Sp2 is a maternally inherited transcription factor required for embryonic development

Abstract

The Sp family of transcription factors is required for the expression of cell cycle- and developmentally regulated genes, and the deregulated expression of a handful of family members is associated with human tumorigenesis. Sp2 is a relatively poorly characterized member of the Sp family that, although widely expressed, exhibits little or no DNA binding or transcriptional activity in human and mouse cell lines. To begin to address the role(s) played by Sp2 in early metazoan development we have cloned and characterized Sp2 from zebrafish (Danio rerio). We report that 1) the intron/exon organization and amino acid sequence of zebrafish Sp2 is closely conserved with its mammalian orthologues, 2) zebrafish Sp2 weakly stimulates an Sp-dependent promoter in vitro and associates with the nuclear matrix in a DNA-independent fashion, 3) zebrafish Sp2 is inherited as a maternal transcript, is transcribed in zebrafish embryos and adult tissues, and is required for completion of gastrulation, and 4) zebrafish lines carrying transgenes regulated by the Sp2 promoter recapitulate patterns of endogenous Sp2 expression.

FIGURE 1.

Syntenic and phylogenetic analysis of zebrafish Sp2. A, a syntenic analysis of genes flanking the Sp2 locus in the zebrafish, mouse, and human genomes is shown. Genes are arrayed on zebrafish chromosome 11 in comparison with their mouse and human orthologues. The chromosomal location of each mouse and human gene is indicated to the right of each gene name. An arrow indicates the Sp2 locus in each species, and syntenic genes are indicated by shaded boxes. B, shown is a phylogenetic analysis of zebrafish Sp2 and mammalian Sp-family members. The predicted zebrafish Sp2 protein sequence was aligned with other Sp family members using ClustalW, and neighbor-joining trees were constructed with 2000 bootstrap replication using MEGA2.1. Bootstrap values less than 50 are not shown. Branch lengths are measured in terms of amino acid substitutions, with the scale indicated below the tree. C, a phylogenetic analysis of zebrafish Sp2 and nine vertebrate Sp2 proteins is shown. Protein sequences were aligned and analyzed as in B.

FIGURE 2.

Sp2 expression in unfertilized zebrafish eggs, developing embryos, and adult tissues. Total RNAs were prepared from unfertilized zebrafish eggs (0 hpf), developing embryos (6–72 hpf), adolescents (6 dpf), and adult (ovary, liver, kidney, spleen, intestine) tissues, reverse-transcribed, and subjected to thermal cycling with gene-specific primers (Sp2-F3 and Sp2-R3). Zebrafish ef1a transcripts were amplified in parallel as an internal control. Amplification reactions in which the template was not included (no template) were performed in parallel as a negative control. Reaction products were resolved on an agarose gel and stained with ethidium bromide.

FIGURE 3.

Expression, transcriptional activity, and subcellular localization of zebrafish Sp2. A, shown is a Western blot of human, mouse, and zebrafish Sp2 proteins expressed in transiently transfected cells. COS-1 cells were transfected with expression vectors carrying human (H, pCMV4-hSp2/flu), mouse (M, pCMV4-mSp2/flu), or zebrafish (Z, pCMV4-ZfSp2/flu) Sp2 cDNAs that had been tagged at their respective carboxyl termini with a 10-amino acid sequence derived from influenza HA. Denatured whole-cell extracts from untransfected (C) and transfected cells were prepared 48 h later and resolved on an acrylamide gel, and ectopically expressed proteins were detected with an anti-HA antibody. Antigen-antibody complexes were developed with an enhanced chemiluminescence kit. In addition to a primary translation product of 85 kDa, a 64-kDa partial-zebrafish Sp2 protein was also detected. B, shown is subcellular localization of zebrafish Sp2. Zebrafish Zf4 cells were transiently transfected with an expression vector that encodes a zebrafish EGFP-Sp2 fusion protein. Left panel, nuclei of transfected cells were identified by 4′,6-diamidino-2-phenylindole-staining (DAPI), and EGFP-Sp2 (Protein) was detected by direct fluorescence microscopy. A merged image (Merge) is also shown. Right panel, transfected cell nuclei were stripped of chromatin via treatment with micrococcal nuclease and ammonium sulfate before staining with 4′,6-diamidino-2-phenylindoleand detection of EGFP-Sp2 (Protein) by direct fluorescence microscopy. C, transient trans-activation of the hamster DHFR promoter by zebrafish Sp2. Zebrafish Zf4 cells were co-transfected with increasing concentrations of a zebrafish Sp2 expression vector (pCMV4-ZfSp2) or empty expression vector (pCMV4) and a DHFR-luciferase reporter gene (DHFR-Lux). The abundance of firefly luciferase activity was quantified relative to levels of Renilla luciferase to account for variations in transfection efficiency. Levels of mean -fold relative trans-activation (±S.E.) are derived from three independent plates of transfected cells. Each plate of transfected cells received a total of 100 ng of DNA. Relative levels of luciferase activity induced by pCMV4 are set equal to one.

FIGURE 4.

Detection of Sp2 expression in developing zebrafish embryos via in situ hybridization. Digoxigenin-labeled probes derived from a portion of the zebrafish Sp2 3′-untranslated region were synthesized in vitro and applied to embryos at various stages of development. A, shown is in situ hybridization with antisense Sp2 probe at 3 hpf. B, shown is in situ hybridization with antisense (left) and sense (right) Sp2 probes at 50–60% epiboly. C, shown is in situ hybridization with antisense (left) and sense (right) Sp2 probes at 90–100% epiboly. D, shown is in situ hybridization with antisense (left and center) and sense (right) Sp2 probes during pharyngula period (24–48 hpf).

FIGURE 5.

Molecular and functional characterization of the zebrafish Sp2 promoter. A, shown is the genomic sequence upstream of the zebrafish Sp2 transcriptional start site as defined by 5′RACE. A 1175-bp genomic fragment immediately adjacent to the zebrafish Sp2 transcriptional start site was cloned and sequenced. A cytosine at −1 relative to the transcriptional start site is underlined. Shaded nucleotides exhibit the greatest homology with sequences upstream of the major human and mouse Sp2 transcriptional start sites (as identified in B). Putative binding sites for trans-acting factors (rectangles indicate predicted CAAT boxes, and an ellipse indicates the predicted TBP-dependent core element for TATA-less promoters) are indicated. Also indicated is a 315-bp proximal genomic fragment (double-spaced sequence) that exhibits intermittent homology with genomic sequences upstream of the human and mouse Sp2 genes, aligned in B, and analyzed for transcriptional activity in C. B, alignment of genomic sequences upstream of the major transcriptional start sites of the human, mouse, and zebrafish Sp2 genes. Sequences were aligned using ClustalW, and identical (black boxes) and similar (gray boxes) nucleotides were annotated using BoxShade 3.21. C, shown is a transient transcription assay using genomic fragments upstream of the zebrafish Sp2 transcriptional start site. Genomic fragments (1175 and 315 bp) upstream of the zebrafish Sp2 transcriptional start site were cloned upstream of the firefly luciferase gene in pGL3-Basic (pGL3) generating pGL3-Sp2 (Sp2) and pGL3-Sp2Δ860 (Sp2-Δ860), respectively. Zebrafish Zf4 cells were transiently transfected with 90 ng of pGL3-Basic, pGL3-Sp2, or pGL3-Sp2Δ860 as well as a Renilla luciferase reporter gene as a control for transfection efficiency. Levels of mean -fold relative trans-activation (±S.E.) are derived from three independent plates of transfected cells. Each plate of transfected cells received a total of 100 ng of DNA.

FIGURE 6.

A genomic fragment from the zebrafish Sp2 locus directs transcription in embryonic transgenic zebrafish. The 1175-bp genomic fragment isolated from the zebrafish Sp2 locus and analyzed in Fig. 5 was cloned upstream of mCherry in plasmid pDB739 and inserted into the zebrafish genome using the TOL2 transposase. Transgenic founders were identified by direct fluorescence microscopy of developing embryos following matings with wild-type animals. mCherry expression in blastula stage embryo (A), at 90% epiboly (B), and during pharyngula (C) and hatching periods (D) is shown. E, amplification of Sp2-related transcripts in developing zebrafish embryos is shown. mCherry (top row) and endogenous Sp2 (bottom row) expression was assessed in unfertilized wild-type eggs (lane 1) and developing Sp2-mCherry transgenic (lanes 2–6) embryos using gene-specific primers and RT-PCR. Lane 2, one-cell stage embryos (10 min post-fertilization). Lane 3, 64-cell stage embryos (2 hpf). Lane 4, 256–1000-cell stage embryos (2.5–3 hpf). Lane 5, blastula high stage embryos (3.5 hpf). Lane 6, 30% epiboly (4.75 hpf).

FIGURE 7.

A genomic fragment from the zebrafish Sp2 locus directs transcription in adolescent and adult transgenic zebrafish. A, shown is expression of the Sp2-mCherry transgene 4 days post-fertilization. Low, uniform levels of mCherry expression are detected with the exception of the kidney. Arrows indicate concentration of mCherry expression within pronephric tubules. B, shown is the coincidence of Sp2-mCherry and CDH17 expression in adolescent zebrafish. Sp2-mCherry and CDH17-EGFP double-transgenic zebrafish were analyzed by confocal fluorescent microscopy. Left, a merged image is shown. Center, a monochrome rendering of Sp2-mCherry expression is shown. Right, a monochrome rendering of CDH17-EGFP expression is shown. Arrows indicate pronephric ducts. C, shown is expression of the Sp2-mCherry transgene in the ovary of an adult zebrafish. Left, brightfield image of developing oocytes is shown. Center, expression of mCherry in developing oocytes is shown. Right, a merged image is shown.

FIGURE 8.

Inactivation of Sp2 in zebrafish embryos leads to developmental arrest during gastrulation. One- to two-cell stage embryos were microinjected with a 1.3-nl mixture containing increasing amounts of a morpholino (Morph2; panels B–E) that spans splice donor sequences flanking the first predicted Sp2 coding exon (exon 2) or a “scrambled” control morpholino (Morph7; Panel F). Microinjected animals were examined microscopically 18 hpf. A, solvent alone. B, 0.5 ng of Morph2. C, 1 ng of Morph2. D, 2 ng of Morph2. E, 4 ng of Morph2. F, 4 ng of Morph7. G, total RNAs prepared from control and morpholino-injected embryos were amplified using gene-specific primers and RT-PCR. Left, RT-PCR reactions were prepared with RNAs from control or Morph2-injected embryos using primers complementary to sequences within the first (Sp2-F8) and third (Sp2-R2) coding exons. These primers produce a 348-bp amplification product. Lane 1, no DNA template. Lane 2, amplification using RNA from uninjected control embryos. Lane 3, amplification using RNA from Morph2-injected embryos. Right, RT-PCR reactions were prepared with RNAs from control or Morph3-injected embryos using primers complementary to sequences within the third (Sp2-F11 and Sp2–12) and fourth (Sp2-R6) coding exons. Lanes 1–3 employed primers Sp2-F11 and Sp2-R6 producing a 617-bp amplification product, whereas lanes 4–6 employed primers Sp2-F12 and Sp2-R6 producing a 379-bp amplification product. Lanes 1 and 4, no DNA template. Lanes 2 and 5, amplification using RNA from uninjected control embryos. Lanes 3 and 6, amplification using RNA from Morph3-injected embryos.