Download Implications on the Role of Cell-Cell and Cell

AMER. ZOOL., 37:195-207 (1997)
Gene Regulation by Thyroid Hormone During Amphibian Metamorphosis:
Implications on the Role of Cell-Cell and Cell-Extracellular
Matrix Interactions1
MELISSA A. STOLOW*, ATSUKO IsmzuYA-OKAf, YUAN SU*, AND YUN-BO SHI* 2
* Laboratory of Molecular Embryology, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Maryland 20892
^Department of Anatomy, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu,
Tochigi 321-02, Japan
SYNOPSIS. Amphibian metamorphosis is the developmental process initiated
by thyroid hormone which transforms a tadpole into a frog. This transformation requires extensive remodeling of almost every tissue in the animal.
One of the more well-studied tadpole tissues that undergoes remodeling is
the small intestine. This tissue requires a shortening in length as well as internal anatomical restructuring to function in the adult frog. Briefly, the tadpole epithelial cells undergo programmed cell death (or apoptosis) and are
replaced by a layer of newly formed adult epithelium. About 20 thyroid
hormone-regulated genes participating in this intestinal remodeling have been
identified. These genes can be divided into several groups based on the proposed functions of their products. One of these groups contains several secreted and/or signaling molecules. Most prominent among these are the Xenopus homologs of the hedgehog and stromelysin-3 genes. Based on the expression profiles and cellular localization, hedgehog appears to be involved
in adult epithelial morphogenesis. Stromelysin-3 may participate in basal
lamina modification which is potentially involved in the apoptosis of the larval epithelium and development of the adult epithelium. Here we will review
in detail the potential roles for these secreted factors as well as the proposed
molecular mechanisms responsible for their physiological functions. Furthermore, we will examine the effect of these proteins on the extracellular environment and how this impacts upon cellular processes involved in intestinal
remodeling.
INTRODUCTION
altered to accommodate the smaller size of
Amphibian metamorphosis is the intricate t h e post-metamorphic frog as well as its
developmental process by which a larval n e w dietary regime (i.e., tadpoles are hertadpole is transformed into a frog (Gilbert bivores, adult frogs are carnivores) (Yoand Frieden, 1981; Gilbert et al., 1996). shizato, 1989; Shi and Ishizuya-Oka, 1996).
intestinal changes are of noticeable
During this process, many morphological These
and biochemical changes occur in the tad- interest because of the multitude of cellular
pole to ensure that almost every tissue is processes required for this transition,
^ t h e tadpole, the intestine is a long
modified for life as a frog. These changes
include among others the loss of the tail and tube-like structure composed of a single
the development of fore- and hind-limbs. In l a y e r o f epithelial cells with a single epiaddition, a major adaptation occurs in the thelial fold, called the typhlosole, where
intestine of the animal. Both the length and mesenchymal tissue is abundant (Fig. 1)
internal structure of the tadpole intestine are (McAvoy and Dixon, 1977; Marshall and
Dixon, 1978; Ishizuya-Oka and Shimozawa, 1987a). During metamorphosis, sevFrom the Symposium Amphibian Metamorphosis: e r a j dramatic changes OCCUr within this Or-
i^T^rsST^^sT^TZ g*n. Fust, the mesenchymal component of
cember 1995, at Washington, D.c.
2
To whom correspondence should be addressed.
the tissue begins to proliferate extensively.
This is SOOn followed by the cellular death
195
196
STAGE
M. A. STOLOW ET AL.
55
FIG. 1. Structural changes in the intestine during amphibian metamorphosis. Schematic cross sections of the
frog intestine are shown at different development stages according to Nieuwkoop and Faber (1956); McAvoy
and Dixon, (1977); and Ishizuya-Oka and Shimozawa, (1987a). During premetamorphosis (stage 55), the larval
tadpole intestine consists of an epithelial layer with a single fold called the typhlosole. As metamorphosis proceeds to the climax at stages 60-63, the larval epithelium undergoes apoptosis (open circles) and is replaced by
proliferating adult epithelium (filled circles). Towards the end of stage 62, apoptotic cells are localized in the
tips of newly forming intestinal folds (not shown), although their number is small. By the end of metamorphosis
(stage 66), the frog intestine has many epithelial folds (E) along with well developed connective tissue (C) and
muscle (M). Shown at the top are the relative levels of the thyroid hormone T3 (3, 5, 3'-triiodothyronine) in the
plasma (Leloup and Buscaglia, 1977).
of the larval epithelium. Concurrently,
groups or islets of proliferating cells, whose
origin is yet unknown, begin to form. These
islets of cells are the precursors of the adult
frog epithelium which will eventually replace all of the larval epithelial cells. As the
transition proceeds, these adult epithelial
cells form a uniform layer which begins to
migrate into folds. By the end of metamorphosis, these folds have increased in height
and number to produce the final morphological structure of the adult intestine, a
structure which is quite similar to that in
higher vertebrates (Shi and Ishizuya-Oka,
1996).
All of these anatomical changes occur in
the intestine because of the regulation of
several cellular processes. These include
programmed cell death (apoptosis) (Wyllie
et al., 1980), cell proliferation, and cell differentiation. Apoptosis occurs in the larval
epithelium so that it can eventually be replaced by the adult frog epithelium. The
connective tissue undergoes extensive cellular proliferation which is necessary for
creating the adult epithelial folds. Thickening of the outer muscle layer also occurs via
cell proliferation (Ishizuya-Oka and Shimozawa, 1987ft, 1992ft; Yoshizato, 1989).
Finally, after the new adult epithelial cells
have proliferated to the appropriate cell
number, they must become fully differentiated to produce the functional epithelial cell
layer. Some epithelial cells, however, remain undifferentiated at the base of the
folds. This population of cells serves as the
GENE REGULATION DURING METAMORPHOSIS
precursors or stem cells for the epithelial
cell layer (McAvoy and Dixon, 1977;
Ishizuya-Oka and Shimozawa, 1987a).
The summation of these cellular events
most likely occurs by two general mechanisms. The first mechanism involves cell
autonomous events. These events occur intracellularly and do not directly impact
neighboring cells in the tissue. An example
of this would be the upregulation of transcription factors or metabolic enzymes required to maintain or alter the state of the
cell. In contrast, the second means of regulation involves extracellular events that occur by a non-cell autonomous mechanism.
These events would include both cell-cell
and cell-extracellular matrix (ECM) interactions. Cellular factors participating in these
types of cell communication would be secreted or cell surface molecules. Cells producing such signaling molecules would directly affect nearby cells and/or their extracellular environment.
Both of these mechanisms are important
and necessary for the life of the cell. However, in this article we will focus on the noncell autonomous events, cell-cell and cellECM interactions, and discuss their impact
on intestinal morphogenesis in Xenopus
laevis during metamorphosis. The sum of
these extracellular events will most likely
play a key role in establishing the final
structure of the adult frog intestine.
EXTRACELLULAR MATRIX
Extracellular forms of communication
are of extreme importance because not
only do they influence the local cellular
environment but they also impact upon
the three-dimensional structure of the tissue and ultimately the entire organism.
Without these types of extracellular interactions, the position and identity of each
individual cell would be adversely affected. Communication with the extracellular environment helps maintain the status
and function of each cell. A critical player
in this communication is the extracellular
matrix (Hay, 1991). Moreover, we believe
that the ECM will be a driving force in remodeling of the frog intestine during metamorphosis. The multitude of cell changes
and movements that occur in this transition
197
will undoubtedly depend upon the influence of interactions between the epithelial
and mesenchymal cells of the intestine with
the ECM.
The extracellular matrix is composed of
many proteins which form a complex networked structure that lies beneath epithelia
and surrounds connective tissue cells (Hay,
1991). In the intestine, the epithelium is
separated from the mesenchyme by a special ECM, the basal lamina, whose components include laminin, entactin, type IV collagen and proteoglycans. The ECM serves
as a structural support medium for the cells
it surrounds but clearly this is not its only
biological function. The interaction between
the cell and the ECM is a dynamic one
which influences cell shape, cell metabolism and ultimately the fate of the cell. Several examples of cell behavior affected by
the ECM are cell-cell and cell-matrix adhesion, cell movement, cell proliferation, and
apoptosis (Hay, 1991; Ruoslahti and Reed,
1994). These behaviors have direct relevance not only to the maintenance of a
cell's status but also to developmental processes and tissue remodeling. Notably, all of
these cellular events occur in the intestine
as it undergoes its dramatic morphological
changes during metamorphosis. Therefore,
it is reasonable to predict that the ECM will
be involved in this particular developmental event.
In addition to the functions described
above, the ECM plays a vital role in promoting the transfer of information between
cell types. For example, the development
of the adult epithelium in the frog intestine
requires the presence of the larval connective tissue (Ishizuya-Oka and Shimozawa,
1992a). Without this mesenchyme, the only
observed morphological change in whole
intestine cultures treated with thyroid hormone (TH) is apoptosis of the primary epithelial cells. The connective tissue is absolutely required for the proliferation and differentiation of the adult epithelial cell layer.
In addition, during natural intestinal metamorphosis, extensive cell-cell contacts are
also present between the epithelium and
the mesenchyme. The cell-cell contacts
probably occur because of modifications
made in the basal lamina separating these
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M. A. STOLOW ET AL.
two cell layers (Ishizuya-Oka and Shimozawa, 19876).
At the molecular level, the interactions
between cells and the ECM can result in
transcriptional regulation of cellular genes
(Jones et al., 1993; Martins-Green and Bissell, 1995). The most prominent example of
this type of regulation was demonstrated by
transfecting a mouse mammary epithelial
cell line with a pJ-casein-CAT fusion gene.
It was shown that the basal lamina which
separates the connective tissue from the epithelium, brings about maximal casein protein expression by causing DNA to transcribe high levels of casein mRNA
(Schmidhauser et al., 1990). This suggests
that the ECM plays an active role in transcriptional events that could potentially alter the metabolic or functional state of the
cell.
GENE REGULATION DURING
METAMORPHOSIS
The transcription of genetic material is a
major biochemical process that is regulated
in order to affect the biological state of the
cell. The activation or repression of subsets
of genes initiates a cascade of events which
ultimately leads to changes in the cell
and/or its surrounding environment. Many
biological events are controlled by this type
of regulation including amphibian metamorphosis (Shi, 1996).
Metamorphosis is initiated by a single
molecular agent, thyroid hormone (TH)
(Gilbert and Frieden, 1981; Gilbert et al.,
1996). The endogenous levels of thyroid
hormone begin to rise around stage 54 during X. laevis development, the period
known as pre-metamorphosis. This profile
of hormone levels correlates exactly with
the timing of the metamorphic transition
(Fig. 1, Leloup and Buscaglia, 1977). The
highest levels of TH occur precisely at the
same stage in development during which
the majority of morphological changes are
taking place in the animal, a period known
as the climax of metamorphosis. This correlation is more than coincidence; it actually represents a cause-and-effect relationship.
Thyroid hormone exerts its effects during
this period by binding to thyroid hormone
receptors (TRs) (Yaoita and Brown, 1990;
Kawahara et al., 1991; Helbing et al.,
1992). TRs are members of the superfamily of steroid hormone receptors. This family of receptors, including the TRs, function
as transcription factors by regulating the
messenger RNA levels of specific genes.
TRs specifically influence gene expression
by binding to the thyroid hormone response
elements (TREs) present in the promoter regions of TH responsive genes (Evans, 1988;
Tsai and O'Malley, 1994). In vivo, this receptor binding occurs most likely as a heterodimeric complex composed of TR and a
member of the 9-cis retinoic acid receptor
family (Yen and Chin, 1994; Tsai and
O'Malley, 1994).
Many of the genes regulated by thyroid
hormone at the transcriptional level have
now been identified (Shi, 1996). This was
achieved by taking advantage of the observation that it is possible to precociously induce metamorphosis in young tadpoles by
adding TH to their rearing water. Differential subtractive hybridization methodologies
have been employed to isolate sets of genes
that are transcriptionally regulated in premetamorphic tadpoles treated with exogenous TH (Wang and Brown, 1991). These
targets of TH action have been isolated from
several tadpole tissues including limb, tail
and intestine (Buckbinder and Brown,
1992; Wang and Brown, 1993; Shi and
Brown, 1993). Genes whose mRNA levels
are altered within the first 24 hr of TH treatment are classified as early response genes.
Late response genes are defined as those
genes whose mRNA levels change after 1
day of TH treatment. Here, we will only describe the results obtained using intestine.
The search for transcriptional targets of
thyroid hormone in the intestine yielded
about 20 unique sequences, all of which
were identified as early response genes
(Shi and Brown, 1993). Within this set, all
but one of the genes were upregulated at
the mRNA level. The one down-regulated
gene has no homology to any other published sequences; therefore, its function is
unknown. Of the remaining sequences,
many have been identified as homologs of
previously cloned genes. These genes can
199
GENE REGULATION DURING METAMORPHOSIS
TABLE 1. Thyroid hormone response genes in the intestine.
Group function
Gene
Reference
Transcription
NF-1"
Zinc finger TFb
bZIPTF*
Cellular metabolism
Nonhepatic arginase
Na + /PO~ 4 cotransporter
Transmembrane protein
Hedgehog
Stromelysin-3
Signaling
Puzianowski-Kuznicka and Shi (1996)
Wang and Brown (1993)
Wang and Brown (1993)
Shi and Brown (1993)
Wang and Brown (1993)
Shi and Brown (1993)
Patterton and Shi (1994)
Shi (1996)
Shi (1996)
Stolow and Shi (1995)
Patterton et al (1995)
"Nuclear factor-1.
b
Zinc finger containing transcription factor.
c
Basic leucine-zipper motif containing transcription factor.
d
Thyroid Hormone Receptor (J.
be divided into three groups based on their
proposed functions (Table 1). The first
group contains several well-characterized
transcription factors. These are nuclear
factor-1 (Puzianowska-Kuznicki and Shi,
1996), zinc finger containing transcription
factor (Wang and Brown, 1993), basic
leucine-zipper motif containing transcription factor and TR@ (Wang and Brown,
1993; Shi and Brown, 1993). These factors
are predicted to regulate transcription of
other downstream genes during the metamorphic transition. The second group is involved in cellular metabolism. Members of
this group include a sodium-phosphate cotransporter (Shi, 1996) and a non-hepatic
arginase (Patterton and Shi, 1994). These
proteins may function to transport phosphates and participate in proline biosynthesis from arginine, respectively.
The third and final group is the class of
secreted and/or signaling molecules. Within
this group are proteins that link the cell to
its extra-cellular environment. One of these
is an unidentified transmembrane protein
which could potentially function as a receptor in intercellular communication (Shi,
1996). The other two members of this group
are Xenopus homologs of the mammalian
hedgehog (Stolow and Shi, 1995) and
stromelysin-3 genes (Patterton et al., 1995).
These genes encode for secreted molecules
and are thus predicted to participate in cellcell and/or cell-ECM interactions. Because
these interactions are the primary focus of
this review, the potential biological function
of each of these proteins in intestinal
remodeling will be discussed in the following sections.
HEDGEHOG SIGNALING IN THE INTESTINE
The hedgehog family of genes contains
many members which have been isolated
from a variety of organisms including
mouse, chicken, zebrafish, and the fruit
fly, Drosophila melanogaster (Mohler and
Vani, 1992; Echelard et al., 1993; Krauss et
al., 1993; Riddle et al., 1993; Tashiro et al.,
1993). The products of these genes have
been shown to be secreted molecules which
play a role in establishing cell position and
identity (Ingham, 1994). In flies, where the
gene was originally cloned, it has been demonstrated that hedgehog is required for
specification of the parasegment boundaries
in the early developing embryo. In addition,
hedgehog is necessary for patterning of the
adult eye, wing and leg (Perrimon, 1994).
Thus, hedgehog has roles both early and late
in development.
The vertebrate homologs of the fly hedgehog gene also appear to function in patterning and cell fate specification. The most
well studied of these vertebrate genes is the
member sonic hedgehog. Sonic hedgehog
has been shown to be important in establishing polarity in the central nervous system and the developing limb as well as influencing differentiation of the somites
(Perrimon, 1995; Bumcrot and McMahon,
1996). During development, all of these organ systems require cell-cell communica-
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M. A. STOLOW ET AL.
B
STAGE
54 58 62 66 F
f
STAGE
54 58 62 66 F
t
FIG. 2. Developmental regulation of thyroid hormone response genes. Intestinal RNA was isolated from the
stages indicated and analyzed by RNA blot hybridization. A. Xenopus hedgehog B. Xenopus stromelysin-3. Ribosomal markers, 28S and 18S, are indicated on the left.
tion to establish patterns within the tissues.
Hedgehog is the proposed signaling molecule responsible for this communication.
This type of cellular signaling could potentially exist in other tissues as well. In this
regard, we and others have recently cloned
the Xenopus homolog of the sonic class of
vertebrate hedgehog genes (Stolow and Shi,
1995; Ekker et al., 1995).
We originally identified Xenopus sonic
hedgehog (Xhh) as a thyroid hormone responsive gene in the intestine during metamorphosis (Shi and Brown, 1993; Stolow
and Shi, 1995). By Northern analysis, Xhh
mRNA was upregulated at the climax of
metamorphosis (stage 62) (Fig. 2A), the period of this transition when most of the morphogenetic changes are occurring (Fig. 1).
Xhh mRNA was also detected before and
after the peak of metamorphosis but at
lower expression levels. This expression
profile was also reproduced when premetamorphic tadpoles were treated with TH to
induce precocious metamorphosis. That is,
the Xhh mRNA was first up-regulated and
then down-regulated in the intestine during
the continuous TH treatment (Stolow and
Shi, 1995).
The spatial and temporal localization of
the Xhh protein was determined by performing immunohistochemistry with Xhh
antibodies on the intestine at different
stages of the metamorphic period (Stolow
and Shi, unpublished results). The temporal
regulation of the protein mimics that of its
mRNA. At the peak of metamorphosis,
stage 62, the epithelial cells surrounding the
lumen of the intestine appeared to contain
the protein. These cells appear to be the proliferating islets of secondary adult epithelium. Furthermore, Xhh seems to be expressed only in the epithelial cells.
These results suggest that in the intestine,
Xenopus hedgehog may participate in epithelial morphogenesis in an autocrine fashion during metamorphosis. The expression
of Xhh in a subset of primary epithelial cells
(Stolow and Shi, unpublished results) could
establish the identity of these cells as precursor stem cells for the secondary adult
epithelium. Currently, the stem cells responsible for the proliferation and formation of
the adult epithelial cell layer are unknown.
The Xhh expressing cells could signal to
each other or cells nearby influencing their
cell identity and fate. These cells would
then be instructed to proliferate and differentiate into adult epithelium. Because Xhh
protein is not detectible by immunohistochemistry toward the end of metamorphosis (data not shown), Xhh is probably
required at high levels during early stages
for the specification of the adult epithelium
but is only needed at very low levels for the
GENE REGULATION DURING METAMORPHOSIS
continued maintenance of the epithelial
layer. Surprisingly, despite all the work that
has been done both genetically and biochemically, the molecule which receives the
hedgehog signal is still unknown. Recent
evidence about the hedgehog protein has
begun to reveal some potential answers to
this problem.
All of the hedgehog proteins, including
Xenopus sonic hedgehog, are auto-proteolytically processed into two fragments, the
amino-terminal (N) and the carboxyterminal (C) domains (Lee et al., 1994;
Bumcrot et al., 1995; Stolow and Shi, unpublished results). Both fragments are secreted into the extracellular environment
but the N domain contains all of the biological activity of the protein (Perrimon,
1995, Bumcrot and McMahon, 1996). This
activity includes both local and long-range
signaling by the protein. The carboxyterminal domain is required for the selfcleavage event. Interestingly, as the protein
is cleaved and secreted the N domain is attached to the cell membrane by unknown
mechanisms (Bumcrot et al., 1995). This attachment to the cell surface produces an environment containing a high local concentration of the hedgehog protein. Functionally, it is believed that this extracellular localization of hedgehog is necessary for the
protein to transmit its short-range signal to
surrounding cells. Release of the protein
from the cell surface would be one means
of producing a long-range signal. What is
not clear is exactly how this signaling is
achieved. Is there a receptor for hedgehog
or is there some other mechanism for its signal transduction?
Recently, Tanaka Hall et al., (1995)
solved the three-dimensional crystal structure of the amino-terminal domain of the
murine sonic hedgehog gene and made
some unexpected observations. They detected the presence of a tetrahedrally coordinated zinc ion that appears to be structurally similar to the zinc coordination sites of
zinc hydrolases such as thermolysin and
carboxypeptidase A. These data suggest that
the mechanism responsible for extracellular
signaling by the hedgehog proteins could
involve the utilization of a potential catalytic site in the N domain.
201
Several possibilities for transmission of
the hedgehog signal include proteolytic activation of a receptor or hydrolysis of membrane lipids to initiate signal transduction.
At the present time, the exact mechanism
is unknown. However, in the tissues where
hedgehog is functioning, including the intestine, one could speculate that hedgehog
might also be cleaving and activating some
signal in the extracellular matrix. This could
be a factor anchored in the ECM or even a
molecular component of the ECM itself.
Any of these types of cell-cell or cell-ECM
interactions could trigger the cascade of
events downstream of hedgehog signaling.
STROMELYSIN-3 AND THE EXTRACELLULAR
MATRIX
Among the thyroid hormone-response
genes that we isolated from the metamorphosing tadpole intestine was the homolog
of the mammalian matrix metalloproteinase
gene, stromelysin-3 (ST3) (Patterton et al.,
1995). ST3 is a member of the large family of matrix metalloproteinases (MMPs)
which includes collagenases, gelatinases,
and stromelysins (Alexander and Werb,
1991; Matrisian, 1992; Birkedal-Hansen et
al., 1993). All of these proteins appear to
play important roles in the modification and
reconstruction of the ECM. The MMPs are
secreted as latent proenzymes which when
activated degrade their target ECM substrates. The activities of the MMPs are regulated by the presence of enzyme inhibitors
within the tissue. The modulation of the levels of the enzymes versus their inhibitors
appears to be a mechanism to regulate ECM
remodeling (Matrisian, 1992).
In mammals, ST3 is considered as a
unique member of the MMPs based on its
sequence homology with the rest of the
family. The expression of ST3 mRNA was
first reported in the stromal cells immediately adjacent to tumor cells in invasive
breast cancers (Basset et al., 1990). Later it
was detected in fibroblastic cells surrounding epithelium undergoing regression such
as the mammary gland during involution after pregnancy and the interdigital region of
limb buds (Lefebvre et al., 1992; Basset et
al., 1990). From these data, it has been suggested that ST3 plays a role in ECM degra-
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M. A. STOLOW ET AL.
dation processes which are common to cell
death and cancer progression.
Recent biochemical studies support the
earlier observation that ST3 is distinct from
any of the other MMPs in its mechanism of
action. Pei and Weiss (1995) reported that
ST3 is secreted from cells not as a latent
proenzyme but in its enzymatically active
form. ST3 is cleaved and activated by an intracellular proteolytic event which occurs
within the constitutive secretory pathway.
This cleavage is performed by the Golgiassociated proteinase, furin. The additional
10 amino acids present between the proand catalytic domains of ST3, compared to
most other MMPs, contain the cleavage recognition site for furin. Cleavage by furin removes the pro-domain allowing ST3 to be
activated.
Once ST3 is secreted as an active enzyme
it is capable of cleaving its target substrate.
The structural similarity between ST3 and
other MMPs suggested that ST3 would degrade ECM components (Basset et al.,
1990, 1993). However, its ECM substrates
have not been discovered. One substrate
that has been identified for ST3 is the serine
proteinase inhibitor, a 1-proteinase inhibitor, a non-ECM protein secreted by breast
cancer cells cultured in vitro (Pei et al,
1994). ST3 rapidly destroys the function of
a 1-proteinase inhibitor (cdPI) by cleaving
the anti-proteinase at a distinct site within
the reactive-site loop. These data suggest
that the class of serine proteinase inhibitors
could act as potential physiological targets
for ST3 at the tumor-stromal cell interface.
Based on current information about alPI,
it has been proposed that ST3-dependent inactivation of this protein could simultaneously affect the proliferative and invasive
activity of neoplastic cells (Pei et al., 1994).
The cloning of the Xenopus homolog of
mammalian ST3 implies an evolutionarily
conserved function for this unique matrix
metalloproteinase. Indeed, in amphibians, it
appears that ST3 may be involved in the
modification of the extracellular matrix as
well as influencing apoptosis of the larval
epithelia during metamorphosis (Patterton
et al., 1995; Ishizuya-Oka et al., unpublished results). By Northern analysis, ST3
mRNA expression is differentially regulated
in tadpole tissues during this transition (Fig.
2B). In the intestine, ST3 mRNA was detected at high levels just prior to the climax
of metamorphosis when primary epithelial
cells are undergoing extensive cell death
and secondary epithelial cells are rapidly
proliferating, hi addition, ST3 was also
found to be highly expressed in the intestine of premetamorphic tadpoles induced to
metamorphose with exogenous TH, but it
was down-regulated again after prolonged
TH treatment, mimicking that during normal development (Patterton et al., 1995).
Interestingly, in both the normal and THinduced metamorphosis, the activation of
the ST3 gene occurred before or at the onset of cell death.
The ST3 mRNA was also found to be
present in other organs where cell death
takes place. Thus, high levels of ST3
expression were also observed during tadpole tail resorption toward the end of metamorphosis, an event requiring large scale
cell death, and low levels of ST3 mRNA
were present at the early stages of hindlimb
development when cell death was presumably occurring during limb morphogenesis
(i.e., digit formation). Together, these data
suggest a correlation between the transcriptional activation of the ST3 gene and subsequent apoptosis in tissue remodeling. This
role for ST3 in frog metamorphosis is thus
similar to the function of mammalian ST3
during development, as its mRNA has also
been found in regions of tissues undergoing apoptosis such as the mammary gland
and during limb digit formation (Lefebvre
et al., 1992; Basset et al., 1990).
At the molecular level, the question of
how ST3 both in frogs and mammals might
initiate or affect apoptosis is unclear. The
connection between the observed cleavage
of the a 1-proteinase inhibitor by human
ST3 and cell death in remodeling tissues is
an enigma. It is not even known if a 1 PI is
present in these tissues undergoing apoptosis. Furthermore, it is quite possible that
there are other substrates for ST3 in these
developmental processes. In addition, recent evidence suggests that there may be another potential function for ST3 during development (Ishizuya-Oka et al., 1996). That
is, ST3 gene expression not only correlates
GENE REGULATION DURING METAMORPHOSIS
with the presence of cell death but with
basal lamina modification in the small intestine of X. laevis during metamorphosis.
The spatio-temporal relationship between
ST3 mRNA expression and structural
changes in the tadpole intestine during
metamorphosis were examined by in situ
hybridization and electron microscopy. At
the cellular level, Xenopus ST3 mRNA was
localized to the fibroblastic-like cells in the
intestine (Fig. 3A) (Patterton et al., 1995;
Ishizuya-Oka et al., 1996). As ST3 expression increased in the fibroblast-like cells located near the lower region of the typhlosole (stage 59) (Fig. 3A), the basal lamina
in this same area began to fold (Fig. 3C).
In contrast, the basal lamina remained thin
in the upper region of the typhlosole where
ST3 expression was low (Fig. 3B). At the
highest mRNA levels of ST3 (stage 61)
(Fig. 3D), the basal lamina attained its
maximum thickness in every region just beneath the epithelium (Fig. 3E). During this
time, cell migration and cell-cell contacts
between the epithelial and connective tissue
were most frequently observed through the
thickest regions of the basal lamina. Concurrently, the epithelium was transformed
from the larval to adult form by apoptosis
and adult cell proliferation. As ST3 levels
decreased, the basal lamina became thinner
(data not shown). These results indicate that
ST3 gene expression precedes structural
modifications of the basal lamina.
In particular, high levels of ST3 mRNA
are present at the time when the basal
lamina is thick but much more penetrable.
Thus, ST3 might degrade some specific
ECM components resulting in a looser
multi-folded ECM, while still leaving the
bulk of the ECM intact. In this regard, it is
interesting to note that transgenic mice created to overexpress stromelysin-1 also have
similar alterations in the ECM (Simpson et
al., 1994; Witty et al., 1995). In contrast,
the degradation of most of the ECM components during metamorphosis is likely mediated by other MMPs after cell death occurs. In fact, at least one such MMP, the putative gelatinase A gene is activated after
cell death is essentially complete (Patterton
et al., 1995). The modification of the ECM
by ST3 is thus likely to influence cell fate
203
as metamorphosis proceeds, by inducing
apoptosis and/or affecting the proliferation
and differentiation of the epithelial cell
layer.
DISCUSSION
The structural changes that occur in the
transformation of the intestine from a larval to adult form are quite dramatic and
must involve the activities of many different factors. Here we have reviewed one
class of molecules involved in this process,
namely those proteins that are secreted into
the extracellular environment. We have focused on two members of this group, Xenopus sonic hedgehog and stromelysin-3,
and tried to present possible functions for
these proteins based on data regarding their
mRNA expression profiles and spatiotemporal localization. The interactions between these proteins and surrounding cells
and/or the ECM appear to be a key component in this tissue remodeling event.
For ease of understanding, we have presented evidence for each of these proteins
individually, but it is likely that hedgehog
and stromelysin-3 do not act alone but
rather employ the activities of other factors
to assist in achieving their downstream effects. For example, ST3 could play a potential role in basal lamina modification and
apoptosis, but it is possible that other molecules in the MMP family may also be necessary for these events. For example, as
mentioned above, the putative gelatinase A
gene is expressed during intestinal remodeling. In addition, several other MMPs also
appear to be involved (Wang and Brown,
1993; Oofusa et al., 1994; Patterton et al.,
1995). These MMPs have different expression profiles than ST3 and each one may
have a distinct role in this transition depending on the tissue specificity and timing of
its expression.
Similarly, it is also possible that Xenopus
hedgehog requires the cooperation of other
factors to achieve its biological function.
Based on the expression profile of Xhh
mRNA and protein in the intestine, we have
suggested that this signaling molecule may
be necessary for establishing the identity
and subsequent proliferation and differentiation of the secondary adult epithelium.
204
M. A. STOLOW ET AL.
CT
E
\
r
CT
FIG. 3. Remodeling of the basal lamina in the intestine during metamorphosis. A. and D. In situ hybridization
using antisense ST3 probe on cross sections of the anterior region of the small intestine. A. At stage 59 the layer
of connective tissue (CT) is thin except for the typhlosole (Ty). Hybridization signals (arrows) are observed in
some cells of the connective tissue near the muscular layer (M), but are weaker in the upper region of the typholosole. X 150 D. Small intestine at stage 61. The basal surface of the epithelium (E) is rugged because of the
growth of adult epithelial primordia (asterisks) into the connective tissue. Most of the connective tissue cells
just beneath the epithelium are positive, i.e., ST3 expressing (arrows). X610 Bars: 20 p.m. L, lumen. B., C. and
E. Electron micrographs of epithelial-connective tissue interface of the small intestine. B. Upper region of the
typhlosole at stage 59 where ST3 expression is weak. The basal lamina (Bl) remains thin. X 12400 C. Bottom
GENE REGULATION DURING METAMORPHOSIS
This might be achieved by direct cell-cell
communication. However, because it has
been proposed that the amino-terminal signaling domain of hedgehog proteins may
act as a protease (Tanaka Hall et al., 1995),
it is interesting to speculate that Xhh might
be cleaving an ECM molecule which allows
for the release of some ECM-anchored
growth factor. This growth factor could act
as a secondary signaling molecule which
would then stimulate proliferation of the
adult epithelium. A second possibility is that
hedgehog could cleave a component of the
basal lamina causing a modification which
would regulate cell-cell communication between the epithelial and connective tissue
layers.
Certainly, many other models for how
these secreted molecules might function in
the larger context of this developmental
event are possible. Due to the nature of this
article, we have kept our discussion of other
genes involved in metamorphosis to a mini• mum. These remaining genes, as well as unidentified ones, will most likely have important roles in coordinating this complex set
of events. The further characterization of
known thyroid hormone response genes, as
well as the cloning and identification of
novel players, will continue to contribute
new information towards solving this multifaceted puzzle. This knowledge will not
only aid in our understanding of amphibian
metamorphosis but will most likely transcend to analogous events in many different systems where cell-cell and cell-ECM
interactions are vital.
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