Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.     

Random movement pattern of the sea urchin Strongylocentrotus droebachiensis

We describe the fine-scale movement of the sea urchin Strongylocentrotus droebachiensis based on analyses of video recordings of undisturbed individuals in the two habitats which mainly differed in food availability, urchin barrens and grazing front. Urchin activity decreased as urchin density increased. Individuals alternated between moving and being stationary and their behaviour did not appear to be affected by either current velocity (within the range from 0 to 15 cm s^− 1) and temperature (2.3 to 6.0 °C). Movement of individuals at each location was compared to that predicted by a random walk model. Mean move length (linear distance between two stationary periods), turning angle and net squared displacement were calculated for each individual. The distribution of turning angles was uniform at each location and there was no evidence of a relationship between urchin density and either move length or urchin velocity. The random model predicted a higher dispersal rate at locations with low urchin densities, such as barrens habitats. However, the movement was sometimes greater or less than predicted by the model, suggesting the influence of local environmental factors. The deviation of individual paths from the model revealed that urchins can be stationary or adopt a local (displacement less than random), random or directional movement. The net daily distance displaced on the barrens, predicted by a random walk model, was similar to the observed movement recorded in our previous study of tagged urchins at one site, but less than that observed at a second site. We postulate that the random dispersal of urchins allows individuals on barrens to reach the kelp zone where food is more abundant although the time required to reach the kelp zone may be considerable (months to years). Urchins decrease their rate of dispersal once they reach the kelp zone so that they likely remain close to this abundant food sources for long periods.     

DNA variations within the sea urchin Otx gene enhancer

We performed both intra- and interspecific comparisons of the Otx gene in the sea urchin to investigate DNA variations within the enhancer elements. Intraspecific comparisons within Hemicentrotus pulcherrimus showed that indel variations were rare within the Otx enhancer, whereas SNP variations were found uniformly within the whole test region. A similar pattern of DNA variation was observed in comparisons between closely related species. On the other hand, both nucleotide substitution and indel variations were at high levels between distant species. Additionally, the regions corresponding to the Otx enhancer in two related species showed substantial activities during development. Our results suggest the possibility that a stabilizing selection has occurred during the evolution of the Otx gene enhancer in the sea urchin that maintains its expression pattern.     

Settlement behaviour and early post-settlement predation of the sea urchin Strongylocentrotus droebachiensis

We examined behaviour of competent larvae of the green sea urchin Strongylocentrotus droebachiensis provided with pebbles encrusted with coralline red algae, a strong settlement inducer, in laboratory experiments. Larvae settled at greater frequencies on upward-facing coralline surfaces and in small gaps between coralline and glass surfaces than expected by a random distribution of settlement. These patterns may be explained by encounter rate with inductive cues. There was no change in settler distributions within ~ 1 week of settlement, indicating no net movement between adjacent microhabitats. In flow, live and recently killed larvae settled or were passively entrapped at greater frequencies on high- than low-rugosity coralline crusts. Recent settlers (0.5-1 mm test diameter) were consumed by small decapod crustaceans and bulldozed by periwinkles. Juveniles became less vulnerable to predation by hermit crabs with increasing size, and reached a growth refuge at ~ 10 mm test diameter. Our laboratory findings suggest that the cryptic distribution of recent settlers is probably not due to microhabitat selection by settling larvae or early post-settlement migration, at least not in response to physical cues such as light or surface texture. Differential rates predation of young juveniles between exposed and cryptic habitats cannot be ruled out as an important determinant of this pattern.     

Effect of dietary history and algal traits on feeding rate and food preference in the green sea urchin Strongylocentrotus droebachiensis

Feeding behaviour is influenced by a variety of factors, including nutritional requirements, the quality of available foods, and environmental conditions. We examined the effect of two factors, food morphology and dietary history, on the feeding rate and preference of the sea urchin Strongylocentrotus droebachiensis. Standardizing food shape and structure did not alter urchins' expected preference for the native kelp Laminaria longicruris over the invasive alga Codium fragile ssp. tomentosoides. However, when foods containing L. longicuris were shaped to mimic the algae, the C. fragile mimic was consumed more rapidly than the kelp mimic. Dietary history had no effect on single diet feeding rate. Urchins feeding on C. fragile consistently consumed twice as much (by mass) as those fed kelp, regardless of their previous diet. Despite higher feeding rates on C. fragile, urchins feeding on this alga were unable to compensate for its low energetic content and ingested less energy. Dietary history had a short-term effect on food preference, with urchins tending to prefer less familiar foods. Our findings suggest that urchins feed on C. fragile at a high rate, due to ease of handling and/or compensatory feeding, and that they do not a have strict preference hierarchy. Rather, food choice appears to reflect active maintenance of a mixed diet.     

Effects of gene regulatory reprogramming on gene expression in human and mouse developing hearts

Lineage-specific regulatory elements underlie adaptation of species and play a role in disease susceptibility. We compared functionally conserved and lineage-specific enhancers by cross-mapping 5042 human and 6564 mouse heart enhancers. Of these, 79 per cent are lineage-specific, lacking a functional orthologue. Heart enhancers tend to cluster and, commonly, there are multiple heart enhancers in a heart locus providing a regulatory stability to the locus. We observed little cross-clustering, however, between lineage-specific and functionally conserved heart enhancers suggesting regulatory function acquisition and development in loci previously lacking heart activity. We also identified 862 human-specific heart enhancers: 417 featuring sequence conservation with mouse (class II) and 445 with neither sequence nor function conservation (class III). Ninety-eight per cent of class III enhancers were deleted from the mouse genome, and we estimated a similar-sized enhancer gain in the human lineage. Human-specific enhancers display no detectable decrease in the negative selection pressure and are strongly associated with genes partaking in the heart regulatory programmes. The loss of a heart enhancer could be compensated by activity of a redundant heart enhancer; however, we observed redundancy in only 15 per cent of class II and III enhancer loci indicating a large-scale reprogramming of the heart regulatory programme in mammals.     

A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.     

Major cis-regulatory elements for rice bidirectional promoter activity reside in the 5′-untranslated regions

Bidirectional promoters are defined as those that regulate adjacent genes organized in a divergent fashion (head to head orientation) and separated by < 1 kb. In order to dissect bidirectional promoter activity in a model plant, deletion analysis was performed for seven rice promoters using promoter-reporter gene constructs, which identified three promoters to be bidirectional. Regulatory elements located in or close to the 5′-untranslated regions (UTR) of one of the genes (divergent gene pair) were found to be responsible for their bidirectional activity. DNA footprinting analysis identified unique protein binding sites in these promoters. Deletion/alteration of these motifs resulted in significant loss of expression of the reporter genes on either side of the promoter. Changes in the motifs at both the positions resulted in a remarkable decrease in bidirectional activity of the reporter genes flanking the promoter. Based on our results, we propose a novel mechanism for the bidirectionality of rice bidirectional promoters.     

R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.     

The 185/333 gene family is a rapidly diversifying host-defense gene cluster in the purple sea urchin Strongylocentrotus purpuratus

The genome of the purple sea urchin contains numerous large gene families with putative immunological functions. One gene family, known as 185/333, is characterized by extraordinary molecular diversity resulting from single nucleotide polymorphisms and the presence or the absence of 27 large blocks of sequences known as elements. The mosaic composition of elements, known as element patterns, that is present within the members of this gene family is encoded entirely in the second of two exons. Many of the elements correspond to one of six types of repeats that are present throughout the genes. The sequence diversity and variation in element patterns led us to investigate the evolution of the 185/333 gene family. The work presented here suggests that the element patterns are the result of both recombination and duplication and/or deletion of intragenic repeats. Each element is composed of a limited number of similar but distinct sequences, and their distribution among the 185/333 genes suggests frequent recombination within this gene family. Phylogenetic analyses of five 185/333 elements and two regions of the intron were performed using two tests: incongruence length difference and incongruence permutation. Results indicated that each pair of sequence segments was incongruent, suggesting that recombination occurs frequently along the length of the genes, including both the intron and the second exon, and that recombination is not restricted to intact elements. Paradoxically, the high level of similarity among the elements indicated that the 185/333 genes appear to be the result of a recent diversification. These results add to the growing body of evidence suggesting that invertebrate immune systems are not simple and static, but are dynamic and highly complex, and may employ group-specific mechanisms for diversification.