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Developmental Biology - Neubüser lab
- Research Projects |
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1. Molecular mechanisms of craniofacial development
The vertebrate face develops from buds of tissue, the facial primordia,
which surround the primitive mouth. Development of the midfacial region
begins with the appearance of the nasal placodes - bilateral ectodermal
thickenings at the ventro-lateral sides of the forebrain - that will
give rise to the olfactory epithelium. Shortly after the placodes become
morphologically apparent the mesenchyme around them starts to grow out
to form the nasal processes. Continued outgrowth depends on interactions
between the epithelium covering these processes and the underlying mesenchyme.
Defined fusions between the initially separated facial primordia then
follow, and give rise to the characteristic shape of the adult face.
For example, the primary palate is formed through fusion between the
oral corners of the medial and lateral nasal processes with the maxialla,
and fusion between the two medial nasal processes in the midline. Errors
in this complex morphogenetic program result in facial malformations,
which are among the most frequent birth defects in humans. We are interested
in the molecular mechanisms that control development of the vertebrate
face.
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FGF8 function during facial development
FGF8 is a member of the fibroblast growth factor family of signaling molecules.
Fgf8 is widely expressed in the ectoderm covering the midfacial area at
early stages of facial development but becomes restricted to a horseshoe
shaped domain of expression around the nasal placodes at later stages (see
figure below - A, B). Mouse embryos in which this gene has been inactivated
in the facial region develop severe facial defects. Such embryos display
midfacial clefts and most derivatives of the first branchial arch are severely
reduced or absent (see figure below - C, D). In early mutant facial mesenchyme
the amount of cell death is increased and cell proliferation is reduced.
Patterning in the remaining tissue is also affected, in particular in the
midfacial area at E9.5 as judged by the analysis of the expression of marker
genes. In addition to the mesenchymal defects, also the development of
the nasal placodes and the surrounding ectoderm is abnormal in Fgf8 mutant
embryos. This includes changes in the expression patterns of ectodermal
signaling molecules. Therefore, altered signaling between the mutant ectoderm
and the underlying mesenchyme is likely to contribute to the defects observed
at later stages.
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Tissue specific inactivation of Fgf8 in the facial
area results in severe facial defects. Facial expression
of Fgf8 at E9.5 (A) and E10.5 (B) . The face of
a wildtype (C) and an Fgf8 mutant embryo (D) at
E16.5. Embryos in which Fgf8 has been inactivated in the facial
area develop a midfacial cleft and show a severe reduction of
the lower jaw and peri-ocular tissue. |
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Identification of genes transcriptionally regulated in facial
mesenchyme in response to FGF signaling
In order to understand how FGF8 controls development of the facial mesenchyme
it is important to identify the genes induced or repressed in response
to FGF8 signaling. We have used an in vitro explant culture system in which
facial mesenchyme is cultured in contact with facial ectoderm, in isolation
or in contact with polymeric beads soaked in FGF8 protein, to identify
such genes.
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Identification of FGF-inducible genes. Facial
mesenchyme was cultured in vitro with FGF or PBS soaked beads.
RNA was isolated from these explants and used to generate a subtracted
(SSH) cDNA-library, enriched for FGF inducible clones. The inserts
of 4400 clones from this library were then arrayed on glass slides.
The resulting microarray was hybridized with probe derived from
RNA isolated from facial mesenchyme cultured with FGF or PBS
soaked beads, labeled with a green or red fluorescent dye (Cy3
or Cy5), respectively. |
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Using a candidate approach, we
have shown that FGF signaling induces the expression of the transcription
factors Pax3 ,Tbx2 ,Erm and Pea3 in facial mesenchyme. To systematically
screen for FGF inducible genes, we have generated a subtracted cDNA-library
from facial mesenchyme cultured in the presence or absence of FGF and have
used this library to produce a customized DNA microarray. This micro-array
was probed with cDNA derived from mesenchyme cultured with or without FGF.
The expression pattern of 200 clones with the strongest differential hybridization
was then analyzed by whole mount in situ hybridization and inducibility
by FGF8 was confirmed. Through this screen we have identified more than
50 genes that are induced in the facial mesenchyme in response to FGF signaling
and we have begun to characterize some of them. We believe that this analysis
will ultimately help to understand the function of FGF signaling during
development at the molecular level.
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2. Development of the sensory
placodes
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3. Feather development
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