PhD thesis

Chapter IV
Submitted manuscript
71
shows 73% -80% sequence similarity
with Cdx/caudal protein sequences
known from other metazoans and 61%
sequence similarity with the Cdx/Xlox
sequence of Nematostella vectensis
(Fig. 2). The TtrCdx segment cloned
by RACE PCR was 1037 base pairs
long and is deposited in GeneBank
under the accession number XXXX.
The transcribed region downstream of
the homeobox ends with a poly-A tail
and shows no similarity to other known
gene sequences.
Not and Cdx expression during
Terebratalia development
Cleavage in Terebratalia transversa
is radial. Within eight hours after
fertilization (hpf), the embryo develops
into a blastula. Gastrulation starts at 18
hpf. The spherical gastrula has a central
blastopore until approximately 26 hpf.
Thereafter, the gastrula elongates and
the blastopore narrows to a slit, moves
anteriorly, and closes at around 42 hpf.
At this stage, the elongated larva starts
to develop its characteristic three–
lobed body, consisting of an anterior
lobe, a mantle lobe, and a pedicle
lobe. At around 65 hpf, the larval setae
start to form and larval development is
complete by approximately 80-96 hpf.
Under our experimental conditions,
larvae settled between 120 and 240
hpf. The expression patterns of TtrNot
and TtrCdx are shown in Figs. 3–5.
Fertilized eggs and early cleavage
stages did not reveal the presence of
a TtrNot RNA transcript (Fig. 3A-C).
Expression of TtrNot starts laterally
on both sides of the blastopore in
the early gastrula stage at 18 hpf
(Fig. 3D). At 22 hpf, the expression
of TtrNot increases in the ectoderm
on both sides of the blastopore and
extends towards the animal pole of the
gastrula (Figs. 3E, F, 5A). These lateral
bands of TtrNot expressing cells fuse
in slightly older stages (i.e., at 24 hpf;
Fig. 3G). Shortly after that, at 26 hpf,
when the archenteron is enlarged, the
band of TtrNot expressing cells on the
animal pole narrows (Figs. 3H, 5B). At
28 hpf the blastopore starts to become
slit-like, the gastrula elongates, and
TtrNot expressing cells are present in
two fields lateral of the blastopore and
close to the future anterior pole of the
larva. The fields of TtrNot expressing
cells are interconnected by a narrow
band of TtrNot expressing cells on the
animal side (Figs. 3I-K, 5C). At around
36 hpf the blastopore starts to close,
the larva elongates further, and TtrNot
expressing cells are only detected
in the animal region of the gastrula,
which later forms the larval apical lobe
(Fig. 3L). Subsequently, the blastopore
closes completely and the larva
differentiates the three characteristic
body lobes. TtrNot is continuously
expressed in a ring of cells in the
apical lobe until the beginning of setae
formation. The area of the apical lobe
where TtrNot is expressed corresponds
to the region that bears the cilia used
for swimming of the larva (Figs. 3M,
N, 5D). Once the larval lobes are fully
established and setae formation has
started (i.e., at around 75 hpf), TtrNot
is no longer detectable (Figs. 3O, 5E).
Likewise, specimens that have settled
and started to metamorphose do not
show any TtrNot expression (Figs. 3P,
5F).
TtrCdx starts to be expressed in
the ectoderm of the gastrula at the
posterior pole of the blastopore (Figs.