Marco Ferg

Automated high-throughput mapping of promoter-enhancer interactions in zebrafish embryos.

Zebrafish embryos offer a unique combination of high-throughput capabilities and the complexity of the vertebrate animal for a variety of phenotypic screening applications. However, there is a need for automation of imaging technologies to exploit the potential of the transparent embryo. Here we report a high-throughput pipeline for registering domain-specific reporter expression in zebrafish embryos with the aim of mapping the interactions between cis-regulatory modules and core promoters. Automated microscopy coupled with custom-built embryo detection and segmentation software allowed the spatial registration of reporter activity for 202 enhancer-promoter combinations, based on images of thousands of embryos. The diversity of promoter-enhancer interaction specificities underscores the importance of the core promoter sequence in cis-regulatory interactions and provides a promoter resource for transgenic reporter studies. The technology described here is also suitable for the spatial analysis of fluorescence readouts in genetic, pharmaceutical or toxicological screens.

Two independent transcription initiation codes overlap on vertebrate core promoters

A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs and recruits general transcription factors to initiate transcription. The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle. Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition. This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression, and determined the positions of H3K4me3-marked promoter-associated nucleosomes. Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (+1) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts.

The TATA-binding protein regulates maternal mRNA degradation and differential zygotic transcription in zebrafish

Early steps of embryo development are directed by maternal gene products and trace levels of zygotic gene activity in vertebrates. A major activation of zygotic transcription occurs together with degradation of maternal mRNAs during the midblastula transition in several vertebrate systems. How these processes are regulated in preparation for the onset of differentiation in the vertebrate embryo is mostly unknown. Here, we studied the function of TATA‐binding protein (TBP) by knock down and DNA microarray analysis of gene expression in early embryo development. We show that a subset of polymerase II‐transcribed genes with ontogenic stage‐dependent regulation requires TBP for their zygotic activation. TBP is also required for limiting the activation of genes during development. We reveal that TBP plays an important role in the degradation of a specific subset of maternal mRNAs during late blastulation/early gastrulation, which involves targets of the miR‐430 pathway. Hence, TBP acts as a specific regulator of the key processes underlying the transition from maternal to zygotic regulation of embryogenesis. These results implicate core promoter recognition as an additional level of differential gene regulation during development.

Genome-wide, whole mount in situ analysis of transcriptional regulators in zebrafish embryos

Transcription is the primary step in the retrieval of genetic information. A substantial proportion of the protein repertoire of each organism consists of transcriptional regulators (TRs). It is believed that the differential expression and combinatorial action of these TRs is essential for vertebrate development and body homeostasis. We mined the zebrafish genome exhaustively for genes encoding TRs and determined their expression in the zebrafish embryo by sequencing to saturation and in situ hybridisation. At the evolutionary conserved phylotypic stage, 75% of the 3302 TR genes encoded in the genome are already expressed. The number of expressed TR genes increases only marginally in subsequent stages and is maintained during adulthood suggesting important roles of the TR genes in body homeostasis. Fewer than half of the TR genes (45%, n=1711 genes) are expressed in a tissue-restricted manner in the embryo. Transcripts of 207 genes were detected in a single tissue in the 24h embryo, potentially acting as regulators of specific processes. Other TR genes were expressed in multiple tissues. However, with the exception of certain territories in the nervous system, we did not find significant synexpression suggesting that most tissue-restricted TRs act in a freely combinatorial fashion. Our data indicate that elaboration of body pattern and function from the phylotypic stage onward relies mostly on redeployment of TRs and post-transcriptional processes.

Highly conserved elements discovered in vertebrates are present in non-syntenic loci of tunicates, act as enhancers and can be transcribed during development

Co-option of cis-regulatory modules has been suggested as a mechanism for the evolution of expression sites during development. However, the extent and mechanisms involved in mobilization of cis-regulatory modules remains elusive. To trace the history of non-coding elements, which may represent candidate ancestral cis-regulatory modules affirmed during chordate evolution, we have searched for conserved elements in tunicate and vertebrate (Olfactores) genomes. We identified, for the first time, 183 non-coding sequences that are highly conserved between the two groups. Our results show that all but one element are conserved in non-syntenic regions between vertebrate and tunicate genomes, while being syntenic among vertebrates. Nevertheless, in all the groups, they are significantly associated with transcription factors showing specific functions fundamental to animal development, such as multicellular organism development and sequence-specific DNA binding. The majority of these regions map onto ultraconserved elements and we demonstrate that they can act as functional enhancers within the organism of origin, as well as in cross-transgenesis experiments, and that they are transcribed in extant species of Olfactores. We refer to the elements as ‘Olfactores conserved non-coding elements’.

The Helix‐Loop‐Helix Protein Id1 Controls Stem Cell Proliferation During Regenerative Neurogenesis in the Adult Zebrafish Telencephalon

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life‐long neurogenesis requires careful management of neural stem cell pools. In a genome‐wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E‐proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis‐promoting signals. Stem Cells 2015;33:892–903

Loss of function of myosin chaperones triggers Hsf1-mediated transcriptional response in skeletal muscle cells

BackgroundMutations in myosin chaperones Unc45b and Hsp90aa1.1 as well as in the Unc45b-binding protein Smyd1b impair formation of myofibrils in skeletal muscle and lead to the accumulation of misfolded myosin. The concomitant transcriptional response involves up-regulation of the three genes encoding these proteins, as well as genes involved in muscle development. The transcriptional up-regulation of unc45b, hsp90aa1.1 and smyd1b is specific to zebrafish mutants with myosin folding defects, and is not triggered in other zebrafish myopathy models.ResultsBy dissecting the promoter of unc45b, we identify a Heat shock factor 1 (Hsf1) binding element as a mediator of unc45b up-regulation in myofibers lacking myosin folding proteins. Loss-of-function of Hsf1 abolishes unc45b up-regulation in mutants with defects in myosin folding.ConclusionsTaken together, our data show that skeletal muscle cells respond to defective myosin chaperones with a complex gene program and suggest that this response is mediated by Hsf1 activation.

Comprehensive expression map of transcription regulators in the adult zebrafish telencephalon reveals distinct neurogenic niches

The zebrafish has become a model to study adult vertebrate neurogenesis. In particular, the adult telencephalon has been an intensely studied structure in the zebrafish brain. Differential expression of transcriptional regulators (TRs) is a key feature of development and tissue homeostasis. Here we report an expression map of 1,202 TR genes in the telencephalon of adult zebrafish. Our results are summarized in a database with search and clustering functions to identify genes expressed in particular regions of the telencephalon. We classified 562 genes into 13 distinct patterns, including genes expressed in the proliferative zone. The remaining 640 genes displayed unique and complex patterns of expression and could thus not be grouped into distinct classes. The neurogenic ventricular regions express overlapping but distinct sets of TR genes, suggesting regional differences in the neurogenic niches in the telencephalon. In summary, the small telencephalon of the zebrafish shows a remarkable complexity in TR gene expression.

Molecular Description of Eye Defects in the Zebrafish Pax6b Mutant, sunrise, Reveals a Pax6b-Dependent Genetic Network in the Developing Anterior Chamber

The cornea is a central component of the camera eye of vertebrates and even slight corneal disturbances severely affect vision. The transcription factor PAX6 is required for normal eye development, namely the proper separation of the lens from the developing cornea and the formation of the iris and anterior chamber. Human PAX6 mutations are associated with severe ocular disorders such as aniridia, Peters anomaly and chronic limbal stem cell insufficiency. To develop the zebrafish as a model for corneal disease, we first performed transcriptome and in situ expression analysis to identify marker genes to characterise the cornea in normal and pathological conditions. We show that, at 7 days post fertilisation (dpf), the zebrafish cornea expresses the majority of marker genes (67/84 tested genes) found also expressed in the cornea of juvenile and adult stages. We also characterised homozygous pax6b mutants. Mutant embryos have a thick cornea, iris hypoplasia, a shallow anterior chamber and a small lens. Ultrastructure analysis revealed a disrupted corneal endothelium. pax6b mutants show loss of corneal epithelial gene expression including regulatory genes (sox3, tfap2a, foxc1a and pitx2). In contrast, several genes (pitx2, ctnnb2, dcn and fabp7a) were ectopically expressed in the malformed corneal endothelium. Lack of pax6b function leads to severe disturbance of the corneal gene regulatory programme.

Gene transcription in the zebrafish embryo: regulators and networks

The precise spatial and temporal control of gene expression is a key process in the development, maintenance and regeneration of the vertebrate body. A substantial proportion of vertebrate genomes encode genes that control the transcription of the genetic information into mRNA. The zebrafish is particularly well suited to investigate gene regulatory networks underlying the control of gene expression during development due to the external development of its transparent embryos and the increasingly sophisticated tools for genetic manipulation available for this model system. We review here recent data on the analysis of cis-regulatory modules, transcriptional regulators and their integration into gene regulatory networks in the zebrafish, using the developing spinal cord as example.