Regulation of the stem cell leukemia (SCL) gene: a tale of two fishes

Abstract

The stem cell leukemia (SCL) gene encodes a tissue-specific basic helix-loop-helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes, a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5' and 3' flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo.

Figure 1

Identification of the pufferfish SCL gene. Comparison of the genomic SCL loci of pufferfish (P), chicken (C), mouse (M), and human (H). Horizontal arrows under each gene indicate the direction of transcription. SCL coding and noncoding exons are shown as red and black boxes, respectively. The position of five enhancer elements that direct expression to endothelium (yellow box), hemopoietic progenitors (gray box), and neural tissue (green, orange and pink boxes) are indicated. SIL and MAP17 flank the SCL gene in human, mouse and chicken but are not found at the pufferfish SCL locus. UPG, unknown pufferfish gene; C3HC4, RING finger-like gene; PDZ, PDZ domain encoding gene; SC, saccular collagen gene; PP2C, protein phosphatase 2C gene; Pr1a, SCL promoter 1a.

Figure 2

Sequence comparisons of pufferfish SCL and SLP-1 with other vertebrate SCL proteins. The bHLH probe (black bar) and zebrafish SCL 3′ probe (gray bar) were used for library screening and Southern blotting.

Figure 3

Expression profile of SCL in pufferfish and zebrafish. RT-PCR analysis of pufferfish SCL (FrSCL) and zebrafish SCL (DrSCL). The pufferfish homologue of the ubiquitously expressed p55 gene (FRp55) and the zebrafish gene elongation factor α (DrEF1) were used as loading controls. 1, spleen; 2, liver; 3, gonads; 4, kidney; 5, gut; 6, gill; 7, brain; 8, heart; 9, muscle; 10, water control.

Figure 4

Expression of the pufferfish SCL gene in transgenic zebrafish embryos. Views of embryos are lateral with anterior left and dorsal top. (a) Diagram of the pufferfish cosmid and the 10.4-kb fragment used to make transgenic zebrafish. (b) Whole-mount in situ analysis showing expression of endogenous zebrafish SCL at 22 hpf. Expression is seen in the HMC, in cell bodies within the ventrolateral region of the spinal cord in the position of motor neurons (SC), and in the cells of the ICM. The ICM consists of round cells that lie between the notochord and the endoderm above the yolk cell extension. (c) A section through the trunk shows expression of endogenous zebrafish SCL in the ICM and in the position of the motor neurons in the spinal cord. (d–k) Expression of the entire pufferfish SCL cosmid at 22 hpf. Varying numbers of cells expressing pufferfish SCL were seen in the ICM (d, e) and the spinal cord (f, g). The oblique lines in d indicates the plane of section corresponding to h, the line in e corresponds to i, and the two lines in f correspond to j and k. Sections confirmed expression in the ICM (h, i) and in the position of primary motor neurons in the spinal cord (h, j, and k). It should be noted that the ICM is formed by cells from the lateral mesoderm that migrate toward the midline, beginning anteriorly and progressing posteriorly. The width of the ICM therefore increases in more posterior regions of the trunk. E, area of ectopic expression. (l–s) Expression of the 10.4-kb construct in 22-hpf embryos. Expression of pufferfish SCL in the ICM (l, m) and the spinal cord (m, n, and o). The oblique line in l indicates the plane of section corresponding to p and the three lines in m correspond to q, r, and s. Expression in the ICM (p, s) and spinal cord (q, r).