1. Zebrafish msxe Expression Analysis
Cassie Wells

2. Outline msxe/MSX1 implication in orofacial clefting
Bioinformatic Analysis
My project goal
Tol2Kit / Gateway Cloning System
Expression Clone Design
Acknowledgements

3. Cleft Lip and/or Palate
The most common congenital facial abnormality
Association studies have linked the gene MSX1 to clefting in humans
Murine Msx1 expression is controlled at the tips of the growing facial prominences by the two highly conserved distal and proximal enhancers, (DE and PE, respectively) Murine Msx1 expression is controlled at the tips of the growing facial prominences by the two highly conserved distal and proximal enhancers, (DE and PE, respectively) (MacKenzie et al., 1997)
Cranial neural crest cells (CNCC) in vertebrates are a class of multipotent ectomesenchymal cells that differentiate into multiple tissues and contribute greatly to jaw formation. Msx1 is expressed within CNCC at multiple stages and plays a critical role during craniofacial development.

4. Bioinformatic Analysis
Schematic diagram of the msxe locus
Bioinformatic analysis revealed two highly conserved enhancer regions (DE and PE), presumably corresponding to the murine enhancers identified by Mackenzie, and a putative promoter

5. Conservation of the DE and Protein Binding Sites
A MSX1: TALE protein binding site was identified within the DE. MSX1/PBX1/dsDNA complex formation was verified on a SDS-PAGE gel and is predicted to be occurring through a large multimeric complex. The UCSC Genome Browser also identified an ACTIVE TCF4 binding site within both the DE and PE via CHiP (Chromatin Immunoprecipitation). Miller (2007) confirmed that there is an active TCF4 binding site within the PE, but did not discuss the binding of TCF4 to the DE. The TALE:MSX1 immediately precedes the TCF4 binding site. Our current hypothesis is as follows: The DE is normally repressed by TCF4. Then along comes Wnt signaling that sends in beta-catenin to ‘derepress’ that part of the DE. This opens up that region and allows the TALE:MSX1 to bind and activate the DE. Generation of a multiple reporter construct will help determine the effect that the binding interactions have on the regulation of MSX1 expression.

6. Project Purpose
My project purpose: To develop the first step, transgenic expression component, of an in vivo functional assay for DNA and protein coding variants at the MSX1 locus.

7. 3 Fragment Recombination
Three PCR Products flanked by specific att sites and three Donor vectors are used in separate BP recombination reactions to generate three entry clones
The three entry clones and a destination vector are used together in a LR recombination reaction to create one expression clone
The segment of interest is isolated via PCR and att sites are added. Recombination with a DONR vector via a BP reaction creates an entry clone. The entry clone is then recombined, along with the other entry clones (p5E and p3E, in this example), with the destination vector. Typically, enhancer-promoter constructs are placed in p5E, the gene of interest in pME, and reporter tags in p3E, although alternative placement strategies are possible.

8. Expression Clone Construct Possibilities
pDestTol2pA2 A B C
A1. Promoter
p5E
A2. DE-Promoter
A3. PE-Promoter
A4. 2.1kb chunk (DE – Prom)
pME
B1. EGFP
B2. msxe
B3. msxe/MSX1 knock-out and rescue
B4. MSX1
B5. mutant MSX1
C1. pA
C2. EGFPpA
p3E
Generation of these basic expression clones are the first step towards understanding the regulation of MSX1. This project yields itself to many future experimental plans, as that will result in a more complete understanding of the interactions at the MSX1 locus and the role that these interactions play in the production of a CL/P phenotype.

9. Acknowledgements Dr. Peter Jezewski (PI) Divakar Prakash Henry Jackson
Dr. Heidi Erlandsen
Institute of Oral Health Research