Vertebrate Development and Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
e-mail: ds4{at}sanger.ac.uk
SUMMARY
The notochord is the defining structure of the chordates, and has essential roles in vertebrate development. It serves as a source of midline signals that pattern surrounding tissues and as a major skeletal element of the developing embryo. Genetic and embryological studies over the past decade have informed us about the development and function of the notochord. In this review, I discuss the embryonic origin, signalling roles and ultimate fate of the notochord, with an emphasis on structural aspects of notochord biology.
Introduction
The notochord is an embryonic midline structure common to all members of
the phylum Chordata (Fig. 1).
In higher vertebrates, the notochord exists transiently and has at least two
important functions. First, the notochord is positioned centrally in the
embryo with respect to both the dorsal-ventral (DV) and left-right (LR) axes.
Here, it produces secreted factors that signal to all surrounding tissues,
providing position and fate information. In this role, the notochord is
important for specifying ventral fates in the central nervous system,
controlling aspects of LR asymmetry, inducing pancreatic fates, controlling
the arterial versus venous identity of the major axial blood vessels and
specifying a variety of cell types in forming somites
(Christ et al., 2004;
Danos and Yost, 1995
;
Fouquet et al., 1997
;
Goldstein and Fishman, 1998
;
Lohr et al., 1997
;
Munsterberg and Lassar, 1995
;
Pourquie et al., 1993
;
Yamada et al., 1993
;
Yamada et al., 1991
). Second,
the notochord plays an important structural role. As a tissue, it is most
closely related to cartilage and is likely to represent a primitive form of
cartilage. Accordingly, the notochord serves as the axial skeleton of the
embryo until other elements, such as the vertebrae, form. In some vertebrate
clades, such as the agnathans (lampreys), and in primitive fish, such as
sturgeons, the notochord persists throughout life. In higher vertebrates,
however, the notochord becomes ossified in regions of forming vertebrae and
contributes to the centre of the intervertebral discs in a structure called
the nucleus pulposis (Linsenmayer et al.,
1986
; Smits and Lefebvre,
2003
; Swiderski and Solursh,
1992
). For the ascidian (tunicate) invertebrate chordates, the
notochord exists during embryonic and larval free-swimming stages, providing
the axial structural support necessary for locomotion
(Satoh, 2003
). Similarly, for
the cephalochordates, the notochord is essential for locomotion and persists
throughout life (Holland et al.,
2004
). Thus, the notochord is essential for normal vertebrate
development; but how does it arise?
Embryonic origins of the notochord
Organiser activities of notochord progenitors
In vertebrates, the notochord arises from the dorsal organiser. Originally
identified by Spemann and Mangold in amphibians, the dorsal organiser is a
region of a vertebrate gastrulae that, when transplanted into prospective
lateral or ventral regions of a host embryo, induces the formation of a second
embryonic axis, while only contributing to notochord and prechordal
mesendoderm (Harland and Gerhart,
1997; Spemann and Mangold,
1924
). In amphibians, this region is the dorsal lip of the
blastopore. In other species, homologous structures have been found: the
embryonic shield of teleost fish, Hensen's node in the chick and the node of
mouse embryos all possess essentially the same activities as Spemann and
Mangold's dorsal organiser (Beddington,
1994
; Oppenheimer,
1936
; Waddington,
1930
). The functions and activities of the dorsal organiser are
complex and have been discussed in detail elsewhere
(Harland and Gerhart, 1997
).
For this discussion, it is useful to consider the relationship between
specification of notochord fate and dorsal organiser activities. First,
however, what are the distinct morphological stages that the dorsal
mesendoderm progresses through on its way to becoming a mature notochord?
The first major transition is from dorsal organiser to chordamesoderm.
During early gastrula stages, the chordamesoderm, which is the direct
antecedent of the notochord, becomes morphologically and molecularly distinct
from other mesoderm. Cellular rearrangements involving the mediolateral
intercalation and convergence of cells towards the dorsal midline, force the
chordamesoderm into an elongated stack of cells. Genetic screens in zebrafish
have identified two loci, floating head (flh) and
bozozok (dharma Zebrafish Information Network), as
being essential for this transition to occur
(Amacher and Kimmel, 1998;
Fekany et al., 1999
;
Solnica-Krezel et al., 1996
;
Talbot et al., 1995
). As
development proceeds, chordamesoderm cells acquire a thick extracellular
sheath and a vacuole. Osmotic pressure within the vacuole acts against the
sheath, gives the notochord its characteristic rod-like appearance, and
provides mechanical properties that are essential for the proper elongation of
embryos and for the locomotion of invertebrate chordates and many vertebrate
species (Adams et al., 1990
;
Koehl et al., 2000
). This
transition, from chordamesoderm to mature notochord, requires a host of loci
that have been identified in zebrafish genetic screens
(Odenthal et al., 1996
;
Stemple et al., 1996
).
|
Mesoderm induction and dorsal specification
One key event during chordate early development is the induction of
mesoderm. Defined today in terms of characteristically expressed genes that
are known to correlate with future mesodermal fate, we now know many of the
molecules involved in mesoderm induction, largely owing to classical
embryological experiments carried out by Pieter Nieuwkoop
(Boterenbrood and Nieuwkoop,
1973; Gerhart,
1999
; Kimelman and Griffin,
2000
). Using frog embryos, Nieuwkoop found that, when left on its
own, the animal pole region, the animal cap, of a frog embryo would form only
a ciliated epidermis; the yolky vegetal cells would form only endoderm. In
combination, however, Nieuwkoop found that the vegetal-derived factors could
convert animal cap cells to a mesoderm fate. In the past 15 years, this assay
has led to the discovery of the signalling pathway that underlies mesoderm
induction (Green and Smith,
1990
; Schier and Shen,
2000
; Smith et al.,
1990
). We now know that Nodal and Nodal-related proteins are
secreted and serve to induce mesoderm formation. Importantly, the response of
animal cap cells to Nodal is graded so that different levels of Nodal
signalling lead to different mesodermal and axial mesendodermal fates. High
levels of Nodal signalling specify the deep gsc-expressing cell
fates, while lower levels specify flh-expressing prospective
chordamesoderm (Gritsman et al.,
2000
).
Nodal ligands employ the TGFß signalling pathway, including receptors
that also serve as activin receptors. Nodal ligands, however, require a
co-receptor of the EGF-CFC family. In zebrafish, the one-eyed pinhead
(oep) locus encodes an EGF-CFC protein that is essential for Nodal
signalling (Gritsman et al.,
1999; Schier et al.,
1997
; Zhang et al.,
1998
). Initially characterised by zygotic loss-of-function,
oep mutant embryos were found to be cyclopean but to possess a
notochord (Schier et al.,
1997
). The oep gene product, however, is also supplied
maternally, and mutants lacking both zygotic and maternal Oep lack all
endoderm and mesoderm, apart from a small amount of muscle that forms in the
tail (Gritsman et al., 1999
).
The type of mesoderm formed in response to inductive signals is dose
dependent. This was first appreciated in Xenopus animal cap
experiments, where different concentrations of activin were found to elicit
different mesodermal fates (Green et al.,
1992
; Green and Smith,
1990
). More recently this dose dependence with respect to Nodal
signalling was revealed when maternal-zygotic oep (MZoep)
mutant zebrafish embryos were compared with zygotic oep mutants
(Gritsman et al., 2000
). The
major defect in zygotic oep mutants, in which low-level Nodal
signalling still occurs, is the lack of gsc-expressing prospective
prechordal mesendoderm, which is the tissue that requires the highest level of
Nodal signalling for proper specification. Indeed, this tissue, which only
transiently expresses gsc in oep mutants, instead turns on
flh expression and becomes chordamesoderm. The maternal supply of Oep
protein is likely to be depleted with time in these embryos, suggesting that
one difference between prechordal and notochord specification is that the
prechordal tissue requires persistent Nodal signalling, while transient Nodal
signalling is sufficient for chordamesoderm specification. Notochord
specification is a stable property of the superficial dorsal organiser. This
is most clearly indicated by the observation that when prospective
chordamesoderm is transplanted anywhere into an equivalently staged host, the
transplanted tissue will form notochord (Saude et al., 2000;
Shih and Fraser, 1996
).
|
Relationship between notochord and cartilage
Consistent with its structural role in vertebrate development, the
notochord shares many features with cartilage. It expresses many genes that
are characteristic of cartilage, such as those that encode type II and type IX
collagen, aggrecan, Sox9 and chondromodulin
(Dietz et al., 1999;
Domowicz et al., 1995
;
Ng et al., 1997
;
Sachdev et al., 2001
;
Zhao et al., 1997
). There is,
however, one clear difference between chondrogenesis and notochord formation.
Chondrocytes normally secrete a highly hyrdrated extracellular matrix, which
gives cartilage its main structural properties
(Knudson and Knudson, 2001
).
By contrast, while notochord cells produce a thick basement membrane sheath,
they retain hydrated materials in large vacuoles
(Adams et al., 1990
;
Coutinho et al., 2004
;
Koehl et al., 2000
;
Parsons et al., 2002
). These
vacuoles allow notochord cells to exert pressure against the sheath walls,
which gives the notochord its structural properties
(Adams et al., 1990
;
Koehl et al., 2000
). There may
be a direct relationship between notochord and cartilage in which cartilage
has evolved to secrete certain materials; by rerouting those materials to an
internal vacuole, notochord cells have co-opted some of the essential
structural properties of cartilage to their needs.
The ultimate fate of the notochord also emphasises the relatedness of
notochord and cartilage. During endochondral bone formation, the type II
collagen-rich extracellular matrix of cartilage is deposited with type X
collagen, which signals the eventual replacement of cartilage by bone
(Linsenmayer et al., 1986;
Schmid et al., 1991
;
Solursh et al., 1986
).
Similarly, during the development of vertebrae, notochord that runs through
the middle of each vertebra first expresses type X collagen and is then
replaced by bone (Linsenmayer et al.,
1986
). Between the vertebrae, the notochord does not express type
X collagen and is not replaced by bone, but becomes the centre of the
intervertebral disc the nucleus pulposis
(Aszodi et al., 1998
;
Smits and Lefebvre, 2003
).
Thus, notochord can become ossified in a fashion similar to cartilage.
Consistent with this view, in mutant mice that lack type II collagen, the
notochord is not replaced by bone, presumably because the type II collagen
network is required for proper deposition of type X collagen.
Evolutionary origins of the notochord
By definition, the notochord arose with the chordates; however, there are
some possible hints to its origin in hemichordates. Hemichordates, along with
echinoderms and chordates, constitute the three monophyletic phyla of the
Deuterostomia (Jefferies,
1991; Peterson et al.,
1999
). Morphologically, several features of the hemichordates
suggest a close relationship with the chordates. For example, in the head of
hemichordates, there is a structure called the stomochord, which bears some
structural resemblance to the notochord. There is, however, little direct
evidence of a notochord in hemichordates. A more definitive answer is likely
to come from an analysis of the expression of canonical notochord genes in
hemichordates (Lowe et al.,
2003
), which might give a clear indication of the relationship
between stomochord and notochord. An analysis of the expression of
brachyury, a gene normally associated with notochord development, in
the hemichordate Ptychodera flava revealed, however, that
brachyury is never expressed in the stomochord
(Peterson et al., 1999
). As
such, an analysis of cartilage-specific genes could also be helpful in
elucidating the relationship between these structures.
In contrast to hemichordates, the ascidians have definitive notochords. A
wide variety of ascidian species have been studied by developmental
biologists, and much attention has been given to the notochord of ascidian
larvae (Satoh, 2003). In one
study, a comparison was drawn between two closely related ascidian species,
Molgula oculata and Molgula occulta; the first sports a
conventional ascidian tail, the other is tailless
(Swalla and Jeffery, 1996
). In
hybrid crosses, the tail of the normally tailless species is restored and a
gene called Manx, which encodes a zinc-finger protein, was identified
by differential gene expression analysis between the two species. The
disruption of Manx expression by antisense oligonucleotides in the
tail-bearing species leads to tail loss
(Swalla and Jeffery, 1996
;
Swalla et al., 1999
). More
recent screens have identified a host of notochord-specific genes and
mutations that affect notochord development in ascidians
(Di Gregorio and Levine, 2002
;
Jeffery, 2002
;
Moody et al., 1999
;
Nakatani et al., 1999
;
Satoh, 2003
).
One process that is important to notochord development is convergent
extension. A variety of studies in vertebrates have implicated the planar cell
polarity (PCP) pathway in the control of convergence and extension during
gastrulation. In zebrafish, for example, embryos in which PCP has been
disrupted exhibit a widened chordamesoderm and a subsequent defect in tail
notochord development (Hammerschmidt et
al., 1996; Heisenberg et al.,
2000
; Jessen et al.,
2002
; Topczewski et al.,
2001
). A mutation that affects ascidian notochord development has
been characterised by positional cloning and was found to encode a homologue
of Prickle, which is a PCP component known to be involved in vertebrate
convergent extension (Carreira-Barbosa et
al., 2003
; Di Jiang et al.,
2005
; Takeuchi et al.,
2003
; Veeman et al.,
2003
).
Roles of the notochord in vertebrate development
Patterning
The notochord has several well-established roles in patterning surrounding
tissues. As these issues have been comprehensively reviewed elsewhere
(Cleaver and Krieg, 2001;
Dodd et al., 1998
;
Hogan and Bautch, 2004
;
Holland et al., 2004
), I only
mention some of the main roles here. Perhaps the best characterised is the
role of the notochord in patterning the neural tube. A series of experiments
involving both the transplantation and the removal of the notochord during
development showed that the notochord can signal the formation of the floor
plate, which is the ventral-most fate of the spinal cord
(Placzek et al., 1991
;
van Straaten et al., 1989
;
Yamada et al., 1991
). Among
the signals secreted by the notochord are the Hedgehog (Hh) proteins. Sonic
Hedgehog, in particular, induces a range of ventral spinal cord fates in a
graded fashion while simultaneously suppressing the expression of
characteristically dorsal genes (Placzek
et al., 1993
; Yamada et al.,
1993
; Yamada et al.,
1991
). Reinforcing and maintaining earlier developmental events,
notochord signals are also involved in establishing LR asymmetry; the ablation
of the notochord in Xenopus gastrulae results in randomisation of
asymmetry (Danos and Yost,
1995
; Lohr et al.,
1997
). In teleosts, notochord-derived Hh signals control the
formation of the horizontal myoseptum, as well as specifying slow-twitch
muscle fates (Barresi et al.,
2000
; Devoto et al.,
1996
). Notochord-derived signals are important for specifying the
formation of the dorsal aorta (Cleaver et
al., 2000
; Fekany et al.,
1999
; Isogai et al.,
2003
; Lawson et al.,
2002
), as well as for normal specification of the cardiac field
(Goldstein and Fishman, 1998
).
Finally, the notochord is important for the normal development of early
endoderm and the pancreas (Cleaver and
Krieg, 2001
; Kim et al.,
1997
).
Structural
Although the patterning roles of the notochord are essential for normal
vertebrate development, the notochord also has an essential structural role.
The notochord is the main axial skeletal element of the chordate early embryo;
without a fully differentiated notochord embryos fail to elongate
(Odenthal et al., 1996;
Saúde et al., 2000
;
Schulte-Merker et al., 1992
;
Schulte-Merker et al., 1994
;
Stemple et al., 1996
;
Talbot et al., 1995
). For many
species, this results in the inability to swim properly, to escape predation
and to feed. Some understanding of this function of the notochord has been
derived from studies of zebrafish mutations
(Driever et al., 1996
;
Haffter et al., 1996
). A
number of loci involved in notochord formation were identified in several
large-scale systematic screens for mutations affecting zebrafish embryogenesis
(Fig. 3) (Odenthal et al., 1996
;
Stemple et al., 1996
). As I
have discussed, a few of the loci identified in these screens have been found
to be important for the formation of the chordamesoderm, highlighting
important signalling roles of the notochord. Most loci, however, have been
found to be profoundly important for structural aspects of notochord
function.
To understand notochord structure, it is helpful to consider the notochord
as part of a mechanical system required for locomotion
(Fig. 4). Zebrafish embryos,
for example, are able to flip their tails within 1 day of fertilisation, and
hatch and swim within 3 days. By analogy, the notochord is like a fire hose,
possessing a strong but flexible sheath that can resist high hydrostatic
pressures. Consider then a situation in which the fire hose is filled with
water balloons, each pushing against the other and against the sheath. In such
an arrangement, the fire hose would be elongated and stiff, but able to bend
in any direction. Finally, with cables running along the top and bottom of the
inflated fire hose, the cables would resist any upward or downward bending of
the hose, and any force acting on the fire hose would deflect it laterally.
For the zebrafish embryo, the equivalent of the fire hose is the
peri-notochordal basement membrane and the water balloons are the vacuolated
notochord cells. Running along the top of the notochord is the floor plate and
along the bottom is the hypochord. Consistent with this structural role, both
the floor plate and the hypochord express a variety of cartilage proteins,
such as type II collagen (Yan et al.,
1995). In addition, mutations in genes such as oep and
cyclops (ndr2 Zebrafish Information Network), which
lead to substantial loss of floor plate, also produce embryos with a profound
downward curvature (Hatta,
1992
; Hatta et al.,
1991
; Schier et al.,
1997
). Thus, although not yet directly established, it is possible
that the floor plate and hypochord act as cables on the respective dorsal and
ventral sides of the notochord.
In zebrafish, the formation of the peri-notochordal basement membrane and
vacuole are each affected by two phenotypic classes of mutations. One class,
originally found to affect both notochord and brain development, comprises the
bashful, grumpy and sleepy loci. These loci have recently
been found to encode zebrafish laminin 1 (bashful), laminin
ß1 (grumpy) and laminin
1 (sleepy), which are
constituents of the basement membrane
(Parsons et al., 2002
) (S. M.
Pollard, PhD thesis, University College London, 2002). The second class of
mutations was originally found to affect notochord and melanophore development
early in embryogenesis, leading eventually to catastrophic cell death
throughout mutant embryos. This class comprises sneezy, happy and
dopey, which encode, respectively, the
, ß and
chains of the coatomer complex, which is an important component of the
secretory pathway and is essential for survival of all eukaryotic cells
(Coutinho et al., 2004
). The
coatomer complex would normally be considered to perform a eukaryotic
housekeeping function. It is surprising that loss of coatomer activity in
zebrafish embryos would lead to a notochord phenotype. One explanation is that
coatomer is maternally supplied, and that the forming notochord is among the
first tissues in which secretory demand exceeds the maternal supply. This loss
of secretory activity leads to the loss of notochord basement membrane and to
a failure of vacuole formation. Together, the zebrafish laminin and coatomer
mutants highlight the importance of notochord structure to its function.
Laminins and notochord structure
Laminins are obligate heterotrimeric protein complexes that consist of an
, ß and
chain, and are integral to basement membranes.
Basement membrane are ubiquitous structures with diverse roles, but they are
usually associated with an epithelium. Typically in a basement membrane,
laminin trimers polymerise to form a meshwork, which is crosslinked to a type
IV collagen meshwork by nidogens
(Colognato and Yurchenco,
2000
; Iozzo, 1998
;
Timpl and Brown, 1996
;
Yurchenco and O'Rear, 1994
).
In addition, associated with basement membranes are heparin-sulphate
proteoglycans (Iozzo, 1998
).
Basement membranes are important for defining boundaries between tissues, for
signalling between cells and, in some situations, for providing a strong
mechanical barrier (Timpl and Brown,
1996
). The latter role is of particular importance in the kidney
glomerulus, where the basement membrane serves as an integral part of the
filtration apparatus while withstanding high hydrostatic pressures
(Miner and Li, 2000
). This is
similar to the role that the peri-notochordal basement membrane plays.
|
|
The positional cloning of the zebrafish bashful, grumpy and
sleepy loci led to the identification of three laminin chains;
1, ß1 and
1 (Parsons et
al., 2002
) (S. M. Pollard, PhD thesis, University College London,
2002). The mutant phenotypes of grumpy and sleepy are
indistinguishable, whereas the bashful phenotype is always weaker.
During the development of grumpy and sleepy mutant embryos,
the notochord phenotype first becomes apparent when vacuoles fail to form
properly and when genes that are characteristically expressed by
chordamesoderm, such as echidna hedgehog, fail to be extinguished, as
they would during normal notochord differentiation. Electron microscopic
analysis of the peri-notochordal basement membrane shows that it is completely
absent in these mutants, and antibody staining shows a complete lack of
laminin 1 immunoreactivity in the most severely affected grumpy and
sleepy mutant embryos. In the weaker bashful mutant embryos,
the peri-notochordal basement membrane is missing only in the anterior
affected regions of the notochord. In the posterior of these embryos, the
notochord is normally differentiated and has laminin 1 immunoreactivity. The
differences between bashful and grumpy or sleepy
are due to a redundancy between laminin
chains. Disruption of laminin
4 expression in bashful mutant embryos, which lack laminin
1, leads to the severe notochord phenotype seen in grumpy and
sleepy mutants, and a complete loss of laminin 1 immunoreactivity (S.
M. Pollard, PhD thesis, University College London, 2002).
These four laminin chains 1,
4, ß1 and
1
participate in the formation of the peri-notochordal basement
membrane, and the chordamesoderm expresses mRNA for each chain. But does the
notochord alone account for the laminin in its basement membrane? This
question has been addressed by an organiser graft assay. In these experiments,
a wild-type dorsal organiser was grafted into the ventral region of a mutant
host embryo, or vice-versa, and the resulting twinned embryos were analysed
for morphological notochord differentiation. Surprisingly, for both
grumpy and sleepy mutant tissues, both type of grafts led to
the same result, a normally differentiated notochord
(Parsons et al., 2002
). An
analysis of laminin immunoreactivity showed that when wild-type organiser is
transplanted into mutant hosts, laminin 1 proteins surround the transplanted
notochord. Thus, laminins can be supplied either by extra notochordal sources
or by the notochord itself.
|
These studies show that formation of a peri-notochordal basement membrane is essential not only to the structure of the notochord, but also for proper notochord differentiation. Indeed, loss of several other genes that are not basement membrane components, per se, but that affect formation of the peri-notochordal basement membrane, also affects the differentiation of the notochord.
Coatomers and notochord structure
Similar to the sleepy and grumpy laminin mutants,
sneezy, happy and dopey mutants are also phenotypically
indistinguishable (Coutinho et al.,
2004). In these mutants, the notochord fails to differentiate, as
measured both by the persistent expression of early marker genes, such as
echidna hedgehog, and by the failure to form normally inflated
vacuoles. As in the laminin mutants, the basement membrane fails to form
properly. Unlike the laminin mutants, however, beginning at about 48 hours
after fertilisation sneezy, happy and dopey mutants undergo
widespread cell death by apoptosis and the embryos disintegrate. By positional
cloning, we identified that sneezy, happy and dopey encode
the zebrafish
, ß and ß' coatomer proteins
(Coutinho et al., 2004
).
Seven coatomer subunits, in combination with the small GTPase Arf1, make up
the coat of COPI vesicles (Bannykh et al.,
1998; Cosson and Letourneur,
1997
; Lowe and Kreis,
1998
; Nickel and Wieland,
1998
). In eukaryotic cells, there are at least three vesicular
coating systems. Clathrin-coated vesicles handle endocytosis and COPII-coated
vesicles mediate the transport of secretory cargo from the endoplasmic
reticulum to the Golgi. COPI-coated vesicles are thought to be largely
responsible for the retrograde transport of enzymes within the Golgi that
maintain its polarity, and for the machinery that transports proteins from the
Golgi back to the rough endoplasmic reticulum (RER).
Electron microscopic analysis revealed two important features of notochord
cells in zebrafish coatomer mutants
(Coutinho et al., 2004).
First, the secretory pathway is blocked, resulting in massive accumulations of
materials within the RER, and disruption of the Golgi apparatus is evident.
Second, the peri-notochordal basement membrane fails to form. Normally, the
peri-notochordal basement membrane has a trilaminar structure
(Fig. 5). The layer closest to
the plasma membrane is a single, darkly stained material with apparent
crosslinks to the underlying plasma membrane. Distal to this layer is a medial
layer of fibres oriented in one direction around the notochord cell, and
distal to this is an outer layer of fibres oriented in an orthogonal
direction. In the coatomer mutants, the outer two layers are not present, but
a distinct inner layer is often observed. Staining with laminin 1 antibody
suggests that the inner layer largely comprises laminin. Using dorsal
organiser grafts of mutant tissue into wild-type embryos, and vice-versa, the
embryonic origin of the outer two layers of this basement membrane was
investigated. Although similar grafting experiments with laminin mutants
showed that laminin could be supplied either by the notochord itself or by
extra-notochordal sources, grafts of coatomer mutant tissue show that both the
RER and basement membrane phenotype are autonomous to the notochord. Thus,
whereas the laminin-rich inner layer may be supplied by surrounding tissues,
the outer layers of the peri-notochordal basement membrane are likely to be
secreted by notochord cells themselves.
The notochord has two features, a thick basement membrane and a vacuole, both of which place unusual demand on the secretory pathway. Coatomer proteins are essential to all cells and both protein and coatomer mRNAs are supplied maternally. During wild-type development, mRNAs encoding coatomer proteins are expressed in chordamesoderm at a high level. Indeed, when observed by in situ hybridisation, coatomer mRNA expression appears to be chordamesoderm-specific and has a similar temporal and spatial pattern of expression to genes such as flh. The high-level of coatomer expression in the developing chordamesoderm is likely to be zygotic, and thus provides some explanation for how mutations in a cell-essential gene could lead to specific early developmental defects.
Conclusions
The notochord is an essential structure for the normal development of chordates. Although much attention has been focussed on its patterning roles, the notochord also has important structural roles. A more complete understanding of the mechanical basis of notochord structure is beginning to emerge from mutational analyses. Given the simplicity of the notochord as an embryonic organ, it is likely to be a useful model of organogenesis, especially for the integration of extracellular matrix formation with cellular differentiation.
ACKNOWLEDGMENTS
I thank Kathy Joubin for critical comments on the manuscript and the Wellcome Trust for support.
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