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Beating hearts can be induced under in vitro conditions when the dorsal blastopore lip (including the zone of Spemann organizer) is treated with Suramin. In contrast, untreated organizer forms dorsal mesodermal derivatives as notochord and somites. When those in vitro produced heart precursor tissues are transplanted ectopically in the posteriortrunk area of early larvae, secondary beating heart structures will be formed. Furthermore, the replacement of the heart primordium of the host embryo by hearttissue induced under in vitro conditions will result in the rescue of the heart anlage. This model could be a valuable tool for the study of the multi-step molecular mechanisms of heart structure induction under in vitro conditions and vasculogenesis after transplantation into the host embryo.
Fig. 1. In vitro induction of heart
structures and transplantation into
larvae. (A) Isolation of dorsal blastopore
lip. The area includes neuroectoderm
(NEU), non involuting mesoderm
(NIMZ), involuting mesoderm (IMZ) and
headendoderm (END) and underlying
mesoderm. (B) The explant was treated
with or without Suramin (150 mM, 4 h)
and cultured in Holtfreter-solution until
siblings reached stage 20 (rescue
experiments, see Fig. 2A) or tailbud
stage (st. 25, Nieuwkoop and Faber,
1956). (C) The slit in the posterior trunk
area of the larva (D) or the extirpation of
the heart anlage (E) were performed
with fine Spemann glass needles,
followed by insertion of the explant
into the anterior (F1, rescue experiment)
or posterior (F2, ectopic transplantation)
trunk area.
Fig. 2. Isolated dorsal blastopore lip treated with or without Suramin.
(A) Dorsal blastopore lip treated with or without Suramin was fixed in
HEMFA when sibling larvae had reached stage 20 (top of the panel). The
pan-endodermal gene marker endodermin can be detected in both series
(one asterisk= view from the side; two asterisks= view from the top. (B)
Whole-mount in situ hybridization of Suramin treated Spemann organizer
(courtesy, Tom Drysdale and Paul Krieg). The heart specific marker can be
identified in all cases. Note that also simultaneously cement gland can be
observed (compare with histology in D). (C) Untreated dorsal blastopore lip
including ectoderm (Spemann organizer) differentiates into notochord (no),
brain structures (b) with a rudimentary eye (ta= tapetum) and cement gland
(cg). (D) Dorsal blastopore lip treated with Suramin (150 mM, 4 h) has
differentiated into a heart tube and mesenthelium.
Fig. 3. Larvae after rescue of the heart anlage or larvae with additional
ectopic hearts. (A) The heartprimordium of this larva was removed at
stage 20 and was replaced by Suramin-treated blastopore lip (rescue
experiment). The larva shows a rhythmic beating heart (h) and an additional
cement gland (cg). (B) Control larva. The heart anlage of this larva was
removed at stage 20. No regeneration of the heart (zone at the white arrow)
took place in this hypertrophic larva with enlarged pronephric area (p). (C)
Same series as in (A), rescue experiment. The 12 day old larvae have a
rhythmic beating heart and are comparable to normal larvae. (D) Same
series as in (B). The 12 day old larvae without heart show severe hypertrophy
with enlarged pronephros (arrow). The heart anlage of these larvae was
removed at stage 20. No beating heart (heart- free zone, see asterisk) could
be identified. (E) Suramin-treated dorsal blastopore lip was implanted in the
posterior trunk area. The larvae (stage 47) showed rhythmic contractions
of the ectopic heart (h). (F) In another larva, also clearly rhythmic beating
heart structures could be observed in addition to the beating normal heart.
A second cement gland has differentiated together with the heart structure.
Fig. 2. Isolated dorsal blastopore lip treated with or without Suramin. o= otolith.
Fig. 4. Comparison of heart structures of the normal larva (stage 47)
with ectopic heart structures of the transplanted in vitro induced heart
structures. (A-D) Normal heart. (E-G) Ectopic heart. (A) Sagittal section of
a normal larva (stage 47). (B) Higher magnification of the section shown in
(A). (C) Transversal section of a stage 47 in the heart region (see arrow head
in (A). (D) Higher magnification of (C). (E) Larva (stage 47) with ectopic heart.
(F) Transversal section of larva shown in (E) in the heart region (arrow b). (G)
Higher magnification of a similar section, shown in (F), but more anterior (see
arrow a) cg, cement gland; b, brain; no, notochord; nh, normal heart; eh,
ectopic heart; ot, otic vesicles; gi, gill area; f, fin; h, heart; neu, neural tube;
so, somites; li, liver.
