We have identified null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. up to 105 individual cells to form a multicellular aggregate. The ligand for chemotaxis is extracellular cAMP that activates a series of second messenger pathways through cAMP, G protein-coupled, serpentine receptors that result in the reorganization of the actin/myosin cytoskeletons and directed movement of cells toward the aggregation center (Parent is preferentially expressed during aggregation and mound formation. null cells are unable to properly polarize or chemotax in cAMP gradients and arrest development at the tight mound stage. The LIM family of proteins, named for the founding members lin-11, isl1, and mec-3, is a group of proteins containing at least one zinc binding LIM domain with the primary structure CX2CX16C23CX2CX16C23CX2C that function as domains for proteinCprotein interactions. LIM domain-containing proteins were discovered as genes required for proper development in two disparate systems: and vulval cell and Tonabersat mechanosensory neuron development, respectively (Way and Chalfie, 1988 ; Freyd LIM domain protein DdLim1, which is required for proper protrusion of lamellipodia during chemotaxis (Sadler null cells are unable to properly organize their actin cytoskeleton or polarize in a chemoattractant gradient, leading to specific defects in cell motility and an inability to properly chemotax and undergo morphogenesis. Our studies provide new insights into the regulatory pathways required for cell sorting during morphogenesis in multicellular organisms. MATERIALS AND METHODS Cell Biology, Development, and Staining Tonabersat Wild-type strain KAx-3 cells were grown and transformed using standard techniques (Nellen strain B/r (Hughes as a Tonabersat food source and developed. Clones were screened for those that were unable to develop past the mound stage. Such strains could be defective in the ability to undergo morphogenesis, such as sorting of the prestalk cells to the anterior, or in cell-type differentiation. interrupted cells were identified as one of the strains that arrested at the mound stage (did not form a tipped aggregate on SM agar plates). Plasmid rescue was performed by digesting genomic DNA isolated from this clonal line with was obtained by using a partial sequence obtained from the rescued REMI plasmid as a probe to screen a ZAP cDNA library described previously (Schnitzler cDNA library. In this screen, we obtained clones containing the AUG initiation codon but never obtained the region encoding the C terminus of the protein and the termination codon. This is probably due to the presence of a high number of codons for lysine and glutamic acid residues near the C terminus, which may act as the binding site for oligo(dT) during library construction, because the sequence is very rich in A residues and mimics the 3 terminal poly(A) of mRNAs. The C terminus was obtained by cloning a genomic fragment, which contained part of the ORF and the UAA termination codon of LIM2 (our unpublished results). All constructs were expressed as stable G418-resistant transformants downstream from the promoter, or the prestalk-specific promoter as described previously (Dynes (Cambridge, UK). For the mutant Y45F, the mutation primer used was (5-CATTTGGATCTTTAAATGTAAGATATGTTGGTGC-3). For the C290S mutant, the mutation primer was (5-CACGTAACTTGTCACCACTACCTGAACTTGAGAAACACTCTGG-3). The mutation primer for C414S was (5-CCAGCGAATGGTACTTGACTAGTGGTACTAGTGAAATGTTCTGG-3). Serping1 The constructs containing these point mutations were sequenced to verify the proper substitutions and determine the absence of any other mutations. For expression of myc-tagged LIM2, PCR was carried out with the sense (5-GTTTTTACTAGTAAAAAAATGGAACAAAAATTAATTTCAGAAGAAGATTTAAATGCAAATAAAAATGTATTATCAG-3) and the antisense (5-CATGACAAGTTTTACCCAC-3) using the cDNA as a template. The resulting fragment was cut with cDNA as a template, were cut with cDNA Tonabersat as a template, and the primers sense (T3 primer) and antisense (5-GTTTTTCTCGAGAAATTAAGCTTCAGCTTTTTTATCACC-3), and cut with null cells expressing each.