First published online 16 September 2003
doi: 10.1242/dev.00750
A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field
Tatiana Hochgreb1,*,
Vania L. Linhares1,2,*,
Diego C. Menezes1,
Allysson C. Sampaio1,
Chao Y. I. Yan3,
Wellington V. Cardoso4,
Nadia Rosenthal5 and
José Xavier-Neto1,
1 Laboratório de Genética e Cardiologia Molecular InCor –
HC.FMUSP 05403-900 São Paulo-SP, Brazil
2 Laboratório de Cardiologia Celular e Molecular IBCCF-UFRJ Rio de
Janeiro-RJ, Brazil
3 Departamento de Histologia e Embriologia, ICB-USP, São Paulo-SP,
Brazil
4 Pulmonary Center – Boston University School of Medicine, Boston, MA,
USA
5 EMBL European Molecular Biology Laboratory Mouse Biology Programme,
Monterotondo-Scalo, Italy
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Fig. 1. A caudorostral wave expands RALDH2 anteriorly. (A) In situ hybridization
indicates that Raldh2 is expressed posterior to Hensen's node (*)
between HH4-6. At HH6-7 spots of RALDH2 blur the otherwise sharp
anteroposterior (AP) boundaries of expression in the lateral mesoderm (white
arrowheads). At HH7+ two faint arches of RALDH2 appear in the
anterior lateral mesoderm (black arrow). These arches intensify at
HH8–-HH8+ and progress anteriorly. At HH9-10
RALDH2 arches join at the midline below the anterior intestinal portal (AIP)
(star). (B) HH6. Strong Raldh2 expression spreads from Hensen's node
in a postero-lateral direction (black arrowhead), whereas faint expression
appears at the anterior border of the lateral mesoderm (white arrowhead). (C)
Grayscale image of B submitted to morphometry. Horizontal bars indicate the
limits of high and low Raldh2 expression and the interval between
them defines anterior expansion of RALDH2. (D) HH8+. Strong arches
of expression form in the anterior lateral mesoderm (black arrows). (E)
Grayscale image of D submitted to morphometry. Upper horizontal bar marks the
anterior limit of Raldh2 expression in the lateral mesoderm. Lower
horizontal bar marks the boundary between the last formed somite and the
unsegmented paraxial mesoderm. The interval between horizontal bars defines
anterior expansion of RALDH2. (F) Expansion of RALDH2 begins slowly between
HH6-7 coinciding with appearance of scattered spots of expression at the
anterior lateral mesoderm. Expansion reaches maximal rates of development
between HH7-8 when faint arches appear at the anterior lateral mesoderm.
Thereafter, it tapers off as RALDH2 arches join at midline below the AIP
between HH9-10. Data are presented as means±s.e.m. AE, anterior
expansion.
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Fig. 2. Evolutionary conservation of the caudorostral wave of RALDH2. RALDH2 in
situ hybridization of mouse embryos from early headfold (EHF) to somite
stages. Note the sharp anteroposterior (AP) edges of RALDH2 expression in the
EHF embryo. Anterior expansion of RALDH2 in the lateral mesoderm is underway
at the 1-somite stage, but a more robust expression of RALDH2 is observed in
embryos at the 2-somite stage. At the 5-somite stage RALDH2 expression
advanced considerably in anterior lateral mesoderm. Black arrows indicate
arches of Raldh2 expression in the lateral mesoderm.
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Fig. 3. Two phases of the relationship between RALDH2 and the cardiac field. (A)
HH7+. GATA4 in situ hybridization (left) was converted to grayscale
image (right) and submitted to morphometry. Anterior and posterior limits of
the cardiac field (chick Gata4 expression) were measured relative to
a horizontal bar transecting the anterior tip of Hensen's node. (B)
HH7+. RALDH2 in situ hybridization (left) was converted to
grayscale image (right) and submitted to morphometry. The anterior limit of
RALDH2 expression in the lateral mesoderm was measured relative to the
anterior tip of Hensen's node. Chick GATA4 and chick RALDH2 parameters above
or below Hensen's node were attributed positive or negative values,
respectively. (C) Two phases of cardiac anteroposterior (AP) signalling by RA.
Phase 1 is characterized by increasing proximity between RALDH2 and
prospective sino-atrial precursors in the posterior extremity of the
cardiogenic plate. Phase 2 is characterized by a progressive encircling of
sino-atrial precursors in the posterior half of the cardiogenic plate by a
field of RALDH2. Data are presented as means±s.e.m.
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Fig. 4. A caudorostral wave of RALDH2 conveys anteroposterior (AP) information to
the cardiac field. (A) HH6 chick RALDH2 in situ hybridization. The anterior
limit of chick Raldh2 expression lies near the anterior tip of
Hensen's node. (B) HH6 chick GATA4 in situ hybridization. The posterior limit
of chick GATA4 lies near the anterior tip of Hensen's node (star). (C) HH6
double chick RALDH2/chick GATA4 in situ hybridization. The domains of chick
Raldh2 (red) and chick Gata4 (blue) converged in the lateral
mesoderm at the level of Hensen's node contacting posterior cardiac precursors
with mesoderm producing RALDH2. (D) HH8 chick RALDH2 expression starts at the
lateral mesoderm from the level of the transition between the last formed
somite and the unsegmented paraxial mesoderm, almost up to the anterior
intestinal portal (AIP) (star). (E) HH8 chick GATA4 expression is in the
lateral mesoderm from the level of somite 2 almost up to the AIP. (F,G) Chick
Raldh2 and chick Gata4 expression in sections respectively
cut from embryos in D and E at the level indicated by the upper bar. (H,I)
Chick Raldh2 and chick Gata4 expression in sections
respectively cut from embryos in D and E at the level indicated by the lower
bar. Below the AIP chick Raldh2 is expressed in splanchnic and
somatic mesoderm (F). Chick Gata4 is expressed in splanchnic mesoderm
and endoderm (G). At the level of somite 2 chick Raldh2 is expressed
in mesoderm (H). Chick Gata4 is expressed in mesoderm (I). Isotopic
(35 S) in situ hybridization for RALDH2 (J) and GATA4 (K) in consecutive
sections indicate that these genes are co-expressed in the splanchnic
mesoderm. Ant Limit, anterior limit; e, endoderm; m, mesoderm; Post Limit,
posterior limit; SOM, somatic mesoderm; SPM, splanchnic mesoderm.
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Fig. 5. Two phases of RA signalling in the mouse cardiac field. A-E (left-side
views) and F-K (frontal views). A-E and G-I are mouse Raldh2 (orange)
and mouse Tbx5 (purple) double in situ hybridizations. (A) Late bud
(LB) stage. Cardiac precursors express mouse Tbx5 and occupy an
anterior position. Mouse Raldh2 is expressed in mesoderm posterior to
the node (*). Separation between cardiac precursors and RALDH2 is maximal. (B)
LB stage. Raldh2 expression expands in the anterior lateral mesoderm.
Mouse Tbx5 expression increases forming a stripe in the lateral
mesoderm oriented in a posterior-direction towards the advancing RALDH2
caudorostral wave. (C) Early headfold stage (EHF). The gap separating cardiac
precursors from RALDH2 is decreased. (D) EHF stage. mouse Raldh2
expression advances to contact the most posterior cardiac precursors. (E) Late
headfold stage (LHF). RALDH2 penetrates the cardiac field and overlaps
posterior cardiac precursors (white arrowhead). (F) LHF stage. Double mouse
Tbx-5 in situ hybridization/lacZ staining in RA-indicator embryos.
Mouse Raldh2 expression takes RA signalling to posterior cardiac
precursors. In this embryo RA signalling overlaps the posterior third of the
cardiac field (bracket). (G-I) Somite stages. Embryos display increasing
overlap between Raldh2 expression and cardiac precursors (black
arrows). (J,K) RALDH2 immunohistochemistry. Arches of RALDH2 expression joined
at the midline in sino-atrial tissue below the anterior intestinal portal
(AIP) (star) in embryos respectively displaying looped and unlooped
hearts.
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Fig. 6. Testing commitment to anteroposterior (AP) fates by reciprocal manipulation
of RA signalling with RA and BMS493, a RA pan-antagonist. All pictures were
taken with the same magnification. Control hearts display a central
ventricular chamber flanked by bilateral limbs formed by posterior precursors.
(A) BMS493 at 10–4 M at stages HH4-7 induced atrophy of the
cardiac inflow compartment and increased the ventricular chamber. Treatment at
stages beyond HH7 failed to affect cardiac morphology, indicating that
posterior precursors commit to their fates between HH7-8. (B) RA at
10–5 to 10–4 M at HH4-7 inhibited
ventricular development. RA treatment beyond HH7 failed to affect chamber
morphology, indicating that ventricular and conotruncal precursors commit to
their fates between stages HH7-8. (C) Amhc1 expression after BMS493
and RA. (D) Codes for dots outlining cardiac structures.
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Fig. 7. The cardiac fate map and RALDH2. (A,C) Grids were superimposed on bright
field/fluorescent overlays of HH7 and HH8 embryos injected with DiI in the
lateral mesoderm, respectively. (B,D) Bright field/fluorescent overlays of
embryos depicted in A and C at HH11+, respectively. (A) DiI
injected in the lateral mesoderm at the level of Hensen's node. (B)
HH11+. DiI injected in A was located in atrium and left sinus
venosus. (C) DiI injected at the anterior lateral mesoderm. (D)
HH11+. DiI injected in C was located in left and right ventricles
(white arrowheads). (E,F) Fate maps of embryos at stages HH7 and HH8
respectively superimposed on typical Raldh2 expression patterns. (E)
HH7. Chick Raldh2 expression predicts the location of prospective
sino-atrial precursors. (F) HH8. Anterior and posterior cardiac precursors
occupy distinct territories, but chick Raldh2 expression no longer
discriminates anterior from posterior cardiac precursors. (G,H) HH7 and HH8
fate maps data grouped as anteroposterior (AP) divisions. (I) The cardiac fate
map at stage HH8 was superimposed on a typical Gata4 expression
pattern.
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Fig. 8. RA inhibition, cardiac fate maps and Amhc1 expression. (A) RA
inhibition by BMS493 10–4 M changed the HH7 fate map.
Anterior precursors (ventricular, conotruncal and vitelline artery) are found
at posterior regions of the cardiac field which otherwise contained only
sino-atrial (posterior) precursors in the absence of BMS493 (compare with B).
(B) Normal HH7 fate map (same data as Fig.
7G). (C) HH7. Bright field/fluorescent overlay of a BMS493-treated
embryo injected at a posterior site in the cardiac field, which is normally
devoid of ventricular precursors. (D) HH11+. Bright
field/fluorescent overlay of the embryo in C. DiI is located in the ventricle.
(E) Scheme of unilateral, topic, BMS493 treatment at HH6. Three agar cylinders
containing BMS493 at 10–4 M (blue circles) were placed on the
endoderm overlying the left lateral mesoderm between Hensen's node and
headfold. (F) HH10. AMHC1 in situ hybridization of a BMS493-treated embryo.
White arrowhead indicates unilateral inhibition of Amhc1 expression
in the left lateral mesoderm.
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© The Company of Biologists Ltd 2003
