Table 1.
Accession numbers of Ftz-F1 ortholog genes used in this study.
Fig 1.
Nucleotide and deduced amino acid sequences of D. magna Ftz-F1.
(A) The DapmaFtz-F1 common region. Black and grey shaded amino acids indicate the putative DNA-binding domain (DBD) and ligand-binding domain (LBD) respectively, based on the alignment of amino acid sequences of signature domains. The Ftz-F1 box (italicized) and the activation factor-2 (AF-2) core (underlined) motifs are indicated. (B) Nucleotide sequence of DapmaFtz-F1 isoform-specific regions. Deduced amino acid sequences starting from the first methionine for each isoform are also indicated. Locations of the primers used in qRT-PCR are emboldened. Numbers on the right indicate the nucleotide and amino acid positions.
Fig 2.
Evolutionary conserved domains of D. magna Ftz-F1.
(A) Schematic diagram of the Ftz-F1 regions that are divided into A/B, C, D and E regions. (B) Alignment of the C region and the Ftz-F1 box (boxed), and (C) alignment of the E region showing the LBD signature domain (boxed) and AF-2 motif (boxed). Identical amino acids are shaded in black whereas amino acids with similar characteristics are colored in red. MeFtz-F1 is the Me. ensis (shrimp) protein; DmFtz-F1 is the Dr. melanogaster (fruit fly) protein; BmFtz-F1 is the B. mori (silkworm) protein; and TcFtz-F1 is the T. castaneum (beetle) protein. MmSf-1 is the steroidogenic factor-1 of Mu. musculus (mouse); and CeNhr-25 is the nuclear hormone receptor-25 of C. elegans (roundworm).
Fig 3.
Phylogenetic tree of the amino acid sequences of the DBD and LBD Ftz-F1 nuclear hormone receptor subfamily.
The percentages of the replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The bar indicates branch length and corresponds to the mean number of the differences (P<0.05) per residue along each branch. Evolutionary distances were computed using the p-distance method.
Fig 4.
Genomic structural organization of (A) the D. magna Ftz-F1 gene and (B) the Dr. melanogaster Ftz-F1 gene. The numbered boxes are exons, and the intervening lines are introns. Colored boxes indicate coding regions; blue represents DBD, whereas orange represents LBD. Empty boxes indicate untranslated regions. Each isoform possesses a unique coding sequence at the 5′end, with black arrows indicating the transcription start site. Scale bars are provided at the top of each diagram for the size in kilobases. Numbers I, II, III, and IV indicate the location of intron splice sites that are conserved between D. magna and Dr. melanogaster. (C) Putative conserved splice sites mapped to the conserved domain of Ftz-F1 from (1) D. magna and (2) Dr. melanogaster. The amino acid sequences shown are from DBD (Number I) and LBD (Numbers II, III, and IV). Red amino acids indicate 10 residues (five residues for pre- and post-introns, respectively) around the intron position assigned as the splice site, whereas further homology up and downstream of the intron is represented in black. Bold amino acid residues are residues shared between two species. Black triangles indicate the location of the intron within the splice site.
Fig 5.
Temporal expression profiles of the DapmaFtz-F1 gene in embryonic developmental stages of D. magna.
(A) DapmaFtz-f1 gene expression levels of the α-isoform and (B) the β-isoform in one embryo of males (blue line) and females (red line). Embryonic development was staged at 0 h (single cell egg), 6 h (late gastrulation stage), 12 h (cephalic-appendage developing stage), 18 h (early thoracic appendage-developing stage), 24 h (after hatching embryo), 30 h (middle carapace-developing stage), 48 h (further developed thoracic appendages and antennae embryo), and 72 h (juvenile Daphnia) after oviposition. Results are shown as copy numbers of transcript per egg. The copy numbers were measured from three independent qPCR amplifications and error bars represent standard error values across samples.
Table 2.
Sexual differences in αFtz-F1 and βFtz-F1 expression in D. magna during embryogenesis.
Ftz-F1 expression was normalized using Ribosomal L32 expression as a reference gene. The fold difference was obtained by normalizing male expression to female expression.
Table 3.
Summary of the siRNA microinjection experiment for phenotype observation.
Excluding the Control_416 siRNA-injected embryos, all Ftz-F1 siRNA-injected embryos developed abnormally, including failure to shed the outer egg membrane and slow development compared to that observed with normal eggs.