A Novel Transcription Factor, zbtb11 Is Critical for Neutrophil Development in Zebrafish

Abstract

Zinc finger and BTB domain containing proteins (BTB-ZF) are transcriptional repressors from a family including members with critical roles in hematopoiesis and oncogenesis (BCL6, PLZF, HIC). In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen for defects in myeloid development, we identified a neutropenic zebrafish mutant with a mutation in zbtb11, a novel BTB-ZF transcription factor, suggesting that zbtb11 is critical for normal neutrophil development.The mutant was found in a gynogenetic haploid ENU screen for defective expression of genes along the developmental pathway from mesoderm to mature neutrophil (deltaC, spi1 and myeloperoxidase/mpx), undertaken to search for novel genetic regulators of myelopoiesis in an unbiased fashion. Since zebrafish are poikilothermic, embryos were screened at 33°C to maximise recovery of temperature dependant alleles; marsanne (man) was one mutant pedigree recovered. man is an early embryonic lethal mutant with normal scl and spi1 expression but markedly reduced numbers of mpx expressing cells. Erythropoiesis is unaffected. Reduced numbers of Sudan Black stained cells demonstrate that man is deficient in neutrophils themselves, not just mpx transcripts. man mutants also develop brain and spinal cord degeneration with hydrocephalus due to apoptosis. man is a temperature sensitive allele i.e. numbers of mpx expressing cells per embryo decrease at higher (restrictive) temperatures and are partially restored at lower (permissive) temperatures:Water temperatureNumber of mpx expressing cells/embryo (mean ± SD, 48hpf)man/man+/?22°C75±1589±1428°C42±1081±1233°C27±1181±22Hence man serves as a conditional allele, allowing manipulation of protein function via manipulation of water temperature. man was mapped to a 40kb region on chromosome 6 containing a single gene, zbtb11. Sequencing of man and WT zbtb11 cDNA revealed a missense mutation (346T->A, 116Cys->Ser) affecting a cysteine residue conserved throughout vertebrate evolution. Pedigree analysis proved this substitution was introduced by ENU mutagenesis and segregated absolutely with the man phenotype in 3863 mutant embryos. Genetic tests implicated this zbtb11 mutation as causing the man phenotype, zbtb11 knockdown with an antisense morpholino oligonucleotide recapitulated the mpx deficiency and other features of the man phenotype. Injection of WT but not mutant zbtb11 mRNA into man embryos completely rescued mpx expression (125 +/−38 mpx positive cells per embryo vs. 67+/−35, p<0.001; uninjected WT 109 +/19, mean +/− SD, 48hpf) and all other aspects of the man phenotype. Since the mutant Zbtb11 protein is inactive in this assay, we hypothesise the amino acid substitution in the N-terminus of the protein compromises function. In view of the temperature sensitive nature of man, this may result from an effect on weak bonding, differentially affecting protein structure at permissive vs. restrictive temperatures. Zebrafish zbtb11 encodes a 1146 amino acid protein with a predicted N-terminal BTB (also known as POZ) domain and 12 C-terminal C2H2 (Kruppel-type) zinc fingers. The BTB domain is a highly conserved, approximately 100 amino acid domain, mediating protein-protein interaction and dimerization. The zebrafish protein is approximately 75% identical to mouse and human ZBTB11 across its BTB domain and zinc fingers. ZBTB11 is expressed physiologically in human hematopoietic cells (Ferrari F et al. BMC Genomics. 2007) and mouse embryonic brain (Gray PA et al. Science. 2004). It appears to be over-expressed in K562 CML cells compared to other acute leukemia and lymphoma lines (Su AI et al. PNAS 2002), suggesting a possible role in oncogenesis. The function of mammalian ZBTB11 is unknown and there are no mammalian models of ZBTB11 dysfunction. The rescue of man by WT zbtb11 forms the basis of a bioassay to test modified Zbtb11 proteins, and a whole animal assay for chemical modifiers of Zbtb11 function. Rescue of man with human ZBTB11 is being attempted to assess functional conservation and to provide a heterologous mammalian bioassay. Temperature-shift experiments exploiting the temperature sensitivity of man should define the time-dependant requirement for Zbtb11 during development. man provides a unique reagent with which to advance understanding of Zbtb11 biochemistry and biology in vivo.