First published online 4 August 2004
doi: 10.1242/dev.01308
Development 131, 4323-4332 (2004)
Published by The Company of Biologists 2004
Retinoic acid signalling specifies intermediate character in the developing telencephalon
Matthew Marklund1,
My Sjödal1,
Blake C. Beehler2,
Thomas M. Jessell3,
Thomas Edlund1,* and
Lena Gunhaga1
1 Umeå Center for Molecular Medicine, Building 6M, 4th floor, Umeå
University, S-901 87 Umeå, Sweden
2 Department of Biochemical Pharmacology, Bristol-Myers Squibb Pharmaceutical
Research Institute, Buffalo, NY 14213, USA
3 Howard Hughes Medical Institute, Department of Biochemistry and Molecular
Biophysics, Columbia University, New York, NY 10032, USA
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Fig. 1. Domains of expression of transcription factors define ventral (V),
intermediate (I) and dorsal (D) subdivisions of the developing telencephalon.
(A) A schematic drawing of a HH stage 22 (E3.5) chick embryo. The dorsal (D)
to ventral (V) line indicates the level of the transverse sections shown in
the corresponding panel. (B) In the telencephalon of a HH stage 22 chick
embryo, MEIS2 was expressed at high levels in the intermediate region. PAX6
was expressed in the dorsal region and in the most dorsal domain of the
intermediate region of the telencephalon. NKX2.1 was expressed exclusively in
the ventral region and EMX1 exclusively in the dorsal region of the
telencephalon (top panel). Below, enlargements depict overlapping (MEIS2/PAX6,
PAX6/EMX1) and largely non-overlapping (MEIS2/NKX2.1, MEIS2/EMX1) regions of
the telencephalon. Yellow staining represents double-labelled cells. (C) The
expression domains of transcription factors in the developing
telencephalon.
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Fig. 2. Telencephalic progenitor cells gradually acquire their intermediate
character. (A) A stage 12 and a stage 14 chick embryo (side view, rostral
towards the right). Black squares indicate intermediate neuroectoderm explant
regions of the embryo. Explants were analysed for expression of transcription
factors after 40-48 hours in culture. (B) Stage 12 intermediate explants
(n=15) generated MEIS2+ (14±7%), PAX6+
(22±7%), NKX2.1+ (7±6%) and EMX1+
(8±6%) cells in different domains. Scale bar: 30 µm. (C-F)
Expression of transcription factors in stage 14 intermediate explants grown
alone, with purified SHH protein, with mFrz8CRD (an antagonist of WNT
signalling) or with conditioned medium containing WNT3A (n=15 for
each). (C) Stage 14 intermediate explants (n=25) grown alone
generated MEIS2+ cells (22±6%), PAX6+ cells
(23±4%) but no NKX2.1+ or EMX1+ cells were
detected. (D) In the presence of SHH, the generation of MEIS2+
cells was blocked, and NKX2.1+ (65±5%) but no
PAX6+ or EMX1+ cells were detected. (E) In the presence
of WNT3A, the generation of MEIS2+ cells was blocked, and
PAX6+ cells (61±3%) and EMX1+ (25±5%) but
no NKX2.1+ or cells were detected. (F) In the presence of mFrz8CRD,
MEIS2+ cells (20±5%) were still generated, while the
generation of PAX6+ cells was blocked. No NKX2.1+ or
EMX1+ cells were detected. Scale bar: 30 µm.
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Fig. 4. RA induces intermediate character in early dorsal telencephalic cells. (A)
A stage 8 chick embryo (dorsal view, rostral is upwards). Black square
indicates prospective dorsal telencephalic neuroectoderm explant region. (B)
Expression of transcription factors in stage 8 dorsal (D, st 8) explants
cultured alone or with all-trans retinoic acid (RA) or a combination of
all-trans retinoic acid and FGF8 for 48-52 hours. Stage 8 D explants cultured
alone (n=20) generated PAX6+ cells (78±4%) but no
MEIS2+, ISL1+, NKX2.1+or EMX1+
cells were detected. Stage 8 D explants (n=20) exposed to all-trans
retinoic acid generated MEIS2+ cells (82±4%) distributed
throughout the explants, while ISL1+ cells (17±3%) and
PAX6+ cells (65±5%) were expressed in complementary regions
in the explant. No NKX2.1+ or EMX1+ cells were detected.
Stage 8 D explants (n=15) exposed to a combination of all-trans
retinoic acid and FGF8 generated PAX6+ cells (63±5%), but no
MEIS2+, NKX2.1 ISL1+ or EMX1+ cells were
detected. Scale bar: 75 µm. (C) Expression of transcription factors in the
ventral-most domain of the prospective intermediate telencephalon in a stage
22 chick embryo. ISL1+ cells were located in the periphery of the
proliferating neuroepithelium, while MEIS2+ cells were situated
throughout the neuroepithelium overlapping with the ISL1+ domain.
No PAX6+ cells were detected in this ventral subdomain of the
intermediate telencephalon.
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Fig. 5. Retinoic acid (RA) induces intermediate character in early ventral
telencephalic cells. (A) A stage 8 chick embryo (dorsal view, rostral is
upwards). Grey square indicates ventral telencephalic neuroectoderm explant
region. (B) Expression of transcription factors in stage 8 ventral (V, st 8)
explants cultured alone or with all-trans retinoic acid for 48-52 hours. Stage
8 V explants (n=15) generated NKX2.1+ cells (>90%) and
ISL1+ cells (19±4%) but no MEIS2+ or
PAX6+ cells. Stage 8 V explants (n=15) exposed to
all-trans retinoic acid generated MEIS2+ cells (23±3%),
ISL1+ cells (17±4%), PAX6+ cells (15±4%)
but no NKX2.1+ or EMX1+ cells. Scale bar: 30 µm.
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Fig. 6. FGF signals inhibit ventral telencephalic cells from responding to RA
signalling. (A) A stage 10 chick embryo (dorsal view, rostral is upwards).
Black square indicates ventral telencephalic neuroectoderm explant region. (B)
Expression of transcription factors in stage 10 ventral (V) explants cultured
alone or with soluble FGFR4 (sFGFR4) for 48-52 hours. Stage 10 V explants
cultured alone (n=15 for each) generated NKX2.1+ cells
(>90%) but no MEIS2+, PAX6+ or EMX1+
cells. In the presence of soluble FGFR4, the generation of NKX2.1+
cells was blocked but MEIS2+ cells (62±3%) and
PAX6+ cells (56±5%) were detected in stage 10 V explants. No
EMX1+ cells were detected. Scale bar: 30 µm. (C) Lateral views
of stage 20-22 chick embryos generated in New Culture from stage 10 grafted
with control or soluble FGFR4 beads. All control embryos showed normal
morphology, while the embryos grafted with soluble FGFR4 had smaller
telencephalic vesicles than did controls. (D) Consecutive transversal sections
of New Culture embryos, showing the expression of transcription factors in the
telencephalon. The broken circle indicates the position of the bead. Embryos
(n=10) grafted with control beads in the prospective ventral
telencephalon expressed NKX2.1+ cells in the ventral region,
Meis2+ cells in the intermediate region and
PAX6+ cells in the dorsal domain of the intermediate region and in
the dorsal region of the telencephalon. EMX1+ cells were located
exclusively in the dorsal region. Embryos (n=10) grafted with soluble
FGFR4 beads in the prospective ventral telencephalon lacked or showed a
severely reduced number of NKX2.1+ cells, in four out of 10 embryos
ventral cells ectopically expressed Meis2, PAX6 was still expressed
in the intermediate and dorsal region of the telencephalon, and the generation
of EMX1+ cells was blocked.
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Fig. 7. RA signalling is required and is sufficient for the specification of
intermediate cells. (A) Lateral views of stage 20-22 chick embryos generated
in New Culture from stage 10 embryos grafted with either control beads (upper
panel) or BMS453 beads (lower panel) into the prospective telencephalon. (B,D)
Consecutive transversal sections of New Culture embryos, showing the
expression of transcription factors in the telencephalon. The broken circle
indicates the position of the bead. (B) Embryos (n=10) grafted with
control beads in the prospective telencephalon (upper panel) expressed
Meis2+ cells in the intermediate region at mRNA level,
PAX6+ cells in the dorsal region and NKX2.1+ in the
ventral region of the telencephalon. Embryos (n=12) grafted with
BMS453 beads into the prospective telencephalon (lower panel) had an
asymmetric expression pattern of Meis2, being expressed only at one
lateral side of the developing telencephalon. EMX1+,
PAX6+ cells were located dorsally, while NKX2.1+ cells
were positioned in the ventral region of the telencephalon. (C) Lateral views
of stage 20-22 chick embryos generated in New Culture from stage 10 embryos
grafted with either control beads (upper panel) or RA beads (lower panel) into
the prospective telencephalon. The heads of the RA-treated embryos were normal
in size but the eyes were enlarged. (D) Embryos (n=10) grafted with
control beads in the prospective telencephalon (upper panel) did not generate
MEIS2+ cells, but generated PAX6+ and EMX1+
cells in the dorsal region and NKX2.1+ in the ventral region of the
telencephalon. Embryos (n=4) grafted with RA beads into the
prospective telencephalon (lower panel) generated a large number of
MEIS2+ cells in the dorsal and intermediate regions of the
telencephalon, a reduced number of PAX6+ cells, no EMX1+
cells and a normal number of NKX.2.1+ cells.
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© The Company of Biologists Ltd 2004
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) (n=25). Stage 14 intermediate
explants (n=25) grown alone generated MEIS2+ cells
(22±6%) and PAX6+ cells (23±4%), but no
NKX2.1+ or EMX1+ cells were detected. In the presence of
BMS453, the generation of MEIS2+ cells was blocked and the
generation of PAX6+ cells was markedly reduced (7±3%). A
small number of NKX2.1+ cells (5±3%) were generated, but no
EMX1+ cells were detected. Scale bar: 30 µm.
