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

Xiaojun Zhao1,*, Ying Yang2,*, David H. A. Fitch2 and Michael A. Herman1,{dagger}

1 Program in Molecular, Cellular and Developmental Biology, Division of Biology, Kansas State University, Manhattan, KS 66506, USA
2 Department of Biology, New York University, New York, NY 10003, USA
* These authors contributed equally



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  		Fig. 1. Cell lineages of the T cell in wild-type and tlp-1 hermaphrodites. (A) Wild-type hermaphrodite T cell lineage. The fates of many different cells in this lineage can be distinguished by nuclear morphology using DIC microscopy (Sulston and Horvitz, 1977Go; Herman and Horvitz, 1994Go). The hyp7 cells T.aa, T.apaa and T.apap join the hypodermal syncytium, but T.apaa has a smaller nucleus and is designated hyp7(sm). PHso1, PHso2, PVW, PHC and PLN have similar nuclear morphologies. x indicates apoptosis. The seam cell (se) is a specialized hypodermal cell. (B) We analyzed T cell lineages on both sides of three ny14, two bx85 and two mh17 mutants (14 total T cell lineages). Arrows indicate that a particular cell was sometimes observed to divide in a subset of the lineages. Six ny14 lineages and one bx85 lineage had the T cell division pattern that is shown in the upper left; however, in two ny14 lineages and one bx85 lineage, the T.app cell divided once to generate two hypodermal cells. Two mh17 lineages had T cell division pattern shown in the upper right; however, in one, the T.pa cell divided to generate two hypodermal cells. Two bx85 lineages had the T cell division pattern shown in the lower left; however, in one, the T.ap cell divided to generate two hypodermal cells. Two mh17 and one bx85 had the T cell division pattern shown in the lower right corner; however, in one of the mh17 lineages, the T.pa cell did not divide and generated a hyp cell.

 


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  		Fig. 2. tlp-1 males have leptoderan-like tails. (A) Adult wild-type males have a round shaped, or peloderan, male tail morphology. (B) tlp-1 males have a pointed, leptoderan, male tail morphology. Scale bar: 10 µm.

 


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  		Fig. 3. Tail hypodermal cell defects in tlp-1 animals. Adherens junctions are labeled by JAM-1::GFP and shown in green. (A,C,E) Wild type. (A) Late L4 male, left-ventral view at a stage when the ray cells begin to cluster and the tail tip cells fuse to form the tail tip syncytium. Cell fusions indicated by punctate adherens staining. (C,E) Adult hermaphrodites, left-ventral and left views, respectively. Note that hyp11 is located dorsally above hyp8. (B,D,F) tlp-1(bx85). (B) Male, ventral view, same stage as that in A, but fusion of tail tip cells has not yet occurred. In addition, only two T-ray primordia are visible on the left side of this animal and all appear to be missing on the right side. (D,F) Hermaphrodites, left view. (D) hyp8 and hyp11 do not appear to be bounded by adherens junctions and could have fused with hyp7; adherens ‘outlines’ of phasmids are not formed or are formed aberrantly. (F) Although the adherens junctions of the phasmid look normal, the tail tip cells are not organized as in wild type, in that at least one extra cell appears in the group of tail tip cells. Scale bar: 20 µm.

 


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  		Fig. 4. tlp-1 cloning and the tlp-1 locus molecular analysis. (A) tlp-1 was mapped to LG IV between unc-26 and egl-23. Tested genomic clones are shown in relative positions below the genetic map. Red lines represent genomic clones that rescued the tlp-1 T cell defect, in which the number of transgenic lines that rescued the T cell defect is indicated (>70% of the transgenic animals were rescued). (B) Sequence analysis of genomic DNA and 1664 bp composite cDNA revealed the intron/exon structure shown. Boxes indicate exons: specifically, black closed regions indicate the open reading frame; red closed portions indicate the 3'-UTR. Position of the SL1 (trans-spliced leader sequence) and the poly(A) tail are shown. The positions of the base changes in the mh17, bx85 are indicated. (C) Schematic of the TLP-1 protein. Positions of the conserved SPLALLA, S/T-rich, Q-rich and zinc-finger domains are indicated. (D) Phylogenetic relationships of Sp-type proteins (larger font) and some other C2H2 zinc finger proteins (smaller font) used to root the Sp protein phylogeny. The names of the 45 protein sequences begin with a three-letter abbreviation of the organism binomen: Rno, Rattus norvegicus; Mmu, Mus musculus; Hsa, Homo sapiens; Dme, Drosophila melanogaster; Dre, Danio rerio; Cel, Caenorhabditis elegans; Sce, Saccharomyces cerevisiae. Only the least ambiguously aligned residues were used in the analyses. Relationships shown are only those supported with some degree of confidence (multifurcations represent ambiguous branching order, not simultaneous divergence). Numbers (1-24) identify branches with such support in at least one of several different kinds of phylogenetic analyses (see Materials and Methods). All proteins deriving from branch 12 uniquely share the SPLALLA motif, which we therefore propose is a shared-derived feature of these proteins. Branch lengths represent the minimum number of amino acid-altering nucleotide substitutions mapped onto the phylogeny by parsimony.

 


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  		Fig. 5. tlp-1 expression. A functional tlp-1 reporter construct is strongly expressed in the nuclei of the posterior intestine cells, weakly expressed in the nuclei of T and its descendent cells, as well as the tail hypodermal cells. (A-G) Fluorescence (upper) and DIC optics (lower). Scale bars: 10 µm. (A) GFP expression in most nuclei of an embryo at approximately 100 minutes when four E cell descendants were present, (B) most nuclei of a gastrulating embryo, (C) the nuclei of posterior cells in an embryo at approximately 270 minutes and (D) the nuclei of posterior cells in a 1.5-fold embryo. (E) GFP expression in the T cell. We also observed that few animals had GFP expression in the hyp9, hyp10 and hyp11 cells. (F) GFP expression in the T.p cell and (G) the T.app cell.

 


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  		Fig. 6. tlp-1 expression responds to the LIN-44 signaling pathway. (A) tlp-1::gfp expression in a lin-44 animal. Fluorescence (upper) and DIC optics (lower). Scale bar: 10 µm. (B) Cell lineages of the T cell in lin-44 and lin-44; tlp-1(mh17) hermaphrodites. We analyzed T cell lineages on both sides of four lin-44; tlp-1(mh17) double mutants. Two had the T cell lineages similar to that of tlp-1 single mutant (Fig. 1). One had the T cell division pattern shown in the upper right corner. One had the T cell division pattern shown third in the lower left corner and four had the T cell division pattern shown in the lower right corner; however, in one, T.app cell divided to generate two neural cells and T.pa divided to four hypodermal cells, and in another T.pa divided to generate four hypodermal cells. Circular arrows indicate positions of inferred polarity reversals. (C) Cell lineages of the T cell in lin-17 and lin-17(n671); tlp-1(mh17) hermaphrodites. lin-17 hermaphrodite T cell lineage is shown on the left, in which T.xx divide once or twice to generate two or four hypodermal cells (Sternberg and Horvitz, 1988Go). We analyzed T cells lineages in six lin-17(n761); tlp-1(mh17) double mutants. In three, there were no further T.xx cell divisions. In six, T.ap divided either once (two lineages) or twice (four lineages) to generate two or four hypodermal cells, respectively. In two, T.pa divided twice to generate four hypodermal cells, however in one of these T.ap also divided twice to generate four hypodermal cells. In the last one, both T.ap and T.pp divided once to generate two hypodermal cells. Arrows indicate that a particular cell was sometimes observed to divide in a subset of the lineages.

 


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  		Fig. 7. Model for TLP-1 function in the T cell lineage. tlp-1 is asymmetrically expressed and functions in the T cell lineage. LIN-44/Wnt signal emanating from the tail hypodermal cells posterior to the T cell is indicated, as are LIN-17/Fz receptors, which are presumably present on the surface of the T cell. TLP-1 expression is indicated by ‘TLP-1’. Hypodermal (hyp) T.a fate is represented by purple shading, neural (n) T.p fate is represented by green shading. Normal T.apa fate is represented by red shading and T.app fate by blue shading. TLP-1 is expressed in the T, T.p and T.app cells in wild-type animals, and affects the divisions of the T.p and T.ap cells. TLP-1 may not be expressed in T.ap, indicated by TLP-1? In lin-44 animals, TLP-1 expression and T cell polarity are reversed. In lin-17 animals, TLP-1 expression and cell polarity are lost.

 

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© The Company of Biologists Ltd 2002