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, 12 (5), 683-98

AFF-1, a FOS-1-regulated Fusogen, Mediates Fusion of the Anchor Cell in C. Elegans

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AFF-1, a FOS-1-regulated Fusogen, Mediates Fusion of the Anchor Cell in C. Elegans

Amir Sapir et al. Dev Cell.

Abstract

Cell fusion is fundamental for reproduction and organ formation. Fusion between most C. elegans epithelial cells is mediated by the EFF-1 fusogen. However, fusion between the anchor cell and the utse syncytium that establishes a continuous uterine-vulval tube proceeds normally in eff-1 mutants. By isolating mutants where the anchor-cell fails to fuse, we identified aff-1. AFF-1 ectopic expression results in fusion of cells that normally do not fuse in C. elegans. The fusogen activity of AFF-1 was further confirmed by its ability to fuse heterologous cells. AFF-1 and EFF-1 differ in their fusogenic activity and expression patterns but share eight conserved predicted disulfide bonds in their ectodomains, including a putative TGF-beta-type-I-Receptor domain. We found that FOS-1, the Fos transcription factor ortholog that controls anchor-cell invasion during nematode development, is a specific activator of aff-1-mediated anchor-cell fusion. Thus, FOS-1 links cell invasion and fusion in a developmental cascade.

Figures

Figure 1
Figure 1. aff-1 Mutants Exhibit AC Fusion Failure Phenotype
Animals in (C)–(H) are in early- to mid-L4 larval stage, and (I) and (J) are gravid adults. In all the panels hereafter, anterior is to the left; ventral down. (A) Schematic view of AC (green) and utse cell precursors (π) before cell fusion. The vulval primordial epithelial cells (blue) invaginate connecting the epidermis (hypodermis) with the uterus. (B) Formation of the utse and cell-cell fusion of AC with π cell daughters connect the uterus through the vulva. (C, E, and G) In wild-type as in eff-1 and aff-1 null alleles the AC is correctly localized on the vulva apex (arrowhead). (D) In wild-type animal, fusion of the AC to the utse syncytium resulted in the formation of a hymen/utse layer (arrow) between the vulval and uterine lumens. (F) In eff-1 mutant, normal AC fusion resulted in hymen formation, indicating that eff-1 is not required for AC fusion. (H) In aff-1 mutant, the AC failed to fuse and was retained at the uterus-vulva junction (arrowhead). (I) Adult animal exhibiting normal development of embryos that are laid from the uterus at the 50–100 cell stage. (J) Strong Egl phenotype of aff-1 mutant hermaphrodite. Embryos complete embryonic development in the uterus, and the larvae hatch inside the mother. The scale bar represents 5 μm in (A)–(H) and 20 μm in (I) and (J).
Figure 2
Figure 2. aff-1 Activity Is Required for AC Fusion
(A) Scheme of aff-1 gene structure with mutations and construct annotations. The first methionine is substituted to isoleucine in ty4 mutation while the tm2214 deletion (red line) introduced a stop codon after alanine 47. The sequence that was used as the template for dsRNA experiments is marked in green and the 8 kb PCR-based rescue fragment in blue. (B) Phenotypic analysis of aff-1 alleles. AC fusion failure and low fertility in ty4 and tm2214 mutants. aff-1 dsRNA phenocopy aff-1 mutant phenotype. ty4 phenotype is rescued by an 8 kb fragment from aff-1. Error bars indicate standard deviation. Asterisks indicate difference from wild-type with statistical significance of p < 0.001 according to unpaired two-tailed t test. (C) Sequence alignment of C. elegans fusogens with their putative homologs from P. pacificus and selected members of the TGF-β type I receptors. The alignment is limited to the structurally defined part of the Hs BRIA sequence. Secondary structures presented under the alignment refer to the solved crystal structure of BRIA. Nine cysteines (pink) are conserved between all the aligned proteins, suggesting that these proteins share a similar structural fold. An additional cysteine followed by asparagine (green) that are part of TGF-β binding domain are not conserved in EFF-1 and AFF-1 proteins. Abbreviations: Ce, C. elegans; Pp, P. pacificus; Dm, D. melanogaster; Xe, X. laevis; Hs, H. sapiens; BRIA, BMP receptor IA extracellular domain; TGF, TGF-βRI. Alignment color code was according to the ClustalX color scheme Jalview software. Accession numbers: Ce AFF-1: EF205023; Pp AFF-1: contig1480; Ce EFF-1: GI:19071563. Pp EFF-1: Contig2476 (http://www.pristionchus.org/cgi-bin/seq_retrieval.pl); Dm Baboon: gi|33589356; Xe ALK4: gi|49903662; TGF Hs: gi|4759226; BRIA Hs: gi|48425316. See Experimental Procedures.
Figure 3
Figure 3. aff-1 Is Required for AC Fusion prior to Cytoplasmic Mixing
AC, arrowhead; utse syncytium, arrows. Nomarski (left) fluorescence (center) and overlaid (right) images of vulval-uterine area in critical intermediates of AC development during L3 to adult. (A) AC invasion in the L3 stage was detected by a cadherin promoter driving GFP expression (cdh-3p::GFP; [Sherwood and Sternberg, 2003]). (B) In aff-1 mutant, AC invasion is normal. (C) In wild-type, cytoplasmic mixing between AC and utse cells is detected by diffusion of the AC marker cdh-3p::GFP to the utse (arrows); VulD represents vulval ring “D.” (D) In aff-1 mutant, cdh-3p::GFP retention in the AC demonstrates that cytoplasmic mixing does not occur (arrowhead). (E) At the vulval “Christmas tree” stage, the AC and utse syncytium form a thin layer between vulva and uterus lumens in wild-type. (F) In aff-1 mutant, this layer is not formed and the unfused AC lies at the vulva-uterus junction (arrowhead). (G) Normal adult vulva after eversion. (H) Unfused AC remains at the apex of the everted aff-1 vulva (arrowhead). All panels are at the same magnification; the scale bar corresponds to 5 μm.
Figure 4
Figure 4. aff-1 Is Required for Fusion Events in Other Tissues
(A–D) Nomarski (center) and the corresponding fluorescence image in selected stages of vulval development of the apical junction marker AJM-1::GFP that marks epithelia cell borders. (A) In wild-type worms, 12 primordial vulval cells are located at the ventral side at late L3 stage. (B) A similar pattern in aff-1 mutants shows that aff-1 does not affect VPCs proliferation. In addition, the fusion of 3° fate VPCs to the epidermis is normal. (C) Fusion of vulval cells results in the formation of vulval rings in wild-type (for example vulD ring, fusion marked with an arrow in inset). vulA represents a single ventral ring (arrow). (D) In aff-1 deletion, the two D cells did not fuse; hence the D ring is unfused (arrow in insert). The A cells fail to fuse before ring formation and two vulA rings form instead of one (arrows). (E–H) Selected stages of seam cell development in wild-type (E and F) and aff-1 mutant worms (G and H) examined by the AJM-1::GFP marker. (E) In wild-type L3 stage, 16 seam cells are on each side of the body, separated by apical junctions (left view). (F) During late L4/early adult, seams undergo cell fusion that results in a long syncytium marked by two parallel lines of AJM-1::GFP; see insert and top of (A). (G) In aff-1 mutant, early seam development is similar to wild-type. (H) During late L4, the seam syncytia did not form, so individual cells are detected and remained unfused in adults. Insert shows detail with unfused apical junctions, arrows. (A, B) and (C, D) are panels with same magnification. The scale bars in (A) and (C) represent 10 μm. (E–H) Scale bar corresponds to 50 μm. V, vulva.
Figure 5
Figure 5. AFF-1 Fuses C. elegans Hypodermal Cells and Heterologous Insect Cells
(A–F) Comma to 1.5-fold stage embryos, anterior to the left and ventral down. (A–D) Confocal projections of embryos from different genetic backgrounds all marked with the apical junction marker AJM-1::GFP. Punctuated staining is due to the background of the GFP. (A) In wild-type embryos, dorsal hypodermal cells undergo fusion (arrowheads). The seam cells do not fuse during embryogenesis (arrows). (B) hsp::aff-1 embryos after heat shock. The disappearance of apical junction between individual cells suggests that AFF-1 causes fusion of the hypodermal cells. (C) eff-1 mutant embryos where most embryonic fusions do not occur. Arrowheads mark some unfused dorsal cells. (D) The lack of eff-1 does not attenuate AFF-1-induced fusion, indicating that AFF-1 acts in an eff-1-independent mechanism. (E and F) Individual frames from time-lapse movies (see Supplemental Data) of control (E) and hsp::aff-1 worms (F) marked by AJM-1::GFP and by eff-1p::GFP that is distributed in the cytoplasm of individual hypodermal cells. The time after heat shock appears on top right. (E) In non-heat-shocked embryos, dorsal fusion is normal while individual seam cells did not fuse (arrows; see Movie S1). (F) In the intermediate step of the heat shock effect (37 min), diffusion of GFP from ventral hypodermal cell to a single seam cell (arrow) concomitant with apical junction removal between these cells indicates that aff-1 is sufficient to induce cell fusion ectopically. In addition, fusion between seams is observed (arrows). The cytoplasmic GFP diffuses through hyp6, hyp7, and seam cells (Movie S2). (G) AFF-1 protein tagged with V5-6XHis epitopes was expressed in Sf9 cells and detected from the cell lysate as a single specific band of apparent MW of 75 kDa by western blot with anti-V5 antibodies. (H) Immunofluorescence with anti-V5 antibodies (red), DAPI staining (blue) on aff-1-expressing cells (green). The lower three cells do not express the construct. (I) AFF-1 protein (red) is distributed at the cell surface and in intracellular puncta. (J–M) AFF-1 expression in a pentanucleate cell. (J) Cell nuclei are marked by DAPI staining (blue) merged with DIC. (K) AFF-1 protein immunostaining (red) and DAPI (blue). (L) Five distinct nuclei (1–5) are detected in the syncytium. (M) AFF-1 protein is localized to the plasma membrane. (N) Ectopic expression of AFF-1 results in multinucleated Sf9 cells 24 hr after transfection. Percentages of multinucleation with respect to aff-1 DNA concentration are shown (filled triangles and blue line). The multinucleation of control cultures transfected with empty vector is marked by an empty triangle. (O) Cell surface AFF-1 induces multinucleation more potently than EFF-1. Percentages of multinucleated cells (empty columns) and surface expression in relative units (gray columns) of empty vector, EFF-1, AFF-1 and a chimera between AFF-1 extracellular domain and EFF-1 transmembrane and cytoplasmic domain (AFF-1::EFF-1cyto; see Supplemental Data). (A–F) Scale bars represent 10 μm; (H and I) 20 μm; (J–M) 10 μm. Error bars represent standard error and stars represent statistical significance of p < 0.05.
Figure 6
Figure 6. Correlation between aff-1 Expression and Its Fusogenic Effect
(A–F) Nomarski (left) aff-1 promoter GFP fusion (aff-1p::GFP) fluorescence signal (center) and overlaid (right) images. (A) At mid-L3, aff-1 is expressed specifically in the AC (arrowhead). (B) AC expression is retained while the vulva invaginates during early L4. (C) Expression of the aff-1 transcriptional reporter is induced in the vulval D ring cells (arrows) and in the AC-utse syncytium as it is formed in late L4 stage (arrowhead). (D) A different focal plane from the same worm shown in (C) where the AC-utse expression is highlighted (arrows). (E) The vulD expression of aff-1 is retained in adult worms (arrows). (F) In lin29(n482) mutants, AC and utse fusion do not occur (Newman et al., 2000). A single AC (arrowhead) is localized adjacent to a single utse cell (arrow). aff-1 signal in utse cells that did not fuse with the AC cell demonstrates that aff-1 is autonomously expressed in the utse cells. (G) Confocal projection of aff-1 expression pattern in L4 larva (dorsolateral view). aff-1 is expressed in VulD ring, the utse, and in the seams. Seam cell expression starts at mid-L4 just before aff-1-dependent fusion. (H) Confocal projection of the H-shaped utse syncytium where aff-1 is expressed (ventral view). In addition, aff-1 expression is detected in two rows of lateral seam cells and in the two uterine ut4 toroids (Ut4). (I) Subcellular localization of AFF-1::GFP protein in the AC during invasion (lateral view of confocal image). The fusion protein was specifically expressed in the AC by the anchor-cell-specific promoter pAC (Kirouac and Sternberg, 2003). AFF-1::GFP protein is localized in intracellular compartments and at the plasma membrane (arrowheads). (J) Similar subcellular localization of AFF-1::GFP was detected at the time of AC fusion in a confocal image (lateral view). (A–F) Scale bar 5 μm; (G) 10 μm; (H) 20 μm.
Figure 7
Figure 7. FOS-1 Controls AC Fusion via aff-1 Expression in utse and AC
(A–C) Nomarski (left), AC marker cdh-3p::GFP (center), and overlaid (right) images of fos-1(ar105) mutant L3 to adult. (A) AC invasion did not occur in fos-1 mutants, indicated by the retention of basement membrane between the gonad and the ectoderm (Sherwood et al., 2005) (arrowhead). (B) In fos-1 mutant L4 larva, the cdh-3p::GFP-labeled AC did not fuse (arrowhead). (C) This fusion failure persists until adulthood (arrowhead). (D and E) Images of fos-1(ar105) mutants. No-marski (left), aff-1 promoter GFP (center), and merged (right). (D) At the stage of AC invasion (L3), AC does not invade in fos-1 mutant (arrowhead; 100%, n = 36) and expresses aff-1 very weakly or is undetectable (97%, n = 33) in comparison to wild-type (see Figure 6A). (E) aff-1 was not detected in the AC or other uterine cells while still retaining expression in VulD ring and in the seam cells (not shown) compared with wild-type (L4; Figures 6B–6D). (F) Schematic representation of FOS-1-mediated regulation of aff-1 RNA (green) and AFF-1 protein (red arrows). FOS-1 controls aff-1 expression during AC invasion. During π/utse differentiation, AFF-1 is expressed in the AC only. After utse AFF-1-independent fusion, the utse syncytium starts expression of aff-1 RNA. Once AC and utse syncytium both express AFF-1, AC-utse fusion occurs. (A)–(E) are with the same magnification. Scale bar represents 5 μm.

Comment in

  • The First Family of Cell-Cell Fusion
    JM White. Dev Cell 12 (5), 667-8. PMID 17488618.
    Our understanding of the developmentally critical process of cell-cell fusion has been greatly advanced by the identification of the first family of cell-cell fusion prot …

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