Epigenetic cues modulating the generation of cell‐type diversity in the cerebral cortex

The cerebral cortex is composed of a large variety of distinct cell‐types including projection neurons, interneurons, and glial cells which emerge from distinct neural stem cell lineages. The vast majority of cortical projection neurons and certain classes of glial cells are generated by radial glial progenitor cells in a highly orchestrated manner. Recent studies employing single cell analysis and clonal lineage tracing suggest that neural stem cell and radial glial progenitor lineage progression are regulated in a profound deterministic manner. In this review we focus on recent advances based mainly on correlative phenotypic data emerging from functional genetic studies in mice. We establish hypotheses to test in future research and outline a conceptual framework how epigenetic cues modulate the generation of cell‐type diversity during cortical development.     

Frequent chloroplast capture among Isodon (Lamiaceae) species in Japan revealed by phylogenies based on variation in chloroplast and nuclear DNA

A recent phylogenetic study based only on chloroplast DNA (cpDNA) variation revealed that populations of an Isodon species are frequently embedded paraphyletically among other Isodon species. This phylogenetic discrepancy between species taxonomy and molecular phylogeny was considered to have resulted from chloroplast DNA captures and/or incomplete lineage sorting. To elucidate which of these factors was mainly responsible for the observed phylogenetic pattern, we performed phylogenetic analyses of multiple populations of Isodon species in Japan using cpDNA variation, three single-copy nuclear genes, and double-digest restriction-site-associated DNA sequencing (ddRAD-seq). Although a species often shared chlorotypes with other species, our phylogenetical analyses based on variation in the three single-copy nuclear genes and the ddRAD-seq data showed that most populations belonging to the same species were monophyletic at the species level, suggesting that chloroplast capture may have frequently occurred between Isodon species. Some populations of an intraspecific taxon were embedded paraphyletically within the species, regardless of the large amount of phylogenetic information in nuclear DNA; this incongruity may have resulted from incomplete lineage sorting.     

Hensen's node regulates avian neural crest differentiation in vitro

Different anteroposterior (AP) regions of the neural crest normally produce different cell types, both in vivo and in vitro. AP differences in neural crest cell fates appear to be specified in part by mechanisms that act prior to neural crest cell migration. We, therefore, examined the possibility that the fates of neural crest cells, like those of neural tube cells, can be regulated by interactions with Hensen's node. Using a transfilter co-culture system, we found that young (stage 3+ to 4) Hensen's node up-regulates the expression of two cranial-specific phenotypes (fibronectin and smooth muscle actin immunoreactivities) in mass cultures of trunk neural crest cells, and down-regulates the expression of a trunk-specific phenotype (melanin synthesis). The changes in phenotype produced by exposure to young Hensen's node were not accompanied by changes in the proliferation of either fibronectin immunoreactive cells or melanocytes. The capacity of Hensen's node to elicit changes in trunk neural crest cell phenotype decreased as the developmental age of the node increased and was lost by stage 6. In addition, old Hensen's node did not stimulate the expression of trunk-specific phenotypes in cranial neural crest cells, suggesting that cranial- and trunk-specific phenotypes are induced by different mechanisms. © 1996 John Wiley & Sons, Inc.     

Flow cytometric sorting of sperm: Influence on fertilization and embryo/fetal development in the rabbit

Viable, intact rabbit sperm, prepared, processed, and flow cytometrically sorted, were used in this study to determine the influence of flow sorting on fertilization and embryo development. In experiment I, flow-sorted or control (unstained and unsorted) sperm were surgically inseminated into the uterine horn of hormonally primed does (10 to 12 does per time point). At 42 hr postsurgical insemination, flushed embryos were assessed for development. Fetal development was determined at day 7, day 14, and day 21 post-surgical insemination. Embryos resulting from does surgically inseminated with control sperm at 42 hr post-insemination were observed to be at the early morula stage of development (>16 cell), whereas embryos from does inseminated with flow sorted sperm were at the 8- to 16-cell stage. No difference was observed between treatments at day 7, 14, or 21, however, there was a significant decrease in fetus number per doe inseminated with flow-sorted sperm over time. In experiment II, mature oocytes were flushed from the oviducts of superovulated does and coincubated in vitro (IVF) with flow-sorted or control rabbit sperm. Oocytes observed at 6 hr post-coincubation exhibited swollen sperm heads in the cytoplasm, demonstrating that fertilization had occurred (2 PN + T). There was a higher percentage of fertilized oocytes by 8 hr post-coincubation for both control (31%) and flow-sorted sperm (31%) when used for IVF. By 10 and 12 hr post-coincubation, little difference was observed in the number of fertilized oocytes between sperm treatments (52% and 66% for control vs. 57 and 54% for flow-sorted, respectively). These studies demonstrate that flow-sorted sperm are capable of fertilizing mature oocytes under in vitro conditions. In addition they show that flow sorting may not negatively influence fertilization events, but likely interferes during early embryonic and fetal development. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Molecular Evolution across Mouse Spermatogenesis

Genes involved in spermatogenesis tend to evolve rapidly, but we lack a clear understanding of how protein sequences and patterns of gene expression evolve across this complex developmental process. We used fluorescence-activated cell sorting (FACS) to generate expression data for early (meiotic) and late (postmeiotic) cell types across 13 inbred strains of mice (Mus) spanning ∼7 My of evolution. We used these comparative developmental data to investigate the evolution of lineage-specific expression, protein-coding sequences, and expression levels. We found increased lineage specificity and more rapid protein-coding and expression divergence during late spermatogenesis, suggesting that signatures of rapid testis molecular evolution are punctuated across sperm development. Despite strong overall developmental parallels in these components of molecular evolution, protein and expression divergences were only weakly correlated across genes. We detected more rapid protein evolution on the X chromosome relative to the autosomes, whereas X-linked gene expression tended to be relatively more conserved likely reflecting chromosome-specific regulatory constraints. Using allele-specific FACS expression data from crosses between four strains, we found that the relative contributions of different regulatory mechanisms also differed between cell types. Genes showing cis-regulatory changes were more common late in spermatogenesis, and tended to be associated with larger differences in expression levels and greater expression divergence between species. In contrast, genes with trans-acting changes were more common early and tended to be more conserved across species. Our findings advance understanding of gene evolution across spermatogenesis and underscore the fundamental importance of developmental context in molecular evolutionary studies.     

Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae

Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.     

RECONNECTING CELL AND ANIMAL LINEAGES: WHAT DO CELL LINEAGES TELL US ABOUT THE EVOLUTION AND DEVELOPMENT OF SPIRALIA?

Cell lineage studies in the clade Eutrochozoa, and especially the Spiralia, remains a rich and relatively untapped source for understanding broad evolutionary developmental problems; including (1) the utility of cell timing formation for phylogenetic hypotheses; (2) the evolution of cell timing changes and its relation to heterochronic patterns; (3) stereotypy or lack thereof in rates of change of cell growth during evolution and its relation to both evolutionary history and current usage; and (4) how mosaic cleavage timing variation may be expected to differ from other groups. A compilation of available cell timing information was made from previous studies where each division was explicitly followed and the total number of cells followed was greater than 24. From that compilation, we performed a series of heuristic and quantitative analyses, including a phylogenetic analysis using cell timing data as characters and analyses of timing variation across all taxa. Our results show that: (1) cell lineage data reconstructs a phylogenetic hypothesis that has similarities, especially among the Mollusca. to the patterns found in morphological and molecular analyses; (2) the mesentoblast (4d) is a unique cell compared to other cell in that it speeds up and slows down relative to other cells in taxa with both unequal and equal cell sizes; (3) some cells that form in the same quartet at the same point in the cell lineage hierarchy have much lower variations than analogous other cells, arguing for architectural constraint or stabilizing selection acting on those cells; and (4) although variation in cell timing generally increases during development, timing of formation of progeny cells in the first quartet has lower variation than the parent cells, arguing that some regulation-like behavior might be present.     

Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B

Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.     

The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.