Drosophila Segmentation Gene Research Articles

Formation of the drosophila larval photoreceptor organ and its neuronal differentiation require continuous Krüppel gene activity

The Drosophila segmentation gene Krüppel ( Kr) is redeployed to play a critical role for the establishment of the larval visual system. Using reporter gene expression conducted by a specific Kr cis-acting element, we were able to trace back the origin of the larval photoreceptor organ, the Bolwig organ, to a single progenitor neuron and to examine Kr function in Bolwig organ development when Kr + activity is absent from embryos due to specific mutations or reduced by neuron-specific and temporally restricted Kr antisense RNA expression. Our results show that Kr is required for neurons to differentiate into Bolwig organs, for fasciculation of the Bolwig nerve, and for this nerve to follow a specific pathway toward the synaptic targets in the larval brain. The transcription factor encoded by Kr is likely to regulate surface molecules necessary for neuronal cell adhesion and recognition in the developing larval visual system.

The Drosophila segmentation gene runt has an extended cis-regulatory region that is required for vital expression at other stages of development

The Drosophila runt gene functions in several developmental pathways during embryogenesis. This gene was initially characterized due to the pivotal role that it plays in the genetic regulatory network that establishes the segmented body pattern. Recently it was found that this X-chromosome-linked gene is one of several dosage-sensitive, X-linked components that is involved in activating the Sex-lethal gene in blastoderm stage female embryos. Finally, this gene is also extensively re-expressed in later stages of embryogenesis in the developing nervous system where it plays an important role in the development of specific neural lineages. We have initiated an analysis of the runt cis-regulatory region in order to investigate runt's roles in these (and other) developmental pathways. Analysis of both the function and the expression patterns of runt genes with truncated cis-regulatory regions indicates that there are multiple elements that make quantitative contributions to runt regulation during segmentation. We find that sequences that are more than 8.5 kb upstream of the runt promoter are necessary for normal expression during the post-blastoderm stages of embryogenesis. Genetic experiments indicate that the post-blastoderm expression of runt is vital to the organism.

Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt

We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.

Identification of Breakpoints in t(8;21) Acute Myelogenous Leukemia and Isolation of a Fusion Transcript, AML1 /ETO, With Similarity to Drosophila Segmentation Gene, runt

Abstract We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5′ sequences of AML1 to 3′ sequences of ETO, oriented telomere to centromere on the der(8) chromosome.

Direct homeodomain-DNA interaction in the autoregulation of the fushi tarazu gene.

A major problem in the elucidation of the molecular mechanisms governing development is the distinction between direct and indirect regulatory interactions among developmental control genes. In vivo studies have indicated that the Drosophila segmentation gene fushi tarazu (ftz) directly or indirectly autoregulates its expression. Here we describe a generally applicable experimental approach which establishes a direct in vivo interaction of the homeodomain protein ftz with the ftz cis-autoregulatory control region. In vitro studies have shown that the DNA-binding specificity of the ftz homeodomain can be changed by a single amino-acid substitution in the recognition helix (Gln 50----Lys). Whereas wild-type ftz homeodomain binds preferentially to a CCATTA motif, the mutant homeodomain (ftzQ50K) recognizes a GGATTA motif. We now find that the in vivo activity of an ftz autoregulatory enhancer element is reduced by mutations of putative ftz-binding sites to GGATTA. This down-regulatory effect is specifically suppressed in vivo by the DNA-binding specificity mutant ftzQ50K. These results establish a direct positive autoregulatory feedback mechanism in the regulation of this homeobox gene.

Homeodomain-independent activity of the fushi tarazu polypeptide in Drosophila embryos.

The Drosophila segmentation gene fushi tarazu (ftz) encodes a homeodomain-containing protein, ftz, that can act as a DNA-binding activator of transcription. In the developing embryo, ftz is expressed in seven stripes which correspond to the even-numbered parasegments. These parasegments are missing in ftz- embryos. When ftz is expressed throughout blastoderm embryos under the control of a heat-shock promoter, the odd-numbered parasegments are lost. This 'anti-ftz' phenotype has been attributed to autoactivation of the endogenous ftz gene by the ectopically expressed protein. Here we show that the same phenotype is induced by ectopic expression of a ftz polypeptide containing a deletion in the homeodomain. Thus, ftz can alter gene expression without binding directly to DNA.

Altering the insertional specificity of a Drosophila transposable element.

Vectors derived from the Drosophila P element transposon are widely used to make transgenic Drosophila. Insertion of most P-element-derived vectors is nonrandom, but they exhibit a broad specificity of target sites. During experiments to identify cis-acting regulatory elements of the Drosophila segmentation gene engrailed, we identified a fragment of engrailed DNA that, when included within a P-element vector, strikingly alters the specificity of target sites. P-element vectors that contain this fragment of engrailed regulatory DNA insert at a high frequency near genes expressed in stripes.

Pax genes, mutants and molecular function.

The paired domain is a conserved DNA binding motif which was first found in Drosophila segmentation gene products. This paired domain is encoded by a well conserved, paired box DNA sequence, also detected in the genomes of other species. The mouse paired box-containing genes are referred to as Pax genes and are expressed in a distinct spatiotemporal pattern during embryogenesis. Pax proteins are able to bind to specific DNA sequences and modulate transcriptional activity. Interestingly, three different Pax genes have already been shown to correspond to some mouse and human mutants, emphasizing their role as developmental control genes.

The Drosophila segmentation gene runt acts as a position-specific numerator element necessary for the uniform expression of the sex-determining gene Sex-lethal.

Female development in Drosophila is established through the activation of the X:A target gene Sex-lethal (Sxl) by an X:A ratio of 1. X-linked zygotic genes, termed numerator elements, comprise part of the X:A ratio and are primarily responsible for the activation of Sxl in females. We demonstrate that the X-linked segmentation gene runt is required for this process and has genetic and molecular properties of a numerator element. Genetically, runt has vital dose-dependent interactions with components of the X:A ratio and alterations in runt activity alter the sexual phenotype of triploid intersexes. Molecularly, loss of runt activity results in a failure to activate appropriately Sxl in the central region of female embryos. We also show that Sxl activation is influenced by the maternal anterior and terminal pattern-forming genes, bicoid (bcd) and torso (tor). These results indicate that the "uniform" activation of Sxl requires input from nonuniformly distributed products. We have demonstrated that runt is one such product and suggest that other genes with nonuniform input exist. runt is distinguished from previously identified regulators of Sxl by its nonuniform role and by the absence of an identifiable helix-loop-helix (HLH) motif, indicating that the activation of Sxl is not controlled solely by HLH proteins.

Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube.

Murine and human sequences homologous to the paired box of the Drosophila segmentation gene paired have been reported previously. Here we describe a zebrafish (Brachydanio rerio) paired box-containing clone, pax[zf-a], which is clearly distinct from reported vertebrate Pax genes. The putative protein encoded by pax[zf-a] contains a paired box and a paired-type homeobox separated by a glycine-rich, acidic linker and a carboxy-terminal end which is remarkably rich in serine, threonine and proline residues. By in situ hybridization to embryonic tissue sections and whole mount embryos, pax[zf-a] transcripts were found within restricted regions of the central nervous system and the eye. In contrast to the murine Pax genes recently characterized, pax[zf-a] is not expressed in the segmented mesoderm. At the 17 h stage, pax[zf-a] expression is detected in a defined area of the diencephalon which circumscribes the presumptive thalamus. This suggests an involvement of pax[zf-a] in pattern formation in the rostral brain. The pax[zf-a] gene is also expressed throughout the hindbrain and spinal cord. This hybridization signal is restricted to a longitudinal column which includes the basal plate. Later in development, at 36 h post-fertilization, pax[zf-a] transcripts are no longer restricted to a specific region of the diencephalon, but are distributed over the entire developing brain.

Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox.

The two mouse genes, En-1 and En-2, that are homologs of the Drosophila segmentation gene engrailed, show overlapping spatially restricted patterns of expression in the neural tube during embryogenesis, suggestive of a role in regional specification. Mice homozygous for a targeted mutation that deletes the homeobox were viable and showed no obvious defects in embryonic development. This may be due to functional redundancy of En-2 and the related En-1 gene product during embryogenesis. Consistent with this hypothesis, the mutant mice showed abnormal foliation in the adult cerebellum, where En-2, and not En-1, is normally expressed.

Repression of the Drosophila fushi tarazu (ftz) segmentation gene.

The striped expression of the Drosophila segmentation gene fushi tarazu in alternate parasegments of the early embryo is controlled by the 740 bp zebra element. Among multiple protein factors that bind to the zebra element, FTZ-F2 behaves as a transcriptional repressor of ftz. Point mutations in the zebra element which disrupt FTZ-F2 binding to DNA cause ectopic expression of zebra-lacZ activity in transformed embryos. The mutant constructs are expressed from the zygotic genome in preblastoderm embryos as early as the third nuclear division cycle. This unprecedented early transcription suggests that ftz requires active repression during initial nuclear division cycles, a novel type of embryonic gene regulation. A putative FTZ-F2 cDNA clone isolated by recognition site screening of an expression library was found to be identical in sequence with the zinc finger protein tramtrack (Harrison and Travers, 1990).

Krox-20: A candidate gene for the regulation of pattern formation in the hindbrain

The mouse gene Krox-20 was isolated on the basis of cross-hybridization with Krüppel, a Drosophila segmentation gene. During recent years, an accumulation of structural, biochemical and expression data has indicated that Krox-20 encodes a transcription factor which may play a key role within a regulatory network involved in pattern formation in the developing hindbrain. The DNA-binding domain of Krox-20 consists of 3 zinc fingers and the protein recognizes a specific GC-rich nucleotide sequence. It can activate the transcription of genes located in the vicinity of this sequence. Krox-20 transcription itself is modulated by growth factors During formation of the vertebrate central nervous system (CNS), the hindbrain is organized into segmental units, called rhombomeres. Prior to the appearance of morphological segmentation, Krox-20 is expressed in 2 stripes within the hindbrain. Later on, the Krox-20 expression domains match with 2 alternate rhombomeres. These data suggest that Krox-20 may regulate aspects of the segmentation process. The observation of segment-specific expression boundaries for homeobox containing genes in the hindbrain suggests that these genes may also be part of the regulatory network governing pattern formation in this region of the CNS. The possible interactions between Krox-20 and homeobox containing genes as well as the search for other members of the network will be discussed.

The Drosophila segmentation gene runt encodes a novel nuclear regulatory protein that is also expressed in the developing nervous system.

Generation of the anterior-posterior body pattern in the Drosophila embryo requires the activity of the segmentation genes. The segmentation gene runt has been classified as one of the primary pair-rule genes because of the pivotal role it plays in regulating the expression of other pair-rule genes. Here, we present the structure of this gene and describe the pattern of runt protein expression during embryogenesis. The deduced protein sequence shows no obvious overall homology with any sequences in the data base. The absence of an identifiable transcription factor motif (e.g., homeo box, zinc finger, leucine zipper, or helix-loop-helix) makes runt different from the other early-acting segmentation proteins. A runt-specific polyclonal antibody was generated and used to demonstrate that the subcellular location of the protein is in the nucleus. Double-staining immunolocalization experiments were used to determine the overlap of the runt protein pattern with the patterns of the pair-rule genes hairy (h), even-skipped (eve), and fushi tarazu (ftz). We found that the patterns of runt and hairy are complementary. Their phasing is shifted anteriorly by two cell diameters with respect to the complementary eve and ftz patterns. Experiments with the runt antibody also indicated that the protein is present throughout embryogenesis and is expressed extensively in the developing central and peripheral nervous system.

Spatially and temporally restricted expression of Pax2 during murine neurogenesis

The expression of the murine paired-box-containing gene, Pax2, is examined in the developing central nervous system by in situ hybridization. Pax2 expression is detected along the boundaries of primary divisions of the neural tube. Initially, Pax2 is expressed in the ventricular zone in two compartments of cells on either side of the sulcus limitans and along the entire rhombencephalon and spinal cord. At later times, Pax2 is restricted to progeny cells that have migrated to specific regions of the intermediate zone. In the eye, Pax2 expression is restricted to the ventral half of the optic cup and stalk and later to the optic disc and nerve. In the ear, expression is restricted to regions of the otic vesicle that form neuronal components. The transient and restricted nature of Pax2 expression suggests that this murine segmentation gene homologue may also establish compartmental boundaries and contribute to the specification of neuronal identity, as do certain Drosophila segmentation genes.

The Drosophila even-skipped promoter is transcribed in a stage-specific manner in vitro and contains multiple, overlapping factor-binding sites.

To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts. We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo. This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei. One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor. Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis. Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis. We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.

The Drosophila Even-Skipped Promoter Is Transcribed in a Stage-Specific Manner In Vitro and Contains Multiple, Overlapping Factor-Binding Sites

To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts. We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo. This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei. One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor. Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis. Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis. We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.

Drosophila Krüppel protein is a transcriptional represser

Krüppel (Kr), one of the zygotically active Drosophila segmentation genes, is expressed in a restricted domain during the blastoderm stage of embryogenesis and is involved in the control of development of the thoracic and abdominal segments of the fly. Kr encodes a polypeptide containing DNA-binding zinc-finger motifs, disruptions of which yield Kr mutants. We have assayed the transcriptional activities of wild-type Kr protein as well as Lac repressor/Kr fusion proteins in HeLa and CV-1 cells. Wild-type Kr and a Lac-Kr chimaeric protein repressed transcription from reporter promoters in which a consensus Kr binding site derived from sequences within the even-skipped promoter had been inserted in an upstream position. We mapped the repression function of Kr to an alanine-rich amino-terminal region of the protein, as a Lac/Kr fusion protein containing only amino acids 26-110 of Kr repressed transcription from a reporter promoter containing upstream lac operators. This demonstrates that the DNA-binding and repression activities of the Kr protein are distinct. These data are consistent with genetic evidence that Kr represses even-skipped and hunchback expression, and suggest that Kr is a negative regulator of transcription in Drosophila.

A sequence-specific DNA-binding protein that activates fushi tarazu segmentation gene expression.

The Drosophila segmentation gene fushi tarazu (ftz) is expressed at the cellular blastoderm stage in a pattern of seven transverse stripes; the stripes lie out of register with the segmental primordia, spanning alternate segmental boundaries. The zebra element, a 740-bp DNA sequence upstream of the ftz translational start, directs striped expression of lacZ when introduced into the fly genome. We have purified to homogeneity a sequence-specific DNA-binding factor, FTZ-F1, that binds to two sites located within the zebra element and to two sites within the ftz protein-coding sequence. FTZ-F1 DNA-binding activity is first detected in extracts of 1.5- to 4-hr embryos, coincident with the time of ftz expression in stripes; the activity then diminishes before reappearing during late embryo, larval, and adult stages. When one of the FTZ-F1-binding sequences in the zebra element is mutated by 2- or 4-base substitutions, the binding to FTZ-F1 is disrupted in vitro, and the intensity of lacZ expression is reduced in transformed embryos, especially in stripes 1, 2, 3, and 6. The results suggest that FTZ-F1 is a transcriptional activator necessary for the proper expression of the ftz gene.

Development(s) in mouse genetics

Introduction After more than 50 years of classical genetics, developmental genetics of the mouse has entered a new phase. The recent advances in molecular biology enable the old questions in developmental biology to be addressed with new methods. Traditionally, mouse genetics has centered around the study of a large number of mutants that either occurred spontaneously or were induced by experimental mutagenesis. The biological questions that were asked were mainly dictated by the nature of the mutants available. Although phenotypic consequences of mutated genes could be analyzed, the nature of the genes themselves remained unknown. Reverse genetics has changed this situation dramatically. We now have many genes at hand whose functions remain to be discovered. A major focus is on the identification and analysis of developmental control genes in vertebrates. Genetic and molecular studies of developmental processes in Drosophila melanogaster have revealed that embryogenesis is under the control of a cascade of genes directing developmental decisions and orchestrating complex processes, such as cell commitment and differentiation. Mutations affecting the formation of metameric units (segmentation genes) or the identity of segments (homeotic genes) facilitated the identification of a gene regulatory network in which a limited number of key regulatory genes control developmental pathways in the fruit fly (Gehring and Hiromi, Annu. Rev. Genet., 20, 147-173, 1986; Akam, Development (Cambridge), 101, 1-22, 1987; Ingham, Nature (London), 335, 25-34, 1988). Is vertebrate embryogenesis, likewise, under the control of similar genes? The cloning of Drosophila homeotic and segmentation genes has identified at least three types of conserved sequence motifs among these developmental control genes. Low stringency cross-hybridization strategies then led to the identification of a large number of vertebrate genes that have also conserved these sequences. The question therefore arises whether the homeobox, paired-box, and zinc-finger containing genes of the mouse play a similar role in development as their counterparts in Drosophila. To answer this question, three approaches are currently being pursued in the functional analysis of candidate mouse development control genes.