Hot off the presses! Apr 19 dev cell

The Apr 19 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:

• Connecting the Dots: Linking Sirtuins and AMPK in Metabolism and Aging
  - dev cell 20(4):e1 (2011)
  The paper "Glucose Restriction Inhibits Skeletal Myoblast Differentiation by Activating SIRT1 through AMPK-Mediated Regulation of Nampt" by Fulco et al. is remarkably integrative. If you think about it, there are only two small molecules in cells most important in providing an indication of energy status: ATP and NAD. These molecules can be sensed by regulatory proteins, such as AMP-kinase (which senses the AMP/ATP ratio) and sirtuins (which require NAD to deacetylate protein substrates). The paper by Fulco shows that AMPK and SIRT1 can be connected in a linear pathway in which glucose restriction triggers AMPK activity, and this activates the gene encoding the NAD synthetic enzyme, Nampt, providing the crucial link to the activation of the sirtuin SIRT1. The net effect of this signaling by ATP and NAD is to activate SIRT1 during energy limitation and restrain differentiation of skeletal myoblasts. This pathway thus elegantly links two critical metabolic regulators! AMPK and sirtuins and provides a framework for how calorie restriction exerts its effects in a particular tissue. This PaperPick refers to "Glucose Restriction Inhibits Skeletal Myoblast Differentiation by Activating SIRT1 through AMPK-Mediated Regulation of Nampt," by M. Fulco, Y. Cen, P. Zhao, E.P. Hoffman, M.W. McBurney, A.A. Sauve, and V. Sartorelli, published in May 2008. Video Abstract (29887 K) Vittorio Sartorelli and Marcella Fulco discuss their identification of a nutrient-sensing pathway that governs the differentiation of skeletal muscle cells.
• Rethinking Embryonic Germ Layers
  - dev cell 20(4):e2 (2011)
  One of the central concepts we teach in developmental biology is that a major early decision made by the embryo is how to allocate cells to the three germ layers: ectoderm (epidermis, neural), mesoderm (muscle, cardiovascular), and endoderm (gut, liver). Using an in vivo lineage-labeling strategy based on random activation of a nonfunctional lacZ (β-galactosidase) gene in the mouse embryo, Tzouanacou et al. show that while the surface ectoderm (epidermis) is set aside early, a bipotential neuromesodermal cell population produces both neural and muscle cells as the body continues to grow out during the somitogenesis stages. This nicely challenges our simple assumptions about how the germ layers are established in early development while providing the basis for a mechanism that coordinates allocation of cells to the spinal cord and the muscles as the embryonic body elongates. This PaperPick refers to "Redefining the Progression of Lineage Segregations during Mammalian Embryogenesis by Clonal Analysis" by E. Tzouanacou, A. Wegener, F.J. Wymeersch, V. Wilson, and J.F. Nicolas, published in September 2009. Video Abstract (26734 K) Elena Tzouanacou and Val Wilson discuss their interests in mammalian cell lineages and how they came to identify one such lineage that violates a strict assumption of germ-layer segregation, as described in their Developmental Cell paper. They also mention recent work that suggests how the behavior of this cell lineage may be regulated to give rise to both mesodermal and neural cell types.
• Rnd-ing up RhoA Activity to Link Neurogenesis with Steps in Neuronal Migration
  - dev cell 20(4):409-410 (2011)
  Neurogenesis is integrated with neuronal migration to ensure proper development of the cerebral cortex. Reporting in Neuron, Pacary et al. (2011) demonstrate that proneural factors activate atypical Rho GTPases Rnd2 and Rnd3 in newborn cortical neurons, leading to compartmentalized modulation of RhoA signaling and differential control of neuronal migration stages.
• Removing the Brakes on Cell Identity
  - dev cell 20(4):411-412 (2011)
  In this issue of Developmental Cell, Dhawan et al. (2011) show that deletion of the Dnmt1 DNA methyltransferase gene in pancreatic insulin-producing cells makes these cells convert into glucagon-producing cells. This suggests that manipulation of a general epigenetic mechanism may be used to redirect cell fates.
• LUMENating Blood Vessels
  - dev cell 20(4):412-414 (2011)
  The acquisition of a lumen is an essential step in vascular morphogenesis. In this issue of Developmental Cell, Xu et al. (2011) show that the small GTPase Rasip is a critical regulator of cytoskeleton dynamics and cell adhesion, which together drive the emergence of vascular lumens.
• iRhoms: ERADicating the Messenger in Growth Control Signaling
  - dev cell 20(4):414-416 (2011)
  iRhoms are inactive rhomboid-like pseudoproteases that lack essential catalytic residues. Although iRhoms are highly conserved in metazoan species, little is known about their function. In a recent issue of Cell, Zettl et al. (2011) show that iRhoms regulate growth factor signaling through endoplasmic reticulum-associated protein degradation (ERAD).
• PP2A Targets SAS-5 in Centriole Assembly
  - dev cell 20(4):416-417 (2011)
  In this issue of Developmental Cell, Kitagawa et al. (2011a) and Song et al. (2011) show that the protein phosphatase PP2A regulates SAS-5 to control centriole duplication. Two paradigms are presented to explain how PP2A regulates SAS-5.
• Pancreatic β Cell Identity Is Maintained by DNA Methylation-Mediated Repression of Arx
  - dev cell 20(4):419-429 (2011)
  Adult pancreatic β cells can replicate during growth and after injury to maintain glucose homeostasis. Here, we report that β cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to α cells. We identified the lineage determination gene aristaless-related homeobox (Arx), as methylated and repressed in β cells, and hypomethylated and expressed in α cells and Dnmt1-deficient β cells. We show that the methylated region of the Arx locus in β cells was bound by methyl-binding protein MeCP2, which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of α cell lineage determination genes to maintain panc! reatic β cell identity.
• LEAFY Target Genes Reveal Floral Regulatory Logic, cis Motifs, and a Link to Biotic Stimulus Response
  - dev cell 20(4):430-443 (2011)
  The transition from vegetative growth to flower formation is critical for the survival of flowering plants. The plant-specific transcription factor LEAFY (LFY) has central, evolutionarily conserved roles in this process, both in the formation of the first flower and later in floral patterning. We performed genome-wide binding and expression studies to elucidate the molecular mechanisms by which LFY executes these roles. Our study reveals that LFY directs an elaborate regulatory network in control of floral homeotic gene expression. LFY also controls the expression of genes that regulate the response to external stimuli in Arabidopsis. Thus, our findings support a key role for LFY in the coordination of reproductive stage development and disease response programs in plants that may ensure optimal allocation of plant resources for reproductive fitness. Finally, motif analyses reveal a possible mechanism for stage-specific LFY recruitment and suggest a role for LFY in ove! rcoming polycomb repression.
• LC3 and GATE-16 N Termini Mediate Membrane Fusion Processes Required for Autophagosome Biogenesis
  - dev cell 20(4):444-454 (2011)
  Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion. We found that both proteins promote tethering and membrane fusion, mediated by the proteins' N-terminal α helices. We further show that short, 10 amino acid long synthetic peptides derived from the N terminus of LC3 or GATE-16 are sufficient to promote membrane fusion. Our data indicate that the fusion activity of LC3 is mediated by positively charged amino acids, whereas the activity of GATE-16 is mediated by hydrophobic interactions. Finally, we demonstrate that LC3 and GATE-16 N termini in general and specific residues needed for the fusion activity are essential for ! the proteins role in autophagosome biogenesis.
• Establishment of Medial Fates along the Proximodistal Axis of the Drosophila Leg through Direct Activation of dachshund by Distalless
  - dev cell 20(4):455-468 (2011)
  The proximodistal (PD) axis of the Drosophila leg is thought to be established by the combined gradients of two secreted morphogens, Wingless (Wg) and Decapentaplegic (Dpp). According to this model, high [Wg+Dpp] activates Distalless (Dll) and represses dachshund (dac) in the distal cells of the leg disc, while intermediate [Wg+Dpp] activates dac in medial tissue. To test this model we identified and characterized a dac cis-regulatory element (dac RE) that recapitulates dac's medial expression domain during leg development. Counter to the gradient model, we find that Wg and Dpp do not act in a graded manner to activate RE. Instead, dac RE is activated directly by Dll and repressed distally by a combination of factors, including the homeodomain protein Bar. Thus, medial leg fates are established via a regulatory cascade in which Wg+Dpp activate Dll and then Dll directly activates dac, with Wg+Dpp as less critical, permissive inputs.
• Hox and Pbx Factors Control Retinoic Acid Synthesis during Hindbrain Segmentation
  - dev cell 20(4):469-482 (2011)
  In vertebrate embryos, retinoic acid (RA) synthesized in the mesoderm by Raldh2 emanates to the hindbrain neuroepithelium, where it induces anteroposterior (AP)-restricted Hox expression patterns and rhombomere segmentation. However, how appropriate spatiotemporal RA activity is generated in the hindbrain is poorly understood. By analyzing Pbx1/Pbx2 and Hoxa1/Pbx1 null mice, we found that Raldh2 is itself under the transcriptional control of these factors and that the resulting RA-deficient phenotypes can be partially rescued by exogenous RA. Hoxa1-Pbx1/2-Meis2 directly binds a specific regulatory element that is required to maintain normal Raldh2 expression levels in vivo. Mesoderm-specific Xhoxa1 and Xpbx1b knockdowns in Xenopus embryos also result in Xraldh2 downregulation and hindbrain defects similar to mouse mutants, demonstrating conservation of this Hox-Pbx-dependent regulatory pathway. These findings reveal a feed-forward mechanism linking Hox-Pbx-dependent RA! synthesis during early axial patterning with the establishment of spatially restricted Hox-Pbx activity in the developing hindbrain.
• Mapping Gene Expression in Two Xenopus Species: Evolutionary Constraints and Developmental Flexibility
  - dev cell 20(4):483-496 (2011)
  Changes in gene expression are thought to be important for morphological evolution, though little is known about the nature or magnitude of the differences. Here, we examine Xenopus laevis and Xenopus tropicalis, two amphibians with very similar development, and ask how their transcriptomes compare. Despite separation for 30–90 million years, there is strong conservation in gene expression in the vast majority of the expressed orthologs. Significant changes occur in the level of gene expression but changes in the timing of expression (heterochrony) were much less common. Differences in level were concentrated in the earliest embryonic stages. Changes in timing were prominently found in pathways that respond to selective features of the environment. We propose that different evolutionary rates across developmental stages may be explained by the stabilization of cell fate determination in the later stages.
• A Genome-Wide Screen Reveals a Role for microRNA-1 in Modulating Cardiac Cell Polarity
  - dev cell 20(4):497-510 (2011)
  Many molecular pathways involved in heart disease have their roots in evolutionarily ancient developmental programs that depend critically on gene dosage and timing. MicroRNAs (miRNAs) modulate gene dosage posttranscriptionally, and among these, the muscle-specific miR-1 is particularly important for developing and maintaining somatic/skeletal and cardiac muscle. To identify pathways regulated by miR-1, we performed a forward genetic screen in Drosophila using wing-vein patterning as a biological assay. We identified several unexpected genes that genetically interacted with dmiR-1, one of which was kayak, encodes a developmentally regulated transcription factor. Additional studies directed at this genetic relationship revealed a previously unappreciated function of dmiR-1 in regulating the polarity of cardiac progenitor cells. The mammalian ortholog of kayak, c-Fos, was dysregulated in hearts of gain- or loss-of-function miR-1 mutant mice in a stress-dependent manner. ! These findings illustrate the power of Drosophila-based screens to find points of intersection between miRNAs and conserved pathways in mammals.
• Dynamics of Core Planar Polarity Protein Turnover and Stable Assembly into Discrete Membrane Subdomains
  - dev cell 20(4):511-525 (2011)
  The core planar polarity proteins localize asymmetrically to the adherens junctions of epithelial cells, where they have been hypothesized to assemble into intercellular complexes. Here, we show that the core proteins are preferentially distributed to discrete membrane subdomains ("puncta"), where they form asymmetric contacts between neighboring cells. Using an antibody internalization assay and fluorescence recovery after photobleaching in prepupal and pupal wings, we have investigated the turnover of two key core proteins, Flamingo and Frizzled, and find that the localization of both within puncta is highly stable. Furthermore, the transmembrane core proteins, Flamingo, Frizzled, and Strabismus, are necessary for stable localization of core proteins to junctions, whereas the cytoplasmic core proteins are required for their concentration into puncta. Thus, we define the distinct roles of specific core proteins in the formation of asymmetric contacts between cells! , which is a key event in the generation of coordinated cellular asymmetry.
• Blood Vessel Tubulogenesis Requires Rasip1 Regulation of GTPase Signaling
  - dev cell 20(4):526-539 (2011)
  Cardiovascular function depends on patent blood vessel formation by endothelial cells (ECs). However, the mechanisms underlying vascular "tubulogenesis" are only beginning to be unraveled. We show that endothelial tubulogenesis requires the Ras interacting protein 1, Rasip1, and its binding partner, the RhoGAP Arhgap29. Mice lacking Rasip1 fail to form patent lumens in all blood vessels, including the early endocardial tube. Rasipl null angioblasts fail to properly localize the polarity determinant Par3 and display defective cell polarity, resulting in mislocalized junctional complexes and loss of adhesion to extracellular matrix (ECM). Similarly, depletion of either Rasip1 or Arhgap29 in cultured ECs blocks in vitro lumen formation, fundamentally alters the cytoskeleton, and reduces integrin-dependent adhesion to ECM. These defects result from increased RhoA/ROCK/myosin II activity and blockade of Cdc42 and Rac1 signaling. This study identifies Rasip1 as a unique,! endothelial-specific regulator of Rho GTPase signaling, which is essential for blood vessel morphogenesis.
• Septin Filament Formation Is Essential in Budding Yeast
  - dev cell 20(4):540-549 (2011)
  Septins are GTP-binding proteins that form ordered, rod-like multimeric complexes and polymerize into filaments, but how such supramolecular structure is related to septin function was unclear. In Saccharomyces cerevisiae, four septins form an apolar hetero-octamer (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11) that associates end-to-end to form filaments. We show that septin filament assembly displays previously unanticipated plasticity. Cells lacking Cdc10 or Cdc11 are able to divide because the now-exposed subunits (Cdc3 or Cdc12, respectively) retain an ability to homodimerize via their so-called G interface, thereby allowing for filament assembly. In such cdc10Δ and cdc11Δ cells, the remaining septins, like wild-type complexes, localize to the cortex at the bud neck and compartmentalize nonseptin factors, consistent with a diffusion barrier composed of continuous filaments in intimate contact with the plasma membrane. Conversely, Cdc10 or Cdc11 mutants that can! not self-associate, but "cap" Cdc3 or Cdc12, respectively, prevent filament formation, block cortical localization, and kill cells.
• PP2A Phosphatase Acts upon SAS-5 to Ensure Centriole Formation in C. elegans Embryos
  - dev cell 20(4):550-562 (2011)
  Centrosome duplication occurs once per cell cycle and ensures that the two resulting centrosomes assemble a bipolar mitotic spindle. Centriole formation is fundamental for centrosome duplication. In Caenorhabditis elegans, the evolutionarily conserved proteins SPD-2, ZYG-1, SAS-6, SAS-5, and SAS-4 are essential for centriole formation, but how they function is not fully understood. Here, we demonstrate that Protein Phosphatase 2A (PP2A) is also critical for centriole formation in C. elegans embryos. We find that PP2A subunits genetically and physically interact with the SAS-5/SAS-6 complex. Furthermore, we show that PP2A-mediated dephosphorylation promotes centriolar targeting of SAS-5 and ensures SAS-6 delivery to the site of centriole assembly. We find that PP2A is similarly needed for the presence of HsSAS-6 at centrioles and for centriole formation in human cells. These findings lead us to propose that PP2A-mediated loading of SAS-6 proteins is critical at the onse! t of centriole formation.
• Protein Phosphatase 2A-SUR-6/B55 Regulates Centriole Duplication in C. elegans by Controlling the Levels of Centriole Assembly Factors
  - dev cell 20(4):563-571 (2011)
  Centrioles play a crucial role in mitotic spindle assembly and duplicate precisely once per cell cycle. In worms, flies, and humans, centriole assembly is dependent upon a key regulatory kinase (ZYG-1/Sak/Plk4) and its downstream effectors SAS-5 and SAS-6. Here we report a role for protein phosphatase 2A (PP2A) in centriole duplication. We find that the PP2A catalytic subunit LET-92, the scaffolding subunit PAA-1, and the B55 regulatory subunit SUR-6 function together to positively regulate centriole assembly. In PP2A-SUR-6-depleted embryos, the levels of ZYG-1 and SAS-5 are reduced and the ZYG-1- and SAS-5-dependent recruitment of SAS-6 to the nascent centriole fails. We show that PP2A physically associates with SAS-5 in vivo and that inhibiting proteolysis can rescue SAS-5 levels and the centriole duplication defect of PP2A-depleted embryos. Together, our findings indicate that PP2A-SUR-6 promotes centriole assembly by protecting ZYG-1 and SAS-5 from degradation.