Hot off the presses! Sep 13 Dev Cell

The Sep 13 issue of the Dev Cell is now up on Pubget (About Dev Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Not miRly a Knockout
  - Dev Cell 21(3):e1 (2011)
  Knockout mice are the gold standard for understanding gene function—or are they? The phenotypes of Egfl7 knockout mice and morphant fish suggested a critical and unexpected role for this secreted protein in angiogenesis, but a highly conserved microRNA, miR-126, was subsequently found in intron 7 of the Egfl7 gene. Wang et al. used gene targeting to knock out miR-126, but not Egfl7, and found that loss of miR-126 alone causes defects in embryonic and postnatal angiogenesis. These observations suggested that miR-126, rather than Egfl7, is the key player in angiogenesis, a result confirmed in a parallel study by Kuhnert et al. (2008), who generated mice specifically lacking Egfl7, but not miR-126, and vice versa. The intimate spatial and transcriptional relationships between miRNAs and protein coding genes suggest that retrospective analysis of other gene-targeted mice may identify additional functions for noncoding RNAs. This PaperPick refers to "The Endothelial-Specific MicroRNA miR-126 Governs Vascular Integrity and Angiogenesis," by S. Wang, A.B. Aurora, B.A. Johnson, X. Qi, J. McAnally, J.A. Hill, J.A. Richardson, R. Bassel-Duby, and E.N. Olson, published in August 2008. Video Abstract
• Revitalizing Our View of Heart Development
  - Dev Cell 21(3):e2 (2011)
  The paper by Kelly et al., from Margaret Buckingham's group at the Pasteur Institute in Paris, changed the way we think about the developing mammalian heart, as well as the origins of congenital heart disease. Prior to this work, classical studies showing that the heart tube grows in part by the transformation of a noncardiac epithelium into cardiomyocytes had lapsed into obscurity. Kelly et al. and other papers have reinstated the notion that heart growth and morphogenesis are highly dynamic and driven by the recruitment of multipotent heart progenitor cells to a primitive scaffold. It was as if a light went on in the heart development field. This and subsequent work, much of it from Buckingham and colleagues, allows us to understand the cellular and molecular nature of heart growth and to see the origins of congenital heart disease in the aberrant behavior of progenitor cell populations. This PaperPick refers to "The Arterial Pole of the Mouse Heart Forms from Fgf10-Expressing Cells in Pharyngeal Mesoderm," by R.G. Kelly, N.A. Brown, and M.E. Buckingham, published in September 2001. Video Abstract
• Between the Sheets: A Molecular Sieve Makes Myelin Membranes
  - Dev Cell 21(3):385-386 (2011)
  Myelin is a lipid-rich, spiraled membrane structure that allows for rapid propagation of action potentials through axons. In this issue, present evidence that myelin basic protein, essential for myelination by oligodendrocytes, regulates the biosynthesis of myelin membranes by restricting diffusion of membrane-bound proteins into compact myelin.
• Aurora Mitochondrialis Drives Fission during Mitosis
  - Dev Cell 21(3):387-388 (2011)
  Mitochondria proliferate by growth and partition during every cell-division cycle. Recently, reported that Aurora A kinase regulates the small GTPase RalA to mediate mitochondrial fission. This work illuminates the molecular mechanism behind mitochondrial inheritance in mammals and extends the functional repertoire of a key mitotic regulator.
• Wt1 Flip-Flops Chromatin in a CTCF Domain
  - Dev Cell 21(3):389-390 (2011)
  CTCF plays diverse roles in nuclear organization and transcriptional regulation. In this issue of Developmental Cell, report a mechanism by which the repressive or active state of chromatin in a domain defined by CTCF can be switched by the Wt1 transcription factor to regulate gene expression.
• Redirecting Traffic in the Nucleus
  - Dev Cell 21(3):390-392 (2011)
  Nuclear spatial organization of genes has emerged as an important determinant of their transcriptional activity. In this issue, show that the Msx1 homeoprotein induces a dramatic redistribution of Ezh2 and H3K27me3 to the nuclear periphery of muscle progenitor cells to repress transcription of developmentally regulated genes.
• Heart Development: Mitochondria in Command of Cardiomyocyte Differentiation
  - Dev Cell 21(3):392-393 (2011)
  Continuous developmental maturation of cardiomyocytes is essential to meet the functional and metabolic demands of the growing heart. A new study () reports that embryonic cardiomyocytes are influenced by mitochondrial maturation, such that closure of the mitochondrial permeability transition pore results in decreased levels of reactive oxygen species, thereby inducing differentiation.
• Tracing Cells for Tracking Cell Lineage and Clonal Behavior
  - Dev Cell 21(3):394-409 (2011)
  Reconstructing the lineage of cells is central to understanding development and is now also an important issue in stem cell research. Technological advances in genetically engineered permanent cell labeling, together with a multiplicity of fluorescent markers and sophisticated imaging, open new possibilities for prospective and retrospective clonal analysis.
• And the Dead Shall Rise: Actin and Myosin Return to the Spindle
  - Dev Cell 21(3):410-419 (2011)
  The spindle directs chromosome partitioning in eukaryotes and, for the last three decades, has been considered primarily a structure based on microtubules, microtubule motors, and other microtubule binding proteins. However, a surprisingly large body of both old and new studies suggests roles for actin filaments (F-actin) and myosins (F-actin-based motor proteins) in spindle assembly and function. Here we review these data and conclude that in several cases the evidence for the participation of F-actin and myosins in spindle function is very strong, and in the situations where it is less strong, there is nevertheless enough evidence to warrant further investigation.
• Transcriptional Activation of Lysosomal Exocytosis Promotes Cellular Clearance
  - Dev Cell 21(3):421-430 (2011)
  Lysosomes are cellular organelles primarily involved in degradation and recycling processes. During lysosomal exocytosis, a Ca2+-regulated process, lysosomes are docked to the cell surface and fuse with the plasma membrane (PM), emptying their content outside the cell. This process has an important role in secretion and PM repair. Here we show that the transcription factor EB (TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity of the PM and promotes their fusion with PM by raising intracellular Ca2+ levels through the activation of the lysosomal Ca2+ channel MCOLN1. Induction of lysosomal exocytosis by TFEB overexpression rescued pathologic storage and restored normal cellular morphology both in vitro and in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis may directly modulate cellular clearance and suggest an alternative therapeutic strategy for disorders associated with intracellular stor! age.
• Lgl1 Activation of Rab10 Promotes Axonal Membrane Trafficking Underlying Neuronal Polarization
  - Dev Cell 21(3):431-444 (2011)
  Directed membrane trafficking is believed to be crucial for axon development during neuronal morphogenesis. However, the underlying mechanisms are poorly understood. Here, we report a role of Lgl1, the mammalian homolog of Drosophila tumor suppressor Lethal giant larvae, in controlling membrane trafficking underlying axonal growth. We find that Lgl1 is associated with plasmalemmal precursor vesicles and enriched in developing axons. Lgl1 upregulation promoted axonal growth, whereas downregulation attenuated it as well as directional membrane insertion. Interestingly, Lgl1 interacted with and activated Rab10, a small GTPase that mediates membrane protein trafficking, by releasing GDP dissociation inhibitor (GDI) from Rab10. Furthermore, Rab10 lies downstream of Lgl1 in axon development and directional membrane insertion. Finally, both Lgl1 and Rab10 are required for neocortical neuronal polarization in vivo. Thus, the Lgl1 regulation of Rab10 stimulates the trafficking ! of membrane precursor vesicles, whose fusion with the plasmalemma is crucial for axonal growth.
• A Size Barrier Limits Protein Diffusion at the Cell Surface to Generate Lipid-Rich Myelin-Membrane Sheets
  - Dev Cell 21(3):445-456 (2011)
  The insulating layers of myelin membrane wrapped around axons by oligodendrocytes are essential for the rapid conduction of nerve impulses in the central nervous system. To fulfill this function as an electrical insulator, myelin requires a unique lipid and protein composition. Here we show that oligodendrocytes employ a barrier that functions as a physical filter to generate the lipid-rich myelin-membrane sheets. Myelin basic protein (MBP) forms this molecular sieve and restricts the diffusion of proteins with large cytoplasmic domains into myelin. The barrier is generated from MBP molecules that line the entire sheet and is, thus, intimately intertwined with the biogenesis of the polarized cell surface. This system might have evolved in oligodendrocytes in order to generate an anisotropic membrane organization that facilitates the assembly of highly insulating lipid-rich membranes.
• Import Oligomers Induce Positive Feedback to Promote Peroxisome Differentiation and Control Organelle Abundance
  - Dev Cell 21(3):457-468 (2011)
  A fundamental question in cell biology is how cells control organelle composition and abundance. Woronin bodies are fungal peroxisomes centered on a crystalline core of the self-assembled HEX protein. Despite using the canonical peroxisome import machinery for biogenesis, Woronin bodies are scarce compared to the overall peroxisome population. Here, we show that HEX oligomers promote the differentiation of a subpopulation of peroxisomes, which become enlarged and highly active in matrix protein import. HEX physically associates with the essential matrix import peroxin, PEX26, and promotes its enrichment in the membrane of differentiated peroxisomes. In addition, a PEX26 mutant that disrupts differentiation produces increased numbers of aberrantly small Woronin bodies. Our data suggest a mechanism where HEX oligomers recruit a key component of the import machinery, which promotes the import of additional HEX. This type of positive feedback provides a basic mechanism for! the production of an organelle subpopulation of distinct composition and abundance.
• The Permeability Transition Pore Controls Cardiac Mitochondrial Maturation and Myocyte Differentiation
  - Dev Cell 21(3):469-478 (2011)
  Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find that closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less-polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox-signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation.
• Dlg3 Trafficking and Apical Tight Junction Formation Is Regulated by Nedd4 and Nedd4-2 E3Â Ubiquitin Ligases
  - Dev Cell 21(3):479-491 (2011)
  The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionar! y diversification of the vertebrate Dlg family in basolateral epithelium formation.
• p63 Mediates an Apoptotic Response to Pharmacological and Disease-Related ER Stress in the Developing Epidermis
  - Dev Cell 21(3):492-505 (2011)
  Endoplasmic reticulum (ER) stress triggers tissue-specific responses that culminate in either cellular adaptation or apoptosis, but the genetic networks distinguishing these responses are not well understood. Here we demonstrate that ER stress induced in the developing zebrafish causes rapid apoptosis in the brain, spinal cord, tail epidermis, lens, and epiphysis. Focusing on the tail epidermis, we uncover an apoptotic response that depends on Puma, but not on p53 or Chop. puma is transcriptionally activated during this ER stress response in a p53-independent manner, and is an essential mediator of epidermal apoptosis. We demonstrate that the p63 transcription factor is upregulated to initiate this apoptotic pathway and directly activates puma transcription in response to ER stress. We also show that a mutation of human Connexin 31, which causes erythrokeratoderma variabilis, induces ER stress and p63-dependent epidermal apoptosis in the zebrafish embryo, thus implicat! ing this pathway in the pathogenesis of inherited disease.
• Dynamic Regulation of Emi2 by Emi2-Bound Cdk1/Plk1/CK1 and PP2A-B56 in Meiotic Arrest of Xenopus Eggs
  - Dev Cell 21(3):506-519 (2011)
  In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Mos and Emi2, an inhibitor of the APC/C ubiquitin ligase. In Xenopus, Cdk1 phosphorylates Emi2 and both destabilizes and inactivates it, whereas Mos recruits PP2A phosphatase to antagonize the Cdk1 phosphorylation. However, how Cdk1 phosphorylation inhibits Emi2 is largely unknown. Here we show that multiple N-terminal Cdk1 phosphorylation motifs bind cyclin B1-Cdk1 itself, Plk1, and CK1ﾎｴ/ﾎｵ to inhibit Emi2. Plk1, after rebinding to other sites by self-priming phosphorylation, partially destabilizes Emi2. Cdk1 and CK1ﾎｴ/ﾎｵ sequentially phosphorylate the C-terminal APC/C-docking site, thereby cooperatively inhibiting Emi2 from binding the APC/C. In the presence of Mos, however, PP2A-B56ﾎｲ/ﾎｵ bind to Emi2 and keep dephosphorylating it, particularly at the APC/C-docking site. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP! 2A phosphatase. Our data also suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation.
• An Ana2/Ctp/Mud Complex Regulates Spindle Orientation in Drosophila Neuroblasts
  - Dev Cell 21(3):520-533 (2011)
  Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to orient the mitotic spindle during NB asymmetric division.
• Repositioning of Aurora B Promoted by Chiasmata Ensures Sister Chromatid Mono-Orientation in Meiosis I
  - Dev Cell 21(3):534-545 (2011)
  During meiosis I, kinetochores of sister chromatids are juxtaposed or fused and mono-orient, while homologous chromosomes that are paired by chiasmata (bivalents) have to biorient. In the absence of chiasmata, biorientation of sister chromatids (univalents), which carries a risk of aneuploidy, has been occasionally detected in several species, including humans. We show in fission yeast that biorientation of fused sister kinetochores predominates during early prometaphase I. Without chiasmata, this undesirable biorientation of univalents persists and eventually evades the spindle assembly checkpoint, provoking abnormal anaphase. When univalents are connected by chiasmata or by an artificial tether, this erroneous attachment is converted to monopolar attachment and stabilized. This stabilization is apparently achieved by a chromosome configuration that brings kinetochores to the outer edge of the bivalent, while bringing Aurora B, a destabilizer of kinetochore-microtubul! e attachment, inward. Our results elucidate how chiasmata favor biorientation of bivalents over that of univalents at meiosis I.
• Reciprocal Repression between Sox3 and Snail Transcription Factors Defines Embryonic Territories at Gastrulation
  - Dev Cell 21(3):546-558 (2011)
  In developing amniote embryos, the first epithelial-to-mesenchymal transition (EMT) occurs at gastrulation, when a subset of epiblast cells moves to the primitive streak and undergoes EMT to internalize and generate the mesoderm and the endoderm. We show that in the chick embryo this decision to internalize is mediated by reciprocal transcriptional repression of Snail2 and Sox3 factors. We also show that the relationship between Sox3 and Snail is conserved in the mouse embryo and in human cancer cells. In the embryo, Snail-expressing cells ingress at the primitive streak, whereas Sox3-positive cells, which are unable to ingress, ensure the formation of ectodermal derivatives. Thus, the subdivision of the early embryo into the two main territories, ectodermal and mesendodermal, is regulated by changes in cell behavior mediated by the antagonistic relationship between Sox3 and Snail transcription factors.
• A Wt1-Controlled Chromatin Switching Mechanism Underpins Tissue-Specific Wnt4 Activation and Repression
  - Dev Cell 21(3):559-574 (2011)
  Wt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. Here, we show in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flanking regions; we term this mode of action "chromatin flip-flop." Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in deve! lopment and disease.
• The Msx1 Homeoprotein Recruits Polycomb to the Nuclear Periphery during Development
  - Dev Cell 21(3):575-588 (2011)
  Control of gene expression during development requires the concerted action of sequence-specific transcriptional regulators and epigenetic modifiers, which are spatially coordinated within the nucleus through mechanisms that are poorly understood. Here we show that transcriptional repression by the Msx1 homeoprotein in myoblast cells requires the recruitment of Polycomb to target genes located at the nuclear periphery. Target genes repressed by Msx1 display an Msx1-dependent enrichment of Polycomb-directed trimethylation of lysine 27 on histone H3 (H3K27me3). Association of Msx1 with the Polycomb complex is required for repression and regulation of myoblast differentiation. Furthermore, Msx1 promotes a dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in myoblast cells and the developing limb in vivo. Our findings illustrate a hitherto unappreciated spatial coordination of transcription factors with the Polycomb complex for appropr! iate regulation of gene expression programs during development.
• HMG Domain Containing SSRP1 Is Required for DNA Demethylation and Genomic Imprinting in Arabidopsis
  - Dev Cell 21(3):589-596 (2011)
  In Arabidopsis, DEMETER (DME) DNA demethylase contributes to reprogramming of the epigenetic state of the genome in the central cell. However, other aspects of the active DNA demethylation processes remain elusive. Here we show that Arabidopsis SSRP1, known as an HMG domain-containing component of FACT histone chaperone, is required for DNA demethylation and for activation and repression of many parentally imprinted genes in the central cell. Although loss of DNA methylation releases silencing of the imprinted FWA-GFP, double ssrp1-3;met1-3 mutants surprisingly showed limited activation of maternal FWA-GFP in the central cell, and only became fully active after several nuclear divisions in the endosperm. This behavior was in contrast to the dme-1;met1 double mutant in which hypomethylation of FWA-GFP by met1 suppressed the DNA demethylation defect of dme-1. We propose that active DNA demethylation by DME requires SSRP1 function through a distinctly different process fr! om direct DNA methylation control.
• Dynamic Regulation of Emi2 by Emi2-Bound Cdk1/Plk1/CK1 and PP2A-B56 in Meiotic Arrest of Xenopus Eggs
  - Dev Cell 21(3):597-598 (2011)