IMP Research Report 2002

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 microarray
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 situhybridization
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
Figure 2: 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.
them. We believe that this analysis will ultimately help
to understand the function of FGF signaling during
development at the molecular level.
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.
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 are using 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 (Figure 3).
Figure 3: 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.
Using a candidate approach, we have shown that FGF
signaling induces the expression of the transcription
factors Pax3, Tbx2, Erm and Pea3 in facial
Contact: neubueser@imp.univie.ac.at
mesenchyme. To systematically screen for FGF
inducible genes, we have generated a subtracted
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