brinker levels regulated by a promoter proximal element support germ cell homeostasis.

A limited BMP signaling range in the stem cell niche of the ovary protects against germ cell tumors and promotes germ cell homeostasis. The canonical repressor of BMP signaling in both the Drosophila embryo and wing disc is the transcription factor Brinker (Brk), yet the expression and potential role of Brk in the germarium has not previously been described. Here, we find that brk expression requires a promoter-proximal element (PPE) to support long-distance enhancer action as well as to drive expression in the germarium. Furthermore, PPE subdomains have different activities; in particular, the proximal portion acts as a damper to regulate brk levels precisely. Using PPE mutants as well as tissue-specific RNA interference and overexpression, we show that altering brk expression within either the soma or the germline affects germ cell homeostasis. Remarkably, we find that Decapentaplegic (Dpp), the main BMP ligand and canonical antagonist of Brk, is upregulated by Brk in the escort cells of the germarium, demonstrating that Brk can positively regulate this pathway.

NaNuTrap: a technique for in vivo cell nucleus labelling using nanobodies.

In vivo cell labelling is challenging in fast developmental processes because many cell types differentiate more quickly than the maturation time of fluorescent proteins, making visualization of these tissues impossible with standard techniques. Here, we present a nanobody-based method, Nanobody Nuclear Trap (NaNuTrap), which works with the existing Gal4/UAS system in Drosophila and allows for early in vivo cell nuclei labelling independently of the maturation time of the fluorescent protein. This restores the utility of fluorescent proteins that have longer maturation times, such as those used in two-photon imaging, for live imaging of fast or very early developmental processes. We also present a more general application of this system, whereby NaNuTrap can convert cytoplasmic GFP expressed in any existing transgenic fly line into a nuclear label. This nuclear re-localization of the fluorescent signal can improve the utility of the GFP label, e.g. in cell counting, as well as resulting in a general increase in intensity of the live fluorescent signal. We demonstrate these capabilities of NaNuTrap by effectively tracking subsets of cells during the fast movements associated with gastrulation.

Coacting enhancers can have complementary functions within gene regulatory networks and promote canalization.

Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, we created deletions of two enhancers, brk5' and brk3', that drive similar but not identical expression of the gene brinker (brk) in early Drosophila embryos. Utilizing both in situ hybridization and quantitative mRNA analysis, we investigated the changes in the gene network state caused by the removal of one or both of the early acting enhancers. brk5' deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3' deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions.

The migrations of Drosophila muscle founders and primordial germ cells are interdependent.

Caudal visceral mesoderm (CVM) cells migrate from posterior to anterior of the Drosophila embryo as two bilateral streams of cells to support the specification of longitudinal muscles along the midgut. To accomplish this long-distance migration, CVM cells receive input from their environment, but little is known about how this collective cell migration is regulated. In a screen we found that wunen mutants exhibit CVM cell migration defects. Wunens are lipid phosphate phosphatases known to regulate the directional migration of primordial germ cells (PGCs). PGC and CVM cell types interact while PGCs are en route to the somatic gonadal mesoderm, and previous studies have shown that CVM impacts PGC migration. In turn, we found here that CVM cells exhibit an affinity for PGCs, localizing to the position of PGCs whether mislocalized or trapped in the endoderm. In the absence of PGCs, CVM cells exhibit subtle changes, including more cohesive movement of the migrating collective, and an increased number of longitudinal muscles is found at anterior sections of the larval midgut. These data demonstrate that PGC and CVM cell migrations are interdependent and suggest that distinct migrating cell types can coordinately influence each other to promote effective cell migration during development.

High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation.

Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identified previously studied Twist-dependent CRMs and robustly predicted new CRM activity in transgenesis, with newly identified Twist-occupied regions supporting diverse spatiotemporal patterns (>74% positive, n = 31). Some, but not all, candidate CRMs require Twist for proper expression in the embryo. The Twist motifs most favored in genome ChIP data (in vivo) differed from those most favored by Systematic Evolution of Ligands by EXponential enrichment (SELEX) (in vitro). Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50 bp of the ChIP summit and, of these, CACATG was most prevalent. Mutagenesis experiments demonstrated that different Twist E-box motif types are not fully interchangeable, suggesting that the ChIP-derived consensus (CABVTG) includes sites having distinct regulatory outputs. Further analysis of position, frequency of occurrence, and sequence conservation revealed significant enrichment and conservation of CABVTG E-box motifs near Twist ChIP-seq signal summits, preferential conservation of ±150 bp surrounding Twist occupied summits, and enrichment of GA- and CA-repeat sequences near Twist occupied summits. Our results show that high resolution in vivo occupancy data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic.

Complex interactions between cis-regulatory modules in native conformation are critical for Drosophila snail expression.

It has been shown in several organisms that multiple cis-regulatory modules (CRMs) of a gene locus can be active concurrently to support similar spatiotemporal expression. To understand the functional importance of such seemingly redundant CRMs, we examined two CRMs from the Drosophila snail gene locus, which are both active in the ventral region of pre-gastrulation embryos. By performing a deletion series in a ∼25 kb DNA rescue construct using BAC recombineering and site-directed transgenesis, we demonstrate that the two CRMs are not redundant. The distal CRM is absolutely required for viability, whereas the proximal CRM is required only under extreme conditions such as high temperature. Consistent with their distinct requirements, the CRMs support distinct expression patterns: the proximal CRM exhibits an expanded expression domain relative to endogenous snail, whereas the distal CRM exhibits almost complete overlap with snail except at the anterior-most pole. We further show that the distal CRM normally limits the increased expression domain of the proximal CRM and that the proximal CRM serves as a `damper' for the expression levels driven by the distal CRM. Thus, the two CRMs interact in cis in a non-additive fashion and these interactions may be important for fine-tuning the domains and levels of gene expression.

A Developmental Program Truncates Long Transcripts to Temporally Regulate Cell Signaling.

Rapid mitotic divisions and a fixed transcription rate limit the maximal length of transcripts in early Drosophila embryos. Previous studies suggested that transcription of long genes is initiated but aborted, as early nuclear divisions have short interphases. Here, we identify long genes that are expressed during short nuclear cycles as truncated transcripts. The RNA binding protein Sex-lethal physically associates with transcripts for these genes and is required to support early termination to specify shorter transcript isoforms in early embryos of both sexes. In addition, one truncated transcript for the gene short-gastrulation encodes a product in embryos that functionally relates to a previously characterized dominant-negative form, which maintains TGF-ß signaling in the off-state. In summary, our results reveal a developmental program of short transcripts functioning to help temporally regulate Drosophila embryonic development, keeping cell signaling at early stages to a minimum in order to support its proper initiation at cellularization.

Autoregulatory feedback controls sequential action of cis-regulatory modules at the brinker locus.

cis-regulatory modules (CRMs) act sequentially to regulate temporal expression of genes, but how the switch from one to the next is accomplished is not well understood. To provide insight, here we investigate the cis-regulatory system controlling brinker (brk) expression in the Drosophila embryo. Two distally located CRMs support expression at different times, while a promoter-proximal element (PPE) is required to support their action. In the absence of Brk protein itself or upon mutagenesis of Brk binding sites within the PPE, the late-acting CRM, specifically, is delayed. This block to late-acting CRM function appears to be removed when the early-acting CRM is also deleted. These results demonstrate that autoregulatory feedback is necessary for the early-acting CRM to disengage from the promoter so that the late-acting CRM may act. Autoregulation may be a commonly used mechanism to control sequential CRM action necessary for dynamic gene expression throughout the course of development.

Molecular phylogeny of songbirds (Passeriformes) inferred from mitochondrial 16S ribosomal RNA gene sequences.

Phylogenetic relationships among the families of passerine birds have been the subject of many debates. These relationships have been investigated by using a number of different character sets, including morphology, proteins, DNA-DNA hybridization, and mitochondrial DNA gene sequences. Our objective was to examine the phylogenetic relationships of a set of passerine songbirds (Oscines) and to test the taxonomic relationships proposed by. We sequenced 1403 aligned bases encompassing the mitochondrial transfer-RNA-Valine and 16S ribosomal RNA genes in 27 species from 14 families (including a Suboscine outgroup). Our results differ in significant ways from the superfamily designations of Sibley and Ahlquist by questioning the monophyly of the Sylvioidea and by placing the Regulidae in the Corvoidea.

Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast.

A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT.