Figure 1.
DTC migration defects of blmp-1 mutants.
(A) Schematic diagram showing the path and direction of DTC migration in phases I, II, and III. The developmental stage in which each migration phase occurs is indicated on the right. The solid line and arrow show, respectively, the path and direction of each migration phase. (B–F) DIC images of adult wild-type (B) and blmp-1(s71) (C–F) gonads. The black line shows the migratory path, starting from the asterisk, the DTC is indicated by the black arrowhead, and the white arrowhead indicates the position at which the DTC initiated dorsal phase II migration. In (C–F), the DTC had a shorter phase I migratory path (distance between the asterisk and white arrowhead) than the wild-type DTC (B); in addition, the DTC in (C) had a longer phase III migration path, that in (D) had a slanted phase II migration path (see text for details), and that in (E) moved in the opposite direction during phase III migration. (F) The DTC had a similar migratory trajectory to that shown in (E), except that it failed to stay on the dorsal side during phase III migration. The gonadal arm shown in B–E is posterior, while that in F is anterior, as the defect was only observed in this arm, as shown in Table S1. The picture in F was flipped 180 degrees so that its migration trajectory could be easily compared to those in B–E. Scale bar 40 µm. (G) Percentage of the indicated blmp-1 mutants and transgenic worms with a DTC migration defect (shown in Figure 1C–F). A: anterior DTC, P: posterior DTC. At least 50 worms were scored for each genotype.
Table 1.
Genetic interactions between blmp-1 and the heterochronic genes lin-29, dre-1, and daf-12 in the timing of the DTC dorsal turn.
Figure 2.
Gene structure of blmp-1 deduced from genomic and cDNA sequences. The boxes indicate exons. The regions encoding the PRDI-BF1-RIZ1 homologous region (PR) domain, nuclear localization signal (NLS), and zinc finger motifs are marked, as is the trans spliced leader SL1. The positions of the blmp-1 mutant alleles, including the region corresponding to the tm548 deletion, are indicated.
Figure 3.
DTCs in the blmp-1 mutant undergo a precocious dorsal turn.
(A) DIC images of wild-type (WT) and blmp-1(s71) posterior gonadal arms during the early L3 (top panels) and late L3 (bottom panels) stages. (a–d) In early L3, when the worms were in the one-P6.p-cell stage (b, d), the wild-type DTC (a) was still in phase I migration, while the blmp-1 DTC (c) had already made a dorsal turn. (e–h) In late L3, when the worms were in the four-P6.p-cell stage (f, h), the wild-type DTC (e) had just begun the dorsal turn, while the blmp-1 DTC (g) had already completed the dorsal phase II migration and undergone centripetal phase III migration. In the left panel, the arrow and dotted line indicate, respectively, the DTC and its migratory path. In the right panel, the arrowhead indicates the P6.p division stage of the same worm shown in the left panel. (B) The percentage of worms with anterior (left) and posterior (right) DTCs that initiated the phase II dorsal turn at the indicated division stage of the P6.p cell (shown in the center) in wild-type (black bars) and blmp-1(s71) (gray bars) worms. At least 33 worms were examined for each genotype.
Figure 4.
BLMP-1 expression pattern and its regulation.
(A) Representative images of a wild-type embryo (a) or larva (b–d) stained with anti-BLMP-1 antibodies. (a) In the 1.5-fold embryo, BLMP-1 is detected in Hyp7, V cells, and P cell precursors. (b–d) In the larva, BLMP-1 is detected in hypodermal and seam cells (b and c), vulval cells (c), and posterior intestinal cells (d). Scale bar 40 µm. (B) BLMP-1 levels in wild-type DTCs at different larval stages revealed by immunostaining with anti-BLMP-1 antibody (a–c) or together with DAPI staining (d–f). (a) BLMP-1 is detected during centrifugal phase I migration in L2. (b, c) No BLMP-1 is detected during dorsal phase II migration in late L3 (b) or during centripetal phase III migration in L4 (c). (d, e, f) The same worms as those in, respectively, a, b, or c stained with DAPI to label nuclei and to examine the developmental stage of the gonad. Scale bar 20 µm. The arrowheads indicate DTCs. (C) The DIC (a, b) and immunostaining (c, d) images of blmp-1(s71) (a, c) and blmp-1(tm548) (b,d) embryos stained by anti-BLMP-1 antibodies. Scale bar 10 µm. (D) Presence of BLMP-1 at the L4 stage in the DTCs of the double mutants lin-29;dre-1 (a) and dre-1;daf-12 (b), as revealed by immunostaining with anti-BLMP-1 antibodies. (c, d) The same worms as those in a and b, respectively, stained with DAPI. Scale bar 20 µm.
Figure 5.
Effects of heterochronic mutations on the transcription of unc-5 and lin-29 in DTCs.
(A) Persistent BLMP-1 expression in DTCs suppresses unc-5 transcription during late larval development. DIC, GFP, and mCherry images at the mid L4 stage of two worms carrying the Pblmp-1::bmp-1::gfp and Punc-5(4.6 kb)::mCherry transgenes. (a–c) A representative worm in which BLMP-1 was normally down-regulated. The DTC has undergone a dorsal turn (a) and does not express BLMP-1::GFP (b), but expresses unc-5. (d–f) A representative worm in which BLMP-1 was still expressed. The DTC has failed to turn dorsalward and is moving centrifugally (d), shows persistent BLMP-1::GFP expression (e), and does not express unc-5 (f). The arrows indicate DTCs and the asterisks the developing vulva. Scale bar 40 µm. (B) The blmp-1 mutation causes precocious unc-5 transcription through precocious expression of lin-29. (a–c) DIC and GFP images of the posterior DTC at the early L3 stage in a wild-type worm (a), a blmp-1(s71) worm (b), and a Plag-2::lin-29-expressing worm (c). All worms carried a Punc-5(4.6 kb)::gfp transgene. Scale bar 20 µm. (C) DIC and GFP images of transgenic animals carrying Plin-29::gfp (a) during late L3 stage. The transgenic animal carrying Plin-29::gfp treated with control vector RNAi (b) or blmp-1 RNAi (c) during the early L2 stage. The arrows indicate DTCs. Scale bars 20 µm.
Figure 6.
blmp-1 is down-regulated at the transcriptional level by lin-29 and daf-12.
(A) Schematic diagram of the Pblmp-1::dgfp transcriptional reporter. (B) DIC (left) and dGFP (right) images of worms carrying the Pblmp-1::dgfp reporter in the indicated genotypes during the early L3 stage (upper panels) or L4 stage (lower panels). Scale bar 10 µm. (C) Percentages of worms with the posterior DTC expressing the Pblmp-1::dgfp transgene (GFP+ DTC) at the indicated developmental stages. More than 20 worms were examined for each genotype at each developmental stage.
Figure 7.
The stability of the GFP::BLMP-1 fusion protein is negatively regulated by dre-1.
(A) Schematic diagram of the Plag-2::gfp::blmp-1 construct. (a–d) DIC (left) and GFP (right) images of wild-type (WT) and dre-1(RNAi) worms carrying the Plag-2::gfp::blmp-1 transgene during early L3 stage (top panels) or L4 stage (bottom panels). (B) Schematic diagram of the Plag-2::gfp construct. (a–d) DIC (left) and GFP (right) images of wild-type (WT; top panels) and dre-1(RNAi) (bottom panels) worms carrying the Plag-2::gfp transgene during early L3 stage (top) or L4 stage (bottom). L3m: L3 molt. In both A and B, the scale bars are 10 µm, the arrows indicate DTCs, and the DTC nuclei are circled in the GFP images. (C) Percentage of worms with DTCs expressing the indicated GFP reporter (GFP+ DTCs) in the transgenic animals at the indicated larva stage. More than 20 animals were examined in each genotype at each larval stage.
Figure 8.
BLMP-1 and DRE-1 are co-immunoprecipitated in human cell cultures, as are their human orthologs PRDI-BF1 and FBXO11.
HEK293T cells were transfected with the indicated plasmids. Expression of the indicated plasmids in the whole cell lysate (WCL) is shown in the bottom panels, with tubulin or GAPDH as the loading control. (A, B) Co-immunoprecipitation of C. elegans Myc-DRE-1 and HA-BLMP-1. (A) Myc-DRE-1 was immunoprecipitated with anti-Myc antibodies and Western blots were probed with anti-HA antibodies to detect HA-BLMP-1 and with anti-Myc antibodies to detect Myc-DRE-1. (B) HA-BLMP-1 was immunoprecipitated with anti-HA antibodies and Western blots were probed with anti-Myc antibodies to detect Myc-DRE-1 and with anti-HA antibodies to detect HA-BLMP-1. (C) Co-immunoprecipitation of C. elegans Myc-DRE-1 and HA-BLMP-1. HA-PRDI-BF1 was immunoprecipitated with anti-HA antibodies and Western blots were probed with anti-Myc antibodies to detect Myc-FBXO11 and with anti-cullin 1 antibodies to detect cullin 1.
Figure 9.
Spatiotemporal regulation of DTC migration.
blmp-1 and lin-42 negatively regulate the timing of the DTC dorsal turn, whereas daf-12, dre-1, and lin-29 function in a redundant fashion to positively regulate the turn. Although daf-12 is activated by DAs (dafachronic acids) and lin-42 inhibits daf-12 and lin-29, they are boxed together for convenience. A double-negative feedback loop between blmp-1 and lin-29 may contribute to the switch-like turning process of DTCs (see text for detail). Gene interactions that occur in phase I migration are shown in green and those that occur in phase II migration are shown in red.