Drosophila Segmentation Gene Research Articles

Spatial control of hairy protein expression during embryogenesis.

We have used a polyclonal antiserum specific for the Drosophila segmentation gene, hairy (h), to analyse its expression during embryogenesis. The pattern of wild-type expression resembles that of h transcription, being expressed in stripes at the blastoderm stage. h is also expressed later in the stomodaeum, proctodaeum, tracheal pits and mesoderm. We demonstrate that h protein stripes show consistent phase relationships to those of the even-skipped (eve) pair-rule gene. We examine h protein patterns in embryos mutant for other segmentation genes, including h itself. We show that lack of h activity appears not to affect h striping, arguing that h expression is not under autoregulatory control. We also show that h activity is not needed for tracheal invagination. Mutations that are rearranged upstream of the h gene cause the loss of specific stripes, indicating that the h promoter includes activating elements that respond to specific spatial cues. Our observations suggest that pair-rule striping may be under redundant control, and we discuss possible implications for hierarchical models of pair-rule gene action.

The Drosophila hairy protein acts in both segmentation and bristle patterning and shows homology to N-myc.

The Drosophila segmentation gene, hairy (h), acts to regulate embryonic segmentation and bristle pattern. We present the DNA sequence of the h gene and of h cDNAs, thereby deducing the organization of the h transcripts. The h gene encodes a 337 amino acid protein that acts in both embryonic segmentation and adult bristle patterning. The h protein includes a domain that shows extensive similarity to a domain of the proto-oncogene N-myc that may be involved in DNA binding and/or protein dimerization. We discuss mechanisms of h action as a transcriptional regulator.

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.

Transcriptional control of Drosophila fushi tarazu zebra stripe expression.

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a characteristic pattern of seven stripes during early embryogenesis. We have used ftz-lacZ fusion genes to determine the effects of deleting relatively small segments of the ftz promoter region necessary for this expression. We find that this regulatory region contains multiple activator and repressor elements. The deletion of one particular activator element results in a preferential loss of expression in the posterior stripes, whereas the deletion of other activator elements causes a general reduction in expression throughout the germ band. The removal of repressor elements results in a loss of repression in the odd-numbered parasegments. We also find that the ftz upstream enhancer element functions primarily in epidermal cells. Our results indicate that ftz transcription is activated in each parasegment through the 'zebra stripe' promoter region and is then inhibited selectively in the odd-numbered parasegments by repressors that bind directly to elements within this promoter region.

A phylogenetic interpretation of the patterns of gene expression in Drosophila embryos

ABSTRACT Two fundamental processes of the development of insect embryos are the generation and the morphological diversification of metameric units. In Drosophila, these processes are under the control of the products of the segmentation (generation) and the homeotic (morphological diversification) genes. Molecular studies of the activity of these genes has revealed spatial and temporal patterns of expression consistent with the requirements inferred from the mutant phenotypes but, in addition, these studies have revealed transient patterns which are difficult to reconcile with those phenotypes. It is possible that these patterns reflect ancestral regulatory elements which are still operational in more primitive insects. The validity of this interpretation can be tested by comparing the embryonic development of long germ band insects like Drosophila melanogaster with that of the more primitive short germ band insects like the locust Schistocerca gregaria and by obtaining and studying locust homologues of Drosophila segmentation and homeotic genes.

Segmentation in leech development.

Segments in glossiphoniid leeches, such as Helobdella triserialis, are the products of sterotyped cell lineages that yield identifiable cells from first cleavage. Cell lines generating segmental tissues are separated from those generating prostomial tissues early in development. Segments arise from five bilateral pairs of longitudinal columns of primary blast cells that are generated by five bilateral pairs of embryonic stem cells called teloblasts. There are four ectodermal cell lines (N, O, P and Q) and one mesodermal cell line (M) on each side of the embryo. In normal development, each cell line generates a segmentally iterated set of identified definitive progeny comprising a mixture of cell types. In the M, O and P cell lines, each blast cell generates one segment's worth of definitive progeny (segmental complement). But the clones of blast cells in each of these three cell lines interdigitate longitudinally with cells of the adjacent clones from the same line, so that the clone of an individual m, o and p blast cell is distributed across more than one segment. Thus, there is no simple clonal basis for morphologically defined segments. In the N and Q cell lines, two blast cells are required to produce one segmental complement of definitive progeny; in each of these two cell lines, two classes of blast cells (nf and ns, qf and qs) are produced in exact alternation. Primary n and q blast cells are about the same size and are produced at the same rate as blast cells for the o and p bandlets, but the longitudinal extent of their clones is roughly half that of the o and p blast cells' clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of the Drosophila segmentation gene runt and analysis of its expression during embryogenesis.

runt is one of the genes required for establishment of the segmented body pattern of the Drosophila embryo. We have isolated DNA sequences containing this gene using P-element transposon tagging. Southern blot analyses of six different DNA rearrangements that are associated with runt mutations revealed a minimal region of 8.5-kb of DNA that was important for function. In germ line transformation experiments, a 14.5-kb segment of DNA that spanned this minimal region provided significant, although not full, levels of runt activity. The runt gene encoded a 2.6-kb poly(A)+ RNA that underwent a series of dynamic changes in its spatial and temporal patterns of accumulation during embryogenesis. The runt RNA was most abundant at the blastoderm stage when it showed the seven stripes of expression characteristic of other Drosophila pair-rule genes.

Region-specific alleles of the Drosophila segmentation gene hairy.

An essential step in the Drosophila segmentation mechanism is the expression of the pair-rule gene hairy in a periodic pattern in the blastoderm. In this paper we describe four regulatory hairy mutations. Using in situ hybridization, we show that these mutations cause alterations in the normal pattern of hairy expression. The new patterns are partial versions of the wild-type pattern and indicate that there are regulatory sequences associated with hairy that respond to distinct cues in different parts of the blastoderm. This suggests that a major function of hairy is to decode a prepattern consisting of complex cues (probably generated by the coordinate and gap classes of segmentation genes) into a simple periodic pattern. We have located the mutations on the DNA map of the hairy gene. They identify a 5' region of approximately less than 20 kb necessary for this decoding function.

The GLI-Kruppel family of human genes.

Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.

The GLI-Kruppel family of human genes.

Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.

Control of neuronal fate by the Drosophila segmentation gene even-skipped.

The central nervous system (CNS) contains a remarkable diversity of cell types. The molecular basis for generating this neuronal diversity is poorly understood. Much is known, however, about the regulatory genes which control segmentation and segment identity during early Drosophila embryogenesis. Interestingly, most of the segmentation and homoeotic genes in Drosophila, as well as many of their vertebrate homologues, are expressed during the development of the nervous system (for example, ref. 3). Are these genes involved in specifying the identity of individual neurons during neurogenesis, just as they specify the identity of cells during segmentation? We previously described the CNS expression of the segmentation gene fushi tarazu (ftz) and showed that ftz CNS expression is involved in the determination of an identified neuron. Here we show that another segmentation gene, even-skipped (eve), is expressed in a different but overlapping subset of neurons. Temperature-sensitive inactivation of the eve protein during neurogenesis alters the fate of two of these neurons. Our results indicate that the nuclear protein products of the eve and ftz segmentation genes are components of the mechanism controlling cell fate during neuronal development.

Characterization of a mouse multigene family that encodes zinc finger structures.

The Drosophila segmentation gene Krüppel encodes multiple tandemly repeated units predicted to form DNA-binding zinc fingers. We have isolated 23 bacteriophages, containing nonoverlapping inserts from a mouse genomic DNA library, on the basis of cross-hybridization under nonstringent conditions to a probe corresponding to the Krüppel finger region. Nucleotide sequence analysis of six phage DNAs indicated that they all contained regions with similarity to Krüppel and potentially encoded zinc finger domains. Within these regions, the level of similarity to Krüppel was particularly high between successive fingers. Northern (RNA) blotting analysis suggested that the mouse sequences belonged to different genes, the expression of some of which was modulated during cell differentiation and development. Hybridization experiments suggested that the similarity between some of the genes extended outside of the finger regions. In conclusion, our data suggest that the mouse genome contains a large family of evolutionarily related genes encoding possible trans-acting factors. These genes are likely to play a regulatory role at the transcriptional level.

Characterization of a Mouse Multigene Family That Encodes Zinc Finger Structures

The Drosophila segmentation gene Krüppel encodes multiple tandemly repeated units predicted to form DNA-binding zinc fingers. We have isolated 23 bacteriophages, containing nonoverlapping inserts from a mouse genomic DNA library, on the basis of cross-hybridization under nonstringent conditions to a probe corresponding to the Krüppel finger region. Nucleotide sequence analysis of six phage DNAs indicated that they all contained regions with similarity to Krüppel and potentially encoded zinc finger domains. Within these regions, the level of similarity to Krüppel was particularly high between successive fingers. Northern (RNA) blotting analysis suggested that the mouse sequences belonged to different genes, the expression of some of which was modulated during cell differentiation and development. Hybridization experiments suggested that the similarity between some of the genes extended outside of the finger regions. In conclusion, our data suggest that the mouse genome contains a large family of evolutionarily related genes encoding possible trans-acting factors. These genes are likely to play a regulatory role at the transcriptional level.

Regulation of the Drosophila segmentation gene hunchback by two maternal morphogenetic centres

Segmentation in the inset embryo is initiated by maternally provided information, which is stored in the developing oocyte. In Drosophila, the genes necessary for this process have been genetically characterized. The anterior segmented region is organized by the bicoid (bcd) gene product. The posterior segmented region is organized by several interacting gene products, among them the oskar (osk) gene product. The first zygotic group of genes, which are thought to respond to the spatial cues provided by the maternal genes, are the gap genes, whose members include hunchback (hb), Krüppel (Kr) and knirps (kni). To elucidate the role played by the maternal genes in expression of the gap gene hb, antibodies were raised against a fusion protein and were used for the cytological localization of the hb gene product in wild-type and mutant embryos. The hb protein is predominantly located in the nucleus. Its spatial expression includes the formation of an anterior-posterior gradient during the early cleavage stages and a strong zygotic expression in the anterior half of the embryo. Analysis of embryos mutant for the maternal genes affecting the anterior-posterior segmentation pattern shows that the formation of the early gradient is controlled by the osk group of genes, whereas efficient activation of the zygotic anterior expression domain is dependent on bcd activity.

Complementary patterns of even-skipped and fushi tarazu expression involve their differential regulation by a common set of segmentation genes in Drosophila.

We have examined the position-dependent expression of two segmentation genes that contain a homeo box, even-skipped (eve) and fushi tarazu (ftz). Products encoded by both genes accumulate in a series of seven transverse stripes along the anterior-posterior body axis of developing embryos. Double-staining experiments show that eve and ftz proteins accumulate in complementary sets of embryonic cells. A total of eight zygotically active segmentation genes are required for the establishment and refinement of the eve and ftz expression patterns during development. Mutations that affect one of the genes were always found to produce a reciprocal alteration in the other. These results suggest that the eve and ftz promoters independently "interpret" the same positional cues to give differential patterns of expression. We propose that a combination of segmentation gene products that activates the expression of eve represses the expression of ftz, and vice versa. This proposal is discussed in the context of a hierarchy of interactions among segmentation genes, whereby the gap genes establish asymmetric patterns of eve and ftz expression in blastoderm-stage embryos, and interactions with other pair-rule genes serve to refine further the complementarity of eve and ftz expression during gastrulation and germ band elongation.

Region-specific expression of mouse homeobox genes in the embryonic mesoderm and central nervous system.

The homeobox is a 180-base-pair sequence characteristically found in homeotic and segmentation genes in Drosophila. Several copies of homeoboxes are also found in the mammalian genome, but it is not known whether these are components of morphogenetic loci in mammals as well. As a step toward understanding the function of mammalian homeoboxes, we have used in situ hybridization to define the spatial pattern of expression of two mouse homeobox genes in the midgestational mouse embryo. The two mouse homeoboxes studied here, Hox 1.2 and Hox 1.4, are located 20 kilobases apart on mouse chromosome 6. Our results demonstrate the following: (i) Hox 1.2 transcripts are localized mainly in the posterior myelencephalon, in the cervical central nervous system (CNS), and in several thoracic prevertebrae; (ii) Hox 1.4 transcripts are localized mainly in the posterior myelencephalon and in the cervical CNS; (iii) within the CNS region expressing Hox 1.4, the level of Hox 1.4 transcripts is higher in the mantle layer than in the ependymal layer and higher in the dorsal than in the ventral area. The specific localization of Hox 1.2 and Hox 1.4 transcripts in the embryonic CNS and the restricted pattern of expression along the rostrocaudal axis are strikingly reminiscent of the expression pattern of Drosophila homeoboxes in the fly embryo and larvae. Despite the different developmental strategies adopted by Drosophila and mammals, functional similarities may exist between Drosophila and mammalian homeobox genes.

Regulation and function of the Drosophila segmentation gene fushi tarazu.

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a pattern of seven stripes at the blastoderm stage. Two cis-acting control elements are required for this expression: the zebra element, which confers the striped pattern by mediating the effects of a subset of segmentation genes; and the upstream element, an enhancer element requiring ftz+ activity for its action. Fusion of the upstream element to a basal promoter results in activation of the heterologous promoter in a ftz-dependent striped pattern, supporting the idea that ftz regulates itself by acting through its enhancer. The upstream element can also confer expression patterns similar to that of the homeotic gene Antennapedia, suggesting that a similar element may play a role in the activation of Antennapedia.

Drosophila Krüppel gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA.

The product of the Drosophila segmentation gene Krüppel was produced in cultured insect cells using the baculovirus expression system. When a cloned Krüppel cDNA sequence was inserted into the viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately equal to 72,000 was observed in the nuclei of infected cells. Antibodies were raised against this protein and used to detect Krüppel in Drosophila embryos. Characterization of the Krüppel protein extracted from infected cells showed that it is tightly bound to the nucleus, it binds to calf thymus DNA-cellulose, and it is phosphorylated. These results support the hypothesis that Krüppel is a regulatory protein that acts by binding DNA.

Finger protein of novel structure encoded by hunchback, a second member of the gap class of Drosophila segmentation genes

The DNA sequence of hunchback, a Drosophila segmentation gene of the gap class, reveals that like Kruppel, the first gap gene analysed, it encodes a protein with finger motifs typical of a class of DNA-binding proteins. Unlike Kruppel, hunchback encodes a maternal transcript, which becomes graded along the longitudinal axis of the embyro. This, together with other features of its pattern of expression in the zygote, suggests specialization among the gap genes.

How to fill a gap in the Drosophila embryo

The ‘gap’ phenotype defines a class of Drosophila segmentation genes required in different domains along the longitudinal axis of the embryo. The domain of gene expression is smaller than the gap seen in the mutant embryo, suggesting that subordinate genes dependent on the gap genes are required to fill the gap. The gap gene Krüppel has been cloned and sequenced. Its product contains sequences predicted to form DNA-binding ‘fingers’, indicating that it may function as a trans-acting factor regulating transcription of other genes.