ABSTRACT
The early transcriptional hierarchy that subdivides the vertebrate hindbrain into seven to eight segments, the rhombomeres (r1-r8), is largely unknown. The Kreisler (MafB, Krml1, Val) gene is earliest gene expressed in an r5/r6-restricted manner and is essential for r5 and r6 development. We have identified the S5 regulatory element that directs early Kreisler expression in the future r5/r6 domain in 0-10 somite stage embryos. variant Hepatocyte Nuclear Factor 1 (vHNF1/HNF1beta/LF-3B) is transiently expressed in the r5/r6 domain of 0-10 somite stage embryos and a vHNF1binding site within this element is essential but not sufficient for r5/r6-specific expression. Thus, early inductive events that initiate Kreisler expression are clearly distinct from later-acting ones that modulate its expression levels. This site and some of the surrounding sequences are evolutionarily conserved in the genomic DNA upstream of the Kreisler gene among species as divergent as mouse, humans, and chickens. This provides the first evidence of a direct requirement for vHNF1 in initiation of Kreisler expression, suggests that the role of vHNF1 is evolutionarily conserved, and indicates that vHNF1 collaborates with other transcription factors, which independently bind to the S5 regulatory region, to establish the r5/r6 domain.
Subject(s)
Gene Expression Regulation, Developmental/physiology , Hepatocyte Nuclear Factor 1/physiology , Homeodomain Proteins/physiology , MafB Transcription Factor/biosynthesis , MafB Transcription Factor/genetics , Rhombencephalon/embryology , Animals , Base Sequence , Binding Sites/genetics , Cell Differentiation/genetics , Conserved Sequence , Enhancer Elements, Genetic , Genetic Variation , Hepatocyte Nuclear Factor 1/biosynthesis , Hepatocyte Nuclear Factor 1/genetics , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , Rhombencephalon/cytology , Rhombencephalon/metabolismABSTRACT
The embryonic hindbrain is subdivided into eight subunits, termed rhombomeres (r1-r8). The Kreisler (Krml1/MafB/val) transcription factor is expressed in and essential for patterning rhombomeres 5 and 6. Here, we have shown that in the chemically induced kreisler(enu) (kr(enu)) allele, a point mutation in the DNA binding domain abolishes or severely reduces Kreisler-dependent transcription. Comparison of kr(enu)/kr(enu) embryos with those homozygous for the classic kreisler (kr) mutation has reconciled past discrepancies and revealed multiple roles of Kreisler in hindbrain segmentation. These analyses demonstrate that Kreisler is required for maintenance and expansion but not initiation of the Krox20 expressing r5 domain. The differences in the "r5-like" phenotype of kr(enu)/kr(enu) and kr/kr mouse embryos, and zebrafish carrying mutations in the Kreisler orthologue valentino (val) suggest that Kreisler performs many of its r5-specific functions by associating with other proteins. By contrast, kr/kr and kr(enu)/kr(enu) mouse and val-/- zebrafish embryos all exhibit indistinguishable defects in r6 specification. Thus, transcriptionally active Kreisler is required for r6 specification. Unlike mouse kr(enu)/kr(enu) and zebrafish val-/- embryos, kr/kr embryos exhibited anterior defects. We determined that the kr chromosomal inversion caused ectopic Kreisler expression in r3 of kr/kr and kr/+ embryos. Hence, Kreisler regulates maintenance and expansion of r5 and specification of r6 but is not required for r3 development.