Molecular cloning and embryonic expression of Xenopus Six homeobox genes

Six genes are vertebrate homologues of the homeobox-containing gene sine oculis, which plays an essential role in controlling Drosophila compound eye development. Here we report the identification and expression patterns of all three subfamilies of Xenopus Six genes. Two Six2 subfamily genes (Six1, Six2) showed very similar expression patterns in cranial ganglia, otic placodes and the eyes. Non-neural expression of Six1 and Six2 was observed with mesodermal head mesenchyme, somites and their derivatives, the muscle anlagen of the embryonic trunk. In addition, Six2 expression was also found with mesenchyme associated with the developing stomach and pronephros. Expression of Six3 subfamily genes (Six3.1, Six3.2, Six6.1, and Six6.2) was restricted to the developing head, where expression was especially observed in derivatives of the forebrain (eyes, optic stalks, the hypothalamus and pituitary gland). Interestingly, expression of all Six3 subfamily members but Six6.2 was also found with the pineal gland primordium and the tegmentum. Expression of Six4 subfamily genes (Six4.1, Six4.2) was present in the developing visceral arches, placodal derivatives (otic vesicle, olfactory system), head mesenchyme and the eye. The observed dynamic expression patterns are largely conserved between lower and higher vertebrates and imply important roles of Six family genes not only in eye formation and myogenesis, but also in the development of the gut, the kidney and of placode-derived structures.     

Eye movements in Daphnia magna

Summary Three types of behavior of the compound eye of Daphnia magna are characterized: flick, a transient rotation elicited by a brief flash of light; fixation, a maintained eye orientation in response to a stationary light stimulus of long-duration; tracking, the smooth pursuit of a moving stimulus. The magnitudes of the flick and fixation responses vary with stimulus position and are generally proportional to stimulus intensity, although at high intensities there is an attenuation of both behaviors. When the stimulus is placed at a position 80° dorsal to the eye axis, there is no response; this area is called the null region. For stationary stimuli in other positions, the direction of the response is such as to bring the stimulus closer to the null region. During tracking, the relative positions of the eye and stimulus change; the eye velocity is approximately half that of the moving stimulus. The regions of the eye in which these behaviors may be induced are different, being largest for flick and smallest for tracking. It is proposed that flick and fixation responses are a means for rotating the eye so that the stimulus is within the area surrounding the null region which is used for tracking.     

Effect of age on sexually dimorphic selenoprotein expression in mice

Clinical data suggest that selenium (Se) supplementation decreases disease predisposition and severity and accelerates recovery in a variety of pathologies. Pre-supplementation Se levels and sex represent important determinants of these Se-dependent health effects. Accordingly, we previously reported on sexually dimorphic expression patterns of Se-dependent glutathione peroxidase 1, type I deiodinase, and selenoprotein P in young mice. In the present study we investigated whether these differences vary with age. The strong sexual dimorphic expression of hepatic type I deiodinase that was observed in young mice vanished both at the mRNA and enzyme activity level by 1 year of age. In contrast, the strong sex-specific differences in renal type I deiodinase mRNA expression were sustained with age. Accordingly, deiodinase enzymatic activities differed in male and female kidneys, largely independent of age [average of 6.8 vs. 15.7 pmol/(min mg) in males vs. females]. In parallel, hepatic Se concentrations and glutathione peroxidase activities increased in female mice compared to male littermates, establishing a new sexual dimorphism in liver. Thus, age represents another important modifier of the dynamic sex- and tissue-specific selenoprotein expression patterns. These data highlight again the unique physiological regulatory mechanisms that have evolved to control Se metabolism according to the actual needs of the organism.     

Calcium limitation in Daphnia magna

The role of ambient calcium concentrations on survival, moulting,growth and egg production was assessed in the cladoceran Daphniamagna. A threshold for survival was found in the range 0.1–0.5mg Ca l^–1, even when ionic strength of the medium waskept constant. Accumulated length and length specific dry weightwas retarded at low Ca (0.5–1.0 mg Ca l^–1) at foodconcentrations above incipient limiting level. For lower foodlevels, the effect of Ca on growth was less clear. The effectof low Ca on growth rate was most manifest during the firstdays after hatching, reflecting the higher Ca demands of theearly juveniles. Age-specific egg production was strongly reducedat Ca concentrations <10 mg Ca l^–1. This was partlyan indirect effect of reduced growth rates, but could also bean effect of higher energetic costs associated with Ca uptakein a Ca-poor medium. The high Ca demands in D.magna may notbe representative of other Daphnia species. Nevertheless, highspecific Ca content seems to be a common property of Daphniaspp. and Ca deficiency could be a major determinant of speciessuccess and community structure among crustacean zooplankton;it also puts constraints on carbon sequestration in the pelagicfood web of softwater lakes.     

Haemoglobin in male Daphnia magna

• 1.1. To define the respiratory function of haemoglobin in male Daphnia magna, the swimming activity, the depression of oxygen uptake by treatment with carbon monoxide and in vivo oxygenation of Hb at various oxygen pressures were investigated.
• 2.2. The P₅₀, values for the purified Hbs from male and female red animals were 2.0 and 2.7 torr in 0.1 M phosphate buffer (pH 7.2) at 20°C, respectively.
• 3.3. The isoelectric focusing patterns of the purified Hbs from male and female red animals showed only small differences in Hb components of high PI values.

     

Molecular cloning and expression of the ilvGEDAY genes from Salmonella typhimurium

The ilvGEDAY genes of Salmonella typhimurium were cloned in Escherichia coli K-12 by in vitro recombination techniques. A single species of recombinant plasmid, designated pDU1, was obtained by selecting for Valr Ampr transformants of strain SK1592. pDU1 was shown to contain a 14-kilobase EcoRI partial digestion product of the S. typhimurium chromosome inserted into the EcoRI site of the pVH2124 cloning vector. The ilvGEDAY genes were found to occupy a maximum length of 7.5 kilobases. Restriction endonuclease analysis of the S. typhimurium ilv gene cluster provided another demonstration of the gene order as well as established the location of ilv Y between ilvA and ilvC. The presence of a ribosomal ribonucleic acid operon on the pDU1 insert, about 3 kilobases from the 5' end of ilvG, was shown by Southern hybridization. The expression of the ilvGEDA operon from pDU1 was found to be elevated, reflecting the increased gene dosage of the multicopy plasmid. A polarity was observed with respect to ilvEDA expression which is discussed in terms of the possible translational effects of the two internal promoter sequences, one located proximal to ilvE and the other located proximal to ilvD.     

Phylogeography of Daphnia magna in Europe

The phylogeographical structure of the water flea Daphnia magna in Europe was analysed using a 609-bp fragment of the cytochrome oxidase subunit I gene. Only moderate overall genetic divergence was detected within Europe. We detected four genetically and, to a large extent, geographically distinct phylogroups within Europe. Our results suggest that these groups recolonized large parts of Europe around 100,000 BP from different refugia. Overall, the pattern suggests a high degree of provincialism with a patchy occurrence of specific lineages, thus confirming the highly subdivided genetic structure usually observed in freshwater zooplankton populations. Although the region around the Mediterranean Sea was only sampled patchily, we obtained strong indications for the occurrence of more divergent genetic lineages in this region. Comparing our European samples to samples from North America and Japan revealed a higher level of differentiation, reflecting limited intercontinental dispersal.     

Thermal avoidance and preference in Daphnia magna

1. Water fleas (Daphnia magna) bred at 23°C were non-responsive to temperatures between 13 and 25°C.

2. At the lower (11°C) and upper limits (30°C) their klinokinetic avoidance behaviour showed a larger intraindividual than interindividual variation.

3. Thermal sensitivity for avoidance responses in D. magna was about 1.5°C.

4. For D. magna bred for one parthenogenetic generation at 14°C heat avoidance temperature was about 8°C lower, and cold avoidance temperature was about 1°C higher than in D. magna from 23°C.

5. In group experiments the animals showed some preference for the acclimation temperature.

6. Cold induced stenothermy and warm induced eurythermy in D. magna were related to the mode of reproduction.

Development of a bicistronic expression system in the branchiopod crustacean Daphnia magna

The viral 2A peptides have recently been used for bicistronic expression in various organisms. In this system, a single mRNA that codes for two proteins flanking the 2A peptide can be translated simultaneously into each protein by ribosomal skipping at this peptide sequence. Here, we tested the function of the Thosea asigna insect virus 2A (T2A) peptide in the branchiopod crustacean Daphnia magna—an emerging model of evolutionary developmental biology. First, we used transgenic Daphnia that expresses a potential bicistronic RNA containing mCherry and histone H2B‐ green fluorescent protein (GFP) open reading frames upstream and downstream of the T2A sequence, respectively. Microscopic observation revealed difference of localization of the two proteins in the cell, homogenous distribution of mCherry and nuclear localization of H2B‐GFP. Second, we changed localization of mCherry from cytoplasm to plasma membrane by attachment of a consensus myristoylation motif in the bicistronic reporter. RNA that codes for this new bicistronic reporter was injected into eggs. At gastrulation stage, we found spectrally distinct fluorescence with enough intensity and resolution to detect membrane localized mCherry and nuclear GFP. These results indicate that the T2A peptide functions in D. magna and T2A‐mediated bicistronic expression would be a promising tool for evo‐devo studies of this species.     

Optimization of mRNA design for protein expression in the crustacean Daphnia magna

The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3′ UTR failed to induce fluorescence. To assess reporter expression, the length of the 3′ UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3′ end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3′ UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3′ UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3′ UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.     

Reproducibility of short-term and reproduction toxicity experiments with Daphnia magna and comparison of the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata in short-term experiments

Short-term toxicity tests with Daphnia magna are reasonably reproducible. The sensitivity of Daphnia pulex, Daphnia cucullata and Daphnia magna proved to be about the same in short-term tests with 15 different chemical compounds.Reproduction toxicity experiments with Daphnia magna can easily be carried out within three weeks. At least duplicate experiments are necessary to arrive at an approximate no-effect level.     

Intersex and male development in Daphnia magna

Sex induction is environmentally stimulated in Daphnia and involves a cue-dependent response for sex determination. Somatic growth was shown to be similar in males and females during juvenile instars, but divergent due to a reduction in male somatic growth, at about the time that females produce ovaries. At this time, males appeared morphologically adult with respect to secondary sex characteristics. Intersex was rare and is unlikely to be important in natural populations. Intersex could be induced in both sexes, and observed more frequently after longer exposure to high temperature, or in the second generation following a temperature change. This indicates an impact on the ability of mothers to determine the sex of the offspring. It may be possible to use intersex characteristics for manipulative investigation of sex-determination mechanisms in Daphnia. Although sex-determination is initiated before birth, intersex occurrence suggests that development of male characters requires an additional process, probably involving hormone activity, during juvenile development and maturation.