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Gastrulation is the process by which the three embryonic germ
layers (mesoderm, endoderm and ectoderm) form from a single-layered
epithelial cell sheet (the epiblast). When gastrulation begins
in the mouse embryo (at ~embryonic day 6.5), epiblast cells near
the prospective posterior end of the embryo begin a process of
convergent migration and delamination into a region called the
primitive streak, where they undergo an epithelial to mesenchymal
transition, and then migrate away from the streak. Both mesoderm
and embryonic endoderm are the descendants of epiblast cells that
have migrated through the streak, whereas embryonic ectoderm (i.e.
the neuroectoderm and surface ectoderm) is descended from cells
that never passed through the streak. The embryonic mesoderm has
varying fates depending on the position at which it emerges from
the streak.
Several FGF family members, which encode secreted proteins, are
expressed at gastrulation stages, in domains consistent with a
role in mesoderm formation or fate determination. We have previously
shown that in Fgf8–/– embryos, epiblast cells
move into the streak and undergo an epithelial to mesenchymal
transition, but most cells then fail to move away from the streak.
Consequently, no embryonic mesoderm- or endoderm-derived tissues
develop, and patterning of the prospective neuroectoderm is greatly
perturbed as a secondary consequence of the lack of normal morphogenetic
movements.
We are currently analyzing the function other FGF genes during
gastrulation using a combinatorial mutational approach.
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The images show sections of a normal (Fgf8+) and an Fgf8
null mutant embryo during gastrulation. In the mutant embryo, cells
are piled up in the primitive streak (right side). The diagram illustrates
the morphogenetic movements in a normal mouse gastrula, with the
green arrows indicating that epiblast cells converge towards the
primtive streak, and the red arrows indicating the pathway that
the nascent mesoderm cells normally take as they move away from
the streak.
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The vertebrate limb develops from an outgrowth (bud) of the lateral
plate mesoderm and its overlying surface ectoderm. As the limb
bud forms, discrete signaling centers develop within it, each
of which will produce molecules essential for normal limb morphogenesis.
The zone of polarizing activity, localized in posterior mesoderm,
produces Sonic hedgehog (SHH). The apical ectodermal ridge (AER),
a specialized epithelium that rims the distal tip of the bud,
produces FGFs and BMPs. The non-ridge ectoderm produces WNT7A.
These secreted signaling molecules are thought to act on cells
in the mesoderm to regulate and coordinate outgrowth and patterning
along all three axes of the limb (proximal/distal, arm to fingertips;
dorsal/ventral, back of hand to palm; anterior/posterior, thumb
to little finger).
Using a conditional gene inactivation approach in mice, we have
been systematically studying the functions of the four FGF genes
that are specifically expressed in the AER, and have concluded
that they play a vital role in ensuring that sufficient progenitor
cells are available to form the normal complement of limb skeletal
elements.
Current projects include an analysis of BMP4 function in limb
development, and an investigation of the cellular mechanisms underlying
the earliest events in the development of the mouse limb bud and
the homologous structure in zebrafish, the pectoral fin. Further
experiments are aimed at testing the hypothesis that SHH and FGFs
act predominantly to regulate cell survival rather than patterning
in the developing limb bud.
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The image shows the pattern of cell death in a normal mouse forelimb
bud (left) and a mutant limb bud in which both Fgf4 and Fgf8
have been conditionally inactivated in the AER.
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A major issue in developmental neurobiology is how the vertebrate
neural tube becomes regionalized and patterned along its axes.
Substantial progress has been made toward understanding the molecular
mechanism of specification and early anterior-posterior (A-P)
patterning of the region that will ultimately form the midbrain,
isthmus, and cerebellum. This region develops from two distinct
portions of the early neural tube, the prospective midbrain and
rostral hindbrain (Mb/Hb). Despite this dual origin, the region
behaves as a single developmental compartment. Its specification
and A-P patterning begin during gastrulation, with the induction
of gene expression that distinguishes this compartment from the
rest of the neuroepithelium and creates asymmetry within it, in
the form of two adjacent, molecularly distinct territories along
the A-P axis. The boundary between these two territories is the
site at which a signaling center known as the isthmic organizer
forms.
We have previously shown that FGF8, which is one of the signals
produced by this organizer, is capable of inducing complete ectopic
midbrains as well as cerebellar tissue in parts of the brain that
do not normally form these structures. These data have suggested
that FGF8 plays a role in patterning and diversifying cells within
the developing Mb/Hb. We are currently investigating the function
of FGF8 signaling in the development of this region using a loss
of function approach.
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The images show an early stage chick embryo in which a bead soaked
in recombinant FGF8 was inserted into the prospective caudal forebrain,
and a section of the brain that subsequently developed, in which
the caudal forebrain was transformed into a mirror-image duplication
of the midbrain (*).
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Formation of the definitive kidney begins at mid-gestation stages,
when a signal from the metanephric mesoderm at the caudal end
of the embryo induces the nearby Wolffian duct to form an outgrowth
known as the ureteric bud (UB). In turn, the UB sends a signal(s)
that stimulates the metanephric mesoderm to condense and develop
into the structures that constitute the functional units (nephrons)
of the kidney. Over time, in response to signals from the metanephric
mesoderm, the UB elongates and branches many times, resulting
in nephrogenesis in regions that are progressively more distant
from the initial site of UB formation. The branched structures
derived from the UB itself will form the collecting system of
the kidney. Thus kidney development is dependent on and coordinated
by reciprocal interactions between the metanephric mesoderm and
the ureteric tree.
We are interested in studying the role of FGF signaling in nephrogenesis,
and also in the molecular mechanism of UB formation. We have recently
discovered that in mutant mouse embryos lacking Slit2 function,
ectopic UBs emerge from the Wolffian duct. By analyzing the molecular
and cellular defects that cause this mutant phenotype, we hope
to answer the question of how SLIT2, a secreted molecule, restricts
the formation of the ureteric bud to its appropriate location
during normal development.
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The image shows normal mouse kidneys in which all the cells derived
from the ureteric bud express a transgene that produces green fluorescent
protein. |
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Studies in Drosophila have demonstrated that the sprouty gene
encodes an FGF-induced negative regulator of FGF signaling that
plays an important role during tracheal morphogenesis and other
developmental processes. Four sprouty-like genes are found in
vertebrates. As in Drosophila, their expression is induced by
FGF signaling, and they function to negatively regulate FGF as
well as other receptor tyrosine kinase signaling.
We are interested in determining the specific functions of the
different Sprouty family members in the mouse, and are using both
conditional loss-of-function and gain-of-function approaches to
investigate this. Studies are currently in progress to determine
the cellular and molecular defects that underlie the phenotypes
observed in Sprouty mutant embryos, which include abnormalities
in gastrointestinal morphogenesis and function. In the long-term,
we plan to determine whether Sprouty genes play roles as tumor
suppressors in preventing mammary or other tumor formation in
the normal mouse.
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last updated Jan 27, 2003
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The image shows Spry2 expression in a chick embryo in which
a bead soaked in recombinant FGF4 protein was inserted into the
region between the limb buds on the right side (top of image), 24
hours earlier. Note that Spry2 is normally expressed in the
mesoderm of all four limb buds, and is also induced in the mesoderm
surrounding the bead.
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