An interview with Mike Levine.

Mike Levine, director of the Lewis-Sigler Institute for Integrative Genomics at Princeton University, is a developmental biologist who has dedicated his career to understanding how gene expression is regulated during development. Some of his most significant research, such as the co-discovery of the homeobox genes and his work on even skipped stripe 2, was performed in Drosophila, but he has since branched out to Ciona intestinalis, which he is using as a model to understand how vertebrate features have evolved. We had a lively chat with Mike at this year's Society for Developmental Biology (SDB) meeting, where he was awarded the Edwin Grant Conklin Medal.

Islet is a key determinant of ascidian palp morphogenesis.

The anterior-most ectoderm of ascidian larvae contains the adhesive papillae, or palps, which play an important role in triggering the metamorphosis of swimming tadpoles. In Ciona intestinalis, the palps consist of three conical protrusions within a field of thickened epithelium that form late in embryogenesis, as tailbuds mature into larvae. The palp protrusions express the LIM-homeodomain transcription factor Islet. Protrusion occurs through differential cell elongation, probably mediated by Islet, as we find that ectopic expression of Islet is sufficient to promote cell lengthening. FGF signaling is required for both Islet expression and palp morphogenesis. Importantly, we show that Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling. We conclude that Islet is a key regulatory factor governing morphogenesis of the palps. It is conceivable that Islet is also essential for the cellular morphogenesis of placode-derived sensory neurons in vertebrates.

Temporal waves of coherent gene expression during Drosophila embryogenesis.

MOTIVATION: Animal development depends on localized patterns of gene expression. Whole-genome methods permit the global identification of differential expression patterns. However, most gene-expression-clustering methods focus on the analysis of entire expression profiles, rather than temporal segments or time windows. RESULTS: In the current study, local clustering of temporal time windows was applied to developing embryos of the fruitfly, Drosophila melanogaster. Large-scale developmental events, involving temporal activation of hundreds of genes, were identified as discrete gene clusters. The time-duration analysis revealed six temporal waves of coherent gene expression during Drosophila embryogenesis. The most powerful expression waves preceded major morphogenetic movements, such as germ band elongation and dorsal closure. These waves of gene expression coincide with the inhibition of maternal transcripts during early development, the specification of ectoderm, differentiation of the nervous system, differentiation of the digestive tract, deposition of the larval cuticle and the reorganization of the cytoskeleton during global morphogenetic events. We discuss the implications of these findings with respect to the gene regulatory networks governing Drosophila development. AVAILABILITY: Data and software are available from the UC Berkeley web resource http://flydev.berkeley.edu/cgi-bin/GTEM/dmap_dm-ag/index_dmap.htm

Transcriptional enhancers in animal development and evolution.

Regulatory DNAs serve as templates to bring weakly interacting transcription factors into close proximity so they can work synergistically to switch genes on and off in time and space. Most of these regulatory DNAs are enhancers that can work over long distances--a million base pairs or more in mammals--to control gene expression. Critical enhancers are sometimes even found within the introns of neighboring genes. This review summarizes well-defined examples of enhancers controlling key processes in animal development. Potential mechanisms of transcriptional synergy are discussed with regard to enhancer structure and contemporary ChIP-sequencing assays, whereby just a small fraction of the observed binding sites represent bona fide regulatory DNAs. Finally, there is a discussion of how enhancer evolution can produce novelty in animal morphology and of the prospects for reconstructing transitions in animal evolution by introducing derived enhancers in basal ancestors.

Combinatorial patterning mechanisms in the Drosophila embryo.

The classical concept of the morphogen gradient proposes that small differences in the levels of a signalling molecule or transcription factor are responsible for producing a continuous spectrum of distinctive cellular identities across a naïve field of cells. In this review, we discuss how the Dorsal gradient controls the dorsal-ventral patterning of the early Drosophila embryo. This gradient extends from the ventral midline of the embryo into dorso-lateral regions, encompassing a cross-sectional field of approximately 20 cells. There is no evidence that these cells acquire distinctive identities due to subtle changes in the nuclear concentrations of the Dorsal protein. Rather, a variety of evidence suggests that the Dorsal gradient generates just three primary thresholds of gene activity. High levels activate gene expression in the presumptive mesoderm, while intermediate and low levels activate gene expression in the ventral and dorsal neurogenic ectoderm, respectively. We discuss how these primary readouts of the gradient establish localized domains of cell signalling, which work in a combinatorial manner with transcriptional networks to produce complex patterns of gene expression and tissue differentiation.

A distinct class of small RNAs arises from pre-miRNA-proximal regions in a simple chordate.

MicroRNAs (miRNAs) have been implicated in various cellular processes. They are thought to function primarily as inhibitors of gene activity by attenuating translation or promoting mRNA degradation. A typical miRNA gene produces a predominant approximately 21-nucleotide (nt) RNA (the miRNA) along with a less abundant miRNA(*) product. We sought to identify miRNAs from the simple chordate Ciona intestinalis through comprehensive sequencing of small RNA libraries created from different developmental stages. Unexpectedly, half of the identified miRNA loci encode up to four distinct, stable small RNAs. The additional RNAs, miRNA-offset RNAs (moRs), are generated from sequences immediately adjacent to the predicted approximately 60-nt pre-miRNA. moRs seem to be produced by RNAse III-like processing, are approximately 20 nt long and, like miRNAs, are observed at specific developmental stages. We present evidence suggesting that the biogenesis of moRs results from an intrinsic property of the miRNA processing machinery in C. intestinalis.

Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of the Ciona embryo.

Mesodermal tissues arise from diverse cell lineages and molecular strategies in the Ciona embryo. For example, the notochord and mesenchyme are induced by FGF/MAPK signaling, whereas the tail muscles are specified autonomously by the localized determinant, Macho-1. A unique mesoderm lineage, the trunk lateral cells, develop from a single pair of endomesoderm cells, the A6.3 blastomeres, which form part of the anterior endoderm, hematopoietic mesoderm and muscle derivatives. MAPK signaling is active in the endoderm descendants of A6.3, but is absent from the mesoderm lineage. Inhibition of MAPK signaling results in expanded expression of mesoderm marker genes and loss of endoderm markers, whereas ectopic MAPK activation produces the opposite phenotype: the transformation of mesoderm into endoderm. Evidence is presented that a specific Ephrin signaling molecule, Ci-ephrin-Ad, is required to establish asymmetric MAPK signaling in the endomesoderm. Reducing Ci-ephrin-Ad activity via morpholino injection results in ectopic MAPK signaling and conversion of the mesoderm lineage into endoderm. Conversely, misexpression of Ci-ephrin-Ad in the endoderm induces ectopic activation of mesodermal marker genes. These results extend recent observations regarding the role of Ephrin signaling in the establishment of asymmetric cell fates in the Ciona notochord and neural tube.

A systems view of Drosophila segmentation.

High-throughput technologies have enabled the systematic identification and characterization of most, or possibly all, of the components governing segmentation in the fruit fly Drosophila. What have we learned?

FoxF is essential for FGF-induced migration of heart progenitor cells in the ascidian Ciona intestinalis.

Heart development requires precise coordination of morphogenetic movements with progressive cell fate specification and differentiation. In ascidian embryos, FGF/MAPK-mediated activation of the transcription factor Ets1/2 is required for heart tissue specification and cell migration. We found that FoxF is one of the first genes to be activated in heart precursors in response to FGF signaling. We identified the FoxF minimal heart enhancer and used a cis-trans complementation test to show that Ets1/2 can interact with the FoxF enhancer in vivo. Next, we found that FoxF function is required downstream and in parallel to the FGF/MAPK/Ets cascade for cell migration. In addition, we demonstrated that targeted expression of a dominant-negative form of FoxF inhibits cell migration but not heart differentiation, resulting in a striking phenotype: a beating heart at an ectopic location within the body cavity of juveniles. Taken together, our results indicate that FoxF is a direct target of FGF signaling and is predominantly involved in the regulation of heart cell migration.

Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae.

The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. We wish to understand how changes in this network produce evolutionary diversity in insect gastrulation. The present study focuses on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene.

Evolution of the ventral midline in insect embryos.

The ventral midline is a source of signals that pattern the nerve cord of insect embryos. In dipterans such as the fruitfly Drosophila melanogaster (D. mel.) and the mosquito Anopheles gambiae (A. gam.), the midline is narrow and spans just 1-2 cells. However, in the honeybee, Apis mellifera (A. mel.), the ventral midline is broad and encompasses 5-6 cells. slit and other midline-patterning genes display a corresponding expansion in expression. Evidence is presented that this difference is due to divergent cis regulation of the single-minded (sim) gene, which encodes a bHLH-PAS transcription factor essential for midline differentiation. sim is regulated by a combination of Notch signaling and a Twist (Twi) activator gradient in D. mel., but it is activated solely by Twi in A. mel. We suggest that the Twi-only mode of regulation--and the broad ventral midline--represents the ancestral form of CNS patterning in Holometabolous insects.

FGF signaling delineates the cardiac progenitor field in the simple chordate, Ciona intestinalis.

Comprehensive gene networks in Ciona intestinalis embryos provide a foundation for characterizing complex developmental processes, such as the initial phases of chordate heart development. The basic helix-loop-helix regulatory gene Ci-Mesp is required for activation of cardiac transcription factors. Evidence is presented that Ci-Ets1/2, a transcriptional effector of receptor tyrosine kinase (RTK) signaling, acts downstream from Mesp to establish the heart field. Asymmetric activation of Ets1/2, possibly through localized expression of FGF9, drives heart specification within this field. During gastrulation, Ets1/2 is expressed in a group of four cells descended from two Mesp-expressing founder cells (the B7.5 cells). After gastrulation, these cells divide asymmetrically; the smaller rostral daughters exhibit RTK activation (phosphorylation of ERK) and form the heart lineage while the larger caudal daughters form the anterior tail muscle lineage. Inhibition of RTK signaling prevents heart specification. Targeted inhibition of Ets1/2 activity or FGF receptor function also blocks heart specification. Conversely, application of FGF or targeted expression of constitutively active Ets1/2 (EtsVp16) cause both rostral and caudal B7.5 lineages to form heart cells. This expansion produces an unexpected phenotype: transformation of a single-compartment heart into a functional multicompartment organ. We discuss these results with regard to the development and evolution of the multichambered vertebrate heart.

GATA factors participate in tissue-specific immune responses in Drosophila larvae.

Drosophila responds to infection by producing a broad range of antimicrobial agents in the fat body and more restricted responses in tissues such as the gut, trachea, and malpighian tubules. The regulation of antimicrobial genes in larval fat depends on linked Rel/NF-kappaB and GATA binding sites. Serpent functions as the major GATA transcription factor in the larval fat body. However, the transcriptional regulation of other tissue-specific responses is less well understood. Here, we present evidence that dGATAe regulates antimicrobial gene expression in the midgut. Regulatory regions for antimicrobial genes Diptericin and Metchnikowin require GATA sites for activation in the midgut, where Grain (dGATAc), dGATAd, and dGATAe are expressed in overlapping domains. Ectopic expression of dGATAe in the larval fat body, where it is normally absent, causes dramatic up-regulation of numerous innate immunity and gut genes, as judged by microarray analysis and in situ hybridization. Ectopic dGATAe also causes a host of symptoms reminiscent of hyperactive Toll (Toll(10b)) mutants, but without apparent activation of Toll signaling. Based on this evidence we propose that dGATAe mediates a Toll-independent immune response in the midgut, providing a window into the first and perhaps most ancient line of animal defense.

Computational models for neurogenic gene expression in the Drosophila embryo.

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. Here, we apply similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. We propose that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo.

The Drosophila microRNA iab-4 causes a dominant homeotic transformation of halteres to wings.

The Drosophila Bithorax Complex encodes three well-characterized homeodomain proteins that direct segment identity, as well as several noncoding RNAs of unknown function. Here, we analyze the iab-4 locus, which produces the microRNAs iab-4-5p and iab-4-3p. iab-4 is analogous to miR-196 in vertebrate Hox clusters. Previous studies demonstrate that miR-196 interacts with the Hoxb8 3' untranslated region. Evidence is presented that miR-iab-4-5p directly inhibits Ubx activity in vivo. Ectopic expression of mir-iab-4-5p attenuates endogenous Ubx protein accumulation and induces a classical homeotic mutant phenotype: the transformation of halteres into wings. These findings provide the first evidence for a noncoding homeotic gene and raise the possibility that other such genes occur within the Bithorax complex. We also discuss the regulation of mir-iab-4 expression during development.

Surfing with the tunicates into the post-genome era.

This year is the centenary of Edward G. Conklin's signal findings in embryology: the elucidation of complete cell lineages and the discovery of localized maternal determinants. Conklin used ascidian embryos to elucidate universal principles in embryology. A century later, ascidians, or sea squirts, have not only entered the post-genome era, but in many ways are leading the way to the promise of a "systems-level" understanding of complex processes such as notochord formation, neurogenesis, and even behavior.

A saturation screen for cis-acting regulatory DNA in the Hox genes of Ciona intestinalis.

A screen for the systematic identification of cis-regulatory elements within large (>100 kb) genomic domains containing Hox genes was performed by using the basal chordate Ciona intestinalis. Randomly generated DNA fragments from bacterial artificial chromosomes containing two clusters of Hox genes were inserted into a vector upstream of a minimal promoter and lacZ reporter gene. A total of 222 resultant fusion genes were separately electroporated into fertilized eggs, and their regulatory activities were monitored in larvae. In sum, 21 separable cis-regulatory elements were found. These include eight Hox linked domains that drive expression in nested anterior-posterior domains of ectodermally derived tissues. In addition to vertebrate-like CNS regulation, the discovery of cis-regulatory domains that drive epidermal transcription suggests that C. intestinalis has arthropod-like Hox patterning in the epidermis.