We have cloned the
pha-2 gene and found that it encodes a homeodomain protein important for the development of the pharynx. Worms with mutated
pha-2 have a shorter and thickened isthmus containing several misplaced cell nuclei, including the M2 and I4 neurons that normally are within the terminal bulb. As a consequence of the morphological defects the mutants exhibit feeding problems and grow slower. By using a gfp-reporter, pPHA2::GFP-E, that is expressed in several pharyngeal cells we have studied the development of the isthmus in detail. At the beginning of the 2-fold embryonic stage, the
pm5 muscle cells, which will form the isthmus, are round shaped and positioned closely together with the
pm4-metacorpus muscle cells. During the 2 to 3-fold transition, the
pm5 cell bodies start to elongate only in an anterior direction and the cell nuclei stay in the area where the terminal bulb is forming. By the end of the 3- fold stage the isthmus has completed its elongation and has developed into a narrow muscular tube free of any cell nuclei. In
pha-2 mutants the development of the pharynx starts properly but, during the 3- fold stage, the
pm5 cell nuclei are mispositioned in the middle of the muscle cells and the elongation of the isthmus appears to be directed both anteriorly and posteriorly. At the late 3-fold stage other cell nuclei become misplaced into the isthmus, which may contribute to isthmus thickening. The forkhead gene
pha-4 is expressed in all cells of the pharyngeal primordium and is thought to regulate most pharyngeal genes. To test if
pha-2 is also a target of
pha-4, we generated transgenic worms carrying two plasmids: pPHA2::GFP-A and a plasmid with a heat-shock promoter fused to the
pha-4 gene. Heat-shocked transgenic worms expressed
pha-2 ectopically in many cells; cells that normally don't express the gene, and therefore we believe that
pha-2 is a downstream target of
pha-4.