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 Developmental Biology - Neubüser lab - Research Projects  
 


   
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.

 
 
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.

Fgf8 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.
 

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.
Identification of FGF-inducible genes 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 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.

2. Development of the sensory placodes

3. Feather development



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