Development

ETTIN (ARF3) physically interacts with KANADI proteins to form a functional complex essential for integument development and polarity determination in Arabidopsis.

Publication Date: 2012 Feb 1 PMID: 22296848
Authors: Kelley, D. - Arreola, A. - Gallagher, T. L. - Gasser, C.
Journal: Development

KANADI (KAN) transcription factors promote abaxial cell fate throughout plant development and are required for organ formation during embryo, leaf, carpel and ovule development. ABERRANT TESTA SHAPE (ATS, or KAN4) is necessary during ovule development to maintain the boundary between the two ovule integuments and to promote inner integument growth. Yeast two-hybrid assays identified ETTIN (ETT, or AUXIN RESPONSE FACTOR 3) as a transcription factor that could physically interact with ATS. ATS and ETT were shown to physically interact in vivo in transiently transformed tobacco epidermal cells using bimolecular fluorescence complementation. ATS and ETT were found to share an overlapping expression pattern during Arabidopsis ovule development and loss of either gene resulted in congenital fusion of the integuments and altered seed morphology. We hypothesize that in wild-type ovules a physical interaction between ATS and ETT allows these proteins to act in concert to define the boundary between integument primordia. We further show protein-protein interaction in yeast between ETT and KAN1, a paralog of ATS. Thus, a direct physical association between ETT and KAN proteins underpins their previously described common role in polarity establishment and organogenesis. We propose that ETT-KAN protein complex(es) constitute part of an auxin-dependent regulatory module that plays a conserved role in a variety of developmental contexts.

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The metalloproteinase inhibitor Reck is essential for zebrafish DRG development.

Publication Date: 2012 Feb 1 PMID: 22296847
Authors: Prendergast, A. - Linbo, T. H. - Swarts, T. - Ungos, J. M. - McGraw, H. F. - Krispin, S. - Weinstein, B. M. - Raible, D. W.
Journal: Development

The neural crest is a migratory, multipotent cell lineage that contributes to myriad tissues, including sensory neurons and glia of the dorsal root ganglia (DRG). To identify genes affecting cell fate specification in neural crest, we performed a forward genetic screen for mutations causing DRG deficiencies in zebrafish. This screen yielded a mutant lacking all DRG, which we named sensory deprived (sdp). We identified a total of four alleles of sdp, all of which possess lesions in the gene coding for reversion-inducing cysteine-rich protein containing Kazal motifs (Reck). Reck is an inhibitor of metalloproteinases previously shown to regulate cell motility. We found reck function to be both necessary for DRG formation and sufficient to rescue the sdp phenotype. reck is expressed in neural crest cells and is required in a cell-autonomous fashion for appropriate sensory neuron formation. In the absence of reck function, sensory neuron precursors fail to migrate to the position of the DRG, suggesting that this molecule is crucial for proper migration and differentiation.

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Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity.

Publication Date: 2012 Feb 1 PMID: 22296846
Authors: Busser, B. W. - Shokri, L. - Jaeger, S. A. - Gisselbrecht, S. S. - Singhania, A. - Berger, M. F. - Zhou, B. - Bulyk, M. L. - Michelson, A. M.
Journal: Development

A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Next, we used protein-binding microarrays to define the sequences that are bound by Slou, Msh and other HD TFs that have mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To understand better the regulatory specificity of an individual FC identity HD, we evaluated the functions of atypical binding sites that are preferentially bound by Slou relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou-binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity.

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Bimodal control of Hoxd gene transcription in the spinal cord defines two regulatory subclusters.

Publication Date: 2012 Jan 25 PMID: 22278926
Authors: Tschopp, P. - Christen, A. J. - Duboule, D.
Journal: Development

The importance of Hox genes in the specification of neuronal fates in the spinal cord has long been recognized. However, the transcriptional controls underlying their collinear expression domains remain largely unknown. Here we show in mice that the correspondence between the physical order of Hoxd genes and their rostral expression boundaries, although respecting spatial collinearity, does not display a fully progressive distribution. Instead, two major anteroposterior boundaries are detected, coinciding with the functional subdivision of the spinal cord. Tiling array analyses reveal two distinct blocks of transcription, regulated independently from one another, that define the observed expression boundaries. Targeted deletions in vivo that remove the genomic fragments separating the two blocks induce ectopic expression of posterior genes. We further evaluate the independent regulatory potential and transcription profile of each gene locus by a tiling array approach using a contiguous series of transgenes combined with locus-specific deletions. Our work uncovers a bimodal type of HoxD spatial collinearity in the developing spinal cord that relies on two separate 'enhancer mini-hubs' to ensure correct Hoxd gene expression levels while maintaining their appropriate anteroposterior boundaries.

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Extracellular leucine-rich repeat proteins are required to organize the apical extracellular matrix and maintain epithelial junction integrity in C. elegans.

Publication Date: 2012 Jan 25 PMID: 22278925
Authors: Mancuso, V. P. - Parry, J. M. - Storer, L. - Poggioli, C. - Nguyen, K. C. - Hall, D. H. - Sundaram, M. V.
Journal: Development

Epithelial cells are linked by apicolateral junctions that are essential for tissue integrity. Epithelial cells also secrete a specialized apical extracellular matrix (ECM) that serves as a protective barrier. Some components of the apical ECM, such as mucins, can influence epithelial junction remodeling and disassembly during epithelial-to-mesenchymal transition (EMT). However, the molecular composition and biological roles of the apical ECM are not well understood. We identified a set of extracellular leucine-rich repeat only (eLRRon) proteins in C. elegans (LET-4 and EGG-6) that are expressed on the apical surfaces of epidermal cells and some tubular epithelia, including the excretory duct and pore. A previously characterized paralog, SYM-1, is also expressed in epidermal cells and secreted into the apical ECM. Related mammalian eLRRon proteins, such as decorin or LRRTM1-3, influence stromal ECM or synaptic junction organization, respectively. Mutants lacking one or more of the C. elegans epithelial eLRRon proteins show multiple defects in apical ECM organization, consistent with these proteins contributing to the embryonic sheath and cuticular ECM. Furthermore, epithelial junctions initially form in the correct locations, but then rupture at the time of cuticle secretion and remodeling of cell-matrix interactions. This work identifies epithelial eLRRon proteins as important components and organizers of the pre-cuticular and cuticular apical ECM, and adds to the small but growing body of evidence linking the apical ECM to epithelial junction stability. We propose that eLRRon-dependent apical ECM organization contributes to cell-cell adhesion and may modulate epithelial junction dynamics in both normal and disease situations.

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Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain.

Publication Date: 2012 Jan 25 PMID: 22278924
Authors: Lahti, L. - Peltopuro, P. - Piepponen, T. P. - Partanen, J.
Journal: Development

The structure and projection patterns of adult mesodiencephalic dopaminergic (DA) neurons are one of the best characterized systems in the vertebrate brain. However, the early organization and development of these nuclei remain poorly understood. The induction of midbrain DA neurons requires sonic hedgehog (Shh) from the floor plate and fibroblast growth factor 8 (FGF8) from the isthmic organizer, but the way in which FGF8 regulates DA neuron development is unclear. We show that, during early embryogenesis, mesodiencephalic neurons consist of two distinct populations: a diencephalic domain, which is probably independent of isthmic FGFs; and a midbrain domain, which is dependent on FGFs. Within these domains, DA progenitors and precursors use partly different genetic programs. Furthermore, the diencephalic DA domain forms a distinct cell population, which also contains non-DA Pou4f1(+) cells. FGF signaling operates in proliferative midbrain DA progenitors, but is absent in postmitotic DA precursors. The loss of FGFR1/2-mediated signaling results in a maturation failure of the midbrain DA neurons and altered patterning of the midbrain floor. In FGFR mutants, the DA domain adopts characteristics that are typical for embryonic diencephalon, including the presence of Pou4f1(+) cells among TH(+) cells, and downregulation of genes typical of midbrain DA precursors. Finally, analyses of chimeric embryos indicate that FGF signaling regulates the development of the ventral midbrain cell autonomously.

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The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos.

Publication Date: 2012 Jan 25 PMID: 22278923
Authors: Kuijk, E. W. - van Tol, L. T. - van de Velde, H. - Wubbolts, R. - Welling, M. - Geijsen, N. - Roelen, B. A.
Journal: Development

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

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S6K links cell fate, cell cycle and nutrient response in C. elegans germline stem/progenitor cells.

Publication Date: 2012 Jan 25 PMID: 22278922
Authors: Korta, D. Z. - Tuck, S. - Hubbard, E. J.
Journal: Development

Coupling of stem/progenitor cell proliferation and differentiation to organismal physiological demands ensures the proper growth and homeostasis of tissues. However, in vivo mechanisms underlying this control are poorly characterized. We investigated the role of ribosomal protein S6 kinase (S6K) at the intersection of nutrition and the establishment of a stem/progenitor cell population using the C. elegans germ line as a model. We find that rsks-1 (which encodes the worm homolog of mammalian p70S6K) is required germline-autonomously for proper establishment of the germline progenitor pool. In the germ line, rsks-1 promotes cell cycle progression and inhibits larval progenitor differentiation, promotes growth of adult tumors and requires a conserved TOR phosphorylation site. Loss of rsks-1 and ife-1 (eIF4E) together reduces the germline progenitor pool more severely than either single mutant and similarly to reducing the activity of let-363 (TOR) or daf-15 (RAPTOR). Moreover, rsks-1 acts in parallel with the glp-1 (Notch) and daf-2 (insulin-IGF receptor) pathways, and does not share the same genetic dependencies with its role in lifespan control. We show that overall dietary restriction and amino acid deprivation cause germline defects similar to a subset of rsks-1 mutant phenotypes. Consistent with a link between diet and germline proliferation via rsks-1, loss of rsks-1 renders the germ line largely insensitive to the effects of dietary restriction. Our studies establish the C. elegans germ line as an in vivo model to understand TOR-S6K signaling in proliferation and differentiation and suggest that this pathway is a key nutrient-responsive regulator of germline progenitors.

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The establishment of asymmetry in Arabidopsis lateral root founder cells is regulated by LBD16/ASL18 and related LBD/ASL proteins.

Publication Date: 2012 Jan 25 PMID: 22278921
Authors: Goh, T. - Joi, S. - Mimura, T. - Fukaki, H.
Journal: Development

In most dicot plants, lateral root (LR) formation, which is important for the construction of the plant root system, is initiated from coordinated asymmetric cell divisions (ACD) of the primed LR founder cells in the xylem pole pericycle (XPP) of the existing roots. In Arabidopsis thaliana, two AUXIN RESPONSE FACTORs (ARFs), ARF7 and ARF19, positively regulate LR formation through activation of the plant-specific transcriptional regulators LATERAL ORGAN BOUNDARIES-DOMAIN 16/ASYMMETRIC LEAVES2-LIKE 18 (LBD16/ASL18) and the other related LBD/ASL genes. The exact biological role of these LBD/ASLs in LR formation is still unknown. Here, we demonstrate that LBD16/ASL18 is specifically expressed in the LR founder cells adjacent to the XPP before the first ACD and that it functions redundantly with the other auxin-inducible LBD/ASLs in LR initiation. The spatiotemporal expression of LBD16/ASL18 during LR initiation is dependent on the SOLITARY-ROOT (SLR)/IAA14-ARF7-ARF19 auxin signaling module. In addition, XPP-specific expression of LBD16/ASL18 in arf7 arf19 induced cell divisions at XPP, thereby restoring the LR phenotype. We also demonstrate that expression of LBD16-SRDX, a dominant repressor of LBD16/ASL18 and its related LBD/ASLs, does not interfere in the specification of LR founder cells with local activation of the auxin response, but it blocks the polar nuclear migration in LR founder cells before ACD, thereby blocking the subsequent LR initiation. Taken together, these results indicate that the localized activity of LBD16/ASL18 and its related LBD/ASLs is involved in the symmetry breaking of LR founder cells for LR initiation, a key step for constructing the plant root system.

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The Her7 node modulates the network topology of the zebrafish segmentation clock via sequestration of the Hes6 hub.

Publication Date: 2012 Jan 25 PMID: 22278920
Authors: Trofka, A. - Schwendinger-Schreck, J. - Brend, T. - Pontius, W. - Emonet, T. - Holley, S. A.
Journal: Development

Using in vitro and in vivo assays, we define a network of Her/Hes dimers underlying transcriptional negative feedback within the zebrafish segmentation clock. Some of the dimers do not appear to be DNA-binding, whereas those dimers that do interact with DNA have distinct preferences for cis regulatory sequences. Dimerization is specific, with Hes6 serving as the hub of the network. Her1 binds DNA only as a homodimer but will also dimerize with Hes6. Her12 and Her15 bind DNA both as homodimers and as heterodimers with Hes6. Her7 dimerizes strongly with Hes6 and weakly with Her15. This network structure engenders specific network dynamics and imparts greater influence to the Her7 node. Computational analysis supports the hypothesis that Her7 disproportionately influences the availability of Hes6 to heterodimerize with other Her proteins. Genetic experiments suggest that this regulation is important for operation of the network. Her7 therefore has two functions within the zebrafish segmentation clock. Her7 acts directly within the delayed negative feedback as a DNA-binding heterodimer with Hes6. Her7 also has an emergent function, independent of DNA binding, in which it modulates network topology via sequestration of the network hub.

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Dusky-like functions as a Rab11 effector for the deposition of cuticle during Drosophila bristle development.

Publication Date: 2012 Jan 25 PMID: 22278919
Authors: Nagaraj, R. - Adler, P. N.
Journal: Development

The morphogenesis of Drosophila sensory bristles is dependent on the function of their actin and microtubule cytoskeleton. Actin filaments are important for bristle shape and elongation, while microtubules are thought to mediate protein and membrane trafficking to promote growth. We have identified an essential role for the bristle cuticle in the maintenance of bristle structure and shape at late stages of bristle development. We show that the small GTPase Rab11 mediates the organized deposition of chitin, a major cuticle component in bristles, and disrupting Rab11 function leads to phenotypes that result from bristle collapse rather than a failure to elongate. We further establish that Rab11 is required for the plasma membrane localization of the ZP domain-containing Dusky-like (Dyl) protein and that Dyl is also required for cuticle formation in bristles. Our data argue that Dyl functions as a Rab11 effector for mediating the attachment of the bristle cell membrane to chitin to establish a stable cuticle. Our studies also implicate the exocyst as a Rab11 effector in this process and that Rab11 trafficking along the bristle shaft is mediated by microtubules.

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SHP-2 acts via ROCK to regulate the cardiac actin cytoskeleton.

Publication Date: 2012 Jan 25 PMID: 22278918
Authors: Langdon, Y. - Tandon, P. - Paden, E. - Duddy, J. - Taylor, J. M. - Conlon, F. L.
Journal: Development

Noonan syndrome is one of the most common causes of human congenital heart disease and is frequently associated with missense mutations in the protein phosphatase SHP-2. Interestingly, patients with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), juvenile myelomonocytic leukemia (JMML) and LEOPARD syndrome frequently carry a second, somatically introduced subset of missense mutations in SHP-2. To determine the cellular and molecular mechanisms by which SHP-2 regulates heart development and, thus, understand how Noonan-associated mutations affect cardiogenesis, we introduced SHP-2 encoding the most prevalent Noonan syndrome and JMML mutations into Xenopus embryos. Resulting embryos show a direct relationship between a Noonan SHP-2 mutation and its ability to cause cardiac defects in Xenopus; embryos expressing Noonan SHP-2 mutations exhibit morphologically abnormal hearts, whereas those expressing an SHP-2 JMML-associated mutation do not. Our studies indicate that the cardiac defects associated with the introduction of the Noonan-associated SHP-2 mutations are coupled with a delay or arrest of the cardiac cell cycle in M-phase and a failure of cardiomyocyte progenitors to incorporate into the developing heart. We show that these defects are a result of an underlying malformation in the formation and polarity of cardiac actin fibers and F-actin deposition. We show that these defects can be rescued in culture and in embryos through the inhibition of the Rho-associated, coiled-coil-containing protein kinase 1 (ROCK), thus demonstrating a direct relationship between SHP-2(N308D) and ROCK activation in the developing heart.

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Frizzled1/2/7 signaling directs beta-catenin nuclearisation and initiates endoderm specification in macromeres during sea urchin embryogenesis.

Publication Date: 2012 Feb PMID: 22274701
Authors: Lhomond, G. - McClay, D. R. - Gache, C. - Croce, J. C.
Journal: Development

In sea urchins, the nuclear accumulation of beta-catenin in micromeres and macromeres at 4th and 5th cleavage activates the developmental gene regulatory circuits that specify all of the vegetal tissues (i.e. skeletogenic mesoderm, endoderm and non-skeletogenic mesoderm). Here, through the analysis of maternal Frizzled receptors as potential contributors to these processes, we found that, in Paracentrotus lividus, the receptor Frizzled1/2/7 is required by 5th cleavage for beta-catenin nuclearisation selectively in macromere daughter cells. Perturbation analyses established further that Frizzled1/2/7 signaling is required subsequently for the specification of the endomesoderm and then the endoderm but not for that of the non-skeletogenic mesoderm, even though this cell type also originates from the endomesoderm lineage. Complementary analyses on Wnt6 showed that this maternal ligand is similarly required at 5th cleavage for the nuclear accumulation of beta-catenin exclusively in the macromeres and for endoderm but not for non-skeletogenic mesoderm specification. In addition, Wnt6 misexpression reverses Frizzled1/2/7 downregulation-induced phenotypes. Thus, the results indicate that Wnt6 and Frizzled1/2/7 are likely to behave as the ligand-receptor pair responsible for initiating beta-catenin nuclearisation in macromeres at 5th cleavage and that event is necessary for endoderm specification. They show also that beta-catenin nuclearisation in micromeres and macromeres takes place through a different mechanism, and that non-skeletogenic mesoderm specification occurs independently of the nuclear accumulation of beta-catenin in macromeres at the 5th cleavage. Evolutionarily, this analysis outlines further the conserved involvement of the Frizzled1/2/7 subfamily, but not of specific Wnts, in the activation of canonical Wnt signaling during early animal development.

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In the absence of BYPASS1-related gene function, the bps signal disrupts embryogenesis by an auxin-independent mechanism.

Publication Date: 2012 Feb PMID: 22274700
Authors: Lee, D. K. - Van Norman, J. M. - Murphy, C. - Adhikari, E. - Reed, J. W. - Sieburth, L. E.
Journal: Development

Development is often coordinated by biologically active mobile compounds that move between cells or organs. Arabidopsis mutants with defects in the BYPASS1 (BPS1) gene overproduce an active mobile compound that moves from the root to the shoot and inhibits growth. Here, we describe two related Arabidopsis genes, BPS2 and BPS3. Analyses of single, double and triple mutants revealed that all three genes regulate production of the same mobile compound, the bps signal, with BPS1 having the largest role. The triple mutant had a severe embryo defect, including the failure to properly establish provascular tissue, the shoot meristem and the root meristem. Aberrant expression of PINFORMED1, DR5, PLETHORA1, PLETHORA2 and WUSCHEL-LIKE HOMEOBOX5 were found in heart-stage bps triple-mutant embryos. However, auxin-induced gene expression, and localization of the PIN1 auxin efflux transporter, were intact in bps1 mutants, suggesting that the primary target of the bps signal is independent of auxin response. Thus, the bps signal identifies a novel signaling pathway that regulates patterning and growth in parallel with auxin signaling, in multiple tissues and at multiple developmental stages.

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The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development.

Publication Date: 2012 Feb PMID: 22274699
Authors: Bedell, V. M. - Person, A. D. - Larson, J. D. - McLoon, A. - Balciunas, D. - Clark, K. J. - Neff, K. I. - Nelson, K. E. - Bill, B. R. - Schimmenti, L. A. - Beiraghi, S. - Ekker, S. C.
Journal: Development

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.

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Programmed reduction of ABC transporter activity in sea urchin germline progenitors.

Publication Date: 2012 Feb PMID: 22274698
Authors: Campanale, J. P. - Hamdoun, A.
Journal: Development

ATP-binding cassette (ABC) transporters protect embryos and stem cells from mutagens and pump morphogens that control cell fate and migration. In this study, we measured dynamics of ABC transporter activity during formation of sea urchin embryonic cells necessary for the production of gametes, termed the small micromeres. Unexpectedly, we found small micromeres accumulate 2.32 times more of the ABC transporter substrates calcein-AM, CellTrace RedOrange, BoDipy-verapamil and BoDipy-vinblastine, than any other cell in the embryo, indicating a reduction in multidrug efflux activity. The reduction in small micromere ABC transporter activity is mediated by a pulse of endocytosis occurring 20-60 minutes after the appearance of the micromeres - the precursors of the small micromeres. Treating embryos with phenylarsine oxide, an inhibitor of endocytosis, prevents the reduction of transporter activity. Tetramethylrhodamine dextran and cholera toxin B uptake experiments indicate that micromeres have higher rates of bulk and raft-associated membrane endocytosis during the window of transporter downregulation. We hypothesized that this loss of efflux transport could be required for the detection of developmental signaling molecules such as germ cell chemoattractants. Consistent with this hypothesis, we found that the inhibition of ABCB and ABCC-types of efflux transporters disrupts the ordered distribution of small micromeres to the left and right coelomic pouches. These results point to tradeoffs between signaling and the protective functions of the transporters.

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{Delta}Np63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation.

Publication Date: 2012 Feb PMID: 22274697
Authors: Romano, R. A. - Smalley, K. - Magraw, C. - Serna, V. A. - Kurita, T. - Raghavan, S. - Sinha, S.
Journal: Development

The transcription factor p63 is important in the development of the skin as p63-null mice exhibit striking defects in embryonic epidermal morphogenesis. Understanding the mechanisms that underlie this phenotype is complicated by the existence of multiple p63 isoforms, including TAp63 and DeltaNp63. To investigate the role of DeltaNp63 in epidermal morphogenesis we generated DeltaNp63 knock-in mice in which the DeltaNp63-specific exon is replaced by GFP. Homozygous DeltaNp63(gfp/gfp) animals exhibit severe developmental anomalies including truncated forelimbs and the absence of hind limbs, largely phenocopying existing knockouts in which all p63 isoforms are deleted. DeltaNp63-null animals show a poorly developed stratified epidermis comprising isolated clusters of disorganized epithelial cells. Despite the failure to develop a mature stratified epidermis, the patches of DeltaNp63-null keratinocytes are able to stratify and undergo a program of terminal differentiation. However, we observe premature expression of markers associated with terminal differentiation, which is unique to DeltaNp63-null animals and not evident in the skin of mice lacking all p63 isoforms. We posit that the dysregulated and accelerated keratinocyte differentiation phenotype is driven by significant alterations in the expression of key components of the Notch signaling pathway, some of which are direct transcriptional targets of DeltaNp63 as demonstrated by ChIP experiments. The analysis of DeltaNp63(gfp/gfp) knockout mice reaffirms the indispensable role of the DeltaN isoform of p63 in epithelial biology and confirms that DeltaNp63-null keratinocytes are capable of committing to an epidermal cell lineage, but are likely to suffer from diminished renewal capacity and an altered differentiation fate.

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Myoblast fusion: lessons from flies and mice.

Publication Date: 2012 Feb PMID: 22274696
Authors: Abmayr, S. M. - Pavlath, G. K.
Journal: Development

The fusion of myoblasts into multinucleate syncytia plays a fundamental role in muscle function, as it supports the formation of extended sarcomeric arrays, or myofibrils, within a large volume of cytoplasm. Principles learned from the study of myoblast fusion not only enhance our understanding of myogenesis, but also contribute to our perspectives on membrane fusion and cell-cell fusion in a wide array of model organisms and experimental systems. Recent studies have advanced our views of the cell biological processes and crucial proteins that drive myoblast fusion. Here, we provide an overview of myoblast fusion in three model systems that have contributed much to our understanding of these events: the Drosophila embryo; developing and regenerating mouse muscle; and cultured rodent muscle cells.

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Patterning embryos with oscillations: structure, function and dynamics of the vertebrate segmentation clock.

Publication Date: 2012 Feb PMID: 22274695
Authors: Oates, A. C. - Morelli, L. G. - Ares, S.
Journal: Development

The segmentation clock is an oscillating genetic network thought to govern the rhythmic and sequential subdivision of the elongating body axis of the vertebrate embryo into somites: the precursors of the segmented vertebral column. Understanding how the rhythmic signal arises, how it achieves precision and how it patterns the embryo remain challenging issues. Recent work has provided evidence of how the period of the segmentation clock is regulated and how this affects the anatomy of the embryo. The ongoing development of real-time clock reporters and mathematical models promise novel insight into the dynamic behavior of the clock.

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Novel Tfap2-mediated control of soxE expression facilitated the evolutionary emergence of the neural crest.

Publication Date: 2012 Feb PMID: 22241841
Authors: Van Otterloo, E. - Li, W. - Garnett, A. - Cattell, M. - Medeiros, D. M. - Cornell, R. A.
Journal: Development

Gene duplication has been proposed to drive the evolution of novel morphologies. After gene duplication, it is unclear whether changes in the resulting paralogs' coding-regions, or in their cis-regulatory elements, contribute most significantly to the assembly of novel gene regulatory networks. The Transcription Factor Activator Protein 2 (Tfap2) was duplicated in the chordate lineage and is essential for development of the neural crest, a tissue that emerged with vertebrates. Using a tfap2-depleted zebrafish background, we test the ability of available gnathostome, agnathan, cephalochordate and insect tfap2 paralogs to drive neural crest development. With the exception of tfap2d (lamprey and zebrafish), all are able to do so. Together with expression analyses, these results indicate that sub-functionalization has occurred among Tfap2 paralogs, but that neo-functionalization of the Tfap2 protein did not drive the emergence of the neural crest. We investigate whether acquisition of novel target genes for Tfap2 might have done so. We show that in neural crest cells Tfap2 directly activates expression of sox10, which encodes a transcription factor essential for neural crest development. The appearance of this regulatory interaction is likely to have coincided with that of the neural crest, because AP2 and SoxE are not co-expressed in amphioxus, and because neural crest enhancers are not detected proximal to amphioxus soxE. We find that sox10 has limited ability to restore the neural crest in Tfap2-deficient embryos. Together, these results show that mutations resulting in novel Tfap2-mediated regulation of sox10 and other targets contributed to the evolution of the neural crest.

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A putative tyrosine phosphorylation site of the cell surface receptor Golden goal is involved in synaptic layer selection in the visual system.

Publication Date: 2012 Feb PMID: 22241840
Authors: Mann, K. - Wang, M. - Luu, S. H. - Ohler, S. - Hakeda-Suzuki, S. - Suzuki, T.
Journal: Development

Golden goal (Gogo) is a cell surface protein that is crucial for proper synaptic layer targeting of photoreceptors (R cells) in the Drosophila visual system. In collaboration with the seven-transmembrane cadherin Flamingo (Fmi), Gogo mediates both temporary and final layer targeting of R-cell axons through its cytoplasmic activity. However, it is not known how Gogo activity is regulated. Here, we show that a conserved Tyr-Tyr-Asp (YYD) tripeptide motif in the Gogo cytoplasmic domain is required for photoreceptor axon targeting. Deleting the YYD motif is sufficient to abolish Gogo function. We demonstrate that the YYD motif is a phosphorylation site and that mutations in the YYD tripeptide impair synaptic layer targeting. Gogo phosphorylation results in axon stopping at the temporary targeting layer, and dephosphorylation is crucial for final layer targeting in collaboration with Fmi. Therefore, both temporary and final layer targeting strongly depend on the Gogo phosphorylation status. Drosophila Insulin-like receptor (DInR) has been reported to regulate the wiring of photoreceptors. We show that insulin signaling is a positive regulator, directly or indirectly, of YYD motif phosphorylation. Our findings indicate a novel mechanism for the regulation of Gogo activity by insulin signaling-mediated phosphorylation. We propose the model that a constant phosphorylation signal is antagonized by a presumably temporal dephosphorylation signal, which creates a permissive signal that controls developmental timing in axon targeting.

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Excitatory glutamate is essential for development and maintenance of the piloneural mechanoreceptor.

Publication Date: 2012 Feb PMID: 22241839
Authors: Woo, S. H. - Baba, Y. - Franco, A. M. - Lumpkin, E. A. - Owens, D. M.
Journal: Development

The piloneural collar in mammalian hairy skin comprises an intricate pattern of circumferential and longitudinal sensory afferents that innervate primary and secondary pelage hairs. The longitudinal afferents tightly associate with terminal Schwann cell processes to form encapsulated lanceolate nerve endings of rapidly adapting mechanoreceptors. The molecular basis for piloneural development, maintenance and function is poorly understood. Here, we show that Nefh-expressing glutamatergic neurons represent a major population of longitudinal and circumferential sensory afferents innervating the piloneural collar. Our findings using a VGLUT2 conditional-null mouse model indicate that glutamate is essential for innervation, patterning and differentiation of NMDAR(+) terminal Schwann cells during piloneural collar development. Similarly, treatment of adult mice with a selective NMDAR antagonist severely perturbed piloneural collar structure and reduced excitability of these mechanosensory neurons. Collectively, these results show that DRG-derived glutamate is essential for the proper development, maintenance and sensory function of the piloneural mechanoreceptor.

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Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression.

Publication Date: 2012 Feb PMID: 22241838
Authors: Ulvklo, C. - Macdonald, R. - Bivik, C. - Baumgardt, M. - Karlsson, D. - Thor, S.
Journal: Development

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. Here, we identify two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers.

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Genetic ablation of Rest leads to in vitro-specific derepression of neuronal genes during neurogenesis.

Publication Date: 2012 Feb PMID: 22241837
Authors: Aoki, H. - Hara, A. - Era, T. - Kunisada, T. - Yamada, Y.
Journal: Development

Rest (RE1-silencing transcription factor, also called Nrsf) is involved in the maintenance of the undifferentiated state of neuronal stem/progenitor cells in vitro by preventing precocious expression of neuronal genes. However, the function of Rest during neurogenesis in vivo remains to be elucidated because of the early embryonic lethal phenotype of conventional Rest knockout mice. In the present study, we have generated Rest conditional knockout mice, which allow the effect of genetic ablation of Rest during embryonic neurogenesis to be examined in vivo. We show that Rest plays a role in suppressing the expression of neuronal genes in cultured neuronal cells in vitro, as well as in non-neuronal cells outside of the central nervous system, but that it is dispensable for embryonic neurogenesis in vivo. Our findings highlight the significance of extrinsic signals for the proper intrinsic regulation of neuronal gene expression levels in the specification of cell fate during embryonic neurogenesis in vivo.

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Jmjd5, an H3K36me2 histone demethylase, modulates embryonic cell proliferation through the regulation of Cdkn1a expression.

Publication Date: 2012 Feb PMID: 22241836
Authors: Ishimura, A. - Minehata, K. - Terashima, M. - Kondoh, G. - Hara, T. - Suzuki, T.
Journal: Development

Covalent modifications of histones play an important role in chromatin architecture and dynamics. In particular, histone lysine methylation is important for transcriptional control during diverse biological processes. The nuclear protein Jmjd5 (also called Kdm8) is a histone lysine demethylase that contains a JmjC domain in the C-terminal region. In this study, we have generated Jmjd5-deficient mice (Jmjd5(Delta)(/)(Delta)) to investigate the in vivo function of Jmjd5. Jmjd5(Delta)(/)(Delta) embryos showed severe growth retardation, resulting in embryonic lethality at the mid-gestation stage. Mouse embryonic fibroblasts (MEFs) derived from Jmjd5 hypomorphic embryos (Jmjd5(neo/neo)) also showed the growth defect. Quantitative PCR analysis of various cell cycle regulators indicated that only Cdkn1a expression was upregulated in Jmjd5(neo/neo) MEFs and Jmjd5(Delta)(/)(Delta) embryos. A knockdown assay with Cdkn1a-specific small interfering RNAs revealed that the growth defect of Jmjd5(neo/neo) MEFs was significantly rescued. In addition, a genetic study using Jmjd5(Delta)(/)(Delta); Cdkn1a(Delta)(/)(Delta) double-knockout mice showed that the growth retardation of Jmjd5(Delta)(/)(Delta) embryos was partially rescued by Cdkn1a deficiency. Chromatin immunoprecipitation analysis showed that increased di-methylated lysine 36 of histone H3 (H3K36me2) and reduced recruitment of endogenous Jmjd5 were detected in the transcribed regions of Cdkn1a in Jmjd5(neo/neo) MEFs. Taken together, these results suggest that Jmjd5 physiologically moderates embryonic cell proliferation through the epigenetic control of Cdkn1a expression.

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The extracellular loops of Smoothened play a regulatory role in control of Hedgehog pathway activation.

Publication Date: 2012 Feb PMID: 22223683
Authors: Carroll, C. E. - Marada, S. - Stewart, D. P. - Ouyang, J. X. - Ogden, S. K.
Journal: Development

The Hedgehog (Hh) signaling pathway plays an instructional role during development, and is frequently activated in cancer. Ligand-induced pathway activation requires signaling by the transmembrane protein Smoothened (Smo), a member of the G-protein-coupled receptor (GPCR) superfamily. The extracellular (EC) loops of canonical GPCRs harbor cysteine residues that engage in disulfide bonds, affecting active and inactive signaling states through regulating receptor conformation, dimerization and/or ligand binding. Although a functional importance for cysteines localized to the N-terminal extracellular cysteine-rich domain has been described, a functional role for a set of conserved cysteines in the EC loops of Smo has not yet been established. In this study, we mutated each of the conserved EC cysteines, and tested for effects on Hh signal transduction. Cysteine mutagenesis reveals that previously uncharacterized functional roles exist for Smo EC1 and EC2. We provide in vitro and in vivo evidence that EC1 cysteine mutation induces significant Hh-independent Smo signaling, triggering a level of pathway activation similar to that of a maximal Hh response in Drosophila and mammalian systems. Furthermore, we show that a single amino acid change in EC2 attenuates Hh-induced Smo signaling, whereas deletion of the central region of EC2 renders Smo fully active, suggesting that the conformation of EC2 is crucial for regulated Smo activity. Taken together, these findings are consistent with loop cysteines engaging in disulfide bonds that facilitate a Smo conformation that is silent in the absence of Hh, but can transition to a fully active state in response to ligand.

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DAZL is essential for stress granule formation implicated in germ cell survival upon heat stress.

Publication Date: 2012 Feb PMID: 22223682
Authors: Kim, B. - Cooke, H. J. - Rhee, K.
Journal: Development

Mammalian male germ cells should be maintained below body temperature for proper development. Here, we investigated how male germ cells respond to heat stress. A short exposure of mouse testes to core body temperature induced phosphorylation of eIF2alpha and the formation of stress granules (SGs) in male germ cells. We observed that DAZL, a germ cell-specific translational regulator, was translocated to SGs upon heat stress. Furthermore, SG assembly activity was significantly diminished in the early male germ cells of Dazl-knockout mice. The DAZL-containing SGs played a protective role against heat stress-induced apoptosis by the sequestration of specific signaling molecules, such as RACK1, and the subsequent blockage of the apoptotic MAPK pathway. Based on these results, we propose that DAZL is an essential component of the SGs, which prevent male germ cells from undergoing apoptosis upon heat stress.

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CycA is involved in the control of endoreplication dynamics in the Drosophila bristle lineage.

Publication Date: 2012 Feb PMID: 22223681
Authors: Salle, J. - Campbell, S. D. - Gho, M. - Audibert, A.
Journal: Development

Endocycles, which are characterised by repeated rounds of DNA replication without intervening mitosis, are involved in developmental processes associated with an increase in metabolic cell activity and are part of terminal differentiation. Endocycles are currently viewed as a restriction of the canonical cell cycle. As such, mitotic cyclins have been omitted from the endocycle mechanism and their role in this process has not been specifically analysed. In order to study such a role, we focused on CycA, which has been described to function exclusively during mitosis in Drosophila. Using developing mechanosensory organs as model system and PCNA::GFP to follow endocycle dynamics, we show that (1) CycA proteins accumulate during the last period of endoreplication, (2) both CycA loss and gain of function induce changes in endoreplication dynamics and reduce the number of endocycles, and (3) heterochromatin localisation of ORC2, a member of the Pre-RC complex, depends on CycA. These results show for the first time that CycA is involved in endocycle dynamics in Drosophila. As such, CycA controls the final ploidy that cells reached during terminal differentiation. Furthermore, our data suggest that the control of endocycles by CycA involves the subnuclear relocalisation of pre-RC complex members. Our work therefore sheds new light on the mechanism underlying endocycles, implicating a process that involves remodelling of the entire cell cycle network rather than simply a restriction of the canonical cell cycle.

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Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways.

Publication Date: 2012 Feb PMID: 22223680
Authors: Dworkin, S. - Darido, C. - Georgy, S. R. - Wilanowski, T. - Srivastava, S. - Ellett, F. - Pase, L. - Han, Y. - Meng, A. - Heath, J. K. - Lieschke, G. J. - Jane, S. M.
Journal: Development

The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.

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Drosophila aPKC is required for mitotic spindle orientation during symmetric division of epithelial cells.

Publication Date: 2012 Feb PMID: 22223679
Authors: Guilgur, L. G. - Prudencio, P. - Ferreira, T. - Pimenta-Marques, A. R. - Martinho, R. G.
Journal: Development

Epithelial cells mostly orient the spindle along the plane of the epithelium (planar orientation) for mitosis to produce two identical daughter cells. The correct orientation of the spindle relies on the interaction between cortical polarity components and astral microtubules. Recent studies in mammalian tissue culture cells suggest that the apically localised atypical protein kinase C (aPKC) is important for the planar orientation of the mitotic spindle in dividing epithelial cells. Yet, in chicken neuroepithelial cells, aPKC is not required in vivo for spindle orientation, and it has been proposed that the polarization cues vary between different epithelial cell types and/or developmental processes. In order to investigate whether Drosophila aPKC is required for spindle orientation during symmetric division of epithelial cells, we took advantage of a previously isolated temperature-sensitive allele of aPKC. We showed that Drosophila aPKC is required in vivo for spindle planar orientation and apical exclusion of Pins (Raps). This suggests that the cortical cues necessary for spindle orientation are not only conserved between Drosophila and mammalian cells, but are also similar to those required for spindle apicobasal orientation during asymmetric cell division.

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p57KIP2 regulates radial glia and intermediate precursor cell cycle dynamics and lower layer neurogenesis in developing cerebral cortex.

Publication Date: 2012 Feb PMID: 22223678
Authors: Mairet-Coello, G. - Tury, A. - Van Buskirk, E. - Robinson, K. - Genestine, M. - Dicicco-Bloom, E.
Journal: Development

During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57(KIP2), an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57(KIP2) regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27(KIP1) controlled IPC proliferation exclusively. Furthermore, p57(KIP2) deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27(KIP1) increased IPC proliferation at E16.5. Consequently, loss of p57(KIP2) increased primarily layer 5-6 neuron production, whereas loss of p27(KIP1) increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57(KIP2) and p27(KIP1) control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation.

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Regulation of DNA replication during development.

Publication Date: 2012 Feb PMID: 22223677
Authors: Nordman, J. - Orr-Weaver, T. L.
Journal: Development

As development unfolds, DNA replication is not only coordinated with cell proliferation, but is regulated uniquely in specific cell types and organs. This differential regulation of DNA synthesis requires crosstalk between DNA replication and differentiation. This dynamic aspect of DNA replication is highlighted by the finding that the distribution of replication origins varies between differentiated cell types and changes with differentiation. Moreover, differential DNA replication in some cell types can lead to increases or decreases in gene copy number along chromosomes. This review highlights the recent advances and technologies that have provided us with new insights into the developmental regulation of DNA replication.

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A taste of TGFbeta in Tuscany.

Publication Date: 2012 Feb PMID: 22223676
Authors: Hata, A. - Brivanlou, A. H.
Journal: Development

The recent FASEB Summer Research Conference entitled 'The TGFbeta Superfamily: Signaling in Development and Disease' was held in August, 2011 in the spectacular setting of Il Ciocco, Lucca, amidst the olive trees in Tuscany, Italy. The organizers assembled an amazing forum, which included 53 speakers and 67 poster presentations from laboratories around the world, to showcase recent advances made in our understanding of the transforming growth factor-beta (TGFbeta) signaling pathway.

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Cilia in vertebrate development and disease.

Publication Date: 2012 Feb PMID: 22223675
Authors: Oh, E. C. - Katsanis, N.
Journal: Development

Through the combined study of model organisms, cell biology, cell signaling and medical genetics we have significantly increased our understanding of the structure and functions of the vertebrate cilium. This ancient organelle has now emerged as a crucial component of certain signaling and sensory perception pathways in both developmental and homeostatic contexts. Here, we provide a snapshot of the structure, function and distribution of the vertebrate cilium and of the pathologies that are associated with its dysfunction.

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Drosophila Polycomb complexes restrict neuroblast competence to generate motoneurons.

Publication Date: 2012 Feb PMID: 22219354
Authors: Touma, J. J. - Weckerle, F. F. - Cleary, M. D.
Journal: Development

Similar to mammalian neural progenitors, Drosophila neuroblasts progressively lose competence to make early-born neurons. In neuroblast 7-1 (NB7-1), Kruppel (Kr) specifies the third-born U3 motoneuron and Kr misexpression induces ectopic U3 cells. However, competence to generate U3 cells is limited to early divisions, when the Eve(+) U motoneurons are produced, and competence is lost when NB7-1 transitions to making interneurons. We have found that Polycomb repressor complexes (PRCs) are necessary and sufficient to restrict competence in NB7-1. PRC loss of function extends the ability of Kr to induce U3 fates and PRC gain of function causes precocious loss of competence to make motoneurons. PRCs also restrict competence to make HB9(+) Islet(+) motoneurons in another neuroblast that undergoes a motoneuron-to-interneuron transition, NB3-1. In contrast to the regulation of motoneuron competence, PRC activity does not affect the production of Eve(+) interneurons by NB3-3, HB9(+) Islet(+) interneurons by NB7-3, or Dbx(+) interneurons by multiple neuroblasts. These findings support a model in which PRCs establish motoneuron-specific competence windows in neuroblasts that transition from motoneuron to interneuron production.

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Bmp signaling regulates a dose-dependent transcriptional program to control facial skeletal development.

Publication Date: 2012 Feb PMID: 22219353
Authors: Bonilla-Claudio, M. - Wang, J. - Bai, Y. - Klysik, E. - Selever, J. - Martin, J. F.
Journal: Development

We performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in cranial neural crest (CNC). Conditional Bmp4 overexpression, using a tetracycline-regulated Bmp4 gain-of-function allele, resulted in facial skeletal changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4-induced genes (BIG) composed predominantly of transcriptional regulators that control self-renewal, osteoblast differentiation and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4 and Bmp7, resulted in complete or partial loss of multiple CNC-derived skeletal elements, revealing a crucial requirement for Bmp signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss-of-function mutants, indicating Bmp-regulated target genes are modulated by Bmp dose. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45gamma and Gata3, that was bound by Smad1/5 in the developing mandible, revealing direct Smad-mediated regulation. These data support the hypothesis that Bmp signaling regulates craniofacial skeletal development by balancing self-renewal and differentiation pathways in CNC progenitors.

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Synchronous and symmetric migration of Drosophila caudal visceral mesoderm cells requires dual input by two FGF ligands.

Publication Date: 2012 Feb PMID: 22219352
Authors: Kadam, S. - Ghosh, S. - Stathopoulos, A.
Journal: Development

Caudal visceral mesoderm (CVM) cells migrate synchronously towards the anterior of the Drosophila embryo as two distinct groups located on each side of the body, in order to specify longitudinal muscles that ensheath the gut. Little is known about the molecular cues that guide cells along this path, the longest migration of embryogenesis, except that they closely associate with trunk visceral mesoderm (TVM). The expression of the fibroblast growth factor receptor (FGFR) heartless and its ligands, pyramus (pyr) and thisbe (ths), within CVM and TVM cells, respectively, suggested FGF signaling may influence CVM cell guidance. In FGF mutants, CVM cells die before reaching the anterior region of the TVM. However, an earlier phenotype observed was that the two cell clusters lose direction and converge at the midline. Live in vivo imaging and tracking analyses identified that the movements of CVM cells were slower and no longer synchronous. Moreover, CVM cells were found to cross over from one group to the other, disrupting bilateral symmetry, whereas such mixing was never observed in wild-type embryos. Ectopic expression of either Pyr or Ths was sufficient to redirect CVM cell movement, but only when the endogenous source of these ligands was absent. Collectively, our results show that FGF signaling regulates directional movement of CVM cells and that native presentation of both FGF ligands together is most effective at attracting cells. This study also has general implications, as it suggests that the activity supported by two FGF ligands in concert differs from their activities in isolation.

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Recruitment of 5' Hoxa genes in the allantois is essential for proper extra-embryonic function in placental mammals.

Publication Date: 2012 Feb PMID: 22219351
Authors: Scotti, M. - Kmita, M.
Journal: Development

The Hox gene family is well known for its functions in establishing morphological diversity along the anterior-posterior axis of developing embryos. In mammals, one of these genes, Hoxa13, is crucial for embryonic survival, as its function is required for the proper expansion of the fetal vasculature in the placenta. Thus, it appears that the developmental strategy specific to placental mammals is linked, at least in part, to the recruitment of Hoxa13 function in developing extra-embryonic tissues. Yet, the mechanism underlying this extra-embryonic recruitment is unknown. Here, we provide evidence that this functional novelty is not exclusive to Hoxa13 but is shared with its neighboring Hoxa11 and Hoxa10 genes. We show that the extra-embryonic function of these three Hoxa genes stems from their specific expression in the allantois, an extra-embryonic hallmark of amniote vertebrates. Interestingly, Hoxa10-13 expression in the allantois is conserved in chick embryos, which are non-placental amniotes, suggesting that the extra-embryonic recruitment of Hoxa10, Hoxa11 and Hoxa13 most likely arose in amniotes, i.e. prior to the emergence of placental mammals. Finally, using a series of targeted recombination and transgenic assays, we provide evidence that the regulatory mechanism underlying Hoxa expression in the allantois is extremely complex and relies on several cis-regulatory sequences.

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crinkled reveals a new role for Wingless signaling in Drosophila denticle formation.

Publication Date: 2012 Feb PMID: 22219350
Authors: Bejsovec, A. - Chao, A. T.
Journal: Development

The specification of the body plan in vertebrates and invertebrates is controlled by a variety of cell signaling pathways, but how signaling output is translated into morphogenesis is an ongoing question. Here, we describe genetic interactions between the Wingless (Wg) signaling pathway and a nonmuscle myosin heavy chain, encoded by the crinkled (ck) locus in Drosophila. In a screen for mutations that modify wg loss-of-function phenotypes, we isolated multiple independent alleles of ck. These ck mutations dramatically alter the morphology of the hook-shaped denticles that decorate the ventral surface of the wg mutant larval cuticle. In an otherwise wild-type background, ck mutations do not significantly alter denticle morphology, suggesting a specific interaction with Wg-mediated aspects of epidermal patterning. Here, we show that changing the level of Wg activity changes the structure of actin bundles during denticle formation in ck mutants. We further find that regulation of the Wg target gene, shaven-baby (svb), and of its transcriptional targets, miniature (m) and forked (f), modulates this ck-dependent process. We conclude that Ck acts in concert with Wg targets to orchestrate the proper shaping of denticles in the Drosophila embryonic epidermis.

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Cdx2 determines the fate of postnatal intestinal endoderm.

Publication Date: 2012 Feb PMID: 22190642
Authors: Stringer, E. J. - Duluc, I. - Saandi, T. - Davidson, I. - Bialecka, M. - Sato, T. - Barker, N. - Clevers, H. - Pritchard, C. A. - Winton, D. J. - Wright, N. A. - Freund, J. N. - Deschamps, J. - Beck, F.
Journal: Development

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.

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Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons.

Publication Date: 2012 Feb PMID: 22190641
Authors: Pan, Y. A. - Choy, M. - Prober, D. A. - Schier, A. F.
Journal: Development

How neurons connect to form functional circuits is central to the understanding of the development and function of the nervous system. In the somatosensory system, perception of sensory stimuli to the head requires specific connections between trigeminal sensory neurons and their many target areas in the central nervous system. Different trigeminal subtypes have specialized functions and downstream circuits, but it has remained unclear how subtype-specific axonal projection patterns are formed. Using zebrafish as a model system, we followed the development of two trigeminal sensory neuron subtypes: one that expresses trpa1b, a nociceptive channel important for sensing environmental chemicals; and a distinct subtype labeled by an islet1 reporter (Isl1SS). We found that Trpa1b and Isl1SS neurons have overall similar axon trajectories but different branching morphologies and distributions of presynaptic sites. Compared with Trpa1b neurons, Isl1SS neurons display reduced branch growth and synaptogenesis at the hindbrain-spinal cord junction. The subtype-specific morphogenesis of Isl1SS neurons depends on the guidance receptor Robo2. robo2 is preferentially expressed in the Isl1SS subset and inhibits branch growth and synaptogenesis. In the absence of Robo2, Isl1SS afferents acquire many of the characteristics of Trpa1b afferents. These results reveal that subtype-specific activity of Robo2 regulates subcircuit morphogenesis in the trigeminal sensory system.

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The genomic regulatory control of skeletal morphogenesis in the sea urchin.

Publication Date: 2012 Feb PMID: 22190640
Authors: Rafiq, K. - Cheers, M. S. - Ettensohn, C. A.
Journal: Development

A central challenge of developmental and evolutionary biology is to understand how anatomy is encoded in the genome. Elucidating the genetic mechanisms that control the development of specific anatomical features will require the analysis of model morphogenetic processes and an integration of biological information at genomic, cellular and tissue levels. The formation of the endoskeleton of the sea urchin embryo is a powerful experimental system for developing such an integrated view of the genomic regulatory control of morphogenesis. The dynamic cellular behaviors that underlie skeletogenesis are well understood and a complex transcriptional gene regulatory network (GRN) that underlies the specification of embryonic skeletogenic cells (primary mesenchyme cells, PMCs) has recently been elucidated. Here, we link the PMC specification GRN to genes that directly control skeletal morphogenesis. We identify new gene products that play a proximate role in skeletal morphogenesis and uncover transcriptional regulatory inputs into many of these genes. Our work extends the importance of the PMC GRN as a model developmental GRN and establishes a unique picture of the genomic regulatory control of a major morphogenetic process. Furthermore, because echinoderms exhibit diverse programs of skeletal development, the newly expanded sea urchin skeletogenic GRN will provide a foundation for comparative studies that explore the relationship between GRN evolution and morphological evolution.

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Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh.

Publication Date: 2012 Feb PMID: 22190639
Authors: Wang, W. - Lian, N. - Ma, Y. - Li, L. - Gallant, R. C. - Elefteriou, F. - Yang, X.
Journal: Development

Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.

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Wnt/beta-catenin signaling directly regulates Foxj1 expression and ciliogenesis in zebrafish Kupffer's vesicle.

Publication Date: 2012 Feb PMID: 22190638
Authors: Caron, A. - Xu, X. - Lin, X.
Journal: Development

Cilia are essential for normal development. The composition and assembly of cilia has been well characterized, but the signaling and transcriptional pathways that govern ciliogenesis remain poorly studied. Here, we report that Wnt/beta-catenin signaling directly regulates ciliogenic transcription factor foxj1a expression and ciliogenesis in zebrafish Kupffer's vesicle (KV). We show that Wnt signaling acts temporally and KV cell-autonomously to control left-right (LR) axis determination and ciliogenesis. Specifically, reduction of Wnt signaling leads to a disruption of LR patterning, shorter and fewer cilia, a loss of cilia motility and a downregulation of foxj1a expression. However, these phenotypes can be rescued by KV-targeted overexpression of foxj1a. In comparison to the FGF pathway that has been previously implicated in the control of ciliogenesis, our epistatic studies suggest a more downstream function of Wnt signaling in the regulation of foxj1a expression and ciliogenesis in KV. Importantly, enhancer analysis reveals that KV-specific expression of foxj1a requires the presence of putative Lef1/Tcf binding sites, indicating that Wnt signaling activates foxj1a transcription directly. We also find that impaired Wnt signaling leads to kidney cysts and otolith disorganization, which can be attributed to a loss of foxj1 expression and disrupted ciliogenesis in the developing pronephric ducts and otic vesicles. Together, our data reveal a novel role of Wnt/beta-catenin signaling upstream of ciliogenesis, which might be a general developmental mechanism beyond KV. Moreover, our results also prompt a hypothesis that certain developmental effects of the Wnt/beta-catenin pathway are due to the activation of Foxj1 and cilia formation.

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alphaE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly.

Publication Date: 2012 Feb PMID: 22190637
Authors: Schepis, A. - Sepich, D. - Nelson, W. J.
Journal: Development

alphaE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of alphaE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of alphaE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of alphaE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of alphaE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of alphaE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and alphaE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and alphaE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.

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Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila.

Publication Date: 2012 Feb PMID: 22190636
Authors: Yamakawa, T. - Yamada, K. - Sasamura, T. - Nakazawa, N. - Kanai, M. - Suzuki, E. - Fortini, M. E. - Matsuno, K.
Journal: Development

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that Pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function resulted in enlargement of the ER that was not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity.

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LACHESIS-dependent egg-cell signaling regulates the development of female gametophytic cells.

Publication Date: 2012 Feb PMID: 22190635
Authors: Volz, R. - von Lyncker, L. - Baumann, N. - Dresselhaus, T. - Sprunck, S. - Gross-Hardt, R.
Journal: Development

In contrast to animals, plant germ cells are formed along with accessory cells in specialized haploid generations, termed gametophytes. The female gametophyte of flowering plants consists of four different cell types, which exert distinct functions in the reproductive process. For successful fertilization, the development of the four cell types has to be tightly coordinated; however, the underlying mechanisms are not yet understood. We have previously isolated the lachesis (lis) mutant, which forms supernumerary gametes at the expense of adjacent accessory cells. LIS codes for the Arabidopsis homolog of the pre-mRNA splicing factor PRP4 and shows a dynamic expression pattern in the maturing female gametophyte. Here, we used LIS as a molecular tool to study cell-cell communication in the female gametophyte. We show that reducing LIS transcript amounts specifically in the egg cell, affects the development of all female gametophytic cells, indicating that cell differentiation in the female gametophyte is orchestrated by the egg cell. Among the defects observed is the failure of homotypic nuclei fusion in the central cell and, as a consequence, a block in endosperm formation. LIS-mediated egg cell signaling, thus, provides a safeguard mechanism that prevents the formation of nurturing tissue in the absence of a functional egg cell.

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Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells.

Publication Date: 2012 Feb PMID: 22190634
Authors: Vandussen, K. L. - Carulli, A. J. - Keeley, T. M. - Patel, S. R. - Puthoff, B. J. - Magness, S. T. - Tran, I. T. - Maillard, I. - Siebel, C. - Kolterud, A. - Grosse, A. S. - Gumucio, D. L. - Ernst, S. A. - Tsai, Y. H. - Dempsey, P. J. - Samuelson, L. C.
Journal: Development

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jkappa binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.

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