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Inhibition of Sonic Hedgehog Signaling with Cyclopamine in Order to Examine the Role of Cell Death in Tail Bud Malformations

Wenxin Wei '02 and Miha Krsmanovic '02
Franklin and Marshall College


Sonic Hedgehog is an essential glycoprotein integral for the proper craniofacial and tail bud development, in addition to the development of many other structures. Cyclopamine has been shown to block Shh, and cause deformations in various parts of the developing embryo, depending on the time of administration, perhaps through the apoptosis of certain cells. Cyclopamine was administered to 20 hour chick embryos to induce tail bud malformations. Dead cells were observed under a fluorescent microscope after staining with propidium iodide. Cyclopamine was observed to induce the apoptosis of clusters of cells in the tail bud region, in addition to massive cell death in the eye region. The massive regions of dead cells shows that the deformations are a result of dying cells, or the lack of proliferation of precursor cells resulting in less growth. Therefore, although Shh is necessary for proper limb patterning, it serves as more of a survival factor in this context. The inhibition of Shh at 20 hours causes cells in the tail bud to stop dividing and die, having implications on the various roles of Shh in early development.

Background and Significance:
Sonic Hedgehog (Shh) is an important glycoprotein essential for the development of diverse tissues during various stages of development (J.M. Britto et. al., 2000). It is integral in controlling the development and patterning of the dorsal ventral axis of the nervous system, neural tube, somites and also the anterior-posterior patterning of the limbs (J.J. Sanz- Ezquerro and C. Tickle, 2000). Shh has been shown to have a role in proper craniofacial and tail bud development.(J.P. Incardona, 1998)
Cyclopamine is a known Shh inhibitor which works by blocking the Shh receptor by interfering with exogenous cholesterol from the yolk, which interacts with the patched or smoothened proteins (J.P. Incardona, 1998). It induces holoprosencephaly in chick embryos when introduced prior to gastrulation, a syndrome characterized by facial and forebrain abnormalities (S.C. Ahlgren, 1999). A study by Ahgren et.al.(1999) demonstrated that inhibition of Shh increased cell death in the neural tube and neural crest, having many implications for later development. In addition, other models for tail bud development suggests that Shh may be required for the proliferation of tail bud cells. In other systems, Shh has been shown to enhance the proliferation of specific cells. To induce tail bud malformation, cyclopamine will be introduced at a later stage of development, at approximately 20 hours (J.P. Incardona, 1998). A staining technique using propidium iodide, which is specific to dead cells, will be utilized to examine whether apoptosis occurred normally in these treated embryos, or if extensive cell death is the cause of the malformations (R.W. Oppenheim, 1999). Propidium iodide (PI) intercalates into double-stranded nucleic acids. PI is excluded by viable cells but can penetrate cell membranes of dying or dead cells. The cyclopamine treated embryo will be compared with a control embryo to observe the specific causes of the malformations.

©Cebra-Thomas, 2001

Last Modified: 3 May 2001

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