The C. elegans tailless/Tlx homolog nhr-67 regulates a stage-specific program of linker cell migration in male gonadogenesis

Abstract

Cell migration is a common event during organogenesis, yet little is known about how migration is temporally coordinated with organ development. We are investigating stage-specific programs of cell migration using the linker cell (LC), a migratory cell crucial for male gonadogenesis of C. elegans. During the L3 and L4 larval stages of wild-type males, the LC undergoes changes in its position along the migratory route, in transcriptional regulation of the unc-5 netrin receptor and zmp-1 zinc matrix metalloprotease, and in cell morphology. We have identified the tailless homolog nhr-67 as a cell-autonomous, stage-specific regulator of timing in LC migration programs. In nhr-67-deficient animals, each of the L3 and L4 stage changes is either severely delayed or never occurs, yet LC development before the early L3 stage or after the mid-L4 stage occurs with normal timing. We propose that there is a basal migration program utilized throughout LC migration that is modified by stage-specific regulators such as nhr-67.

Fig. 1.

Linker cell migration. By the late L1 stage, the linker cell (LC; pink) has been specified and is positioned to become the leader cell in the male gonad (blue). The LC migrates from the early L2 through the mid-L4 stage. The red arrows indicate the LC migratory path during each of these larval stages. It begins by migrating anteriorly on the ventral bodywall, then turning from the ventral to dorsal side during the L2 molt. It migrates posteriorly during the L3 and L4 stages. In the mid-L3 stage, it performs a second turn from the dorsal back down to the ventral bodywall. In all figures, anterior (A) is left; posterior (P) is right; dorsal (D) is top; ventral (V) is bottom.

Fig. 2.

nhr-67 is required for gonad migration during the L3 and L4 stages. (A-C) Comparison of gonad migration in wild-type and nhr-67(RNAi) animals. The gonads of wild-type (Aa,Ba,Ca) and nhr-67(RNAi) (Ab,Bb,Cb) animals are shown at the early L3 stage (A), the L3 molt (B), and the late L4 stage (C). (A) The gonads appear identical in the early L3 stage. The LCs are indicated by arrows and the gonads are outlined in yellow. (B) By the L3 molt, the gonad has turned from the dorsal to the ventral side in the wild type (Ba) but not in the nhr-67(RNAi) animal (Bb). Also, the gonad migration is slower in the nhr-67(RNAi) animal. (C) By the late L4 stage, in the wild-type animal the gonad has completed its migration, having reached the cloaca (Ca, arrowhead). In the nhr-67(RNAi) animal, the gonad does not complete its migration, but does finally turn ventrally (Cb). (D) Summary of the wild-type versus nhr-67 gonadal characteristics for the early L3 stage, L3 molt, and late L4 stage. (E,F) Nomarski images (top) and fluorescence images (bottom) of gonads in an early L3 stage (E) and an early/mid-L4 stage (F) wild-type animal. nhr-67::GFP is expressed in the LC (arrow). Scale bars: 20 μm.

Fig. 3.

nhr-67 negatively regulates unc-5 in the L3 stage and positively regulates zmp-1 in the L4 stage. (A) unc-5::GFP expression in the LC during the L3 and L4 stages remains constant in wild-type animals (top row), but becomes progressively stronger in nhr-67(RNAi) animals (bottom row). (B) Summary of the levels of unc-5::GFP fluorescence in the LC of wild-type and nhr-67(RNAi) animals. (C) Nomarski image of an animal overexpressing UNC-5 in the LC during the L3 and L4 stages. In this nhr-67::unc-5-expressing animal, the LC continues to migrate on the dorsal bodywall instead of turning ventrally in the mid-L3 stage. (D) The LC expresses zmp-1::GFP during the L4 stage in wild-type animals. The gonad is outlined in yellow and the LC is indicated by an arrow. Nomarski, top; fluorescence, bottom. (E) In nhr-67(RNAi) animals, zmp-1::GFP is not expressed in the L4 stage LC. (F) Summary of the expression of fluorescent markers in the LC of wild-type versus nhr-67(RNAi) animals in the L4 stage. Genes that are expressed throughout LC migration, such as lag-2, him-4, mig-2 and gon-1, are not regulated by nhr-67. By contrast, zmp-1 and unc-5, which have specific L3 and L4 stage expression, are regulated by nhr-67. Scale bars: 20 μm.

Fig. 4.

Changes in LC morphology at L3 and L4 stages in wild-type and nhr-67(RNAi) animals. (A) A time series of LC migration during the L3 and L4 stages showing that the LC shape becomes increasingly polarized over time. In the left column are overlays of Nomarski and fluorescence images of the male gonad (outlined in yellow) expressing the LC marker lag-2::YFP (arrow). Enlarged Nomarski (middle column) and fluorescence (right column) images show LC shape at each stage. (a,b) The LC shape is round in the L3 stage, but changes from vertically (a) to horizontally (b) oblong. (c) In the early L4 stage, the LC leading edge develops a point. (d,e) During the L4 stage, the LC becomes increasingly elongated. (f) The LC completes its migration once it reaches the U.lp/U.rp cell (arrowhead) and becomes rounded as it is engulfed by U.lp/U.rp. Scale bars: 20 μm (left) and 10 μm (middle and right). (B) In mid/late L4 stage nhr-67(RNAi) animals, the LC still has a round morphology. (C) Quantification of LC shape during the L3 and L4 stages in wild-type and nhr-67(RNAi) animals. P-value calculated by Mann-Whitney U test.

Fig. 5.

Temporal cues are primarily responsible for changes in the LC at the L4 stage. (A-D) The LC in a mid-L4 stage him-4 mutant (A,B) has a polarized shape similar to that of wild-type males (not shown), but different from a similarly positioned LC in an nhr-67(RNAi) male (C,D) of the same stage. (E,F) An L4 stage him-4 mutant correctly expresses zmp-1::GFP in the LC, even though the LC is abnormally positioned near the pharynx (Nomarski, left; fluorescence, right). The LC also has the appropriate elongated morphology of the L4 stage. (G,H) Confocal image of L4 stage male posterior body and a high-magnification image of LC show MIG-2::GFP to be membrane localized to the ventral, adherent side (arrow). (I,J) Nomarski image of nhr-67(RNAi) male showing cytoplasmic and membrane localization of MIG-2::GFP (arrow) in the LC without polarization to the adherent side. Scale bars: 20 μm in E,I; 10 μm in J.

Fig. 6.

nhr-67 regulates changes in the LC at the L4 stage independently of those at the L3 stage. Animals with a hybrid gonadal phenotype consisting of the L3 stage nhr-67 gonadal defect but a wild-type L4 stage gonad. (A) Nomarski image of a late L4 stage animal showing a gonad with the early characteristics of an nhr-67(RNAi) animal: the LC fails to turn from the dorsal to the ventral side in the mid-L3 stage and the LC (arrow) migrates slowly. (B) However, the same LC has wild-type zmp-1::GFP expression. Nomarski, top; fluorescence, bottom. Scale bars: 20 μm in A; 10 μm in B.

Fig. 7.

Model for LC migration in C. elegans. (A) LC migration consists of two levels of regulation: a basal migration program and stage-specific modifiers such as nhr-67. The basic shape of the gonad during the L1 through L4 stages is indicated in black. The basal migration program (dark blue) is implemented at the start of LC migration in the L1-to-L2 molt and continues until LC death. The genes expressed throughout LC migration and used in the basal program include lag-2, mig-2, gon-1 and him-4. nhr-67 regulates changes in LC migration from the early L3 stage through to the mid/late L4 stage (red). There are likely to be other stage-specific regulators (purple and light blue) because LC development up until the L3 stage and in the late L4 stage occurs normally in nhr-67-deficient males. In the absence of nhr-67-dependent migration, the basal migration program continues and the LC migrates but remains in its early L3 stage form for an extended time. (B) nhr-67-dependent migration can be dissected into specific components. nhr-67 negatively regulates unc-5, but positively regulates zmp-1 expression, polarization of LC shape and migration speed.