, 17 177 Stockholm, Sweden.
2 Division of Molecular Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
3 Division of Developmental Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
View larger version (84K):
[in a new window]
|
Fig. 1. Expression of proneural bHLH genes in the developing ventral midbrain in
relation to tyrosine hydroxylase (Th), Lmx1b and
Nurr1 expression. (A-Q) Coronal sections of the
ventral midbrain. ISH at E11.5 and E13.5 revealed Th mRNA in DA
neurons (A,F) and Lmx1b mRNA expression in both DA progenitors and
neurons in zone 1 of the VM (B,G). The expression of the proneural bHLH genes
was restricted to the ventricular zone, with the exception of Ngn2,
which is seen also in the IZ (P,Q). Ngn1 mRNA was expressed only in
zone 2 (C), Ngn2 was expressed in zones 1 and 2 (D,) and
Mash1 was expressed in zones 1, 2 and 3 at different levels (E). At
E11.5, Mash1 and Ngn2 protein were expressed in a `salt-and pepper' pattern in
the VZ with some double-labelled cells (K-M). Mash1 (N) and Ngn2 (O) are
co-expressed with some Sox2+ progenitors. In addition, Ngn2 is also
expressed in Sox2– cells (O). Nurr1 and Ngn2 proteins are
co-expressed in some cells in the IZ of Ngn2KIGFP/+ embryos at
E11.5 (P,Q). A summary of all the expression data at E11.5 is shown in
R. Scale bar: 100 µm.
|
|
View larger version (71K):
[in a new window]
|
Fig. 2. Loss of DA neurons in Ngn2 null mutant mice. An almost
complete ablation of DA neurons was detected at E11.5 as assessed by Th
(A-C) and Pitx3 (D,E) immunostaining. The expression of the
pan-neuronal marker, ßIII tubulin, together with Toto nuclear labeling,
revealed a complete loss of neuronal cell bodies in the midline region of
Ngn2 mutant mice at E11.5 (F). Arrows point to Toto-labeled
nuclei and asterisks denote the absence of cell bodies, with only ßIII
tubulin+ processes present. At E14.5, Toto/Tuj1 and MAP2/Hoechst
expression revealed a reduction of the size of the marginal zone in mutant
embryos, indicating a severely reduced number of neurons (G,H). A
severe reduction in number of Th+ cells (I-K) and in Pitx3
staining (L,M) was also detected. Note that Th+ cells in
mutant embryos are localized lateral to the midline.
***P<0.001 and **P<0.01, as
assessed by Student's t-test with Welch's correction (n=4-5
in A,H). Scale bar: 100 µm in B-E,G,H,J-M; 10 µm in F.
|
|
View larger version (125K):
[in a new window]
|
Fig. 3. Mash1 and Ngn1 do not contribute significantly to the loss of
Th+ DA neurons in Ngn2 mutant mice. Mice double mutant for
Ngn2 and Ngn1 do not show any further loss of Th+
cells at E14.5, compared with Ngn2 single mutants (A-C),
indicating that only Ngn2 is required for the development of midbrain
DA neurons. A severe reduction in the number of Th+ cells is
detected at E11.5 in Mash1;Ngn2 double mutant mice (D-F), but
the defect is not more severe than in Ngn2 single mutant mice (see
Fig. 2). Consistently, no
alteration in the number or distribution of Th+ cells is detected
in Mash1 single mutant mice at E11.5 (G-I) or E14.5
(J-L). *P<0.01, as assessed by Student's
t-test with Welch's correction (D) or one-way ANOVA, with
Bonferroni's correction for multiple comparisons (G,J). n=3-6. Scale
bar: 100 µm.
|
|
View larger version (92K):
[in a new window]
|
Fig. 4. Loss of Ngn2 results in a decrease in number of Nurr1+ cells
at E11.5. (A-D) No change in BrdU incorporation (A,B) or in
Sox2+/Ki67+ double labeled cells (C,D) was found in
Ngn2–/– mice compared with wild types at
E11.5. (E-G) A simultaneous decrease in number of Nurr1+
cells was detected in the intermediate and marginal zones (E, n=5;
**P<0.01, Student's t-test with Welch's
correction). (H,I) GFP+/Nurr1+ cells were also
missing directly under the ventral midline in Ngn2Gfp/Gfp
embryos. (J-M) The expression of Hes5 (J,K) and Dll1
(L,M), two markers of proneural activity, was also reduced in the ventricular
zone. Scale bar: 100 µm.
|
|
View larger version (43K):
[in a new window]
|
Fig. 5. Radial glia cells continue to accumulate in Ngn2 mutant mice,
but the number of Nurr1+ cells partially recovers at E14.5.
(A-D) By E13.5, Hes5 (A,B) and Dll1 (C,D) expression are significantly
recovered in zone 1 VZ. (E,F) Cresyl violet staining highlights the
enlarged ventricular zone in Ngn2 null mice at E14.5. (G-J)
This cyto-architectural change is mirrored by the accumulation of radial glia
cells, marked by RC2 /GLAST (G,H) or Sox2/GLAST (I,J) double staining.
(K-M) At E14.5, the reduction in the number of Nurr1+ cells
in Ngn2 mutants was less pronounced than at E11.5 (see
Fig. 4G), indicating a partial
rescue of the block in differentiation of DA neuron precursors (K, n=3;
**P<0.01, Student's t-test with Welch's
correction). Scale bar: 100 µm.
|
|
View larger version (79K):
[in a new window]
|
Fig. 6. Partial rescue of the loss of Nurr1+ and Th+ cells
in Ngn2 mutant mice by endogenous or overexpressed Mash1.
(A) At E11.5, Mash1 is transiently downregulated in zone 1 of
Ngn2–/– mice (arrow), but has partially
recovered by E13.5. (B) Substitution of Ngn2 expression by
Mash1 (Ngn2KIMash1/KIMash1, abbreviated to
Ngn2M/M) did not rescue the reduction in number of
Th+ cells observed in Ngn2 mutants at E11.5.
(C,E-G) However, Mash1 overexpression partially rescues the
loss of Th+ DA neurons at E14.5. (D,H-J) The greater loss of
Nurr1+ cells in Ngn2;Mash1 double mutants than in
Ngn2 single mutants indicates that endogenous Mash1 partially rescues
the loss of Nurr1+ precursors at E14.5 (B-D, n=3-6;
***P<0.001, one-way ANOVA, with Bonferroni's correction
for multiple comparisons). Scale bar: 100 µm in A; 20 µm in E-J.
|
|
View larger version (56K):
[in a new window]
|
Fig. 7. Further but incomplete rescue of Th expression at E17.5, and a schematic
model of the expression and function of proneural bHLH in the development of
midbrain DA neurons. (A-D) The number of Th+
neurons is further increased in Ngn2 and
Ngn2KIMash1/KIMash1 mutants, but remains low at 9% in
Ngn2;Mash1 double mutants. (E) We show that Ngn2 is expressed
in VZ cells that express Sox2 and radial glia markers. Ngn2 is required for
the differentiation of Sox2+ ventricular zone cells into
Nurr1+ DA precursors, as deletion of Ngn2 results in a
decrease in the number of Nurr1+ cells (step 1). This function is
partially rescued by endogenous Mash1 recovered at E13.5 or by Mash1 expressed
under the control of the Ngn2 promoter. Ngn2 is expressed by
Nurr1+ cells in the IZ and a second possible function of Ngn2 is to
promote the differentiation of Nurr1+ precursors into DA neurons
(step 2). This step could be partially rescued by a substitution of Ngn2 by
Mash1 (Ngn2KIMash1/KIMash1). Thus, our results suggest
that Mash1 can also partially rescue the number of DA neurons. Scale bar: 100
µm.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2006
