A Fully Synthetic Transcriptional Platform for a Multicellular Eukaryote

Regions of genomic DNA called enhancers encode binding sites for transcription factor proteins. Binding of activators and repressors increase and reduce transcription, respectively, but it is not understood how combinations of activators and repressors generate precise patterns of transcription during development. Here, we explore this problem using a fully synthetic transcriptional platform in Drosophila consisting of engineered transcription factor gradients and artificial enhancers. We found that binding sites for a transcription factor that makes DNA accessible are required together with binding sites for transcriptional activators to produce a functional enhancer. Only in this context can changes in the number of activator binding sites mediate quantitative control of transcription. Using an engineered transcriptional repressor gradient, we demonstrate that overlapping repressor and activator binding sites provide more robust repression and sharper expression boundaries than non-overlapping sites. This may explain why this common motif is observed in many developmental enhancers. http://www.cell.com/cell-reports/fulltext/S2211-1247(16)31715-6

Moving Reading List

For those of you who follow this list, I'll be moving it over to twitter--it's far easier to manage: https://twitter.com/JustinMCrocker

The landscape of RNA polymerase II transcription initiation in C. elegansreveals promoter and enhancer architectures

RNA polymerase transcription initiation sites are largely unknown inCaenorhabditis elegans. The initial 5′ end of most protein-coding transcripts is removed by trans-splicing, and noncoding initiation sites have not been investigated. We characterized the landscape of RNA Pol II transcription initiation, identifying 73,500 distinct clusters of initiation. Bidirectional transcription is frequent, with a peak of transcriptional pairing at 120 bp. We assign transcription initiation sites to 7691 protein-coding genes and find that they display features typical of eukaryotic promoters. Strikingly, the majority of initiation events occur in regions with enhancer-like chromatin signatures. Based on the overlap of transcription initiation clusters with mapped transcription factor binding sites, we define 2361 transcribed intergenic enhancers. Remarkably, productive transcription elongation across these enhancers is predominantly in the same orientation as that of the nearest downstream gene. Directed elongation from an upstream enhancer toward a downstream gene could potentially deliver RNA polymerase II to a proximal promoter, or alternatively might function directly as a distal promoter. Our results provide a new resource to investigate transcription regulation in metazoans.

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality
Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ~4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared withDrosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor–binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.

Inferring chromatin-bound protein complexes from genome-wide binding assays

Inferring chromatin-bound protein complexes from genome-wide binding assays [METHOD]

Genome-wide binding assays can determine where individual transcription factors bind in the genome. However, these factors rarely bind chromatin alone, but instead frequently bind to cis-regulatory elements (CREs) together with other factors thus forming protein complexes. Currently there are no integrative analytical approaches that can predict which complexes are formed on chromatin. Here, we describe a computational methodology to systematically capture protein complexes and infer their impact on gene expression. We applied our method to three human cell types, identified thousands of CREs, inferred known and undescribed complexes recruited to these CREs, and determined the role of the complexes as activators or repressors. Importantly, we found that the predicted complexes have a higher number of physical interactions between their members than expected by chance. Our work provides a mechanism for developing hypotheses about gene regulation via binding partners, and deciphering the interplay between combinatorial binding and gene expression.

Frequency of TERT promoter mutations in human cancers

Frequency of TERT promoter mutations in human cancers

by João Vinagre

Article

Reactivation of telomerase has been implicated in human tumorigenesis. Here, somatic mutations in the TERT promoter are reported in cancers of the central nervous system, bladder, follicular cell-derived thyroid and melanoma, thus demonstrating that TERT promoter mutations are a frequent event in human cancer.

Nature Communications doi: 10.1038/ncomms3185

Authors: João Vinagre, Ana Almeida, Helena Pópulo, Rui Batista, Joana Lyra, Vasco Pinto, Ricardo Coelho, Ricardo Celestino, Hugo Prazeres, Luis Lima, Miguel Melo, Adriana Gaspar da Rocha, Ana Preto, Patrícia Castro, Ligia Castro, Fernando Pardal, José Manuel Lopes, Lúcio Lara Santos, Rui Manuel Reis, José Cameselle-Teijeiro, Manuel Sobrinho-Simões, Jorge Lima, Valdemar Máximo, Paula Soares

Local features determine cis-regulatory function [Genetics]

by White, M. A., Myers, C. A., Corbo, J. C., Cohen, B. A.

Transcription factors (TFs) recognize short sequence motifs that are present in millions of copies in large eukaryotic genomes. TFsmust distinguish their target binding sites from a vast genomic excess of spurious motif occurrences; however, it is unclear whether functional sites are distinguished from nonfunctional motifs by local primary sequence features...

DNA-binding site changes in forkhead evolution [Evolution]

DNA-binding site changes in forkhead evolution [Evolution]

by Nakagawa, S., Gisselbrecht, S. S., Rogers, J. M., Hartl, D. L., Bulyk, M. L.

The evolution of transcriptional regulatory networks entails the expansion and diversification of transcription factor (TF) families. The forkhead family of TFs, defined by a highly conserved winged helix DNA-binding domain (DBD), has diverged into dozens of subfamilies in animals, fungi, and related protists. We have used a combination of maximum-likelihood...

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design

by Robin Smith

Background: Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. Results: We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. Conclusions: This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.

An Epigenetic Trap Stabilizes Singular Olfactory Receptor Expression

An Epigenetic Trap Stabilizes Singular Olfactory Receptor Expression

David B. Lyons, William E. Allen, Tracie Goh, Lulu Tsai, Gilad Barnea, Stavros Lomvardas. The molecular mechanisms regulating olfactory receptor (OR) expression in the mammalian nose are not yet understood. Here, we identify the transient expression of histone demethylase LSD1 and the ....

SET1 and p300 Act Synergistically, through Coupled Histone Modifications, in Transcriptional Activation by p53

SET1 and p300 Act Synergistically, through Coupled Histone Modifications, in Transcriptional Activation by p53

Zhanyun Tang, Wei-Yi Chen, Miho Shimada, Uyen T.T. Nguyen, Jaehoon Kim, Xiao-Jian Sun, Toru Sengoku, Robert K. McGinty, Joseph P. Fernandez, Tom W. Muir, Robert G. Roeder. The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with....

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis

Publication date: 15 August 2013
Source:Developmental Biology, Volume 380, Issue 2
Author(s): Heather Marlow , David Q. Matus , Mark Q. Martindale
The primary axis of cnidarians runs from the oral pole to the apical tuft and defines the major body axis of both the planula larva and adult polyp. In the anthozoan cnidarian Nematostella vectensis, the primary oral–aboral (O–Ab) axis first develops during the early embryonic stage. Here, we present evidence that pharmaceutical activators of canonical wnt signaling affect molecular patterning along the primary axis of Nematostella. Although not overtly morphologically complex, molecular investigations in Nematostella reveal that the O–Ab axis is demarcated by the expression of differentially localized signaling molecules and transcription factors that may serve roles in establishing distinct ectodermal domains. We have further characterized the larval epithelium by determining the position of a nested set of molecular boundaries, utilizing several newly characterized as well as previously reported epithelial markers along the primary axis. We have assayed shifts in their position in control embryos and in embryos treated with the pharmacological agents alsterpaullone and azakenpaullone, Gsk3β inhibitors that act as canonical wnt agonists, and the Wnt antagonist iCRT14, following gastrulation. Agonist drug treatments result in an absence of aboral markers, a shift in the expression boundaries of oral markers toward the aboral pole, and changes in the position of differentially localized populations of neurons in a dose-dependent manner, while antagonist treatment had the opposite effect. These experiments are consistent with canonical wnt signaling playing a role in an orally localized wnt signaling center. These findings suggest that in Nematostella, wnt signaling mediates O–Ab ectodermal patterning across a surprisingly complex epithelium in planula stages following gastrulation in addition to previously described roles for the wnt signaling pathway in endomesoderm specification during gastrulation and overall animal–vegetal patterning at earlier stages of anthozoan development.

Deciphering the onychophoran ‘segmentation gene cascade’: Gene expression reveals limited involvement of pair rule gene orthologs in segmentation, but a highly conserved segment polarity gene network

Deciphering the onychophoran ‘segmentation gene cascade’: Gene expression reveals limited involvement of pair rule gene orthologs in segmentation, but a highly conserved segment polarity gene network

Publication date: Available online 20 July 2013
Source:Developmental Biology
Author(s): Ralf Janssen , Graham E. Budd
The hallmark of the arthropods is their segmented body, although origin of segmentation, however, is unresolved. In order to shed light on the origin of segmentation we investigated orthologs of pair rule genes (PRGs) and segment polarity genes (SPGs) in a member of the closest related sister-group to the arthropods, the onychophorans. Our gene expression data analysis suggests that most of the onychophoran PRGs do not play a role in segmentation. One possible exception is the even-skipped (eve) gene that is expressed in the posterior end of the onychophoran where new segments are likely patterned, and is also expressed in segmentation-gene typical transverse stripes in at least a number of newly formed segments. Other onychophoran PRGs such as runt (run), hairy/Hes (h/Hes) and odd-skipped (odd) do not appear to have a function in segmentation at all. Onychophoran PRGs that act low in the segmentation gene cascade in insects, however, are potentially involved in segment-patterning. Most obvious is that from the expression of the pairberry (pby) gene ortholog that is expressed in a typical SPG-pattern. Since this result suggested possible conservation of the SPG-network we further investigated SPGs (and associated factors) such as Notum in the onychophoran. We find that the expression patterns of SPGs in arthropods and the onychophoran are highly conserved, suggesting a conserved SPG-network in these two clades, and indeed also in an annelid. This may suggest that the common ancestor of lophotrochozoans and ecdysozoans was already segmented utilizing the same SPG-network, or that the SPG-network was recruited independently in annelids and onychophorans/arthropods.

Differential and redundant functions of gooseberry and gooseberry neuro in the central nervous system and segmentation of the Drosophila embryo

Differential and redundant functions of gooseberry and gooseberry neuro in the central nervous system and segmentation of the Drosophila embryo

Publication date: Available online 22 July 2013
Source:Developmental Biology
Author(s): Haihuai He , Markus Noll
The gooseberry locus of Drosophila consists of two homologous Pax genes, gooseberry neuro (gsbn) and gooseberry (gsb). Originally characterized by genetics as a single segment-polarity gene, its role in segmentation has been enigmatic, as only deficiencies uncovering both genes showed a strong segmentation phenotype while mutants of gsb did not. To solve this conundrum and assay for differential roles of gsbn and gsb, we have obtained by homologous recombination for the first time null mutants of either gene as well as a deficiency inactivating only gsbn and gsb. Our analysis shows that (i) gsbn null mutants are subviable while all surviving males and most females are sterile; (ii) gsb and gsbn share overlapping functions in segmentation and the CNS, in which gsbn largely, but not completely depends on the transcriptional activation by the product of gsb; (iii) as a consequence, in the absence of gsbn, gsb becomes haploinsufficient for its function in the CNS, and gsbn−/−gsb−/+ mutants die as larvae. Such mutants display defects in the proper specification of the SNa branch of the segmental nerve, which appears intact in gsbn−/− mutants. Lineage analysis in the embryonic CNS showed that gsbn is expressed in the entire lineage derived from NB5-4, which generates 4 or 5 motoneurons whose axons are part of the SNa branch and all of which except one also express BarH1. Analysis of gsbn−/−gsb−/+ clones originating from NB5-4 further suggests that gsb and gsbn specify the SNa fate and concomitantly repress the SNc fate in this lineage and that their products activate BarH1 transcription. Specification of the SNa fate by Gsb and Gsbn occurs mainly at the NB and GMC stage. However, the SNa mutant phenotype can be rescued by providing Gsbn as late as at the postmitotic stage. The hierarchical relationship between gsb and gsbn, the haploinsufficiency of gsb in gsbn mutants, and their redundant roles in the epidermis and CNS are discussed. A model is proposed how selection for both genes occurred after their duplication during evolution.

Female Behaviour Drives Expression and Evolution of Gustatory Receptors in Butterflies

Female Behaviour Drives Expression and Evolution of Gustatory Receptors in Butterflies

by Adriana D. Briscoe et al.

by Adriana D. Briscoe, Aide Macias-Muñoz, Krzysztof M. Kozak, James R. Walters, Furong Yuan, Gabriel A. Jamie, Simon H. Martin, Kanchon K. Dasmahapatra, Laura C. Ferguson, James Mallet, Emmanuelle Jacquin-Joly, Chris D. Jiggins

Secondary plant compounds are strong deterrents of insect oviposition and feeding, but may also be attractants for specialist herbivores. These insect-plant interactions are mediated by insect gustatory receptors (Grs) and olfactory receptors (Ors). An analysis of the reference genome of the butterfly Heliconius melpomene, which feeds on passion-flower vines (Passiflora spp.), together with whole-genome sequencing within the species and across the Heliconius phylogeny has permitted an unprecedented opportunity to study the patterns of gene duplication and copy-number variation (CNV) among these key sensory genes. We report in silico gene predictions of 73 Gr genes in the H. melpomene reference genome, including putative CO2, sugar, sugar alcohol, fructose, and bitter receptors. The majority of these Grs are the result of gene duplications since Heliconius shared a common ancestor with the monarch butterfly or the silkmoth. Among Grs but not Ors, CNVs are more common within species in those gene lineages that have also duplicated over this evolutionary time-scale, suggesting ongoing rapid gene family evolution. Deep sequencing (∼1 billion reads) of transcriptomes from proboscis and labial palps, antennae, and legs of adult H. melpomene males and females indicates that 67 of the predicted 73 Gr genes and 67 of the 70 predicted Or genes are expressed in these three tissues. Intriguingly, we find that one-third of all Grs show female-biased gene expression (n = 26) and nearly all of these (n = 21) are Heliconius-specific Grs. In fact, a significant excess of Grs that are expressed in female legs but not male legs are the result of recent gene duplication. This difference in Gr gene expression diversity between the sexes is accompanied by a striking sexual dimorphism in the abundance of gustatory sensilla on the forelegs of H. melpomene, suggesting that female oviposition behaviour drives the evolution of new gustatory receptors in butterfly genomes.

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

Luke A. Gilbert, Matthew H. Larson, Leonardo Morsut, Zairan Liu, Gloria A. Brar, Sandra E. Torres, Noam Stern-Ginossar, Onn Brandman, Evan H. Whitehead, Jennifer A. Doudna, Wendell A. Lim, Jonathan S. Weissman, Lei S. Qi. 

The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 pro....

Evolution of the pair rule gene network: Insights from a centipede

Evolution of the pair rule gene network: Insights from a centipede

Publication date: Available online 26 June 2013
Source:Developmental Biology
Author(s): Jack Green , Michael Akam
Comparative studies have examined the expression and function of homologues of the Drosophila melanogaster pair rule and segment polarity genes in a range of arthropods. The segment polarity gene homologues have a conserved role in the specification of the parasegment boundary, but the degree of conservation of the upstream patterning genes has proved more variable. Using genomic resources we identify a complete set of pair rule gene homologues from the centipede Strigamia maritima, and document a detailed time series of expression during trunk segmentation. We find supportive evidence for a conserved hierarchical organisation of the pair rule genes, with a division into early- and late-activated genes which parallels the functional division into primary and secondary pair rule genes described in insects. We confirm that the relative expression of sloppy-paired and paired with respect to wingless and engrailed at the parasegment boundary is conserved between myriapods and insects; suggesting that functional interactions between these genes might be an ancient feature of arthropod segment patterning. However, we find that the relative expression of a number of the primary pair rule genes is divergent between myriapods and insects. This corroborates suggestions that the evolution of upper tiers in the segmentation gene network is more flexible. Finally, we find that the expression of the Strigamia pair rule genes in periodic patterns is restricted to the ectoderm. This suggests that any direct role of these genes in segmentation is restricted to this germ layer, and that mesoderm segmentation is either dependent on the ectoderm, or occurs through an independent mechanism.

Size-dependent regulation of dorsal–ventral patterning in the early Drosophila embryo

Size-dependent regulation of dorsal–ventral patterning in the early Drosophila embryo

Publication date: Available online 22 June 2013
Source:Developmental Biology
Author(s): Mayra Garcia , Marcos Nahmad , Gregory T. Reeves , Angelike Stathopoulos

How natural variation in embryo size affects patterning of the Drosophila embryo dorsal–ventral (DV) axis is not known. Here we examined quantitatively the relationship between nuclear distribution of the Dorsal transcription factor, boundary positions for several target genes, and DV axis length. Data were obtained from embryos of a wild-type background as well as from mutant lines inbred to size select embryos of smaller or larger sizes. Our data show that the width of the nuclear Dorsal gradient correlates with DV axis length. In turn, for some genes expressed along the DV axis, the boundary positions correlate closely with nuclear Dorsal levels and with DV axis length; while the expression pattern of others is relatively constant and independent of the width of the Dorsal gradient. In particular, the patterns of snail (sna) and ventral nervous system defective (vnd) correlate with nuclear Dorsal levels and exhibit scaling to DV length; while the pattern of intermediate neuroblasts defective (ind) remains relatively constant with respect to changes in Dorsal and DV length. However, in mutants that exhibit an abnormal expansion of the Dorsal gradient which fails to scale to DV length, only sna follows the Dorsal distribution and exhibits overexpansion; in contrast, vnd and ind do not overexpand suggesting some additional mechanism acts to refine the dorsal boundaries of these two genes. Thus, our results argue against the idea that the Dorsal gradient works as a global system of relative coordinates in this system and suggest that individual targets respond to changes in embryo size in a gene-specific manner.

Temporal regulation of single-minded target genes in the ventral midline of the Drosophila central nervous system

Temporal regulation of single-minded target genes in the ventral midline of the Drosophila central nervous system

Publication date: 15 August 2013
Source:Developmental Biology, Volume 380, Issue 2
Author(s): Joung-Woo Hong , Kye Won Park , Michael S. Levine
Differentiation of a specific organ or tissue requires sequential activation of regulatory genes. However, little is known about how serial gene expression is temporally regulated. Here, we present evidence that differential expression of single-minded (sim) target genes can be attributed, in part, to the number of Sim and Tango (Tgo) heterodimer binding sites within their enhancer regions. The Sim, termed a master regulator, directs ventral midline differentiation of Drosophila central nervous system (CNS). According to data on the onset timing of ventral midline gene expression, sim target genes are classified into at least 2 groups (early and late). The sim and rhomboid (rho) genes are activated during early midline differentiation whereas orthodenticle (otd), CG10249, and slit (sli) genes undergo activation during later stages of midline differentiation. Germline transformation and in situ hybridization with transgenic embryos demonstrate that enhancers activating sim and rho expression contain 4 Sim–Tgo binding sites whereas only 1 Sim–Tgo binding site is found in an enhancer of sli. A mutagenized version of the rho enhancer lacking either 1, 2, or 3 Sim–Tgo binding sites mediated progressively more delayed expression of a lacZ reporter gene in the ventral midline. In contrast, a modified sli enhancer displayed progressively earlier onset of lacZ expression when 1, 2, or 3 more Sim–Tgo binding sites were added. Taken together, these results suggest that the number of Sim–Tgo-binding sites is decisive in determining the timing of gene expression in the developing ventral midline. We also discuss a combinatorial model accounting for the sequential expression of sim target genes.

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis

Publication date: 15 August 2013
Source:Developmental Biology, Volume 380, Issue 2
Author(s): Heather Marlow , David Q. Matus , Mark Q. Martindale
The primary axis of cnidarians runs from the oral pole to the apical tuft and defines the major body axis of both the planula larva and adult polyp. In the anthozoan cnidarian Nematostella vectensis, the primary oral–aboral (O–Ab) axis first develops during the early embryonic stage. Here, we present evidence that pharmaceutical activators of canonical wnt signaling affect molecular patterning along the primary axis of Nematostella. Although not overtly morphologically complex, molecular investigations in Nematostella reveal that the O–Ab axis is demarcated by the expression of differentially localized signaling molecules and transcription factors that may serve roles in establishing distinct ectodermal domains. We have further characterized the larval epithelium by determining the position of a nested set of molecular boundaries, utilizing several newly characterized as well as previously reported epithelial markers along the primary axis. We have assayed shifts in their position in control embryos and in embryos treated with the pharmacological agents alsterpaullone and azakenpaullone, Gsk3β inhibitors that act as canonical wnt agonists, and the Wnt antagonist iCRT14, following gastrulation. Agonist drug treatments result in an absence of aboral markers, a shift in the expression boundaries of oral markers toward the aboral pole, and changes in the position of differentially localized populations of neurons in a dose-dependent manner, while antagonist treatment had the opposite effect. These experiments are consistent with canonical wnt signaling playing a role in an orally localized wnt signaling center. These findings suggest that in Nematostella, wnt signaling mediates O–Ab ectodermal patterning across a surprisingly complex epithelium in planula stages following gastrulation in addition to previously described roles for the wnt signaling pathway in endomesoderm specification during gastrulation and overall animal–vegetal patterning at earlier stages of anthozoan development.