TLP-1 is an asymmetric cell fate determinant that responds to Wnt signals and controls male tail tip morphogenesis in C. elegans

Xiaojun Zhao 1 Ying Yang 0 David H. A. Fitch 0 Michael A. Herman ) 1 0 Department of Biology, New York University , New York, NY 10003 , USA 1 Program in Molecular, Cellular and Developmental Biology, Division of Biology, Kansas State University , Manhattan, KS 66506 , USA SUMMARY We have isolated mutations defining a new gene, tlp-1, that affect asymmetric cell fates and morphogenesis during the development of the C. elegans tail. tlp-1 mutations cause defects in the specification of asymmetric cell fates in the descendants of the T blast cell, whose polarity is controlled by Wnt signaling and cause abnormal male tail development leading to the formation of a posterior protrusion reminiscent of leptoderan, or pointy tailed, nematode species. In wild-type C. elegans males, which have a peloderan or rounded tail, retraction of the tail tip hypodermis involves a temporally ordered set of cell fusions and changes in cell shape that appear to be heterochronically delayed in tlp-1 males, suggesting that subtle changes in these events can bring about evolutionary changes in morphology. tlp-1 encodes a C2H2 zinc-finger protein that is a member of the Sp family of transcription factors. A TLP-1::GFP fusion protein is expressed in the Two fundamental questions in developmental biology are understanding how morphogenesis is controlled and how changes in morphogenesis during evolution lead to the elaboration of different forms. For morphogenesis to occur properly during development, many cellular processes must be correctly controlled; among these are the establishment of different cell fates through asymmetric cell divisions, cell polarity and cell fusion. Changes in any of these processes can lead to changes in morphology that over time could lead to the genesis of different forms. The polarities of specific cells in the C. elegans tail are controlled by Wnt signaling. The conserved Wnt signaling pathway is one of the major signaling pathways controlling animal development (reviewed by Cadigan and Nusse, 1997; Wodarz and Nusse, 1998). Through genetic and molecular studies, the identification of the components that function in a canonical signaling pathway, as well as the probable order of their action, has been determined. In short, Wnt signals are short-range secreted signals that act through Frizzled (Fz) nuclei of many cells during early embryogenesis and then becomes restricted primarily to posterior cells. At hatching, it is expressed in several head neurons, the posterior intestine cells, tail hypodermal cells, the T cells and specific T-cell descendents in a pattern that suggests TLP-1 may be asymmetrically expressed during the divisions of the T cell lineage. Furthermore, the asymmetry of TLP-1 expression and function appears to be controlled by Wnt signals that control T cell polarity. These results suggest that tlp-1 encodes a transcription factor required for cellular asymmetry that functions downstream of Wnt signals that control cell polarity, as well as in cell fusion and patterning in the C. elegans tail. receptors at the cell surface. Through the action of Dishevelled inside the cell, b -catenin is stabilized, causing it to accumulate in the cytoplasm and the nucleus, where it interacts with Tcf factors to activate target genes. Mutations in the Wnt gene lin-44 cause the polarities of certain cells in the C. elegans tail the B, TL and TR cells to be reversed (Herman and Horvitz, 1994; Herman et al., 1995). LIN-44 is expressed by the epidermal cells at the tip of the developing tail, which are posterior to the cells whose polarities are affected by lin-44 mutations. LIN-44 function is required within the tail tip cells, suggesting that LIN-44 is secreted by these cells and affects the polarity of more anterior asymmetric cell divisions (Herman et al., 1995). Mutations in the Frizzled-related gene lin-17 cause a loss of polarity in the same cells in which lin-44 mutations cause a reversal of polarity (Sternberg and Horvitz, 1988; Sawa et al., 1996), suggesting that LIN-17 serves as the receptor for LIN-44. The Wnt pathway that controls the polarity of the TL and TR cells shares some components with the canonical Wnt pathway, such as the requirement for the Tcf homolog, POP-1, but may not require other components such as b -catenin (Herman, 2001), suggesting that it is a novel Wnt pathway. The defects in cell polarity observed in lin-44 and lin-17 mutants lead to defects in morphogenesis as evidenced by the formation of abnormal male tail structures in both mutants (Sternberg and Horvitz, 1988; Herman and Horvitz, 1994). Another cellular function that often affects morphogenesis is cell fusion. In vertebrates, the formation of multinucleated cells, or syncytia, is involved in the formation of skeletal muscles, bones and, in mammals, the placenta. In C. elegans, one third of all the somatic nuclei in the adult animal are part of one of a few syncytia that are formed in reproducible spatial and temporal patterns (Shemer and Podbilewicz, 2000). For example, a detailed analysis of male tail development has revealed that a specific temporal order of cell fusions and cell shape changes lead to the formation of the male tail. C. elegans male tail tip morphogenesis begins after the bursal ray cell divisions are complete (Nguyen et al., 1999). The tail tip cells then lose adhesion with the cuticle and recede anteriorly. This tail tip retraction is accompanied by changes in cell shape, position and cell contacts. The retraction of the tail tip causes the adult male tail to be blunt ended, or peloderan, where the bursal fan extends posteriorly and envelopes the tail tip. By contrast, these changes in cell shape, position and cell contacts do not occur in hermaphrodites, and the pointed larval shape is retained in the adult. In other nematode species, such as Oscheius myriophila, the male tail tip cells also fail to retract during morphogenesis of the tail, causing the adult male tail to be pointed, or leptoderan, and protrude beyond the posterior edge of the adult fan (Nguyen et al., 1999). To further understand the roles cell polarity and cell fusions play in morphogenesis, we have isolated mutations identifying a new gene, tlp-1 (T cell lineage defective and leptoderan tail). We show that tlp-1 is required for the specification of asymmetric cell fates in the descendants of the T cell, as well as for cell fusions and cell shape changes of the tail tip hypodermal cells that lead to drastic changes in male tail morphology. tlp-1 encodes a nuclear-localized C2H2 zincfinger protein and is asymmetrically expressed during the divisions of the T cell lineage. Expression studies and cell lineage analysis in double mutants suggest that tlp-1 is expressed in response to lin-44 and lin-17 genes in the control of T cell polarity. Our results suggest that tlp-1 encodes a transcription factor required for morphogenesis of the C. elegans tail by regulating asymmetric cell fates i (...truncated)