First published online September 2, 2003
doi: 10.1242/10.1242/dev.00704
The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans
Tatjana Piotrowski1,2,*,
,
Dae-gwon Ahn3,*,
Thomas F. Schilling4,
Sreelaja Nair4,
Ilya Ruvinsky5,
Robert Geisler6,
Gerd-Jörg Rauch6,
Pascal Haffter6,
Leonard I. Zon7,
Yi Zhou7,
Helen Foott7,
Igor B. Dawid1 and
Robert K. Ho3
1 National Institutes of Health, NICHD, LMG, Bldg. 6B, 9000 Rockville Pike,
Bethesda, MD 20892, USA
2 University of Utah, Department of Neurobiology and Anatomy, 401 MREB, Salt
Lake City, UT 84132, USA
3 University of Chicago, Department of Organismal Biology and Anatomy, Chicago,
IL 60637, USA
4 University of California, Irvine, Department of Developmental and Cell
Biology, Irvine, CA 92697, USA
5 Princeton University, Department of Molecular Biology, Princeton, NJ 08544,
USA
6 Max-Planck-Institute for Developmental Biology, Spemannstr.35, 72076,
Tübingen, Germany
7 Howard Hughes Medical Institute, Division of Hematology/Oncology, Children's
Hospital, Boston, MA 02115, USA
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Fig. 1. Craniofacial and aortic arch phenotype in vgo (tu285
allele, AB* background). (A) Alcian Blue cartilage preparation of a 5 dpf
wild-type larva, ventral view. (B) Schematic drawing of the cartilages in A
(blue, pharyngeal skeleton; red, neurocranium). (C) Dissected pharyngeal
cartilages of a 5 dpf vgo larva. The cartilages in the mandibular (m,
pq) and hyoid (ch, hm) arches are drastically reduced and the pharyngeal
arches 3-7 (cb1-5) are completely absent. (D) Ventral view of the dissected
neurocranium in a vgo larva. The mesodermally derived parachordalia
(pc) are malformed and the anterior pole of the notochord (nc) extends almost
to the point where the trabeculae (t) fuse. cb1-5, ceratobranchial cartilages
1-5; ch, ceratohyal cartilage; hm, hyomandibula; m, Meckel's cartilage of
mandibular arch; nc, notochord; oa, occipital arch; pq, palatoquadrate; pc,
parachordalia; t, trabeculae. (E-G) Aortic arches of 2.5 dpf larvae visualized
with fluorescent microbeads. (E) Wild-type larva. At this stage, five aortic
arches are visible (arrows). (F,G) vgo mutants showing variable
reductions of the aortic arches. (F) Only one interrupted aortic arch is
present (arrow). (G) Only three aortic arches formed but are much smaller in
diameter than wild-type aortic arches (arrows).
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Fig. 2. Linkage between tbx1 and vgotm208. (A) Amino
acid sequence alignment of zebrafish tbx1 and other T-box genes.
Residues identical to zebrafish tbx1 are in gray. Only T-domain
sequences are shown. (B) Physical and genetic maps of linkage group 5 (LG5)
showing map positions of tbx1 and vgotm208. (C)
Sequence analysis of tbx1 in wild-type (wt),
vgotm208 and vgotu285 mutants. Box
diagrams represent conceptual products of translation of tbx1 cDNAs
from wild type, vgotm208 and vgotu285.
T-box region is shown by hatched boxes. Note that the A to T transition in
vgotm208 eliminates the AlwNI recognition
sequence (CAGNNNCTG).
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Fig. 3. Expression of vgo/tbx1 during zebrafish embryogenesis. (A) Animal
pole view. (B-E,G) Dorsal views and (F,H-L) lateral views. (A) In a shield
stage embryo (6 hpf), expression is confined to the hypoblast cells of the
shield (arrow) and flanking region. (B) Six-somite stage (12 hpf). Expressing
cells are now organized into the cranial paraxial mesoderm (thin lateral
stripe, marked by an arrowhead) and a more medial group of mesenchyme cells
consisting of prospective pharyngeal endodermal cells and cells of parachordal
mesoderm (arrow). (C) Ten-somite stage (14 hpf). New expression commences
within the otic placode (arrow). (D) Twenty-somite stage (19 hpf).
vgo/tbx1-expressing cells are now organized into the primordia of the
pharyngeal arches (p1-p7). OV, otic vesicle. (E) 27 hpf. vgo/tbx1
expression within the mesodermal core and endodermal epithelia of individual
arches. Anterior towards the left. (F) Lateral view of vgo/tbx1
expression in a 30 hpf embryo. In the anterior arches, the mesodermal core is
in focus. In the posterior arches, the endodermal pouches are visible. (G)
Horizontal section through the pharyngeal region of a 36 hpf embryo. The
endodermal pouches (e) and mesodermal cores (m) of the arches are clearly
vgo/tbx1 positive. (H,I) tbx1 is expressed in pharyngeal
arch muscles. (H) At 48 hpf, vgo/tbx1 is expressed in most of the
pharyngeal arch muscles that also express myod (I). (J-L) High
magnification views of vgo/tbx1 expression within the otic vesicle.
(J) 24 hpf. (K) 48 hpf. (L) 72 hpf. Expression is initially found throughout
the otic vesicle except for the anteroventral corner (arrow in J). Strong
expression is maintained in the developing cristae (asterisks in K,L) and
semicircular canals (arrows in K,L). ah, adductor hyoideus; am, adductor
mandibulae; ih, interhyoideus; hh, hyohyoideus; ima, intermandibularis
anterior; imp, intermandibularis posterior; sh, sternohyoideus; tv,
transversus ventralis.
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Fig. 4. Cell transplantation experiments between wild-type and vgo/tbx1
larvae (tm208 allele). (A,B) Ventral views of 3 dpf vgo/tbx1
larvae stained with Alcian Blue, showing the rescue of cartilages (arrows) in
the vicinity of transplanted Tar*-expressing wild-type cells (black
staining). (A) p6 is rescued only on the left side of the embryo and is absent
on the contralateral side. (B) m and ch cartilages are rescued on the left
side, judged by their shape and cell number compared with the contralateral
side. (C) Lateral view of left ear of a 3 dpf vgo/tbx1 larva.
Transplanted wild-type cells (in red fluorescence) contributed to the
developing semicircular canals (arrows). (D,E) The same larva as in C at 5
dpf. Wild-type cells were able to partially restore ear size and the presence
of semicircular canals (arrow) in the left ear (E), whereas the right ear,
which did not receive any wild-type cells, remained small (D) and without
semicircular canals. cb1-5, ceratobranchial cartilages 1-5; ch, ceratohyal
cartilage; hm, hyomandibula; m, Meckel's cartilage of mandibular arch; nc,
notochord; oa, occipital arch; pq, palatoquadrate; pc, parachordalia; t,
trabeculae.
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Fig. 5. Expression of vgo/tbx1 pathway genes in mutants. Ventral views
with anterior towards the left in all panels. (A,B) Expression of
edn1 in wild-type (A) and vgo/tbx1 (B) 27 hpf embryos. (C,D)
Expression of tbx1 in 25 hpf wild-type (C) and vgo/tbx1 (D)
embryos. In vgo/tbx1 embryos, tbx1 mRNA is still present in
cells ventral to the otic vesicle (Fig. 5D, arrows). At this stage, the otic
vesicle in vgo embryos no longer express tbx1. (E,F)
edn1 expression in 25 hpf wild-type (E) and vgo/tbx1 (F)
embryos. In vgo/tbx1 embryos, edn1 expression is very much
reduced in cells immediately anterior to the otic vesicle. The downregulation
of edn1 in the arches is not due to the absence of cells, as
tbx1-expressing cells are present (D). (G,H) Expression of
hand2 in 34 hpf wild-type (G) and vgo/tbx1 (H) embryos.
(I,J) Expression of crestin in 20-somite wild-type (I) and
vgo/tbx1 (J) embryos. Absence of hand2 expression in the
posterior arches of vgo/tbx1 embryos is not caused by the absence of
neural crest cells, as revealed by the pan-neural crest marker
crestin in vgo/tbx1 embryos (J). Arrows indicate neural
crest cells ventral and posterior to the otic vesicle.
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Fig. 6. Expression analysis of tbx1, edn1 and fgf8 in mutants.
(A,B) Expression of vgo/tbx1 in 29 hpf suc (A) and 28 hpf
han (B) embryos. (C) Expression of edn1 in han
embryos. (D) fgf8 expression in the mandibular arch of
vgo/tbx1 (30 hpf). (E,F) vgo/tbx1 expression in 36 hpf
wild-type (E) and ace (F) embryos.
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Fig. 7. Rescue of edn1 expression by transplanted endodermal cells in
vgo/tbx1 embryos. Transplanted cells in brown (labeled with biotin)
and edn1 expression in blue. (A) Expression of edn1 in a 27
hpf wild-type control embryo. (B) vgo/tbx1 host embryo in which
transplanted wild-type cells induced upregulation of edn1 (arrows).
(C) vgo/tbx1 host embryo, in which Tar*-injected endodermal cells
contributed to the pharynx on only one side. On the control side,
edn1 expression is weak and disorganized. The side that received
endodermal cells (brown cells, arrows) shows much stronger expression of
edn1, which includes cells located several cell diameters away from
the transplanted cells (arrowheads point at pigment cells). Flat mounts in
dorsal views with anterior towards the left in all panels.
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Fig. 8. Model of the genetic pathway regulating development of the anterior and
posterior pharyngeal arches in zebrafish. (A) edn1 expression in 27
hpf wild-type embryo and (B) schematic drawing of the tbx1 pathway in
an arch primordium (dark blue, pharyngeal arch epithelium and mesodermal core;
light blue, neural crest cells). Outlined in black is the hyoid arch,
exemplifying the distribution of edn1- and tbx1-positive
cells within a developing arch. (C) Genetic pathway regulating development of
the anterior and posterior arches and zebrafish mutants analyzed in this study
with the expression of tbx1, edn1 and hand2 in these
mutants. Dash indicates `no data'. Mandibular (p1) and hyoid (p2) arches:
tbx1 regulates edn1 within the arch epithelium and the
mesodermal core. Within these arches, edn1 is also possibly regulated
independently by another gene, probably an Fgf. edn1 in turn
regulates the expression of hand2 in the neural crest-derived cells
surrounding the mesodermal core of the arch. In posterior arches (p3-7),
tbx1 is likely to be the major regulator of edn1. Downstream
of edn1, hand2 expression is likely to be controlled by
edn1, as well as by tbx1 via an as yet unknown signaling
molecule (`?').
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© The Company of Biologists Ltd 2003
