The epithelial polarity genes frazzled and GUK-holder adjust morphogen gradients to coordinate changes in cell position with cell fate specification.

Morphogenetic gradients specify distinct cell populations within tissues. Originally, morphogens were conceived as substances that act on a static field of cells, yet cells usually move during development. Thus, the way cell fates are defined in moving cells remains a significant and largely unsolved problem. Here, we investigated this issue using spatial referencing of cells and 3D spatial statistics in the Drosophila blastoderm to reveal how cell density responds to morphogenetic activity. We show that the morphogen decapentaplegic (DPP) attracts cells towards its peak levels in the dorsal midline, whereas dorsal (DL) stalls them ventrally. We identified frazzled and GUK-holder as the downstream effectors regulated by these morphogens that constrict cells and provide the mechanical force necessary to draw cells dorsally. Surprisingly, GUKH and FRA modulate the DL and DPP gradient levels and this regulation creates a very precise mechanism of coordinating cell movement and fate specification.

The KRÜPPEL-like transcription factor DATILÓGRAFO is required in specific cholinergic neurons for sexual receptivity in Drosophila females.

Courtship is a widespread behavior in which one gender conveys to the other a series of cues about their species identity, gender, and suitability as mates. In many species, females decode these male displays and either accept or reject them. Despite the fact that courtship has been investigated for a long time, the genes and circuits that allow females to generate these mutually exclusive responses remain largely unknown. Here, we provide evidence that the Krüppel-like transcription factor datilógrafo (dati) is required for proper locomotion and courtship acceptance in adult Drosophila females. dati mutant females are completely unable to decode male courtship and almost invariably reject males. Molecular analyses reveal that dati is broadly expressed in the brain and its specific removal in excitatory cholinergic neurons recapitulates the female courtship behavioral phenotype but not the locomotor deficits, indicating that these are two separable functions. Clonal analyses in female brains identified three discrete foci where dati is required to generate acceptance. These include neurons around the antennal lobe, the lateral horn, and the posterior superior lateral protocerebrum. Together, these results show that dati is required to organize and maintain a relatively simple excitatory circuit in the brain that allows females to either accept or reject courting males.

Formation of the BMP activity gradient in the Drosophila embryo.

The dorsoventral axis of the Drosophila embryo is patterned by a gradient of bone morphogenetic protein (BMP) ligands. In a process requiring at least three additional extracellular proteins, a broad domain of weak signaling forms and then abruptly sharpens into a narrow dorsal midline peak. Using experimental and computational approaches, we investigate how the interactions of a multiprotein network create the unusual shape and dynamics of formation of this gradient. Starting from observations suggesting that receptor-mediated BMP degradation is an important driving force in gradient dynamics, we develop a general model that is capable of capturing both subtle aspects of gradient behavior and a level of robustness that agrees with in vivo results.

High-resolution mapping of the Drosophila fourth chromosome using site-directed terminal deficiencies.

For more than 80 years, the euchromatic right arm of the Drosophila fourth chromosome (101F-102F) has been one of the least genetically accessible regions of the fly genome despite the fact that many important genes reside there. To improve the mapping of genes on the fourth chromosome, we describe a strategy to generate targeted deficiencies and we describe 13 deficiencies that subdivide the 300 kb between the cytological coordinates 102A6 and 102C1 into five discrete regions plus a 200-kb region from 102C1 to 102D6. Together these deficiencies substantially improve the mapping capabilities for mutant loci on the fourth chromosome.

3D Clumped Cell Segmentation Using Curvature Based Seeded Watershed.

Image segmentation is an important process that separates objects from the background and also from each other. Applied to cells, the results can be used for cell counting which is very important in medical diagnosis and treatment, and biological research that is often used by scientists and medical practitioners. Segmenting 3D confocal microscopy images containing cells of different shapes and sizes is still challenging as the nuclei are closely packed. The watershed transform provides an efficient tool in segmenting such nuclei provided a reasonable set of markers can be found in the image. In the presence of low-contrast variation or excessive noise in the given image, the watershed transform leads to over-segmentation (a single object is overly split into multiple objects). The traditional watershed uses the local minima of the input image and will characteristically find multiple minima in one object unless they are specified (marker-controlled watershed). An alternative to using the local minima is by a supervised technique called seeded watershed, which supplies single seeds to replace the minima for the objects. Consequently, the accuracy of a seeded watershed algorithm relies on the accuracy of the predefined seeds. In this paper, we present a segmentation approach based on the geometric morphological properties of the 'landscape' using curvatures. The curvatures are computed as the eigenvalues of the Shape matrix, producing accurate seeds that also inherit the original shape of their respective cells. We compare with some popular approaches and show the advantage of the proposed method.

Variation in the dorsal gradient distribution is a source for modified scaling of germ layers in Drosophila.

Specification of germ layers along the dorsoventral axis by morphogenetic gradients is an ideal model to study scaling properties of gradients and cell fate changes during evolution. Classical anatomical studies in divergent insects (e.g., flies and grasshoppers) revealed that the neuroectodermal size is conserved and originates similar numbers of neuroblasts of homologous identity. In contrast, mesodermal domains vary significantly in closely related Drosophila species. To further investigate the underlying mechanisms of scaling of germ layers across Drosophila species, we quantified the Dorsal (Dl)/NF-κB gradient, the main morphogenetic gradient that initiates separation of the mesoderm, neuroectoderm, and ectoderm. We discovered a variable range of Toll activation across species and found that Dl activates mesodermal genes at the same threshold levels in melanogaster sibling species. We also show that the Dl gradient distribution can be modulated by nuclear size and packing densities. We propose that variation in mesodermal size occurs at a fast evolutionary rate and is an important mechanism to define the ventral boundary of the neuroectoderm.

A novel genetic tool for clonal analysis of fourth chromosome mutations.

The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FRT). Here we introduce a method that overcomes these limitations and allows for the generation of single Minute haplo-4 clones of any fourth chromosome mutant gene in tissues of developing and adult flies.

An analysis of genetic changes during the divergence of Drosophila species.

BACKGROUND: It has been long appreciated that speciation involves changes in body plans and establishes genetic, reproductive, developmental and behavioral incompatibilities between populations. However, little is still known about the genetic components involved in these changes or the sequence and scale of events that lead to the differentiation of species. PRINCIPAL FINDINGS: In this paper, we investigated the genetic changes in three closely related species of Drosophila by making pair-wise comparisons of their genomes. We focused our analysis on the modern relatives of the alleles likely to be segregating in pre-historic populations at the time or after the ancestor of D. simulans became separated from the ancestor of D. melanogaster. Some of these genes were previously implicated in the genetics of reproduction and behavior while the biological functions of others are not yet clear. CONCLUSIONS: Together these results identify different classes of genes that might have participated in the beginning of segregation of these species millions of years ago in Africa.

Upright imaging of Drosophila embryos.

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

crossveinless defines a new family of Twisted-gastrulation-like modulators of bone morphogenetic protein signalling.

The Twisted gastrulation (Tsg) proteins are modulators of bone morphogenetic protein (BMP) activity in both vertebrates and insects. We find that the crossveinless (cv) gene of Drosophila encodes a new tsg-like gene. Genetic experiments show that cv, similarly to tsg, interacts with short gastrulation (sog) to modulate BMP signalling. Despite this common property, Cv shows a different BMP ligand specificity as compared with Tsg, and its expression is limited to the developing wing. These findings and the presence of two types of Tsg-like protein in several insects suggest that Cv represents a subgroup of the Tsg-like BMP-modulating proteins.

Molecular mapping of deletion breakpoints on chromosome 4 of Drosophila melanogaster.

As part of our effort to induce and identify mutations in all genes on chromosome 4 of Drosophila melanogaster, we have mapped the breakpoints of eight chromosome 4 deficiencies relative to the predicted genes along this chromosome. Although the approximate locations of Df(4)G, Df(4)C3, Df(4)M101-62f, Df(4)M101-63a, Df(4)J2, Df(4)O2, Df(4)C1-10AT, and Df(4)B2-2D are known (some from cytological observations and others predicted from P element locations), the extents of these deletions have not been mapped with respect to the predicted genes identified by the Drosophila Genome Project. Polymerase chain reaction primers were designed to amplify the predicted exons of all chromosome 4 genes, and homozygous embryos for each deficiency were identified and their DNA used to test for the presence or absence of these exons. By testing for the inability to amplify various exons along the length of the chromosome, we were able to determine which predicted genes are missing in each deficiency. The five deficiencies, Df(4)G, Df(4)C3, Df(4)C1-10AT, and Df(4)B2-20 (all terminal deletions), and Df(4)M101-62f (a proximal interstitial deletion), enabled us to partition the gene-containing, right arm of chromosome 4 into five regions. Region A [uncovered by Df(4)M101-62f] contains the proximal-most 21 genes; region B [uncovered by Df(4)B2-2D] contains the next 12 genes; region C [uncovered by Df(4)B2-2D and Df(4)C1-10AT] contains the next 17 genes; region D [uncovered by Df(4)B2-2D, Df(4)C1-10AT, and Df(4)C3] contains the next 21 genes; and region E [uncovered by Df(4)B2-2D, Df(4)C1-10AT, Df(4)C3, and Df(4)G] contains the distal-most ten genes. By using Df(4)M101-62f, Df(4)B2-2D, Df(4)C1-10AT, Df(4)C3, and Df(4)G in complementation tests, we can assign newly induced recessive lethal mutations to one of the five regions on chromosome 4. This will substantially reduce the amount of DHPLC analysis required to match each mutation to a predicted transcript on chromosome 4.