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Embryonic
Development Lab
Embryonic
Development Lab staff
Genes
Regulating Pattern Formation During Embryonic Development
Research: Our laboratory has identified several new developmental control
genes(transcription factors) that appear to be involved in regulating
the formation of and the pattern of structures in the primary embryonic
axis and the limb axis. We are analyzing their normal function and potential
role in oncogenesis. Many of the same regulatory and signaling cascades
appear to operate during both primary embryonic induction and establishment
of secondary embryonic fields, such as the limb. Analyzing the role
that such transcriptional regulators play in different developmental
contexts may offer additional insights into the sorts of basic processes
that they govern in the cell. Furthermore, key processes in development,
including differential cell proliferation, programmed cell death, migration,
and cell-cell interactions, are recapitulated in a pathologic manner
during oncogenesis and metastasis, suggesting an aberrant regulation
or "reactivation" of such processes. Understanding how these
events are triggered and regulated will be invaluable in deciphering
tumor biology and ultimately help to identify new ways to intercept
cellular targets that drive tumor cell behavior.
The major events
of gastrulation include inductive interactions that establish and pattern
the embryonic axis, and morphogenetic movements yielding the germ layers
and shaping the embryo. We have isolated several novel homeobox and
T-box genes involved in mesoderm formation during gastrulation, and
we are exploring their roles in regulating mesodermal cell fate and
behavior, with particular emphasis on the coordination of organized
morphogenetic movements. Both gain of function experiments in chick
embryos and generation of null mutant mouse embryos are being used for
functional analyses. Chick tail development is a continuation of gastrulation
and we have demonstrated that the tail tip retains true Spemann-type
organizer activity (induces a secondary embryonic axis) including neural
induction, cell recruitment into paraxial mesoderm, and induction of
gastrulation-like morphogenetic movements to produce elongated mesodermal
outgrowths. Tail bud grafts to extraembryonic membranes provide an accessible
and controlled experimental system to analyze inductive activities in
this tissue removed from the context of other endogenous signals in
the embryo.
Because the major
events of gastrulation occur over a short time span and require very
dynamic regulation of gene expression, a second focus of the laboratory
is to decipher the mechanisms mediating these rapid expression changes.
Several features of one of the organizer-specific homeobox genes we
are studying (Gnot1) may provide new clues to expose how such regulation
works at the posttranscriptional level. Both the abundance and localization
(nuclear to cytoplasmic transport) of this gene are highly regulated
in the embryo.
Limb development
is an intensively studied and excellent model for vertebrate pattern
formation that offers a solid conceptual framework to interpret new
results. In this system, patterning is tightly linked to differential
growth regulation; 5' members of the Hoxd homeobox cluster appear to
regulate anterior-posterior (e.g., thumb to little finger) pattern of
limb skeletal elements as downstream targets of Sonic Hedgehog signaling.
Using a transgenic model, we have found that some of the 5' Hoxd genes
also play a role in establishing and/or maintaining these polarizing
signals in the posterior limb through a positive feedback loop with
Sonic Hedgehog. Hoxd genes also appear to play key roles in regulating
proliferation of chondrogenic elements in the limb, and we have identified
the c-Fos oncogene as a possible target of Hoxd genes, but generally
the effector target genes through which they act remain elusive. Considerably
less is known regarding the relative importance of Hoxd genes at these
later (fetal) stages of cartilage proliferation and potentially under
pathologic conditions. We are analyzing the Hoxd-12 and Hoxd-13 as prototypic
examples of 5' Hoxd gene function, with a major emphasis on identifying
direct targets at the molecular level and on learning more about late
roles in proliferating cartilage and potential contribution to neoplastic
processes using an inducible transgenic model system.
An area of emerging
interest is elucidating how limb initiation and position along the body
axis are regulated. Both retinoids and FGF's play critical roles in
this as well as in other inductive events during embryonic development.
We are developing new tools to evaluate dynamic changes in retinoid
distribution and to analyze FGF signaling at localized sites in the
embryo. In this context, we have evidence that the transcription factor
T, or Brachyury, may also play a role in the relay of FGF signals from
the embryonic midline to the periphery that is thought to initiate limb
budding. Intriguingly, in some systems, T has been shown to participate
in positive feedback loops with FGF's. As part of a collaborative effort,
we are developing dominate-negative FGF receptors that can be applied
in soluble form to antagonize FGF signals. These will serve as useful
tools to document FGF relay sites in the embryo and as an adjunct to
evaluate the possible roles of T as an intracellular component of an
FGF relay that initiates limb budding.
Retinoids are clearly
important at multiple points in development and have been implicated
in positioning both sites of limb initiation and limb polarizing regions.
It remains uncertain whether these events are mediated by localized
regions of higher RA or differential tissue sensitivities to RA. We
are interested in developing methods to evaluate RA sources in situ
during early stages of development when the embryo is rapidly changing.
In a collaborative effort, we have developed a rapid in situ assay using
a chimeric glucocorticoid/retinoic acid receptor/ GFP fusion protein.
The chimeric protein demonstrates RA-dependent nuclear translocation,
providing for a rapid read-out assay by confocal fluorescence imaging
of cultured live embryo slices on transfected monolayers expressing
the chimera.
Recent Publications
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Knezevic V,
et al. Development 1997 ; 124 : 4523-36
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Knezevic V,
et al . Development 1997 ; 124 :411-9
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Knezevic V,
et al. Development 1998 ; 125 :1791-801
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Celli G, et
al. EMBO J 1998 ; 17 :1642-55
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Mackem S, et
al. Cell Tissue Res(Rev) 1999; 296:27-31
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Mackem S, et
al. J Biol Chem 2001;276:45501-504
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Knezevic V and
Mackem S, Genesis 2001;30:264-273
Collaborators
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