54
Xenopus
nature of the inducers, the competence of the ectoderm, and
the timing of induction. The conversations about these questions would be clarifed when the molecules responsible for
neural induction and transformation were identif ed.
5.3. INSIGHT INTO SIGNALING PATHWAYS
CONTROLLING ANTERIOR-POSTERIOR
EMBRYONIC PATTERNING FROM
MOLECULAR BIOLOGICAL STUDIES
Experimental embryology studies relied on morphological
and histological features to assess the AP characteristics
of the neural structures. The analyses were therefore performed long after the initial AP patterning event occurred.
A high degree of expertise was required to distinguish structural differences in the neural and the associated tissues,
especially since the induced structures were often disorganized. To overcome these issues, tissues from different species were used, and inductive experiments were conducted
in explants and implants. These experimental limitations
restricted further precision and quantitative understanding
of the mechanisms controlling AP neural patterning.
The application of molecular biological techniques to
embryological studies from the 1980s greatly facilitated
investigation of developmental principles on all fronts,
including issues concerning AP specifcation. By then, Xenopus
laevis had become the most widely used amphibian model
(Gurdon and Hopwood, 2000). Xenopus not only possesses
all the advantages of the other amphibian species, such as
accessibility to microsurgery, but also the adult Xenopus
frogs are responsive to gonadotrophin hormone so that they
can be induced to spawn throughout the year. The number
of eggs can range from hundreds to thousands from a single
female in a day, and the rate of embryonic development is
rapid, with the embryos reaching the tadpole stage in three
to four days. Molecular studies were thus implemented
heavily in this species, and enormous progress was made on
the molecular control of embryonic patterning in a relatively
short time.
One important aspect that aided molecular analyses of
AP neural induction was the cloning of molecular markers
that not only revealed neural identity but also distinguished
anterior from posterior neural tissues. Gene expression that
specifcally marked cement gland, forebrain, midbrain,
hindbrain, and spinal cord made it possible to uncover events
involved in early AP neural patterning prior to tissue differentiation and formation of specifc structures (Blitz and Cho,
1995; Bradley et al., 1993; Brivanlou and Harland, 1989;
Papalopulu et al., 1991a ; Saha and Grainger, 1992; Sharpe
et al., 1987; Sharpe and Gurdon, 1990; Sive et al., 1989).
Using these markers, some of the classical experiments performed in other amphibian species were re-examined using
Xenopus. Examples include the discovery of regional differences of the organizer (dorsal lip) that had distinct ability to
induce different AP neural markers (Stewart and Gerhart,
1990; Vodicka and Gerhart, 1995; Zoltewicz and Gerhart,
1997), progressive conversion of anterior to more posterior
cell fates in the nascent neural plate underlaid by post-involuted mesoderm (Sive et al., 1989), and regional differences
in dorsal mesoderm in inducing AP neural markers at gastrula and neurula stages (Sharpe and Gurdon, 1990). These
position-specifc markers also allowed examination of different signaling pathways that control AP neural patterning.
Several pathways have been identifed that have the ability
to convert anterior to more posterior neural tissues, with
their antagonists expressed in the anterior region to promote
development of the anterior structures.
5.3.1. RETINOIC ACID SIGNALING
Retinoic acid (RA) was known for its teratogenic effects on
mammals and could induce limb defects and microcephaly
(Conlon, 1995). The impact of RA on head reduction was
particularly intriguing and enticed several groups to investigate its action on nervous system development. Using
exogenously supplied RA on whole embryos, early neural
explants, or conjugates of competent ectoderm with dorsal
mesoderm, it was demonstrated that RA reduced expression
of genes characteristic of anterior structures, such as cement
gland, olfactory pits, eyes, forebrain, and midbrain, and
increased expression or anteriorly shifted the expression of
posterior neural genes (Durston et al., 1989; Lloret-Vilaspasa
et al., 2010; Papalopulu et al., 1991b; Ruiz i Altaba and
Jessell, 1991b; Sive et al., 1990). The response to RA peaked
at gastrula and gradually diminished during early neurula
stages. RA not only affected general AP neural patterning
but also infuenced the differentiation of neurons at different
AP positions (Papalopulu and Kintner, 1996 ). Measurement
of endogenous RA in Xenopus embryos using HPLC or an
RA-responsive reporter construct indicated that RA was
present at gastrula and neurula stages when neural tissues
were patterned along the AP axis, and a posterior-to-anterior
concentration gradient of RA existed at early neurula stages
(Chen et al., 1994; Durston et al., 1989; Yelin et al., 2005).
RA did not seem to affect neural induction but could transform anterior to more posterior neural tissues. Mesodermal
tissues could be altered similarly to express more posterior
characters (Ruiz i Altaba and Jessell, 1991a). These results
supported the notion that RA might potentially function as
a posteriorizing factor in the “activation-transformation”
model proposed by Nieuwkoop.
To complement the gain-of-function experiments applying ectopic RA, loss-of-function studies were performed
using dominantly interfering receptor constructs for the RA
receptors, RAR or RXR, both of which act as RA-dependent
transcription activators. In addition to using explant experiments, dominant-negative (DN) receptors were often introduced into one side of the early Xenopus embryos so that
the expression of a battery of AP neural markers could be
compared with that on the control, uninjected side in the
same embryo. These studies confrmed that blocking RA
signaling directly impacted the level and/or the AP position of the region-specifc neural markers (Blumberg et al.,
Xenopus
nature of the inducers, the competence of the ectoderm, and
the timing of induction. The conversations about these questions would be clarifed when the molecules responsible for
neural induction and transformation were identif ed.
5.3. INSIGHT INTO SIGNALING PATHWAYS
CONTROLLING ANTERIOR-POSTERIOR
EMBRYONIC PATTERNING FROM
MOLECULAR BIOLOGICAL STUDIES
Experimental embryology studies relied on morphological
and histological features to assess the AP characteristics
of the neural structures. The analyses were therefore performed long after the initial AP patterning event occurred.
A high degree of expertise was required to distinguish structural differences in the neural and the associated tissues,
especially since the induced structures were often disorganized. To overcome these issues, tissues from different species were used, and inductive experiments were conducted
in explants and implants. These experimental limitations
restricted further precision and quantitative understanding
of the mechanisms controlling AP neural patterning.
The application of molecular biological techniques to
embryological studies from the 1980s greatly facilitated
investigation of developmental principles on all fronts,
including issues concerning AP specifcation. By then, Xenopus
laevis had become the most widely used amphibian model
(Gurdon and Hopwood, 2000). Xenopus not only possesses
all the advantages of the other amphibian species, such as
accessibility to microsurgery, but also the adult Xenopus
frogs are responsive to gonadotrophin hormone so that they
can be induced to spawn throughout the year. The number
of eggs can range from hundreds to thousands from a single
female in a day, and the rate of embryonic development is
rapid, with the embryos reaching the tadpole stage in three
to four days. Molecular studies were thus implemented
heavily in this species, and enormous progress was made on
the molecular control of embryonic patterning in a relatively
short time.
One important aspect that aided molecular analyses of
AP neural induction was the cloning of molecular markers
that not only revealed neural identity but also distinguished
anterior from posterior neural tissues. Gene expression that
specifcally marked cement gland, forebrain, midbrain,
hindbrain, and spinal cord made it possible to uncover events
involved in early AP neural patterning prior to tissue differentiation and formation of specifc structures (Blitz and Cho,
1995; Bradley et al., 1993; Brivanlou and Harland, 1989;
Papalopulu et al., 1991a ; Saha and Grainger, 1992; Sharpe
et al., 1987; Sharpe and Gurdon, 1990; Sive et al., 1989).
Using these markers, some of the classical experiments performed in other amphibian species were re-examined using
Xenopus. Examples include the discovery of regional differences of the organizer (dorsal lip) that had distinct ability to
induce different AP neural markers (Stewart and Gerhart,
1990; Vodicka and Gerhart, 1995; Zoltewicz and Gerhart,
1997), progressive conversion of anterior to more posterior
cell fates in the nascent neural plate underlaid by post-involuted mesoderm (Sive et al., 1989), and regional differences
in dorsal mesoderm in inducing AP neural markers at gastrula and neurula stages (Sharpe and Gurdon, 1990). These
position-specifc markers also allowed examination of different signaling pathways that control AP neural patterning.
Several pathways have been identifed that have the ability
to convert anterior to more posterior neural tissues, with
their antagonists expressed in the anterior region to promote
development of the anterior structures.
5.3.1. RETINOIC ACID SIGNALING
Retinoic acid (RA) was known for its teratogenic effects on
mammals and could induce limb defects and microcephaly
(Conlon, 1995). The impact of RA on head reduction was
particularly intriguing and enticed several groups to investigate its action on nervous system development. Using
exogenously supplied RA on whole embryos, early neural
explants, or conjugates of competent ectoderm with dorsal
mesoderm, it was demonstrated that RA reduced expression
of genes characteristic of anterior structures, such as cement
gland, olfactory pits, eyes, forebrain, and midbrain, and
increased expression or anteriorly shifted the expression of
posterior neural genes (Durston et al., 1989; Lloret-Vilaspasa
et al., 2010; Papalopulu et al., 1991b; Ruiz i Altaba and
Jessell, 1991b; Sive et al., 1990). The response to RA peaked
at gastrula and gradually diminished during early neurula
stages. RA not only affected general AP neural patterning
but also infuenced the differentiation of neurons at different
AP positions (Papalopulu and Kintner, 1996 ). Measurement
of endogenous RA in Xenopus embryos using HPLC or an
RA-responsive reporter construct indicated that RA was
present at gastrula and neurula stages when neural tissues
were patterned along the AP axis, and a posterior-to-anterior
concentration gradient of RA existed at early neurula stages
(Chen et al., 1994; Durston et al., 1989; Yelin et al., 2005).
RA did not seem to affect neural induction but could transform anterior to more posterior neural tissues. Mesodermal
tissues could be altered similarly to express more posterior
characters (Ruiz i Altaba and Jessell, 1991a). These results
supported the notion that RA might potentially function as
a posteriorizing factor in the “activation-transformation”
model proposed by Nieuwkoop.
To complement the gain-of-function experiments applying ectopic RA, loss-of-function studies were performed
using dominantly interfering receptor constructs for the RA
receptors, RAR or RXR, both of which act as RA-dependent
transcription activators. In addition to using explant experiments, dominant-negative (DN) receptors were often introduced into one side of the early Xenopus embryos so that
the expression of a battery of AP neural markers could be
compared with that on the control, uninjected side in the
same embryo. These studies confrmed that blocking RA
signaling directly impacted the level and/or the AP position of the region-specifc neural markers (Blumberg et al.,