Regulatory interactions and role in cell type specification of the Malpighian tubules by the cut, Krüppel, and caudal genes of Drosophila.

Krüppel and caudal genes are both required for normal segmentation of the embryo, and the developmental regulatory gene cut is necessary for the normal specification of external sensory organs. These three genes are also expressed in the Malpighian tubules before and during differentiation. Two of the genes, Krüppel and cut, are known to be required for development of the tubules. We report that the absence of maternal and zygotic caudal function reduces their normal growth and elongation. Normal Krüppel function, which is known to be required for caudal expression, is also required for cut expression, while cut and caudal are expressed independently of each other. Cell type transformations of Malpighian tubules were studied by examining the effects of mutations on the expression of markers specific to Malpighian tubules, hindgut, or midgut of normal embryos. Loss of Krüppel activity confers hindgut characteristics on those cells that normally form the Malpighian tubules with all markers tested. Loss of cut function alters the expression of some markers but not others. The pathway of tissue specific gene regulation, apparently, branches beyond Krüppel to form at least a cut and a caudal branch.     

Kruppel is a gap gene in the intermediate germband insect Oncopeltus fasciatus and is required for development of both blastoderm and germband-derived segments

Segmentation in long germband insects such as Drosophila occurs essentially simultaneously across the entire body. A cascade of segmentation genes patterns the embryo along its anterior-posterior axis via subdivision of the blastoderm. This is in contrast to short and intermediate germband modes of segmentation where the anterior segments are formed during the blastoderm stage and the remaining posterior segments arise at later stages from a posterior growth zone. The biphasic character of segment generation in short and intermediate germ insects implies that different formative mechanisms may be operating in blastoderm-derived and germband-derived segments. In Drosophila, the gap gene Krüppel is required for proper formation of the central portion of the embryo. This domain of Krüppel activity in Drosophila corresponds to a region that in short and intermediate germband insects spans both blastoderm and germband-derived segments. We have cloned the Krüppel homolog from the milkweed bug, Oncopeltus fasciatus (Hemiptera, Lygaeidae), an intermediate germband insect. We find that Oncopeltus Krüppel is expressed in a gap-like domain in the thorax during the blastoderm and germband stages of embryogenesis. In order to investigate the function of Krüppel in Oncopeltus segmentation, we generated knockdown phenotypes using RNAi. Loss of Krüppel activity in Oncopeltus results in a large gap phenotype, with loss of the mesothoracic through fourth abdominal segments. Additionally, we find that Krüppel is required to suppress both anterior and posterior Hox gene expression in the central portion of the germband. Our results show that Krüppel is required for both blastoderm-derived and germband-derived segments and indicate that Krüppel function is largely conserved in Oncopeltus and Drosophila despite their divergent embryogenesis.     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interactions of Krüppel with other terminal genes. The gap genes tailless and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

The giant gene of Drosophila encodes a b-ZIP DNA-binding protein that regulates the expression of other segmentation gap genes.

The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Krüppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Krüppel and knirps segmentation gap genes. The analysis of the interactions between Krüppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Krüppel is in fact mediated by a negative effect of Krüppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Krüppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.     

Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krüppel

We have identified early embryo proteins related to the segmentation gene Krüppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krüppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krüppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krüppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krüppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krüppel function may involve post-translational modification of proteins.     

Mutually repressive interactions between the gap genes giant and Krüppel define middle body regions of the Drosophila embryo

The gap genes play a key role in establishing pair-rule and homeotic stripes of gene expression in the Drosophila embryo. There is mounting evidence that overlapping gradients of gap gene expression are crucial for this process. Here we present evidence that the segmentation gene giant is a bona fide gap gene that is likely to act in concert with hunchback, Krüppel and knirps to initiate stripes of gene expression. We show that Krüppel and giant are expressed in complementary, non-overlapping sets of cells in the early embryo. These complementary patterns depend on mutually repressive interactions between the two genes. Ectopic expression of giant in early embryos results in the selective repression of Krüppel, and advanced-stage embryos show cuticular defects similar to those observed in Krüppel- mutants. This result and others suggest that the strongest regulatory interactions occur among those gap genes expressed in nonadjacent domains. We propose that the precisely balanced overlapping gradients of gap gene expression depend on these strong regulatory interactions, coupled with weak interactions between neighboring genes.     

Zinc finger protein gene complexes on mouse chromosomes 8 and 11

Two murine homologs of the Drosophila Krüppel gene, a member of the gap class of developmental control genes that encode a protein with zinc fingers, were mapped to mouse chromosomes 8 and 11 by using somatic cell hybrids and an interspecific backcross. Surprisingly, both genes were closely linked to two previously mapped, Krüppel-related zinc finger protein genes, suggesting that they are part of gene complexes.     

PAR-1 kinase phosphorylates Dlg and regulates its postsynaptic targeting at the Drosophila neuromuscular junction

Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, we show that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Krüppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits.     

Plasticity within the lateral somatic mesoderm of Drosophila embryos

Each of 30 Drosophila larval somatic muscles has its individual shape, insertion sites and innervation. From the very beginning, the formation of individual muscles is controlled by a set of muscle identity genes. The four lateral transverse muscles (LT1-LT4) are thought to be specified by the combinatorial activity of Krüppel (Kr), apterous (ap) and muscle specific homeobox (msh) genes whilst the activity of the ladybird (lb) genes is required for proper formation of the neighbouring segmental border muscle (SBM). We have recently shown that ectopic expression of lb changes the identity of Kr-expressing lateral muscle precursors and recruits them to form enlarged or duplicated SBMs. Here we report that loss of msh function leads to a similar transformation resulting in the overproduction of SBMs. Inversely, in msh gain of function embryos, the prospective SBM myoblasts change their identity resulting in the formation of enlarged lateral transverse muscles. These data indicate a key role for the msh and lb genes in the specification and diversification of myoblast lineages from the lateral domain, and reveal a plasticity of cell fate within the somatic mesoderm of Drosophila.     

A multigene family encoding several "finger" structures is present and differentially active in mammalian genomes

Mouse genomic DNA contains multiple copies of sequences homologous to the Drosophila "Krüppel," a member of the "gap" class of developmental control genes of the fruit fly. The most interesting aspect of the homologous region is that, like Xenopus TFIIIA, it contains multiple finger-like folded domains capable of binding to nucleic acids. We have isolated six individual phages from a mouse genomic library on the basis of their DNA homology to Krüppel finger-coding probes, and describe here the DNA sequence and expression of two such clones containing finger-like structures. Upon differentiation of mouse teratocarcinoma cell line F9 with retinoic acid and cAMP, the expression of both genes was drastically reduced, and in one instance was undetectable. Each of the several other eukaryotic DNAs analyzed contained multiple copies of homologous genes with putative finger structures, indicating the presence of a finger-containing multigene family in higher organisms.