Development

Activation of the skeletogenic gene regulatory network in the early sea urchin embryo.

Publication Date: 2010 Feb 24 PMID: 20181745
Authors: Sharma, T. - Ettensohn, C. A.
Journal: Development

The gene regulatory network (GRN that underlies the development of the embryonic skeleton in sea urchins is an important model for understanding the architecture and evolution of developmental GRNs. The initial deployment of the network is thought to be regulated by a derepression mechanism, which is mediated by the products of the pmar1 and hesC genes. Here, we show that the activation of the skeletogenic network occurs by a mechanism that is distinct from the transcriptional repression of hesC. By means of quantitative, fluorescent whole-mount in situ hybridization, we find that two pivotal early genes in the network, alx1 and delta, are activated in prospective skeletogenic cells prior to the downregulation of hesC expression. An analysis of the upstream regulation of alx1 shows that this gene is regulated by MAPK signaling and by the transcription factor Ets1; however, these inputs influence only the maintenance of alx1 expression and not its activation, which occurs by a distinct mechanism. By altering normal cleavage patterns, we show that the zygotic activation of alx1 and delta, but not that of pmar1, is dependent upon the unequal division of vegetal blastomeres. Based on these findings, we conclude that the widely accepted double-repression model is insufficient to account for the localized activation of the skeletogenic GRN. We postulate the existence of additional, unidentified repressors that are controlled by pmar1, and propose that the ability of pmar1 to derepress alx1 and delta is regulated by the unequal division of vegetal blastomeres.

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Runx1 and Runx2 cooperate during sternal morphogenesis.

Publication Date: 2010 Feb 24 PMID: 20181744
Authors: Kimura, A. - Inose, H. - Yano, F. - Fujita, K. - Ikeda, T. - Sato, S. - Iwasaki, M. - Jinno, T. - Ae, K. - Fukumoto, S. - Takeuchi, Y. - Itoh, H. - Imamura, T. - Kawaguchi, H. - Chung, U. I. - Martin, J. F. - Iseki, S. - Shinomiya, K. I. - Takeda, S.
Journal: Development

Chondrocyte differentiation is strictly regulated by various transcription factors, including Runx2 and Runx3; however, the physiological role of Runx1 in chondrocyte differentiation remains unknown. To examine the role of Runx1, we generated mesenchymal-cell-specific and chondrocyte-specific Runx1-deficient mice [Prx1 Runx1(f/f) mice and alpha1(II Runx1(f/f) mice, respectively] to circumvent the embryonic lethality of Runx1-deficient mice. We then mated these mice with Runx2 mutant mice to obtain mesenchymal-cell-specific or chondrocyte-specific Runx1; Runx2 double-mutant mice [Prx1 DKO mice and alpha1(II DKO mice, respectively]. Prx1 Runx1(f/f) mice displayed a delay in sternal development and Prx1 DKO mice completely lacked a sternum. By contrast, alpha1(II Runx1(f/f) mice and alpha1(II DKO mice did not show any abnormal sternal morphogenesis or chondrocyte differentiation. Notably, Runx1, Runx2 and the Prx1-Cre transgene were co-expressed specifically in the sternum, which explains the observation that the abnormalities were limited to the sternum. Histologically, mesenchymal cells condensed normally in the prospective sternum of Prx1 DKO mice; however, commitment to the chondrocyte lineage, which follows mesenchymal condensation, was significantly impaired. In situ hybridization analyses demonstrated that the expression of alpha1(II collagen (Col2a1 - Mouse Genome Informatics, Sox5 and Sox6 in the prospective sternum of Prx1 DKO mice was severely attenuated, whereas Sox9 expression was unchanged. Molecular analyses revealed that Runx1 and Runx2 induce the expression of Sox5 and Sox6, which leads to the induction of alpha1(II collagen expression via the direct regulation of promoter activity. Collectively, these results show that Runx1 and Runx2 cooperatively regulate sternal morphogenesis and the commitment of mesenchymal cells to become chondrocytes through the induction of Sox5 and Sox6.

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Polycystin-dependent fluid flow sensing targets histone deacetylase 5 to prevent the development of renal cysts.

Publication Date: 2010 Feb 24 PMID: 20181743
Authors: Xia, S. - Li, X. - Johnson, T. - Seidel, C. - Wallace, D. P. - Li, R.
Journal: Development

Polycystin 1 and polycystin 2 are large transmembrane proteins, which, when mutated, cause autosomal dominant polycystic kidney disease (ADPKD, a highly prevalent human genetic disease. The polycystins are thought to form a receptor-calcium channel complex in the plasma membrane of renal epithelial cells and elicit a calcium influx in response to mechanical stimulation, such as fluid flow across the apical surface of renal epithelial cells. The functional role of the polycystins in mechanosensation remains largely unknown. Here, we found that myocyte enhancer factor 2C (MEF2C and histone deacetylase 5 (HDAC5, two key regulators of cardiac hypertrophy, are targets of polycystin-dependent fluid stress sensing in renal epithelial cells in mice. We show that fluid flow stimulation of polarized epithelial monolayers induced phosphorylation and nuclear export of HDAC5, which are crucial events in the activation of MEF2C-based transcription. Kidney-specific knockout of Mef2c, or genetrap-inactivation of a MEF2C transcriptional target, MIM, resulted in extensive renal tubule dilation and cysts, whereas Hdac5 heterozygosity or treatment with TSA, an HDAC inhibitor, reduced cyst formation in Pkd2(-/-) mouse embryos. These findings suggest a common signaling motif between myocardial hypertrophy and maintenance of renal epithelial architecture, and a potential therapeutic approach to treat ADPKD.

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A role for juvenile hormone in the prepupal development of Drosophila melanogaster.

Publication Date: 2010 Feb 24 PMID: 20181742
Authors: Riddiford, L. M. - Truman, J. W. - Mirth, C. K. - Shen, Y. C.
Journal: Development

To elucidate the role of juvenile hormone (JH in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1 in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met(27) larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis.

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Double bromodomain protein BET-1 and MYST HATs establish and maintain stable cell fates in C. elegans.

Publication Date: 2010 Feb 24 PMID: 20181741
Authors: Shibata, Y. - Takeshita, H. - Sasakawa, N. - Sawa, H.
Journal: Development

The maintenance of cell fate is important for normal development and tissue homeostasis. Epigenetic mechanisms, including histone modifications, are likely to play crucial roles in cell-fate maintenance. However, in contrast to the established functions of histone methylation, which are mediated by the polycomb proteins, the roles of histone acetylation in cell-fate maintenance are poorly understood. Here, we show that the C. elegans acetylated-histone-binding protein BET-1 is required for the establishment and maintenance of stable fate in various lineages. In most bet-1 mutants, cells adopted the correct fate initially, but at later stages they often transformed into a different cell type. By expressing BET-1 at various times in development and examining the rescue of the Bet-1 phenotype, we showed that BET-1 functions both at the time of fate acquisition, to establish a stable fate, and at later stages, to maintain the established fate. Furthermore, the disruption of the MYST HATs perturbed the subnuclear localization of BET-1 and caused bet-1-like phenotypes, suggesting that BET-1 is recruited to its targets through acetylated histones. Our results therefore indicate that histone acetylation plays a crucial role in cell-fate maintenance.

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H-, N- and Kras cooperatively regulate lymphatic vessel growth by modulating VEGFR3 expression in lymphatic endothelial cells in mice.

Publication Date: 2010 Mar PMID: 20179099
Authors: Ichise, T. - Yoshida, N. - Ichise, H.
Journal: Development

Mammalian Ras, which is encoded by three independent genes, has been thought to be a versatile component of intracellular signalling. However, when, where and how Ras signalling plays essential roles in development and whether the three Ras genes have overlapping functions in particular cells remain unclear. Here, we show that the three Ras proteins dose-dependently regulate lymphatic vessel growth in mice. We find that lymphatic vessel hypoplasia is a common phenotype in Ras compound knockout mice and that overexpressed normal Ras in an endothelial cell lineage selectively causes lymphatic vessel hyperplasia in vivo. Overexpression of normal Ras in lymphatic endothelial cells leads to sustained MAPK activation, cellular viability and enhanced endothelial network formation under serum-depleted culture conditions in vitro, and knockdown of endogenous Ras in lymphatic endothelial cells impairs cell proliferation, MAPK activation, cell migration and endothelial network formation. Ras overexpression and knockdown result in up- and downregulation of vascular endothelial growth factor receptor (VEGFR) 3 expression, respectively, in lymphatic endothelial cells in vitro. The close link between Ras and VEGFR3 in vitro is consistent with the result that Ras knockout and transgenic alleles are genetic modifiers in lymphatic vessel hypoplasia caused by Vegfr3 haploinsufficiency. Our findings demonstrate a cooperative function of the three Ras proteins in normal development, and also provide a novel aspect of VEGFR3 signalling modulated by Ras in lymphangiogenesis.

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Endothelial beta1 integrins regulate sprouting and network formation during vascular development.

Publication Date: 2010 Mar PMID: 20179098
Authors: Malan, D. - Wenzel, D. - Schmidt, A. - Geisen, C. - Raible, A. - Bolck, B. - Fleischmann, B. K. - Bloch, W.
Journal: Development

beta1 integrins are important regulators of vascular differentiation and development, as their endothelial-specific deletion results in embryonic lethality. In the present study, we investigated the molecular mechanisms underlying the prominent vascular abnormalities that occur in the absence of beta1 integrins. Because of the early embryonic lethality of knockout mice, we studied endothelial cell and vessel development in beta1-integrin-deficient murine embryonic stem cells to gain novel insights into the role of beta1 integrins in vasculo-angiogenesis. We found that vessel development was strongly defective in the mutant embryoid bodies (EBs), as only primitive and short sprouts developed from clusters of vascular precursors in beta1 integrin(-/-) EBs, whereas complex network formation of endothelial tubes was observed in wild-type EBs. The vascular defect was due to deficient beta1 integrin expression in endothelial cells, as its endothelial-specific re-expression rescued the phenotype entirely. The mechanism responsible for defective vessel formation was found to be reduced endothelial cell maturation, migration and elongation. Moreover, the lower number of endothelial cells in beta1 integrin(-/-) EBs was due to an increased apoptosis versus proliferation rate. The enhanced apoptosis and proliferation of beta1 integrin(-/-) endothelial cells was related to the elevation of peNOS and pAKT signaling molecules, respectively. Our data demonstrate that endothelial beta1 integrins are determinants of vessel formation and that this effect is mediated via different signaling pathways.

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Interplay of auxin, KANADI and Class III HD-ZIP transcription factors in vascular tissue formation.

Publication Date: 2010 Mar PMID: 20179097
Authors: Ilegems, M. - Douet, V. - Meylan-Bettex, M. - Uyttewaal, M. - Brand, L. - Bowman, J. L. - Stieger, P. A.
Journal: Development

Class III HD-ZIP and KANADI gene family members have complementary expression patterns in the vasculature and their gain-of-function and loss-of-function mutants have complementary vascular phenotypes. This suggests that members of the two gene families are involved in the establishment of the spatial arrangement of phloem, cambium and xylem. In this study, we have investigated the role of these two gene families in vascular tissue differentiation, in particular their interactions with the plant hormone auxin. We have analyzed the vasculature of plants that have altered expression levels of Class III HD-ZIP and KANADI transcription factors in provascular cells. Removal of either KANADI or Class III HD-ZIP expression in procambium cells led to a wider distribution of auxin in internal tissues, to an excess of procambium cell recruitment and to increased cambium activity. Ectopic expression of KANADI1 in provascular cells inhibited procambium cell recruitment due to negative effects of KANADI1 on expression and polar localization of the auxin efflux-associated protein PIN-FORMED1. Ectopic expression of Class III HD-ZIP genes promoted xylem differentiation. We propose that Class III HD-ZIP and KANADI transcription factors control cambium activity: KANADI proteins by acting on auxin transport, and Class III HD-ZIP proteins by promoting axial cell elongation and xylem differentiation.

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SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification.

Publication Date: 2010 Mar PMID: 20179096
Authors: Hattori, T. - Muller, C. - Gebhard, S. - Bauer, E. - Pausch, F. - Schlund, B. - Bosl, M. R. - Hess, A. - Surmann-Schmitt, C. - von der Mark, H. - de Crombrugghe, B. - von der Mark, K.
Journal: Development

SOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.

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LIN28 alters cell fate succession and acts independently of the let-7 microRNA during neurogliogenesis in vitro.

Publication Date: 2010 Mar PMID: 20179095
Authors: Balzer, E. - Heine, C. - Jiang, Q. - Lee, V. M. - Moss, E. G.
Journal: Development

LIN28 is an RNA-binding protein that is expressed in many developing tissues. It can block let-7 (Mirlet7) microRNA processing and help promote pluripotency. We have observed LIN28 expression in the developing mouse neural tube, colocalizing with SOX2, suggesting a role in neural development. To better understand its normal developmental function, we investigated LIN28 activity during neurogliogenesis in vitro, where the succession of neuronal to glial cell fates occurs as it does in vivo. LIN28 expression was high in undifferentiated cells, and was downregulated rapidly upon differentiation. Constitutive LIN28 expression caused a complete block of gliogenesis and an increase in neurogenesis. LIN28 expression was compatible with neuronal differentiation and did not increase proliferation. LIN28 caused significant changes in gene expression prior to any effect on let-7, notably on Igf2. Furthermore, a mutant LIN28 that permitted let-7 accumulation was still able to completely block gliogenesis. Thus, at least two biological activities of LIN28 are genetically separable and might involve distinct mechanisms. LIN28 can differentially promote and inhibit specific fates and does not function exclusively by blocking let-7 family microRNAs. Importantly, the role of LIN28 in cell fate succession in vertebrate cells is analogous to its activity as a developmental timing regulator in C. elegans.

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Retinoic acid orchestrates fibroblast growth factor signalling to drive embryonic stem cell differentiation.

Publication Date: 2010 Mar PMID: 20179094
Authors: Stavridis, M. P. - Collins, B. J. - Storey, K. G.
Journal: Development

Embryonic stem (ES) cells fluctuate between self-renewal and the threshold of differentiation. Signalling via the fibroblast growth factor (Fgf)/Erk pathway is required to progress from this dynamic state and promote mouse ES cell differentiation. Retinoic acid also induces differentiation in many cellular contexts, but its mechanism of action in relation to Fgf/Erk signalling in ES cells is poorly understood. Here, we show for the first time that endogenous retinoid signalling is required for the timely acquisition of somatic cell fate in mouse ES cells and that exposure to retinoic acid advances differentiation by a dual mechanism: first increasing, but in the long-term decreasing, Fgf signalling. Rapid retinoid induction of Fgf8 and downstream Erk activity on day 1 in differentiation conditions may serve to ensure loss of self-renewal. However, more gradual repression of Fgf4 by retinoic acid is accompanied by an overall reduction in Erk activity on day 2, and the acquisition of neural and non-neural fates is now advanced by inhibition of Fgf signalling. So, although blocking Fgf/Erk activity is known to promote ES cell self-renewal, once cells have experienced a period of such signals, subsequent inhibition of Fgf signalling has the opposite effect and drives differentiation. We further show in the embryo that retinoid repression of Fgf signalling promotes neural differentiation onset in an analogous step in the extending embryonic body axis and so identify attenuation of Fgf signalling by retinoic acid as a conserved fundamental mechanism driving differentiation towards somatic cell fates.

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IGF signaling between blastema and wound epidermis is required for fin regeneration.

Publication Date: 2010 Mar PMID: 20179093
Authors: Chablais, F. - Jazwinska, A.
Journal: Development

In mammals, the loss of a limb is irreversible. By contrast, urodele amphibians and teleost fish are capable of nearly perfect regeneration of lost appendages. This ability depends on direct interaction between the wound epithelium and mesenchymal progenitor cells of the blastema. It has been known for decades that contact between the wound epithelium and the underlying blastema is essential for successful regeneration. However, the underlying mechanisms are poorly understood. Here, we show that upon amputation the blastema induces expression of the ligand Igf2b, which then activates IGF signaling specifically in cells of the adjacent apical epithelium. Inhibition of IGF signaling by either morpholino antisense technology, or by specific chemical inhibitors of Igf1 receptor function NVP-AEW541 and NVP-ADW742, impairs fin regeneration. At the cellular level, this block in regeneration is reflected by a lack of the distinctive basal epithelium, increased apoptosis in the wound epidermis and reduced proliferation of blastema cells. Furthermore, induction of the blastemal and wound epidermal markers cannot be supported in the absence of IGF signaling. These data provide evidence that Igf2b expressed in the blastema promotes the properties of the adjacent wound epidermis, which subsequently are necessary for blastema function. Thus, IGF signaling upregulated upon fin amputation represents a signal from the blastema to the wound epithelium, a crucial step in appendage regeneration.

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Maternal control of early mouse development.

Publication Date: 2010 Mar PMID: 20179092
Authors: Li, L. - Zheng, P. - Dean, J.
Journal: Development

The hiatus between oocyte and embryonic gene transcription dictates a role for stored maternal factors in early mammalian development. Encoded by maternal-effect genes, these factors accumulate during oogenesis and enable the activation of the embryonic genome, the subsequent cleavage stages of embryogenesis and the initial establishment of embryonic cell lineages. Recent studies in mice have yielded new findings on the role of maternally provided proteins and multi-component complexes in preimplantation development. Nevertheless, significant gaps remain in our mechanistic understanding of the networks that regulate early mammalian embryogenesis, which provide an impetus and opportunities for future investigations.

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On growth and form: a Cartesian coordinate system of Wnt and BMP signaling specifies bilaterian body axes.

Publication Date: 2010 Mar PMID: 20179091
Authors: Niehrs, C.
Journal: Development

The regulation of body axis specification in the common ancestor of bilaterians remains controversial. BMP signaling appears to be an ancient program for patterning the secondary, or dorsoventral, body axis, but any such program for the primary, or anteroposterior, body axis is debated. Recent work in invertebrates indicates that posterior Wnt/beta-catenin signaling is such a mechanism and that it evolutionarily predates the cnidarian-bilaterian split. Here, I argue that a Cartesian coordinate system of positional information set up by gradients of perpendicular Wnt and BMP signaling is conserved in bilaterians, orchestrates body axis patterning and contributes to both the relative invariance and diversity of body forms.

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Alterations in phosphorus, calcium and PTHrP contribute to defects in dental and dental alveolar bone formation in calcium-sensing receptor-deficient mice.

Publication Date: 2010 Mar PMID: 20150282
Authors: Sun, W. - Sun, W. - Liu, J. - Zhou, X. - Xiao, Y. - Karaplis, A. - Pollak, M. R. - Brown, E. - Goltzman, D. - Miao, D.
Journal: Development

To determine whether the calcium-sensing receptor (CaR) participates in tooth formation and dental alveolar bone development in mandibles in vivo, we examined these processes, as well as mineralization, in 2-week-old CaR-knockout (CaR(-/-)) mice. We also attempted to rescue the phenotype of CaR(-/-) mice by genetic means, in mice doubly homozygous for CaR and 25-hydroxyvitamin D 1alpha-hydroxylase [1alpha(OH)ase] or parathyroid hormone (Pth). In CaR(-/-) mice, which exhibited hypercalcemia, hypophosphatemia and increased serum PTH, the volumes of teeth and of dental alveolar bone were decreased dramatically, whereas the ratio of the area of predentin to total dentin and the number and surface of osteoblasts in dental alveolar bone were increased significantly, as compared with wild-type littermates. The normocalcemia present in CaR(-/-);1alpha(OH)ase(-/-) mice only slightly improved the defects in dental and alveolar bone formation observed in the hypercalcemic CaR(-/-) mice. However, these defects were completely rescued by the additional elimination of hypophosphatemia and by an increase in parathyroid hormone-related protein (PTHrP) expression in the apical pulp, Hertwig's epithelial root sheath and mandibular tissue in CaR(-/-); Pth(-/-) mice. Therefore, alterations in calcium, phosphorus and PTHrP contribute to defects in the formation of teeth and alveolar bone in CaR-deficient mice. This study indicates that CaR participates in the formation of teeth and in the development of dental alveolar bone in mandibles in vivo, although it appears to do so largely indirectly.

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Syntabulin, a motor protein linker, controls dorsal determination.

Publication Date: 2010 Mar PMID: 20150281
Authors: Nojima, H. - Rothhamel, S. - Shimizu, T. - Kim, C. H. - Yonemura, S. - Marlow, F. L. - Hibi, M.
Journal: Development

In amphibian and teleost embryos, the dorsal determinants (DDs) are believed to be initially localized to the vegetal pole and then transported to the prospective dorsal side of the embryo along a microtubule array. The DDs are known to activate the canonical Wnt pathway and thereby promote the expression of genes that induce the dorsal organizer. Here, by identifying the locus of the maternal-effect ventralized mutant tokkaebi, we show that Syntabulin, a linker of the kinesin I motor protein, is essential for dorsal determination in zebrafish. We found that syntabulin mRNA is transported to the vegetal pole during oogenesis through the Bucky ball (Buc)-mediated Balbiani body-dependent pathway, which is necessary for establishment of animal-vegetal (AV) oocyte polarity. We demonstrate that Syntabulin is translocated from the vegetal pole in a microtubule-dependent manner. Our findings suggest that Syntabulin regulates the microtubule-dependent transport of the DDs, and provide evidence for the link between AV and dorsoventral axis formation.

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The cytolinker Pigs is a direct target and a negative regulator of Notch signalling.

Publication Date: 2010 Mar PMID: 20150280
Authors: Pines, M. K. - Housden, B. E. - Bernard, F. - Bray, S. J. - Roper, K.
Journal: Development

Gas2-like proteins harbour putative binding sites for both the actin and the microtubule cytoskeleton and could thus mediate crosstalk between these cytoskeletal systems. Family members are highly conserved in all metazoans but their in vivo role is not clear. The sole Drosophila Gas2-like gene, CG3973 (pigs), was recently identified as a transcriptional target of Notch signalling and might therefore link cell fate decisions through Notch activation directly to morphogenetic changes. We have generated a null mutant in CG3973 (pigs): pigs(1) mutants are semi-viable but adult flies are flightless, showing indirect flight muscle degeneration, and females are sterile, showing disrupted oogenesis and severe defects in follicle cell differentiation, similar to phenotypes seen when levels of Notch/Delta signalling are perturbed in these tissues. Loss of Pigs leads to an increase in Notch signalling activity in several tissues. These results indicate that Gas2-like proteins are essential for development and suggest that Pigs acts downstream of Notch as a morphogenetic read-out, and also as part of a regulatory feedback loop to relay back information about the morphogenetic state of cells to restrict Notch activation to appropriate levels in certain target tissues.

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The HMX/NKX homeodomain protein MLS-2 specifies the identity of the AWC sensory neuron type via regulation of the ceh-36 Otx gene in C. elegans.

Publication Date: 2010 Mar PMID: 20150279
Authors: Kim, K. - Kim, R. - Sengupta, P.
Journal: Development

The differentiated features of postmitotic neurons are dictated by the expression of specific transcription factors. The mechanisms by which the precise spatiotemporal expression patterns of these factors are regulated are poorly understood. In C. elegans, the ceh-36 Otx homeobox gene is expressed in the AWC sensory neurons throughout postembryonic development, and regulates terminal differentiation of this neuronal subtype. Here, we show that the HMX/NKX homeodomain protein MLS-2 regulates ceh-36 expression specifically in the AWC neurons. Consequently, the AWC neurons fail to express neuron type-specific characteristics in mls-2 mutants. mls-2 is expressed transiently in postmitotic AWC neurons, and directly initiates ceh-36 expression. CEH-36 subsequently interacts with a distinct site in its cis-regulatory sequences to maintain its own expression, and also directly regulates the expression of AWC-specific terminal differentiation genes. We also show that MLS-2 acts in additional neuron types to regulate their development and differentiation. Our analysis describes a transcription factor cascade that defines the unique postmitotic characteristics of a sensory neuron subtype, and provides insights into the spatiotemporal regulatory mechanisms that generate functional diversity in the sensory nervous system.

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A conditional mutation in Arabidopsis thaliana separase induces chromosome non-disjunction, aberrant morphogenesis and cyclin B1;1 stability.

Publication Date: 2010 Mar PMID: 20150278
Authors: Wu, S. - Scheible, W. R. - Schindelasch, D. - Van Den Daele, H. - De Veylder, L. - Baskin, T. I.
Journal: Development

The caspase family protease, separase, is required at anaphase onset to cleave the cohesin complex, which joins sister chromatids. However, among eukaryotes, separases have acquired novel functions. Here, we show that Arabidopsis thaliana radially swollen 4 (rsw4), a temperature-sensitive mutant isolated previously on the basis of root swelling, harbors a mutation in At4g22970, the A. thaliana separase. Loss of separase function in rsw4 at the restrictive temperature is indicated by the widespread failure of replicated chromosomes to disjoin. Surprisingly, rsw4 has neither pronounced cell cycle arrest nor anomalous spindle formation, which occur in other eukaryotes upon loss of separase activity. However, rsw4 roots have disorganized cortical microtubules and accumulate the mitosis-specific cyclin, cyclin B1;1, excessive levels of which have been associated with altered microtubules and morphology. Cyclin B1;1 also accumulates in certain backgrounds in response to DNA damage, but we find no evidence for aberrant responses to DNA damage in rsw4. Our characterization of rsw4 leads us to hypothesize that plant separase, in addition to cleaving cohesin, regulates cyclin B1;1, with profound ramifications for morphogenesis.

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The Trithorax group protein Ash2l and Saf-A are recruited to the inactive X chromosome at the onset of stable X inactivation.

Publication Date: 2010 Mar PMID: 20150277
Authors: Pullirsch, D. - Hartel, R. - Kishimoto, H. - Leeb, M. - Steiner, G. - Wutz, A.
Journal: Development

Mammals compensate X chromosome gene dosage between the sexes by silencing of one of the two female X chromosomes. X inactivation is initiated in the early embryo and requires the non-coding Xist RNA, which encompasses the inactive X chromosome (Xi) and triggers its silencing. In differentiated cells, several factors including the histone variant macroH2A and the scaffold attachment factor SAF-A are recruited to the Xi and maintain its repression. Consequently, in female somatic cells the Xi remains stably silenced independently of Xist. Here, we identify the Trithorax group protein Ash2l as a novel component of the Xi. Ash2l is recruited by Xist concomitantly with Saf-A and macroH2A at the transition to Xi maintenance. Recruitment of these factors characterizes a developmental transition point for the chromatin composition of the Xi. Surprisingly, expression of a mutant Xist RNA that does not cause gene repression can trigger recruitment of Ash2l, Saf-A and macroH2A to the X chromosome, and can cause chromosome-wide histone H4 hypoacetylation. This suggests that a chromatin configuration is established on non-genic chromatin on the Xi by Xist to provide a repressive compartment that could be used for maintaining gene silencing. Gene silencing is mechanistically separable from the formation of this repressive compartment and, thus, requires additional pathways. This observation highlights a crucial role for spatial organization of chromatin changes in the maintenance of X inactivation.

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The bHLH/PAS transcription factor singleminded 2s promotes mammary gland lactogenic differentiation.

Publication Date: 2010 Mar PMID: 20150276
Authors: Wellberg, E. - Metz, R. P. - Parker, C. - Porter, W. W.
Journal: Development

We have previously demonstrated that the bHLH/PAS transcription factor, singleminded 2s (Sim2s), is required for proper mammary ductal morphogenesis and luminal epithelial differentiation. Furthermore, loss of Sim2s in breast cancer cells resulted in downregulation of epithelial markers and acquisition of a basal-like phenotype. The objective of this study was to further define the role of Sim2s in mammary differentiation. We found that Sim2s is developmentally regulated throughout mammary gland development with highest expression during lactation. Mammary glands from nulliparous mice expressing Sim2s driven by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter were morphologically indistinguishable from wild-type mice but displayed hallmarks of precocious lactogenic differentiation. These included elevated expression of the milk protein genes Wap and Csn2, and apical localization of the lactation marker Npt2b. Consistent with the in vivo results, Sim2s enhanced prolactin-mediated Csn2 expression in HC11 and CIT3 mouse mammary epithelial cells, and downregulation of Sim2s by shRNA in HC11 cells inhibited Csn2 expression. Chromatin immunoprecipitation (ChIP) analyses of the Csn2 gene found that Sim2s associates with the Csn2 promoter and re-ChIP experiments showed that Sim2s interacted with the RNA II polymerase (RNAPII) complex. Together, these data demonstrate, for the first time, that Sim2s is required for establishing and maintaining mammary gland differentiation.

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giant is a bona fide gap gene in the intermediate germband insect, Oncopeltus fasciatus.

Publication Date: 2010 Mar PMID: 20147384
Authors: Liu, P. Z. - Patel, N. H.
Journal: Development

Drosophila undergoes a form of development termed long germ segmentation, where all segments are specified nearly simultaneously so that by the blastoderm stage, the entire body plan has been determined. This mode of segmentation is evolutionarily derived. Most insects undergo short or intermediate germ segmentation, where only anterior segments are specified early, and posterior segments are sequentially specified during germband elongation. These embryological differences imply that anterior and posterior segments might rely upon different molecular mechanisms. In Drosophila, embryos mutant for giant show a gap in the anterior as well fusions of several abdominal segments. In Tribolium, a short germ beetle, giant is required for segmental identity, but not formation, in gnathal segments and also for segmentation of the entire abdomen. This raises the possibility that giant might not act as a gap gene in short and intermediate germ insects. Oncopeltus fasciatus is an intermediate germ insect that is an outgroup to the clade containing Drosophila and Tribolium. We cloned the Oncopeltus homolog of giant and determined its expression and function during segmentation. We find that Oncopeltus giant is a canonical gap gene in the maxillary and labial segments and also plays a gap-like role in the first four abdominal segments. Our results suggest that giant was a bona fide gap gene in the ancestor of these insects with this role being lost in the lineage leading towards Tribolium. This highlights the conservation of anterior patterning and evolutionary plasticity of the genetic regulation controlling posterior segmentation, even in short and intermediate germ insects.

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Signaling via Alk5 controls the ontogeny of lung Clara cells.

Publication Date: 2010 Mar PMID: 20147383
Authors: Xing, Y. - Li, C. - Li, A. - Sridurongrit, S. - Tiozzo, C. - Bellusci, S. - Borok, Z. - Kaartinen, V. - Minoo, P.
Journal: Development

Clara cells, together with ciliated and pulmonary neuroendocrine cells, make up the epithelium of the bronchioles along the conducting airways. Clara cells are also known as progenitor or stem cells during lung regeneration after injury. The mechanisms of Clara cell differentiation are largely unknown. Transforming growth factor beta (TGFbeta)is a multifunctional molecule with roles in normal development and disease pathogenesis. In this study, we deleted the TGFbeta type I receptor Alk5 in the embryonic lung epithelium using Gata5-Cre mice. Absence of Alk5 blocked Clara cell differentiation but had no effect on ciliated or pulmonary neuroendocrine cells. Hairy/Enhancer of Split-1, which is expressed in Clara cell putative ;progenitors' was found to be a downstream target of Alk5 in vivo and in vitro. Loss of Alk5-mediated signaling also stimulated Pten gene expression and inhibited ERK phosphorylation in vivo. Using lung epithelial cells, we show that Alk5-regulated Hes1 expression is stimulated through Pten and the MEK/ERK and PI3K/AKT pathways. Thus, the signaling pathway by which TGFbeta/ALK5 regulates Clara cell differentiation may entail inhibition of Pten expression, which in turn activates ERK and AKT phosphorylation.

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lethal giant larvae is required with the par genes for the early polarization of the Drosophila oocyte.

Publication Date: 2010 Mar PMID: 20147382
Authors: Fichelson, P. - Jagut, M. - Lepanse, S. - Lepesant, J. A. - Huynh, J. R.
Journal: Development

Most cell types in an organism show some degree of polarization, which relies on a surprisingly limited number of proteins. The underlying molecular mechanisms depend, however, on the cellular context. Mutual inhibitions between members of the Par genes are proposed to be sufficient to polarize the C. elegans one-cell zygote and the Drosophila oocyte during mid-oogenesis. By contrast, the Par genes interact with cellular junctions and associated complexes to polarize epithelial cells. The Par genes are also required at an early step of Drosophila oogenesis for the maintenance of the oocyte fate and its early polarization. Here we show that the Par genes are not sufficient to polarize the oocyte early and that the activity of the tumor-suppressor gene lethal giant larvae (lgl) is required for the posterior translocation of oocyte-specific proteins, including germline determinants. We also found that Lgl localizes asymmetrically within the oocyte and is excluded from the posterior pole. We further demonstrate that phosphorylation of Par-1, Par-3 (Bazooka) and Lgl is crucial to regulate their activity and localization in vivo and describe, for the first time, adherens junctions located around the ring canals, which link the oocyte to the other cells of the germline cyst. However, null mutations in the DE-cadherin gene, which encodes the main component of the zonula adherens, do not affect the early polarization of the oocyte. We conclude that, despite sharing many similarities with other model systems at the genetic and cellular levels, the polarization of the early oocyte relies on a specific subset of polarity proteins.

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Cadherin-7 and cadherin-6B differentially regulate the growth, branching and guidance of cranial motor axons.

Publication Date: 2010 Mar PMID: 20147381
Authors: Barnes, S. H. - Price, S. R. - Wentzel, C. - Guthrie, S. C.
Journal: Development

Cadherin-7 (Cad7) and cadherin-6B (Cad6B) are expressed in early and late phases of cranial motoneuron development, respectively. Cad7 is expressed by cranial motoneurons soon after they are generated, as well as in the environment through which their axons extend. By contrast, Cad6B is expressed by mature cranial motoneurons. We demonstrate in chick that these cadherins play distinct roles in cranial motor axon morphology, branching and projection. Using in vitro approaches, we show that Cad7 enhances motor axon outgrowth, suppresses the formation of multiple axons and restricts interstitial branching, thus promoting the development of a single unbranched axon characteristic of differentiating motoneurons. Conversely, Cad6B in vitro promotes motor axon branching, a characteristic of mature motoneurons. In vivo gain- and loss-of-function experiments for these cadherins yielded phenotypes consistent with this interpretation. In particular, a loss of cadherin-mediated interactions in vivo led to dysregulation of the cranial motoneuron normal branching programme and caused axon navigation defects. We also demonstrate that Cad6B functions via the phosphatidylinositol 3-kinase pathway. Together, these data show that Cad7 and Cad6B differentially regulate cranial motoneuron growth, branching and axon guidance.

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Epithelial relaxation mediated by the myosin phosphatase regulator Mypt1 is required for brain ventricle lumen expansion and hindbrain morphogenesis.

Publication Date: 2010 Mar PMID: 20147380
Authors: Gutzman, J. H. - Sive, H.
Journal: Development

We demonstrate that in the zebrafish hindbrain, cell shape, rhombomere morphogenesis and, unexpectedly, brain ventricle lumen expansion depend on the contractile state of the neuroepithelium. The hindbrain neural tube opens in a specific sequence, with initial separation along the midline at rhombomere boundaries, subsequent openings within rhombomeres and eventual coalescence of openings into the hindbrain ventricle lumen. A mutation in the myosin phosphatase regulator mypt1 results in a small ventricle due to impaired stretching of the surrounding neuroepithelium. Although initial hindbrain opening remains normal, mypt1 mutant rhombomeres do not undergo normal morphological progression. Three-dimensional reconstruction demonstrates cell shapes within rhombomeres and at rhombomere boundaries are abnormal in mypt1 mutants. Wild-type cell shape requires that surrounding cells are also wild type, whereas mutant cell shape is autonomously regulated. Supporting the requirement for regulation of myosin function during hindbrain morphogenesis, wild-type embryos show dynamic levels of phosphorylated myosin regulatory light chain (pMRLC). By contrast, mutants show continuously high pMRLC levels, with concentration of pMRLC and myosin II at the apical side of the epithelium, and myosin II and actin concentration at rhombomere boundaries. Brain ventricle lumen expansion, rhombomere morphology and cell shape are rescued by inhibition of myosin II function, indicating that each defect is a consequence of overactive myosin. We suggest that the epithelium must ;relax', via activity of myosin phosphatase, to allow for normal hindbrain morphogenesis and expansion of the brain ventricular lumen. Epithelial relaxation might be a widespread strategy to facilitate tube inflation in many organs.

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Sequential requirement of Sox4 and Sox11 during development of the sympathetic nervous system.

Publication Date: 2010 Mar PMID: 20147379
Authors: Potzner, M. R. - Tsarovina, K. - Binder, E. - Penzo-Mendez, A. - Lefebvre, V. - Rohrer, H. - Wegner, M. - Sock, E.
Journal: Development

The highly related transcription factors Sox4 and Sox11 are expressed in the developing sympathetic nervous system. In the mouse, Sox11 appears first, whereas Sox4 is prevalent later. Using mouse mutagenesis and overexpression strategies in chicken, we studied the role of both SoxC proteins in this tissue. Neither Sox4 nor Sox11 predominantly functioned by promoting pan-neuronal or noradrenergic differentiation of sympathetic neurons as might have been expected from studies in neuronal precursors of the central nervous system. The transcriptional network that regulates the differentiation of sympathetic neurons remained intact and expression of noradrenergic markers showed only minor alterations. Instead, Sox11 was required in early sympathetic ganglia for proliferation of tyrosine hydroxylase-expressing cells, whereas Sox4 ensured the survival of these cells at later stages. In the absence of both Sox4 and Sox11, sympathetic ganglia remained hypoplastic throughout embryogenesis because of consecutive proliferation and survival defects. As a consequence, sympathetic ganglia were rudimentary in the adult and sympathetic innervation of target tissues was impaired leading to severe dysautonomia.

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The PXY-CLE41 receptor ligand pair defines a multifunctional pathway that controls the rate and orientation of vascular cell division.

Publication Date: 2010 Mar PMID: 20147378
Authors: Etchells, J. P. - Turner, S. R.
Journal: Development

Controlling the orientation of cell division is fundamental to the development of complex body plans. This is particularly apparent in plants, where development is determined by differential growth that results solely from changes in cell expansion and orientation of the cell division plane. Despite the fundamental importance of cell division orientation to plant development, the mechanisms regulating this process remain almost completely unknown. During vascular development, the meristematic cambial cells divide down their long axis in a highly orientated manner to generate clear files of cells. The receptor kinase PXY has previously be shown to be essential for this orientation. Here, we demonstrate that the division plane is determined by the interactions of PXY and its peptide ligand, CLE41. PXY is expressed within dividing meristematic cells of the procambium, whereas CLE41 localises to the adjacent phloem cells. Altering the pattern of CLE41 expression leads to a loss of cell division orientation and a dramatic loss of ordered vascular tissue development. By contrast, increasing phloem-specific expression of CLE41 results in more cell divisions, but the orientation of cell division is retained, leading to both increased and well-ordered vascular development. We demonstrate that PXY signalling is down-regulated by CLE41. This feedback mechanism is crucial in integrating the different roles of PXY signalling in controlling xylem differentiation, regulating the rate of vascular cell division and determining the orientation of cell division. Parallels with animal systems indicate that localised signalling from adjacent cells is a general mechanism for defining the plane of cell division.

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COUP-TFs regulate eye development by controlling factors essential for optic vesicle morphogenesis.

Publication Date: 2010 Mar PMID: 20147377
Authors: Tang, K. - Xie, X. - Park, J. I. - Jamrich, M. - Tsai, S. - Tsai, M. J.
Journal: Development

Transcriptional networks, which are initiated by secreted proteins, cooperate with each other to orchestrate eye development. The establishment of dorsal/ventral polarity, especially dorsal specification in the optic vesicle, is poorly understood at a molecular and cellular level. Here, we show that COUP-TFI (Nr2f1) and COUP-TFII (Nr2f2) are highly expressed in the progenitor cells in the developing murine eye. Phenotype analysis of COUP-TFI and COUP-TFII single-gene conditional knockout mouse models suggests that COUP-TFs compensate for each other to maintain morphogenesis of the eye. However, in eye-specific COUP-TFI/TFII double-knockout mice, progenitor cells at the dorso-distal optic vesicle fail to differentiate appropriately, causing the retinal pigmented epithelium cells to adopt a neural retina fate and abnormal differentiation of the dorsal optic stalk; the development of proximo-ventral identities, neural retina and ventral optic stalk is also compromised. These cellular defects in turn lead to congenital ocular colobomata and microphthalmia. Immunohistochemical and in situ hybridization assays reveal that the expression of several regulatory genes essential for early optic vesicle development, including Pax6, Otx2, Mitf, Pax2 and Vax1/2, is altered in the corresponding compartments of the mutant eye. Using ChIP assay, siRNA treatment and transient transfection in ARPE-19 cells in vitro, we demonstrate that Pax6 and Otx2 are directly regulated by COUP-TFs. Taken together, our findings reveal novel and distinct cell-intrinsic mechanisms mediated by COUP-TF genes to direct the specification and differentiation of progenitor cells, and that COUP-TFs are crucial for dorsalization of the eye.

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FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst.

Publication Date: 2010 Mar PMID: 20147376
Authors: Yamanaka, Y. - Lanner, F. - Rossant, J.
Journal: Development

Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.

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Transcriptional control of stem cell maintenance in the Drosophila intestine.

Publication Date: 2010 Mar PMID: 20147375
Authors: Bardin, A. J. - Perdigoto, C. N. - Southall, T. D. - Brand, A. H. - Schweisguth, F.
Journal: Development

Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. Here we find that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and identify genes of the Enhancer of split complex [E(spl)-C] as the major targets of this repression. In addition, we find that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. We propose that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation.

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Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos.

Publication Date: 2010 Mar PMID: 20147374
Authors: Kawaguchi, J. - Nichols, J. - Gierl, M. S. - Faial, T. - Smith, A.
Journal: Development

Neural crest is a source of diverse cell types, including the peripheral nervous system. The transcription factor Sox10 is expressed throughout early neural crest. We exploited Sox10 reporter and selection markers created by homologous recombination to investigate the generation, maintenance and expansion of neural crest progenitors. Sox10-GFP-positive cells are produced transiently from mouse embryonic stem (ES) cells by treatment with retinoic acid in combination with Fgf8b and the cytokine leukaemia inhibitory factor (Lif). We found that expression of Sox10 can be maintained using noggin, Wnt3a, Lif and endothelin (NWLE). ES cell-derived Sox10-GFP-positive cells cultured in NWLE exhibit molecular markers of neural crest progenitors. They differentiate into peripheral neurons in vitro and are able to colonise the enteric network in organotypic gut cultures. Neural crest cells purified from embryos using the Sox10 reporter also survive in NWLE, but progressively succumb to differentiation. We therefore applied selection to eliminate differentiating cells. Sox10-selected cells could be clonally expanded, cryopreserved, and multiplied for over 50 days in adherent culture. They remained neurogenic in vitro and in foetal gut grafts. Generation of neural crest from mouse ES cells opens a new route to the identification and validation of determination factors. Furthermore, the ability to propagate undifferentiated progenitors creates an opportunity for experimental dissection of the stimuli and molecular circu that govern neural crest lineage progression. Finally, the demonstration of robust enteric neurogenesis provides a system for investigating and modelling cell therapeutic approaches to neurocristopathies such as Hirschsprung's disease.

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Making a difference together: reciprocal interactions in C. elegans and zebrafish asymmetric neural development.

Publication Date: 2010 Mar PMID: 20147373
Authors: Taylor, R. W. - Hsieh, Y. W. - Gamse, J. T. - Chuang, C. F.
Journal: Development

Brain asymmetries are thought to increase neural processing capacity and to prevent interhemispheric conflict. In order to develop asymmetrically, neurons must be specified along the left-right axis, assigned left-side versus right-side identities and differentiate appropriately. In C. elegans and zebrafish, the cellular and molecular mechanisms that lead to neural asymmetries have recently come to light. Here, we consider recent insights into the mechanisms involved in asymmetrical neural development in these two species. Although the molecular details are divergent, both organisms use iterative cell-cell communication to establish left-right neuronal identity.

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Echinoid regulates Flamingo endocytosis to control ommatidial rotation in the Drosophila eye.

Publication Date: 2010 Mar PMID: 20110316
Authors: Ho, Y. H. - Lien, M. T. - Lin, C. M. - Wei, S. Y. - Chang, L. H. - Hsu, J. C.
Journal: Development

Planar cell polarity (PCP) refers to a second polarity axis orthogonal to the apicobasal axis in the plane of the epithelium. The molecular link between apicobasal polarity and PCP is largely unknown. During Drosophila eye development, differentiated photoreceptors form clusters that rotate independently of the surrounding interommatidial cells (ICs). Here, we demonstrate that both Echinoid (Ed), an adherens junction-associated cell adhesion molecule, and Flamingo (Fmi), a PCP determinant, are endocytosed via a clathrin-mediated pathway in ICs. Interestingly, we found that Ed binds the AP-2 adaptor and is required for the internalization of Fmi into ICs. Loss of ed led to increased amounts of Fmi on the cell membrane of non-rotating ICs and also to the misrotation of photoreceptor clusters. Importantly, overexpression of fmi in ICs alone was sufficient to cause misrotation of the adjacent photoreceptor clusters. Together, we propose that Ed, when internalized by AP-2, undergoes co-endocytosis with, and thereby decreases, Fmi levels on non-rotating ICs to permit correct rotation of ommatidial clusters. Thus, co-endocytosis of Ed and Fmi provides a link between apicobasal polarity and PCP.

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Differential requirement of Salvador-Warts-Hippo pathway members for organ size control in Drosophila melanogaster.

Publication Date: 2010 Mar PMID: 20110315
Authors: Milton, C. C. - Zhang, X. - Albanese, N. O. - Harvey, K. F.
Journal: Development

The Salvador-Warts-Hippo (SWH) pathway contains multiple growth-inhibitory proteins that control organ size during development by limiting activity of the Yorkie oncoprotein. Increasing evidence indicates that these growth inhibitors act in a complex network upstream of Yorkie. This complexity is emphasised by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue lacking the core SWH pathway components salvador, warts or hippo is highly overgrown and resistant to developmental apoptosis, whereas tissue lacking fat or expanded is not. Here we explore the relative contribution of SWH pathway proteins to organ size control by determining their temporal activity profile throughout Drosophila melanogaster eye development. We show that eye tissue lacking fat, expanded or discs overgrown displays elevated Yorkie activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. Fat and Expanded do possess Yorkie-repressive activity in the pupal eye, but loss of fat or expanded at this stage of development can be compensated for by Merlin. Fat appears to repress Yorkie independently of Dachs in the pupal eye, which would contrast with the mode of action of Fat during larval development. Fat is more likely to restrict Yorkie activity in the pupal eye together with Expanded, given that pupal eye tissue lacking both these genes resembles that of tissue lacking either gene. This study highlights the complexity employed by different SWH pathway proteins to control organ size at different stages of development.

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SIX1 acts synergistically with TBX18 in mediating ureteral smooth muscle formation.

Publication Date: 2010 Mar PMID: 20110314
Authors: Nie, X. - Sun, J. - Gordon, R. E. - Cai, C. L. - Xu, P. X.
Journal: Development

Dysfunction of the ureter often leads to urine flow impairment from the kidney to the bladder, causing dilation of the ureter and/or renal pelvis. Six1 is a crucial regulator of renal development: mutations in human SIX1 cause branchio-oto-renal (BOR) syndrome and Six1(-/-) mice exhibit renal agenesis, although the ureter is present. It remains unclear whether Six1 plays a role in regulating ureter morphogenesis. We demonstrate here that Six1 is differentially expressed during ureter morphogenesis. It was expressed in undifferentiated smooth muscle (SM) progenitors, but was downregulated in differentiating SM cells (SMCs) and had disappeared by E18.5. In Six1(-/-) mice, the ureteral mesenchymal precursors failed to condense and differentiate into normal SMCs and showed increased cell death, indicating that Six1 is required for the maintenance and normal differentiation of SM progenitors. A delay in SMC differentiation was observed in Six1(-/-) ureters. A lack of Six1 in the ureter led to hydroureter and hydronephrosis without anatomical obstruction when kidney formation was rescued in Six1(-/-) embryos by specifically expressing Six1 in the metanephric mesenchyme, but not the ureter, under control of the Eya1 promoter. We show that Six1 and Tbx18 genetically interact to synergistically regulate SMC development and ureter function and that their gene products form a complex in cultured cells and in the developing ureter. Two missense mutations in SIX1 from BOR patients reduced or abolished SIX1-TBX18 complex formation. These findings uncover an essential role for Six1 in establishing a functionally normal ureter and provide new insights into the molecular basis of urinary tract malformations in BOR patients.

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Slowdown promotes muscle integrity by modulating integrin-mediated adhesion at the myotendinous junction.

Publication Date: 2010 Mar PMID: 20110313
Authors: Gilsohn, E. - Volk, T.
Journal: Development

The correct assembly of the myotendinous junction (MTJ) is crucial for proper muscle function. In Drosophila, this junction comprises hemi-adherens junctions that are formed upon arrival of muscles at their corresponding tendon cells. The MTJ mainly comprises muscle-specific alphaPS2betaPS integrin receptors and their tendon-derived extracellular matrix ligand Thrombospondin (Tsp). We report the identification and functional analysis of a novel tendon-derived secreted protein named Slowdown (Slow). Homozygous slow mutant larvae exhibit muscle or tendon rupture, sluggish larval movement, partial lethality, and the surviving adult flies are unable to fly. These defects result from improper assembly of the embryonic MTJ. In slow mutants, Tsp prematurely accumulates at muscle ends, the morphology of the muscle leading edge changes and the MTJ architecture is aberrant. Slow was found to form a protein complex with Tsp. This complex is biologically active and capable of altering the morphology and directionality of muscle ends. Our analysis implicates Slow as an essential component of the MTJ, crucial for ensuring muscle and tendon integrity during larval locomotion.

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