Research Highlights
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Studying the Developing Palate Cleft palate is one of the most common human birth defects, often resulting in difficulties in breathing, feeding, speech and dental development. Despite the frequency of cleft palate and other facial clefts, the molecular mechanisms underlying these malformations are not well understood. Drs. Kathy Svoboda, Douglas Benson and L-Bruno Ruest lead efforts to discover the root molecular causes of faulty palate development such that these problems might be prevented before birth. In the meantime, Drs. Emet Schneiderman and Kenneth Salyer are focused on the postnatal treatment of cleft patients; they are initiating clinical studies with an international consortium of cleft centers to identify the surgical procedures that best facilitate normal post-natal growth. This issue of the BMS Newsletter highlights the molecular research conducted in the Svoboda and Benson laboratories. How the hard palate forms While a child is still in utero, projections of mesenchymal tissue surrounded by a thin sheet of epithelium project vertically on each side of the tongue. As the fetus grows, these palatal shelves elevate over the tongue and grow to meet at the midline. There, the medial edge epithelium (MEE) of each adheres to the other. The MEE cells then undergo epithelial to mesenchymal transition (EMT) while they migrate into the mesenchyme. They ultimately undergo apoptosis, leaving a confluent fused palate of mesenchyme (Figure 1). Previous work found that EMT and fusion in mammals depends on local synthesis of transforming growth factor beta 3 (TGFĂ&#x;3). Blockage of TGFĂ&#x;3 in mammalian palates abrogates fusion, and addition of exogenous TGFĂ&#x;3 to chicken palates (which do not make their own and do not normally fuse) causes them to fuse. The Svoboda Lab discovered that the
TGFĂ&#x;3 in this process signals to the transcription factor Twist1 through phosphatidylinositol-3 kinase (PI3K).
Figure 1. Sequence of palate EMT and fusion. The Benson Lab recently noted the expression of members of the Eph family of receptor tyrosine kinases (RTKs) and their membrane-bound ligands, the ephrins, in palate MEE before and during fusion. Mutations of at least one ephrin, ephrin-B1, are linked to human syndromes that include cleft palate. They therefore ASKED WHETHER %PH EPHRIN SIGNALING plays a role in palatal EMT and fusion. Ephrins are unique among RTK ligands in that they can also function as receptors, transducing signals into the ephrin-bearing cell upon Eph binding from other cells (Figure 2).
Ephrin signaling controls palatal fusion independent of TGFĂ&#x;3 Embryonic chicken palates were cultured in the presence of recombinant proteins comprised of either the extracellular domain of ephrinB2 (to activate Eph forward signaling) or EphB2 (to activate ephrin-B reverse signaling) fused to human IgG Fc. Activation of signaling for both pathways requires clustering of the proteins, accomplished by preincubation with anti-Fc antibody. When applied in unclustered form, these proteins still bind their targets and act as competitive inhibitors to block signal activation. Dr. Symone San Miguel (Benson Lab) and Dr. Maria Serrano (Svoboda Lab) found that activation of ephrin-B reverse signaling by incubation with clustered EphB2 protein caused fusion as efficiently as TGFĂ&#x;3, and moreover, that it did so without the action of TGFĂ&#x;3 (Figure 3). Indeed, addition of unclustered Eph protein to block ephrin reverse signaling abrogated the ability of TGFĂ&#x;3 to cause fusion. They also incubated embryonic
A
Figure 2. Forward and reverse ephrin signaling.
This process is called “reverse signaling�, and is essential for diverse developmental events involving cell migration and tissue segregation. Thus, these studies examined contributions of both forward and reverse signaling using ex vivo palate fusion model systems.
Figure 3. Eph and ephrin effects on palate fusion. (A) Palatal shelves were dissected from eight day old chicken embryos and cultured in contact on a support for 72 h in the presence of specific treatments, as indicated. continued page 5