Species-specific genetic markers for identification of early life-history stages of Cape hakes, Merluccius capensis and Merluccius paradoxus in the southern Benguela Current

The correct identification of fish egg and larval stages is crucial for inferring spawning areas and subsequent dispersal routes for marine fishes. The authors use species-specific mtDNA polymerase chain reaction to estimate proportions of Merluccius capensis and Merluccius paradoxus eggs and larvae and to elucidate early life stage distribution patterns in the southern Benguela system.     

Towards therapeutic application of ocular stem cells

The first example of cell therapy using cultured stem cells dates back to 1981, when it was demonstrated that human epidermis could be grown in the laboratory and transplanted onto burnt patients to reconstitute a functional epidermis [Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 1979;76(11):5665-8; Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1981;1:75-8; Gallico 3rd GG, O'Connor NEMJ, Compton CC, Kehinde O, Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311(7):448-51]. This was the onset of regenerative medicine, which is now being developed also in many other fields including ophthalmology. Emerging cell therapies for the restoration of sight have focused on two areas of the eye that are critical for visual function, the cornea and the retina. The relatively easy access of the cornea, the homogeneity of the cells forming the different layers of the corneal epithelium and the improvement of cell culture protocols are leading to considerable success in corneal epithelium restoration. Rebuilding the entire cornea is however still far from reality. The restoration of the retina has recently been achieved in different animal models of retinal degeneration using immature photoreceptors, and two other promising strategies have been demonstrated: transplantation of endothelial precursors to rescue retinal vessels and neurons, and transplantation of retinal pigmented epithelial cells to preserve vision over the long term. The relevance of these approaches will be discussed in function of the disease targeted.     

Molecular phylogeny, scale evolution and taxonomy of centrohelid heliozoa

Heliozoa are ubiquitous, unicellular phagotrophs with slender radiating axopodia for trapping prey. We sequenced 18S rRNA genes from 35 cultured centrohelid heliozoa (18 studied by electron microscopy) and 28 environmental libraries (18 freshwater, 10 marine), yielding 97 new sequences, this exceeding described species. Phylogenetic analyses show two major groups and that ancestral centrohelids probably had inner plate-like tangential and distinct outer radial silica scales, the latter diverging early into contrasting scale types seen in extant Pterocystis/Choanocystis and Acanthocystis/Raphidiophryidae. Scales were lost at least thrice. Pterocystis is paraphyletic, as was the classical family Acanthocystidae; Heterophrys was polyphyletic. Using scale morphology and rRNA sequences, we establish new families Pterocystidae (Pterocystis, Raineriophrys, Chlamydaster), Marophryidae (type Marophrys (Heterophrys) marina gen. et comb. nov.) and Choanocystidae, new suborders Pterocystina (Pterocystidae, Choanocystidae, Heterophryidae) and Acanthocystina (Acanthocystidae, Raphidiophryidae, Marophryidae), and ten new Pterocystis, Acanthocystis and Choanocystis species. Most clades are exclusively freshwater or exclusively marine; evolutionary transitions between these habitats have been rare.     

Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata

Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of this huge eukaryotic group. Multiple protein sequences will be needed fully to resolve basal bikont phylogeny. Nonetheless, our results suggest that combined SSU+LSU rDNA phylogenies can help to resolve several ambiguous regions of the eukaryotic tree and identify key taxa for subsequent multi-gene analyses.     

Space‐time tradeoffs in the development of precipitation‐based isoscape models for determining migratory origin

Precipitation stable isotope patterns over continental scales provide a fundamental tool for tracking origins of migratory species. Hydrogen isotopes from rain and environmental waters are assimilated into animal tissues and may thereby reveal the location where tissues were synthesized. Predictive isotopic maps (or isoscapes) of stable hydrogen isotope values in precipitation (δ²H_p) are typically generated by time‐averaging observations from a global network of stations that have been sampled irregularly in space and time. We previously demonstrated that restricting the temporal range in δ²H_p isoscapes to biologically relevant time frames did not improve predictions of geographic origin for two migratory species in North America and Europe; rather, it decreased the accuracy of assignment. Here, we examined whether the reduction in assignment accuracy stemmed from a decrease in the number of sampling stations available to support isoscape development for shorter time periods. Multiple regression models were used to predict the hydrogen isotope composition in precipitation using isotopic measurements from each station along with a suite of independent variables. The reduction in the number of stations with δ²H_p measurements used to estimate isoscape model parameters did not alter the accuracy and precision of assignments consistently. We also examined accuracy across a range of reduced station numbers and found that mean accuracy was affected only at very low numbers of stations, indicating that the spatial isotopic patterns in precipitation that are useful for assignment applications can be characterized with data from relatively limited data stations. The number and spatial distribution of stations may have more influence when geostatistical models are used to generate isoscapes, as they incorporate spatial correlation in the dataset. The results can be used to guide future research in understanding how data availability and constraints in creating δ²H_p isoscapes may affect predictions of geographic origins.     

TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor–activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named “TRP channel–associated factors” (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.     

Msx genes define a population of mural cell precursors required for head blood vessel maturation

Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.     

Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo

Mice deficient in α-sarcoglycan (Sgca-null mice) develop progressive muscular dystrophy and serve as a model for human limb girdle muscular dystrophy type 2D. Sgca-null mice suffer a more severe myopathy than that of mdx mice, the model for Duchenne muscular dystrophy. This is the opposite of what is observed in humans and the reason for this is unknown. In an attempt to understand the cellular basis of this severe muscular dystrophy, we isolated clonal populations of myogenic progenitor cells (MPCs), the resident postnatal muscle progenitors of dystrophic and wild-type mice. MPCs from Sgca-null mice generated much smaller clones than MPCs from wild-type or mdx dystrophic mice. Impaired proliferation of Sgca-null myogenic precursors was confirmed by single fiber analysis and this difference correlated with Sgca expression during MPC proliferation. In the absence of dystrophin and associated proteins, which are only expressed after differentiation, SGCA complexes with and stabilizes FGFR1. Deficiency of Sgca leads to an absence of FGFR1 expression at the membrane and impaired MPC proliferation in response to bFGF. The low proliferation rate of Sgca-null MPCs was rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted into dystrophic muscle, Sgca-null MPCs exhibited reduced engraftment. The reduced proliferative ability of Sgca-null MPCs explains, at least in part, the severity of this muscular dystrophy and also why wild-type donor progenitor cells engraft efficiently and consequently ameliorate disease.