cis-acting control elements for Krüppel expression in the Drosophila embryo.

Krüppel (Kr), a gap gene of Drosophila, shows complex spatial patterns of expression during the different stages of embryogenesis. In order to identify cis-acting sequences required for normal Kr gene expression, we analysed the expression patterns of fusion gene constructs in transgenic embryos. In these constructs, bacterial lacZ expression was placed under the control of Kr sequences in front of a basal promoter. We identified cis-acting Kr control units which drive beta-galactosidase expression in 10 known locations of Kr expression in early and late embryos. More than one cis-regulatory element drives the expression in the anterior domain at the blastoderm stage, in the nervous system, the midline precursor cells and in the amino-serosa. In addition, two cis-acting elements direct the first zygotic expression of Kr in a striped subpattern within the central region of the blastoderm embryo. Both elements respond to alterations in the activities of maternal organizer genes known to be required for Kr expression in establishing the thoracic and anterior abdominal segments in the wild-type embryo.     

A Caenorhabditis elegans wee1 homolog is expressed in a temporally and spatially restricted pattern during embryonic development

A wee1 homolog, wee-1.1, is expressed in both a temporally and spatially restricted pattern during early Caenorhabditis elegans embryogenesis, and is undetectable throughout the remainder of embryogenesis. The wee-1.1 message appears to be zygotically expressed in the somatic founder cell E of the 12-cell embryo. This expression disappears when the E blastomere divides for the first time. The wee-1.1 message then appears transiently in the nuclei of the eight great-granddaughter cells of the AB somatic founder cell, just before these cells divide in the 16-cell embryo. Following this division, the wee-1.1 mRNA is no longer detectable throughout the remainder of embryogenesis. The expression of wee-1.1 in the E blastomere and in the AB progeny appears to be restricted to nuclei in prophase and metaphase of the cell cycle. Analysis of the wee-1.1 mRNA expression pattern in maternal-effect lethal mutants suggests that this expression pattern is restricted to cells of the E and AB fates in the early embryo. This mRNA expression pattern is restricted to a 10-15-min span of embryonic development and may be regulating the timing of crucial cell divisions at this early stage of development.     

Interactions of Drosophila Ultrabithorax Regulatory Regions with Native and Foreign Promoters

The Ultrabithorax (Ubx) gene of the Drosophila bithorax complex is required to specify parasegments 5 and 6. Two P-element ``enhancer traps' have been recovered within the locus that contain the bacterial lacZ gene under the control of the P-element promoter. The P insertion that is closer to the Ubx promoter expresses lacZ in a pattern similar to that of the normal Ubx gene, but also in parasegment 4 during embryonic development. Two deletions have been recovered that remove the normal Ubx promoter plus several kilobases on either side, but retain the lacZ reporter gene. The lacZ patterns from the deletion derivatives closely match the normal pattern of Ubx expression in late embryos and imaginal discs. The lacZ genes in the deletion derivatives are also negatively regulated by Ubx and activated in trans by Contrabithorax mutations, again like the normal Ubx gene. Thus, the deleted regions, including several kilobases around the Ubx promoter, are not required for long range interactions with Ubx regulatory regions. The deletion derivatives also stimulate transvection, a pairing-dependent interaction with the Ubx promoter on the homologous chromosome.     

Pag-3, a Caenorhabditis Elegans Gene Involved in Touch Neuron Gene Expression and Coordinated Movement

Mutations in a newly identified gene, pag-3, cause ectopic expression of touch neuron genes mec-7, mec-7lacZ and mec-4lacZ in the lineal sisters of the ALM touch neurons, the BDU neurons. pag-3 mutants also show a reverse kinker uncoordinated phenotype. The first pag-3 allele was isolated in a screen for mutants with altered immunofluorescence staining patterns. Two additional pag-3 alleles were identified in a noncomplementation screen of 38,000 haploid genomes. All of the pag-3 alleles were recessive to wild type and cause the same phenotypes. Two-factor crosses, deficiency mapping and three-factor crosses located pag-3 to the right arm of the X chromosome between unc-3 and unc-7. Because recessive mutations in pag-3 result in expression of several touch cell specific genes in the BDU neurons, pag-3(+) must directly or indirectly suppress expression of these genes in the BDU neurons. Although pag-3 mutants did not show mec-3lacZ expression in their BDU neurons, expression of mec-7lacZ and mec-4lacZ in the BDU neurons of pag-3 mutants required mec-3(+).     

The role of the NK-homeobox gene slouch (S59) in somatic muscle patterning.

In the Drosophila embryo, a distinct class of myoblasts, designated as muscle founders, prefigures the mature pattern of somatic body wall muscles. Each founder cell appears to be instrumental in generating a single larval muscle with a defined identity. The NK homeobox gene S59 was the first of a growing number of proposed 'identity genes' that have been found to be expressed in stereotyped patterns in specific subsets of muscle founders and their progenitor cells and are thought to control their developmental fates. In the present study, we describe the effects of gain- and loss-of-function experiments with S59. We find that a null mutation in the gene encoding S59, which we have named slouch (slou), disrupts the development of all muscles that are derived from S59-expressing founder cells. The observed phenotypes upon mutation and ectopic expression of slouch include transformations of founder cell fates, thus confirming that slouch (S59) functions as an identity gene in muscle development. These fate transformations occur between sibling founder cells as well as between neighboring founders that are not lineage-related. In the latter case, we show that slouch (S59) activity is required cell-autonomously to repress the expression of ladybird (lb) homeobox genes, thereby preventing specification along the lb pathway. Together, these findings provide new insights into the regulatory interactions that establish the somatic muscle pattern.     

A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo

In the chick embryo, two secreted factors have recently be shown to cooperate in inducing the first axial structure, the primitive streak: cWnt8C (normally expressed around the circumference of the embryo, in the marginal zone) and the TGF beta superfamily member cVg1 (expressed in the posterior part of the marginal zone) (Development 128 (2001) 2915). Misexpression of Vg1 in the anterior marginal zone induces an ectopic primitive streak and recapitulates the morphological changes associated with normal primitive streak formation. Here, we analyse the time-course of appearance and disappearance of expression of 12 genes (cVg1, Lef1, Nodal, FGF8, cWnt8C, cBra, cNot1, goosecoid, HNF3 beta, Chordin, Otx2 and Sox3, whose normal expression is also polarized at early stages of development) in response to cVg1 misexpression in the anterior marginal zone. We show that a hierarchy of gene expression accompanies induction of the ectopic axis, reminiscent of the order in which the same genes begin to be expressed in the normal embryo.     

Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.     

MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis.

The yeast MER1 gene is required for the production of viable meiotic products and for meiotic recombination. Cytological analysis of chromosome spreads from a mer1 mutant indicates that the MER1 gene product is also required for normal chromosome pairing. mer1 strains make axial elements, precursors to the synaptonemal complex; however, the chromosomes in most nuclei do not become fully synapsed. The DNA sequence of the MER1 coding region was determined; the MER1 open reading frame encodes a 270-amino-acid protein with a molecular mass of 31.1 kilodaltons. The MER1 protein shows limited sequence similarity to calmodulin. Expression of the MER1 gene was examined by RNA blot hybridization analysis and through the construction and analysis of mer1::lacZ fusion genes. Expression of the MER1 gene is meiotically induced and required the IME1 gene product. Thus, expression of the MER1 gene early in meiosis is required for proper chromosome pairing and meiotic recombination.     

SEMAPHORE1 functions during the regulation of ancestrally duplicated knox genes and polar auxin transport in maize

The expression of class 1 knotted1-like homeobox (knox) genes affects numerous plant developmental processes, including cell-fate acquisition, lateral organ initiation, and maintenance of shoot apical meristems. The SEMAPHORE1 gene product is required for the negative regulation of a subset of maize knox genes, the duplicated loci rough sheath 1 and gnarley1 (knox4). Recessive mutations in semaphore1 result in the ectopic expression of knox genes in leaf and endosperm tissue. Genetic analyses suggest that SEMAPHORE1 may regulate knox gene expression in a different developmental pathway than ROUGH SHEATH2, the first-identified regulator of knox gene expression in maize. Mutations at semaphore1 are pleiotropic, disrupting specific domains of the shoot. However, unlike previously described mutations that cause ectopic knox gene expression, semaphore1 mutations affect development of the embryo, endosperm, lateral roots, and pollen. Moreover, polar transport of the phytohormone auxin is significantly reduced in semaphore1 mutant shoots. The data suggest that many of the pleiotropic semaphore1 phenotypes result from defective polar auxin transport (PAT) in sem1 mutant shoots, and support models correlating down-regulated knox gene expression and PAT in maize shoots.     

Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI.

In Saccharomyces cerevisiae, the TPI gene product, triosephosphate isomerase, makes up about 2% of the soluble cellular protein. Using in vitro and in vivo footprinting techniques, we have identified four binding sites for three factors in the 5' noncoding region of TPI: a REB1-binding site located at positions -401 to -392, two GCR1-binding sites located at positions -381 to -366 and -341 to -326, and a RAP1-binding site located at positions -358 to -346. We tested the effects of mutations at each of these binding sites on the expression of a TPI::lacZ gene fusion which carried 853 bp of the TPI 5' noncoding region integrated at the URA3 locus. The REB1-binding site is dispensable when material 5' to it is deleted; however, if the sequence 5' to the REB1-binding site is from the TPI locus, expression is reduced fivefold when the site is mutated. Because REB1 blocks nucleosome formation, the most likely function of its binding site in the TPI controlling region is to prevent the formation of nucleosomes over the TPI upstream activation sequence. Mutations in the RAP1-binding site resulted in a 10-fold reduction in expression of the reporter gene. Mutating either GCR1-binding site alone had a modest effect on expression of the fusion. However, mutating both GCR1-binding sites resulted in a 68-fold reduction in the level of expression of the reporter gene. A LexA-GCR1 fusion protein containing the DNA-binding domain of LexA fused to the amino terminus of GCR1 was able to activate expression of a lex operator::GAL1::lacZ reporter gene 116-fold over background levels. From this experiment, we conclude that GCR1 is able to activate gene expression in the absence of REB1 or RAP1 bound at adjacent binding sites. On the basis of these results, we suggest that GCR1 binding is required for activation of TPI and other GCR1-dependent genes and that the primary role of other factors which bind adjacent to GCR1-binding sites is to facilitate of modulate GCR1 binding in vivo.     

Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium

The Salmonella typhimurium metE and metR genes share a common control region, with overlapping, divergently transcribed promoters. A double gene fusion was constructed in which the metE promoter directs expression of the Escherichia coli lacZ gene and the metR promoter directs expression of the E. coli galK gene. By using an E. coli strain lysogenized with a lambda bacteriophage carrying the metE-lacZ metR-galK double fusion (lambda Elac.Rgal), two classes of cis-acting mutations were isolated that increase metR-galK expression. The first class of mutations causes a simultaneous decrease in metE-lacZ expression by disrupting the normal MetR-mediated activation of the metE promoter. The mutations are located within a region extending from 17 to 34 base pairs upstream of the -35 region of the metE promoter. Gel mobility shift assays and DNaseI protection experiments demonstrated that the MetR protein specifically binds to a 24-base-pair region encompassing these mutations. The second class of mutations increases metR-galK expression by directly altering the promoter consensus sequences of the metE and metR promoters.     

Expression of the homeotic gene mab-5 during Caenorhabditis elegans embryogenesis.

mab-5 is a member of a complex of homeobox-containing genes evolutionarily related to the Antennapedia and bithorax complexes of Drosophila melanogaster. Like the homeotic genes in Drosophila, mab-5 is required in a particular region along the anterior-posterior body axis, and acts during postembryonic development to give cells in this region their characteristic identities. We have used a mab-5-lacZ fusion integrated into the C. elegans genome to study the posterior-specific expression of mab-5 during embryogenesis. The mab-5-lacZ fusion was expressed in the posterior of the embryo by 180 minutes after the first cleavage, indicating that the mechanisms responsible for the position-specific expression of mab-5-lacZ act at a relatively early stage of embryogenesis. In embryos homozygous for mutations in the par genes, which disrupt segregation of factors during early cleavages, expression of mab-5-lacZ was no longer localized to the posterior. This suggests that posterior-specific expression of mab-5 depends on the appropriate segregation of developmental factors during early embryogenesis. After extrusion of any blastomere of the four-cell embryo, descendants of the remaining three cells could still express the mab-5-lacZ fusion. In these partial embryos, however, the fusion was often expressed in cells scattered throughout the embryo, suggesting that cell-cell interactions and/or proper positioning of early blastomeres are required for mab-5 expression to be localized to the posterior.