Francesca Pentimalli

Technology: (Truly) on and off at the flick of a switch

of the lungs, the organs of our body harbour an assortment of variously sized tubes. A study now reports that the fundamental property of every biological tube — that of having a single cavity, or lumen — is genetically controlled. Michel Bagnat and colleagues focused on the largest tube in the body, the gut. A mutation in the zebrafish tcf2 (transcription factor 2, hepatic) gene causes the gut to have not one, but many lumens. The wild-type gut normally develops through the fusion of more than one adjacent lumen, and so the authors suggest that tcf2 mutations interrupt the normal coalescence process. But how does tcf2 mediate its function? A DNA microarray experiment pulled out a candidate effector, claudin15 (cldn15): cldn15 transcripts are absent from the gut of tcf22169 mutants, and knocking down cldn15 has the same consequence as tcf2 inactivation. Claudins form pores that mediate the movement of ions across an epithelium. cldn15 could therefore regulate the passage of ions across the developing gut wall, and the ensuing influx of fluid into the tube cavity might somehow regulate lumen coalescence. This hypothesis was supported in an in vitro lumen assay: when ion-impermeable cells were made to express cldn15, they became ‘leaky’ to ions, and the lumen of many cysts expanded. Similarly, treating cells with compounds that favour the accumulation of fluid in the lumen led to the formation of a single larger lumen, which was generated by a process that mimics the lumen coalescence that occurs during development. Finally, the authors showed that disruption of electrochemical gradient formation in vivo produced a phenotype that was similar to that of tcf22169 mutants. Tube development is often associated with changes in the adhesive properties of cells. Although these more conventional mechanisms might also be operating here, this work points firmly to the involvement of an electrochemical gradient and the physical pressure that is exerted by an influx of fluid: the latter would promote both tube expansion and generate the force required to fuse adjacent lumens.

Developmental biology: Unleashing regenerative potential

Whitehead Institute for Biomedical Research, Cambridge, USA. The potential to replace damaged tissues, organs and appendages is obviously of great biomedical interest, but, unfortunately, not all organisms have extensive regenerative abilities. New work shows that the Wnt–β-catenin pathway is crucial to the regeneration of appendages in several vertebrates. Remarkably, the regenerationpromoting properties of β-catenin function are not restricted to organisms that are naturally able to regrow appendages. This molecule can also promote limb regeneration in the chick embryo, in which limb regeneration was not previously thought to occur. The authors first showed in axolotls, Xenopus laevis and zebrafish that interfering with the Wnt pathway through infection with adenoviruses expressing antagonists such as Axin1 or Dkk1 caused defects in limb or fin regeneration. Consistent with this,

Unleashing regenerative potential: Developmental biology

Whitehead Institute for Biomedical Research, Cambridge, USA. The potential to replace damaged tissues, organs and appendages is obviously of great biomedical interest, but, unfortunately, not all organisms have extensive regenerative abilities. New work shows that the Wnt–β-catenin pathway is crucial to the regeneration of appendages in several vertebrates. Remarkably, the regenerationpromoting properties of β-catenin function are not restricted to organisms that are naturally able to regrow appendages. This molecule can also promote limb regeneration in the chick embryo, in which limb regeneration was not previously thought to occur. The authors first showed in axolotls, Xenopus laevis and zebrafish that interfering with the Wnt pathway through infection with adenoviruses expressing antagonists such as Axin1 or Dkk1 caused defects in limb or fin regeneration. Consistent with this,

Unleashing regenerative potential

Whitehead Institute for Biomedical Research, Cambridge, USA. The potential to replace damaged tissues, organs and appendages is obviously of great biomedical interest, but, unfortunately, not all organisms have extensive regenerative abilities. New work shows that the Wnt–β-catenin pathway is crucial to the regeneration of appendages in several vertebrates. Remarkably, the regenerationpromoting properties of β-catenin function are not restricted to organisms that are naturally able to regrow appendages. This molecule can also promote limb regeneration in the chick embryo, in which limb regeneration was not previously thought to occur. The authors first showed in axolotls, Xenopus laevis and zebrafish that interfering with the Wnt pathway through infection with adenoviruses expressing antagonists such as Axin1 or Dkk1 caused defects in limb or fin regeneration. Consistent with this,

RNA world: MicroRNAs: unicellular organisms also have their share

URLs MicroRNAs (miRNAs) — a class of small RNAs with a role in the regulation of gene expression — had until now been found only in multicellular organisms. Now, Wang, Qi and colleagues provide the first report of miRNAs in a unicellular organism, the green alga Chlamydomonas reinhardtii, casting shadow on previous suggestions that miRNAs might have helped to drive evolution to a multicellular state. To characterize the small-RNA pathways in C. reinhardtii, the authors cloned and sequenced the whole 18–28-nucleotide RNA fraction extracted during algal vegetative growth. More than 4,000 unique small RNAs perfectly matched genomic sequences, indicating an unexpected complexity of putative RNAi processes in this organism. Two hundred small RNAs were predicted to derive from genomic sequences — either intronic or intragenic — that can potentially form the hairpin structures typical of miRNA precursors. About 20 of them were annotated as miRNAs on the basis of the presence of a corresponding sequenced miRNA* — that is, the strand pairing to a mature miRNA in the opposite arm of the precursor, the presence of which indicates that a miRNA is indeed processed from its stem-loop-structured precursor. The others were considered to be miRNA candidates. The expression of some of these miRNAs was confirmed by northern blot analysis. Using plant miRNA-prediction criteria, the authors identified more than 600 putative miRNA target sites among the annotated proteincoding transcripts and ESTs that are available for C. reinhardtii. Most of these lie in coding sequences — and some in untranslated regions — of genes involved in various processes. As in plants, the alga miRNAs seem to function by inducing cleavage of their target mRNAs, rather than by inhibiting translation, which is the common mechanism in animals. In fact, several miRNAs were shown to induce cleavage of their predicted targets, both in vitro and in vivo, with a pattern that is characteristic of the RNA-induced silencing complex-mediated cleavage. Although further studies are necessary to investigate miRNA functions in C. reinhardtii, the authors suggest a role in gametogenesis because few randomly selected miRNAs changed expression pattern during this process. The discovery of miRNAs in a unicellular organism poses interesting evolutionary questions. Although miRNAs of C. reinhardtii and higher plants have common features that suggest the existence of a conserved machinery for miRNA production and function, they do not show sequence homology. So, did miRNAs evolve independently in unicellular and multicellular organisms, or are C. reinhardtii miRNAs evolutionary intermediates? Francesca Pentimalli

MicroRNAs: unicellular organisms also have their share: RNA world

URLs MicroRNAs (miRNAs) — a class of small RNAs with a role in the regulation of gene expression — had until now been found only in multicellular organisms. Now, Wang, Qi and colleagues provide the first report of miRNAs in a unicellular organism, the green alga Chlamydomonas reinhardtii, casting shadow on previous suggestions that miRNAs might have helped to drive evolution to a multicellular state. To characterize the small-RNA pathways in C. reinhardtii, the authors cloned and sequenced the whole 18–28-nucleotide RNA fraction extracted during algal vegetative growth. More than 4,000 unique small RNAs perfectly matched genomic sequences, indicating an unexpected complexity of putative RNAi processes in this organism. Two hundred small RNAs were predicted to derive from genomic sequences — either intronic or intragenic — that can potentially form the hairpin structures typical of miRNA precursors. About 20 of them were annotated as miRNAs on the basis of the presence of a corresponding sequenced miRNA* — that is, the strand pairing to a mature miRNA in the opposite arm of the precursor, the presence of which indicates that a miRNA is indeed processed from its stem-loop-structured precursor. The others were considered to be miRNA candidates. The expression of some of these miRNAs was confirmed by northern blot analysis. Using plant miRNA-prediction criteria, the authors identified more than 600 putative miRNA target sites among the annotated proteincoding transcripts and ESTs that are available for C. reinhardtii. Most of these lie in coding sequences — and some in untranslated regions — of genes involved in various processes. As in plants, the alga miRNAs seem to function by inducing cleavage of their target mRNAs, rather than by inhibiting translation, which is the common mechanism in animals. In fact, several miRNAs were shown to induce cleavage of their predicted targets, both in vitro and in vivo, with a pattern that is characteristic of the RNA-induced silencing complex-mediated cleavage. Although further studies are necessary to investigate miRNA functions in C. reinhardtii, the authors suggest a role in gametogenesis because few randomly selected miRNAs changed expression pattern during this process. The discovery of miRNAs in a unicellular organism poses interesting evolutionary questions. Although miRNAs of C. reinhardtii and higher plants have common features that suggest the existence of a conserved machinery for miRNA production and function, they do not show sequence homology. So, did miRNAs evolve independently in unicellular and multicellular organisms, or are C. reinhardtii miRNAs evolutionary intermediates? Francesca Pentimalli

Complete Darwin on the web

mapping genetic determinants of complex human disease, but questions have been raised by some about how universally HapMap data can be used. Two papers represent the most thorough investigation of this concern so far, and conclude that HapMap data will be valuable for mapping studies in human populations around the world. The HapMap project has characterized haplotype structures across the genome for four human populations with the goal of enabling genome-wide sets of SNPs to be picked for whole-genome association studies. The general principle is simple — if two or more SNPs are in strong linkage disequilibrium (LD), just one of these variants (known as a tagSNP) needs to be genotyped to capture information on all of them. But are haplotype structures similar enough in populations other than those covered by the HapMap to allow successful mapping studies? This is one question addressed in the study by Conrad, Jakobsson and Coop et al., who looked at SNP variation across the genome in 927 people from 52 populations. Although they found marked differences in the extent of LD, they also revealed correlations in the positioning of recombination hot spots between different populations. Furthermore, there was extensive haplotype sharing between the HapMap populations and the 52 populations that this study assessed — good news for mapping studies using tagSNPs. As expected, haplotype sharing with the HapMap was generally correlated to geographical closeness to a HapMap population. Consistent with this, the best tagging of common variants from non-HapMap populations was achieved using tagSNPs from the nearest HapMap sample. Some populations were more difficult to tag than others, notably African populations, in which the extent of LD is reduced. The authors also describe how tagging can be improved in the case of some admixed populations by combining tagSNP sets from different HapMap populations. De Bakker, Burtt and Graham et al. also looked at how well HapMap tagSNPs cover common variants in other populations, testing the approach on 11 non-HapMap samples. Furthermore, they carried out simulations of whole-genome association mapping using these tags specifically to determine how powerful such studies are likely to be. Good coverage and statistical power of greater than 80% were achieved using HapMap tagSNPs for non-HapMap populations. These authors also showed how more effective studies could be carried out by combining tagSNPs from different groups. For a non-HapMap African-American population, power was increased to 80–90% by using some tags from the Caucasian HapMap set, rather than just using a set from the African population that was sampled by the HapMap. Altogether, these studies confirm the potential of the HapMap, combined with information about the history of individual populations, as a powerful tool for mapping common variants in human populations. Louisa Flintoft

Developmental biology: Homeobox genes: eyeing the clock

URLs Homeobox genes are best known for their crucial role in early animal development. New work by Decembrini et al. now provides evidence that homeobox proteins can work as effectors of a cellular clock during retinal cell differentiation. Differentiation of the progenitor precursor cells into retinal neurons requires the activation of homeobox genes according to a precise, evolutionarily conserved time schedule. To gain insight into the mechanisms that guarantee the tight temporal coordination between cell birth and cell-fate specification, Decembrini and colleagues studied the spatiotemporal expression patterns of three homeobox genes that are essential to drive the differention of photoreceptor cells (Xotx5b) or bipolar cells (Xotx2 and Xvsx1) in the Xenopus retina. In the early progenitor cells, Xotx5b, Xotx2 and Xvsx1 are transcribed but not translated, indicating the existence of some post-transcriptional regulatory mechanism. This translational inhibition is due to the presence of cis-regulatory sequences in the 3′ UTRs of these genes, which might be targets for regulatory microRNAs — a computational prediction identified several such putative targets. Regardless of the mechanism of inhibition, there is a striking correlation between the translational onset of each homeobox message and the generation of the retinal cells in which the event takes place. Interestingly, to be efficiently translated, the mRNAs of Xotx5b, Xotx2 and Xvsx1 require cell-cycle progression. In fact, blocking cellcycle progression, for example, by hydroxyurea or Xgadd-45γ, results in a reduced translation of the homeobox mRNAs and a decreased differentiation of photoreceptors and bipolar cells. Is this reduction in retinal cells a direct consequence of the low levels of the homeobox proteins? Indeed, this seems to be the case, because the overexpression of Xotx5b or Xvsx1 coding sequences can reverse the effects of the cell-cycle inhibitor, increasing the population of photoreceptors or bipolar cells, respectively. By contrast, transcripts that also contain the 3′ UTR are much less effective in rescuing retinal cell differentiation. So, these results seem to indicate the existence of a cell-cycledependent cellular clock that sets the time when retinal cells are generated; the homeobox proteins are likely to function as downstream effectors of this cellular clock, because their ectopic expression can bypass the blockade of the cell cycle. The authors shortened the cell cycle of late progenitor cells by transfecting regulators such as E2F or cdk2/cyclinA2 to show that the retinal cellular clock might function by measuring cell-cycle length rather than the time spent cycling. They suggest that the lengthening of the cell cycle that is observed during retinogenesis would allow the progenitors to translate enough homeobox proteins to differentiate further. The translational inhibitors, which are likely to be part of the clock machinery, as well as the molecular nature of the other clock components, remain to be identified. Francesca Pentimalli