Transient photoreception in the hindbrain is permissive to the life history transition of hatching in Atlantic halibut.

In nonmammalian vertebrates, photoreception takes place in the deep brain already early in development, but knowledge is lacking about the functions of these nonvisual photoreceptive systems. Prior to hatching, Atlantic halibut has a transient bilateral cluster of photoreceptive cells in the hindbrain. The cluster is imbedded in a neuronal network projecting to the narrow belt of hatching glands in the yolk sac. In halibut, hatching is inhibited in light and activated by transfer to darkness and c-fos analysis during hatching shows that the hindbrain cluster and hatching glands have neural activation. Unexpectedly, the hindbrain cluster expresses dual photopigments, vertebrate ancient opsin and melanopsin. Evolutionarily, these opsins are believed to belong to different classes of photopigments found in rhabdomeric and ciliary photoreceptors. The concept that an organism develops transient light sensitivity to target critical aspects of life history transitions as hatching provides a fascinating landscape to investigate the timing of other biological events.

Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX, SOX4, and IRX3.

Genome-wide association studies identified noncoding SNPs associated with type 2 diabetes and obesity in linkage disequilibrium (LD) blocks encompassing HHEX-IDE and introns of CDKAL1 and FTO [Sladek R, et al. (2007) Nature 445:881-885; Steinthorsdottir V, et al. (2007) Nat. Genet 39:770-775; Frayling TM, et al. (2007) Science 316:889-894]. We show that these LD blocks contain highly conserved noncoding elements and overlap with the genomic regulatory blocks of the transcription factor genes HHEX, SOX4, and IRX3. We report that human highly conserved noncoding elements in LD with the risk SNPs drive expression in endoderm or pancreas in transgenic mice and zebrafish. Both HHEX and SOX4 have recently been implicated in pancreas development and the regulation of insulin secretion, but IRX3 had no prior association with pancreatic function or development. Knockdown of its orthologue in zebrafish, irx3a, increased the number of pancreatic ghrelin-producing epsilon cells and decreased the number of insulin-producing beta-cells and glucagon-producing alpha-cells, thereby suggesting a direct link of pancreatic IRX3 function to both obesity and type 2 diabetes.

Maternal 3'UTRs: from egg to onset of zygotic transcription in Atlantic cod.

BACKGROUND: Zygotic transcription in fish embryos initiates around the time of gastrulation, and all prior development is initiated and controlled by maternally derived messenger RNAs. Atlantic cod egg and embryo viability is variable, and it is hypothesized that the early development depends upon the feature of these maternal RNAs. Both the length and the presence of specific motifs in the 3'UTR of maternal RNAs are believed to regulate expression and stability of the maternal transcripts. Therefore, the aim of this study was to characterize the overall composition and 3'UTR structure of the most common maternal RNAs found in cod eggs and pre-zygotic embryos. RESULTS: 22229 Sanger-sequences were obtained from 3'-end sequenced cDNA libraries prepared from oocyte, 1-2 cell, blastula and gastrula stages. Quantitative PCR revealed that EST copy number below 9 did not reflect the gene expression profile. Consequently genes represented by less than 9 ESTs were excluded from downstream analyses, in addition to sequences with low-quality gene hits. This provided 12764 EST sequences, encoding 257 unique genes, for further analysis. Mitochondrial transcripts accounted for 45.9-50.6% of the transcripts isolated from the maternal stages, but only 12.2% of those present at the onset of zygotic transcription. 3'UTR length was predicted in nuclear sequences with poly-A tail, which identified 191 3'UTRs. Their characteristics indicated a more complex regulation of transcripts that are abundant prior to the onset of zygotic transcription. Maternal and stable transcripts had longer 3'UTR (mean 187.1 and 208.8 bp) and more 3'UTR isoforms (45.7 and 34.6%) compared to zygotic transcripts, where 15.4% had 3'UTR isoforms and the mean 3'UTR length was 76 bp. Also, diversity and the amount of putative polyadenylation motifs were higher in both maternal and stable transcripts. CONCLUSIONS: We report on the most pronounced processes in the maternally transferred cod transcriptome. Maternal stages are characterized by a rich abundance of mitochondrial transcripts. Maternal and stable transcripts display longer 3'UTRs with more variation of both polyadenylation motifs and 3'UTR isoforms. These data suggest that cod eggs possess a complex array of maternal RNAs which likely act to tightly regulate early developmental processes in the newly fertilized egg.

Exonic remnants of whole-genome duplication reveal cis-regulatory function of coding exons.

Using a comparative genomics approach to reconstruct the fate of genomic regulatory blocks (GRBs) and identify exonic remnants that have survived the disappearance of their host genes after whole-genome duplication (WGD) in teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs) with predicted target genes. These elements demonstrate evolutionary separation of overlapping protein-coding and regulatory information after WGD in teleosts. We present evidence that the corresponding mammalian exons are still under both coding and non-coding selection pressure, are more conserved than other protein coding exons in the host gene and several control sets, and share key characteristics with highly conserved non-coding elements in the same regions. Their dual function is corroborated by existing experimental data. Additionally, we show examples of human exon remnants stemming from the vertebrate 2R WGD. Our findings suggest that long-range cis-regulatory inputs for developmental genes are not limited to non-coding regions, but can also overlap the coding sequence of unrelated genes. Thus, exonic regulatory elements in GRBs might be functionally equivalent to those in non-coding regions, calling for a re-evaluation of the sequence space in which to look for long-range regulatory elements and experimentally test their activity.

Multiple transcription factor codes activate epidermal wound-response genes in Drosophila.

Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes-Ddc, ple, msn, and kkv-that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound-response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view.

Treatment with small interfering RNA affects the microRNA pathway and causes unspecific defects in zebrafish embryos.

MicroRNAs (miRNAs) are generated from primary transcripts through sequential processing by two RNase III enzymes, Drosha and Dicer, in association with other proteins. This maturation is essential for their function as post-transcriptional regulators. Notably, Dicer is also a component of RNA-induced silencing complexes, which incorporate either miRNA or small interfering RNA (siRNA) as guides to target specific mRNAs. In zebrafish, processed miRNAs belonging to the miR-430 family have previously been shown to promote deadenylation and degradation of maternal mRNAs during early embryogenesis. We show that injection of one-cell-stage zebrafish embryos with siRNA causes a significant reduction in the endogenous levels of processed miR-430 and other miRNAs, leading to unspecific developmental defects. Coinjection of siRNA with preprocessed miR-430 efficiently rescued development. This indicates that the abnormalities generally observed in siRNA-treated zebrafish embryos could be due to inhibition of miR-430 processing and/or activity. Our results also suggest that the miRNA pathway in mammals, under some experimental or therapeutic conditions, may be affected by siRNA.

Cholecystokinin mRNA in Atlantic herring, Clupea harengus--molecular cloning, characterization, and distribution in the digestive tract during the early life stages.

The mRNA of the peptide hormone cholecystokinin (CCK) was isolated from juvenile Atlantic herring, Clupea harengus, by RT-PCR. The open reading frame encodes a 137 amino acid-long precursor protein. The peptide sequence of herring CCK-8, DYMGWMDF, is identical to that of higher vertebrates and elasmobranchs, and contains methionine in the sixth position from the C-terminus, which has not been reported previously in teleosts. Expression analysis by in situ hybridization shows that positive endocrine-like cells were mainly located in the pyloric caeca and to a less extent in the rectum of the juvenile. A few positive cells were also found in the pyloric portion of the stomach and the intestine. CCK cells were present in all the larvae examined from the day of hatching onwards. Although the CCK cells were scattered throughout the whole midgut, no signals were detected in either the foregut or the hindgut. Since herring larvae have a straight gut, the distribution pattern of CCK cells seems to be reflected in the anatomy of the gut.

Isolation and characterization of two teleost melanopsin genes and their differential expression within the inner retina and brain.

Melanopsin is a newly discovered photopigment that is believed to be involved in the regulation of circadian rhythms in tetrapods. Here we describe the characterization of the first two teleost melanopsins (opn4a and opn4b) isolated from Atlantic cod (Gadus morhua). These two teleost genes belong to a subgroup of melanopsins that also include members from Xenopus, chicken, and Takifugu. In situ hybridization revealed that opn4a and opn4b are differentially expressed within the retina and brain. In the larval and adult retina, both melanopsins are expressed in a subset of cells in the inner retina, resembling amacrine and ganglion cells. In addition, opn4a is expressed in the horizontal cells, indicating a separate task for this gene. In the brain, the two melanopsins are separately expressed in two major retinal and extraretinal photosensitive integration centers, namely, the suprachiasmatic nucleus (opn4a) and the habenula (opn4b). The expression of opn4a in the suprachiasmatic nucleus in cod is similar to the melanopsin expression found in Xenopus. This suggests a conserved role for this opsin and an involvement in mediation of nonvisual photoreceptive tasks, such as entraining circadian rhythms and/or hypophysiotrophic systems. The differential expression of opn4b in the habenula suggests that this gene plays a role similar to that of opn4a, in that it is also situated in an area that integrates photic inputs from the pineal as well as other brain regions. Thus, the habenula may be an additional region that mediates photic cues in teleosts.

Molecular evidence that only two opsin subfamilies, the blue light- (SWS2) and green light-sensitive (RH2), drive color vision in Atlantic cod (Gadus morhua).

Teleosts show a great variety in visual opsin complement, due to both gene duplication and gene loss. The repertoire ranges from one subfamily of visual opsins (scotopic vision) including rod opsin only retinas seen in many deep-sea species to multiple subfamilies of visual opsins in some pelagic species. We have investigated the opsin repertoire of Atlantic cod (Gadus morhua) using information in the recently sequenced cod genome and found that despite cod not being a deep sea species it lacks visual subfamilies sensitive towards the most extreme parts of the light spectra representing UV and red light. Furthermore, we find that Atlantic cod has duplicated paralogs of both blue-sensitive SWS2 and green-sensitive RH2 subfamilies, with members belonging to each subfamily linked in tandem within the genome (two SWS2-, and three RH2A genes, respectively). The presence of multiple cone opsin genes indicates that there have been duplication events in the cod ancestor SWS2 and RH2 opsins producing paralogs that have been retained in Atlantic. Our results are supported by expressional analysis of cone opsins, which further revealed an ontogenetic change in the array of cone opsins expressed. These findings suggest life stage specific programs for opsin regulation which could be linked to habitat changes and available light as the larvae is transformed into an early juvenile. Altogether we provide the first molecular evidence for color vision driven by only two families of cone opsins due to gene loss in a teleost.

Exorhodopsin and melanopsin systems in the pineal complex and brain at early developmental stages of Atlantic halibut (Hippoglossus hippoglossus).

The complexity of the nonvisual photoreception systems in teleosts has just started to be appreciated, with colocalization of multiple photoreceptor types with unresolved functions. Here we describe an intricate expression pattern of melanopsins in early life stages of the marine flat fish Atlantic halibut (Hippoglossus hippoglossus), a period when the unpigmented brain is directly exposed to environmental photons. We show a refined and extensive expression of melanopsins in the halibut brain already at the time of hatching, long before the eyes are functional. We detect melanopsin in the habenula, suprachiasmatic nucleus, dorsal thalamus, and lateral tubular nucleus of first feeding larvae, suggesting conserved functions of the melanopsins in marine teleosts. The complex expression of melanopsins already at larval stages indicates the importance of nonvisual photoreception early in development. Most strikingly, we detect expression of both exorhodopsin and melanopsin in the pineal complex of halibut larvae. Double-fluorescence labeling showed that two clusters of melanopsin-positive cells are located lateral to the central rosette of exorhodopsin-positive cells. The localization of different photopigments in the pineal complex suggests that two parallel photoreceptor systems may be active. Furthermore, the dispersed melanopsin-positive cells in the spinal cord of halibut larvae at the time of hatching may be primary sensory cells or interneurons representing the first example of dispersed high-order photoreceptor cells. The appearance of nonvisual opsins early in the development of halibut provides an alternative model for studying the evolution and functional significance of nonvisual opsins.

Gene expression profiling of Atlantic cod (Gadus morhua) embryogenesis using microarray.

Atlantic cod (Gadus morhua) is a fish species of high importance, as a key species in a range of Northern ecosystems, in fisheries, and as an emerging species in aquaculture. So far, little is known about the transcriptional activity during early developmental stages of Atlantic cod. Hence, we decided to use a cDNA microarray covering 7,000 genes to analyze the temporal activity of the transcriptome during cod embryogenesis. Twelve different embryonic time points were selected, covering major developmental stages and processes such as maternally derived mRNA, blastula, gastrula, segmentation, hatching, and first-feeding larval stage. The microarray analysis revealed a highly dynamic transcriptional profile, showing for the first time the differential expression of thousands of known and unknown genes during Atlantic cod embryogenesis. These initial findings will serve as an important baseline for future in-depth studies of candidate genes involved in development, reproductive control, disease resistance, growth, nutrient digestion, and metabolism.

miR-9 controls the timing of neurogenesis through the direct inhibition of antagonistic factors.

The timing of commitment and cell-cycle exit within progenitor populations during neurogenesis is a fundamental decision that impacts both the number and identity of neurons produced during development. We show here that microRNA-9 plays a key role in this process through the direct inhibition of targets with antagonistic functions. Across the ventricular zone of the developing zebrafish hindbrain, miR-9 expression occurs at a range of commitment stages. Abrogating miR-9 function transiently delays cell-cycle exit, leading to the increased generation of late-born neuronal populations. Target protection analyses in vivo identify the progenitor-promoting genes her6 and zic5 and the cell-cycle exit-promoting gene elavl3/HuC as sequential targets of miR-9 as neurogenesis proceeds. We propose that miR-9 activity generates an ambivalent progenitor state poised to respond to both progenitor maintenance and commitment cues, which may be necessary to adjust neuronal production to local extrinsic signals during late embryogenesis.

Identification and characterization of two zebrafish nectin-1 genes that are differentially expressed in the developing eye and brain.

Nectins are cell adhesion molecules of the immunoglobulin type that play important roles in the development of the nervous system. We have characterized two paralogous zebrafish nectin-1 genes, nectin-1a and nectin-1b, that differ in expression. Nectin-1a expression is first found in the anterior neural keel and later in the optic cup. In the retina, nectin-1a appears in the outer part and extends inwards, while nectin-1b starts in the inner part and spreads outwards. Only nectin-1a was detected in the cornea, the lens, and in the region of photoreceptor cell differentiation in the retina. Both genes were expressed in ganglion cells and inner nuclear neurons. In the brain, nectin-1a was restricted to the epiphysis and a cluster of cells in the posterior hindbrain, whereas nectin-1b was found in several brain areas. Zebrafish may, therefore, be a useful model for identifying different functions of nectin-1 in the developing eye and nervous system.

Genomic regulatory blocks underlie extensive microsynteny conservation in insects.

Insect genomes contain larger blocks of conserved gene order (microsynteny) than would be expected under a random breakage model of chromosome evolution. We present evidence that microsynteny has been retained to keep large arrays of highly conserved noncoding elements (HCNEs) intact. These arrays span key developmental regulatory genes, forming genomic regulatory blocks (GRBs). We recently described GRBs in vertebrates, where most HCNEs function as enhancers and HCNE arrays specify complex expression programs of their target genes. Here we present a comparison of five Drosophila genomes showing that HCNE density peaks centrally in large synteny blocks containing multiple genes. Besides developmental regulators that are likely targets of HCNE enhancers, HCNE arrays often span unrelated neighboring genes. We describe differences in core promoters between the target genes and the unrelated genes that offer an explanation for the differences in their responsiveness to enhancers. We show examples of a striking correspondence between boundaries of synteny blocks, HCNE arrays, and Polycomb binding regions, confirming that the synteny blocks correspond to regulatory domains. Although few noncoding elements are highly conserved between Drosophila and the malaria mosquito Anopheles gambiae, we find that A. gambiae regions orthologous to Drosophila GRBs contain an equivalent distribution of noncoding elements highly conserved in the yellow fever mosquito Aëdes aegypti and coincide with regions of ancient microsynteny between Drosophila and mosquitoes. The structural and functional equivalence between insect and vertebrate GRBs marks them as an ancient feature of metazoan genomes and as a key to future studies of development and gene regulation.

RNAi and microRNAs: from animal models to disease therapy.

The discovery of the phenomenon of RNA interference (RNAi) and its existence in mammals quickly suggested a great potential for use in disease therapy. Rapid advances have been made in the development of RNAi-based technologies and promising results have been obtained from studies on mammalian cell culture systems and animal in vivo models. However, the progress in our understanding of the RNAi pathway and the related function of microRNAs (miRNAs) have also raised concerns regarding various types of side effects that may restrict the use of this technology in human therapy. At the same time, our new knowledge about the functional roles of miRNAs as regulators of many cellular processes, including proliferation, differentiation, development, and neuronal function, is revolutionizing cell biology and will have a major impact on medical research. In this review, we focus on the discoveries that have been made in animal models and how this insight can be translated to human medicine and disease therapy. In this connection, we will particularly discuss the challenges associated with the efforts to develop RNAi-based therapeutics.