As cells differentiate, they lose the ability to be converted into other cell types; however, this terminally differentiated state is not necessarily irreversible. For instance, ectopic expression of transcription factors (TFs) like MyoD, the master regulator for skeletal myogenesis, can convert other differentiated cells into muscle (Weintraub et al.,1989). The efficiency of conversion by master regulators, however, is dependent on cellular context. While certain cells like fibroblasts convert with high efficiency, others do not. We hypothesize that some cells are refractory to reprogramming because of factors that inhibit an ectopically expressed master regulator from exerting its activity. In order to find factors that inhibit reprogramming, we have conducted a pilot RNAi screen and are following it with a clonal genetic screen. To study these reprogramming efforts, we use neuronal terminal selectors that are essential for inducing specific neuronal types. One of these factors,
che-1, a Zn-finger TF, is required for the terminal differentiation of ASE neurons, a pair of gustatory neurons in the head of the worm (Hobert,2008). Similar to other master regulatory factors, the ability of ectopically expressed
che-1 to induce ASE neuron fate in other cell types is limited. Transgenic animals containing the
che-1 gene driven by a heat shock inducible promoter and a GFP reporter driven by the promoter of an ASE marker are being used for the screen. Under non heat-shocked conditions, these animals express GFP in one ASE neuron. Upon heat shock, CHE-1 is ubiquitously expressed, however GFP is only turned on in the ASE and a few other neurons, indicating that CHE-1 is not sufficient to induce ASE-like fate. We are screening for factors that when knocked down or mutated, would allow CHE-1 to induce expression of the ASE marker in other cell types. This method has already turned out to be successful, as we have discovered that knockdown of
lin-53, a histone chaperone and a component of histone modifying complexes, allows ectopic CHE-1 to reprogram mitotic germ cells into ASE like neurons based on morphology, marker expression and antibody staining of fate specific proteins (Tursun et al.,2010). Here I will be presenting data for other candidates isolated from these screens. Some of the observed phenotypes include expression of an ASE marker in different areas of the germline, muscle, pharynx and hypodermis. Initial characterized of these putative mutant phenotypes will include scoring for the expression of more ASE and pan-neuronal markers and for the loss of tissue specific markers.