Expression of Fluorescent Proteins in <em>Branchiostoma lanceolatum</em> by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Institut Pasteur

CNRS, UPMC, UMR 7622, INSERM ERL U1156

In zebrafish, skeletal muscle precursors can adopt at least three distinct fates: fast, non-pioneer slow, or pioneer slow muscle fibers. Slow muscle fibers develop from adaxial cells and depend on Hedgehog signaling. We analyzed when precursors become committed to their fates and the step(s) along their differentiation pathway affected by Hedgehog. Unexpectedly, we find that embryos deficient in Hedgehog signaling still contain postmitotic adaxial cells that differentiate into fast muscle fibers instead of slow. We show that by the onset of gastrulation, slow and fast muscle precursors are already spatially segregated but uncommitted to their fates until much later, in the segmental plate when slow precursors become independent of Hedgehog. In contrast, pioneer and non-pioneer slow muscle precursors share a common lineage from the onset of gastrulation. Our results demonstrate that slow muscle precursors form independently of Hedgehog signaling and further provide direct evidence for a multipotent muscle precursor population whose commitment to the slow fate depends on Hedgehog at a late stage of development when postmitotic adaxial cells differentiate into slow muscle fibers.

Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors.

&#x82E5;&#x8981;&#x6807;&#x8BC6;&#x6240;&#x9700;&#x7684;&#x8111;&#x53D1;&#x80B2;&#x548C; somites &#x7684;&#x57FA;&#x56E0;&#xFF0C;&#x6211;&#x4EEC;&#x6267;&#x884C;&#x8BD5;&#x70B9;&#x5C4F;&#x5E55;&#x7684;&#x96CC;&#x6838;&#x53D1;&#x80B2;&#x5355;&#x500D;&#x4F53;&#x7684;&#x6591;&#x9A6C;&#x9C7C;&#x80DA;&#x80CE;&#x4EA7;&#x751F;&#x75C5;&#x6BD2;&#x63D2;&#x5165;&#x8BF1;&#x53D8;&#x7C73;&#x7684;&#x6BCD;&#x4EB2;&#x3002;&#x6211;&#x4EEC;&#x63CF;&#x8FF0;&#x4E86;&#x4E00;&#x79CD;&#x6709;&#x6548;&#x7684;&#x65B9;&#x6CD5;&#xFF0C;&#x627E;&#x51FA;&#x65B0;&#x7684;&#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x548C;&#x53D7;&#x5F71;&#x54CD;&#x7684;&#x57FA;&#x56E0;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x4E00;&#x4E2A;&#x5C0F;&#x89C4;&#x6A21;&#x7684;&#x5C4F;&#x5E55;&#xFF0C;&#x786E;&#x5B9A;&#x6240;&#x9700;&#x7684;&#x5927;&#x8111;&#x53D1;&#x80B2;&#xFF0C;&#x5305;&#x62EC;&#x65B0;&#x7B49;&#x4F4D;&#x57FA;&#x56E0; nagie &#x5F00;&#x5143;&#x3001; pou5f1&#x3001; &#x6838;&#x7CD6;&#x4F53;&#x86CB;&#x767D; L36&#xFF0C;&#x548C; n-&#x9499;&#x7C98;&#x86CB;&#x767D;&#x3001; &#x5C42;&#x7C98;&#x8FDE;&#x86CB;&#x767D; g1 &#x57FA;&#x56E0;&#x6240;&#x9700;&#x7684;&#x6B63;&#x5E38;&#x9AA8;&#x9ABC;&#x808C;&#x8089;&#x7EA4;&#x7EF4;&#x7EC4;&#x7EC7;&#x548C;&#x4F53;&#x8282;&#x7F16;&#x7EC4;&#x7684;&#x65B0;&#x7B49;&#x4F4D;&#x57FA;&#x56E0;&#x7684;&#x4E94;&#x4E2A;&#x57FA;&#x56E0;&#x7684;&#x7ED3;&#x679C;&#x3002;

&#x6591;&#x9A6C;&#x9C7C;&#x7684;&#x8054;&#x5408;&#x7684;&#x5355;&#x500D;&#x4F53;&#x548C;&#x63D2;&#x5165;&#x7A81;&#x53D8;&#x5C4F;&#x5E55;&#x3002;

To identify genes required for development of the brain and somites, we performed a pilot screen of gynogenetic haploid zebrafish embryos produced from mothers mutagenized by viral insertion. We describe an efficient method to identify new mutations and the affected gene. In addition, we report the results of a small-scale screen that identified five genes required for brain development, including novel alleles of nagie oko, pou5f1, ribosomal protein L36, and n-cadherin, as well as a novel allele of the laminin g1 gene that is required for normal skeletal muscle fiber organization and somite patterning.

Combined haploid and insertional mutation screen in the zebrafish.

Pour identifier les g&#xE8;nes n&#xE9;cessaires au d&#xE9;veloppement du cerveau et les somites, nous avons effectu&#xE9; un &#xE9;cran pilote d&#x26;#39;embryons de poisson z&#xE8;bre gynog&#xE9;n&#xE9;tiques haplo&#xEF;des produites &#xE0; partir de m&#xE8;res mutagen&#xE8;se par insertion virale. Nous d&#xE9;crivons une m&#xE9;thode efficace pour identifier de nouvelles mutations et le g&#xE8;ne affect&#xE9;. En outre, nous pr&#xE9;sentons les r&#xE9;sultats d&#x26;#39;un &#xE9;cran de petite &#xE9;chelle qui a identifi&#xE9; cinq g&#xE8;nes n&#xE9;cessaires au d&#xE9;veloppement du cerveau, y compris de nouveaux all&#xE8;les de nagie oko, pou5f1, prot&#xE9;ine ribosomique L36, et le n-cadh&#xE9;rine, ainsi que d&#x26;#39;un nouvel all&#xE8;le de la laminine g1 g&#xE8;ne qui est n&#xE9;cessaire pour l&#x26;#39;organisation normale des fibres musculaires squelettiques et les motifs somite.

Combin&#xE9; &#xE9;cran Mutation haplo&#xEF;des et insertionnelle dans le poisson-z&#xE8;bre

Kombiniert haploiden und Insertionsmutation Bildschirm im Zebrafisch.

Per identificare i geni necessari per lo sviluppo del cervello e somiti, abbiamo eseguito una schermata pilota di embrioni di zebrafish aploide gynogenetic prodotte dalle madri mutagenized di inserimento virale. Descriviamo un metodo efficiente per identificare nuove mutazioni e il gene alterato. Inoltre, si riportano i risultati di uno schermo su piccola scala che ha identificato cinque geni necessari per lo sviluppo del cervello, tra cui nuovi alleli nagie oko, pou5f1, proteina ribosomiale L36, e n-caderina, cos&igrave; come un romanzo allele del gene g1 laminina che &egrave; necessario per l&#39;organizzazione della fibra muscolare scheletrica normale e patterning somite.

Schermo combinato aploidi e inserzionale mutazione in the zebrafish.

&#x8133;&#x3068;&#x4F53;&#x7BC0;&#x306E;&#x958B;&#x767A;&#x306B;&#x5FC5;&#x8981;&#x306A;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x540C;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x633F;&#x5165;&#x306B;&#x3088;&#x3063;&#x3066;&#x5909;&#x7570;&#x6BCD;&#x89AA;&#x304B;&#x3089;&#x751F;&#x6210;&#x96CC;&#x6027;&#x767A;&#x751F;&#x534A;&#x6570;&#x4F53;&#x306E;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x80DA;&#x306E;&#x30D1;&#x30A4;&#x30ED;&#x30C3;&#x30C8;&#x306E;&#x753B;&#x9762;&#x3092;&#x884C;&#x3063;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x65B0;&#x3057;&#x3044;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3001;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x540C;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x52B9;&#x7387;&#x7684;&#x306A;&#x65B9;&#x6CD5;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x52A0;&#x3048;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;nagie OKO&#x3001;pou5f1&#x3001;&#x30EA;&#x30DC;&#x30BD;&#x30FC;&#x30E0;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;L36&#x3001;&#x304A;&#x3088;&#x3073;N-&#x30AB;&#x30C9;&#x30D8;&#x30EA;&#x30F3;&#x306E;&#x65B0;&#x898F;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50;&#x3068;&#x540C;&#x69D8;&#x306B;&#x3001;&#x30E9;&#x30DF;&#x30CB;&#x30F3;G1&#x306E;&#x65B0;&#x898F;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x542B;&#x3080;&#x3001;&#x8133;&#x306E;&#x767A;&#x9054;&#x306B;&#x5FC5;&#x8981;&#x306A;5&#x3064;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x540C;&#x5B9A;&#x3057;&#x305F;&#x5C0F;&#x898F;&#x6A21;&#x306E;&#x753B;&#x9762;&#x306E;&#x7D50;&#x679C;&#x3092;&#x5831;&#x544A;&#x3059;&#x308B;&#x901A;&#x5E38;&#x306E;&#x9AA8;&#x683C;&#x7B4B;&#x7DDA;&#x7DAD;&#x306E;&#x7D44;&#x7E54;&#x3068;&#x4F53;&#x7BC0;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x306B;&#x5FC5;&#x8981;&#x3068;&#x3055;&#x308C;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x3002;

&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x3067;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x305F;&#x534A;&#x6570;&#x4F53;&#x304A;&#x3088;&#x3073;&#x633F;&#x5165;&#x5909;&#x7570;&#x753B;&#x9762;

&#xB450;&#xB1CC;&#xC640; somites&#xC758; &#xAC1C;&#xBC1C;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xC720;&#xC804;&#xC790;&#xB97C; &#xC2DD;&#xBCC4; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC6B0;&#xB9AC; &#xC5B4;&#xBA38;&#xB2C8; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC0BD;&#xC785; &#xD558; &#xC5EC; mutagenized&#xC5D0;&#xC11C; &#xC0DD;&#xC0B0; &#xD558;&#xB294; gynogenetic haploid zebrafish &#xD0DC;&#xC544;&#xC758; &#xD30C;&#xC77C;&#xB7FF; &#xD654;&#xBA74; &#xC218;&#xD589;. &#xC6B0;&#xB9AC;&#xB294; &#xC0C8;&#xB85C;&#xC6B4; &#xBCC0;&#xC774; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xB294; &#xC720;&#xC804;&#xC790;&#xB97C; &#xC2DD;&#xBCC4; &#xD558;&#xB294; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xBC29;&#xBC95; &#xC124;&#xBA85;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; nagie oko, pou5f1, ribosomal &#xB2E8;&#xBC31;&#xC9C8; L36, &#xBC0F; n cadherin &#xC18C;&#xC124; &#xB300;&#xB9BD; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xACE8;&#xACA9; &#xADFC;&#xC721; &#xC12C;&#xC720; &#xC870;&#xC9C1; &#xBC0F; somite &#xD328;&#xD134;&#xD654; laminin g 1 &#xC720;&#xC804;&#xC790;&#xC758; &#xC0C8;&#xB85C;&#xC6B4; &#xB300;&#xB9BD; &#xC720;&#xC804;&#xC790;&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xB450;&#xB1CC; &#xBC1C;&#xB2EC;&#xC5D0; &#xD544;&#xC694;&#xD55C; 5 &#xAC1C;&#xC758; &#xC720;&#xC804;&#xC790;&#xB97C; &#xD655;&#xC778; &#xD558;&#xB294; &#xC18C;&#xADDC;&#xBAA8; &#xD654;&#xBA74; &#xACB0;&#xACFC; &#xBCF4;&#xACE0;.

&#xACB0;&#xD569; &#xB41C; zebrafish haploid insertional &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xD654;&#xBA74;.

Para identificar los genes necesarios para el desarrollo del cerebro y somitas, se realiz&#xF3; un piloto de la pantalla de los embriones de pez cebra ginogen&#xE9;ticos haploides producidos a partir de las madres mutagenizadas mediante la inserci&#xF3;n viral. Se describe un m&#xE9;todo eficaz para identificar nuevas mutaciones y el gen afectado. Adem&#xE1;s, se reportan los resultados de una pantalla peque&#xF1;a escala que identifica cinco genes necesarios para el desarrollo del cerebro, incluyendo los nuevos alelos de nagie oko, pou5f1, la prote&#xED;na ribosomal L36, y N-cadherina, as&#xED; como un alelo de la novela de g1 laminina gen que se requiere para el normal organizaci&#xF3;n del esqueleto de la fibra muscular y el patr&#xF3;n somito.

Combinado mutaci&#xF3;n haploide y en la inserci&#xF3;n de la pantalla en el pez cebra

The cephalochordate amphioxus (Branchiostoma sp.) is an important animal model for studying the evolution of chordate developmental mechanisms. Obtaining amphioxus embryos is a key step for these studies. It has been shown that an increase of 3-4 degrees C in water temperature triggers spawning of the European amphioxus (Branchiostoma lanceolatum) in captivity, however, very little is known about the natural spawning behavior of this species in the field. In this work, we have followed the spawning behavior of the European amphioxus during two spawning seasons (2004 and 2005), both in the field and in captivity. We show that animals in the field spawn approximately from mid-May through early July, but depending on the year, they show different patterns of spawning. Thus, even if temperature has a critical role in the induction of the spawning in captivity, it is not the major factor in the field. Moreover, we report some improvements on the methodology for inducing spawning in captivity (e.g. in maintenance, light cycle control and induction of spawning in a laboratory without running sea water system). These studies have important implications for amphioxus animal husbandry and for improving laboratory techniques to develop amphioxus as an experimental animal model.

Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum).

In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues, such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analysed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmp-responding cells are excluded from the central domain and that this pattern is modulated by laminins, but not by Hedgehog signalling. Knockdown of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos. Last, extracellular matrix-associated heparan sulfate proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that laminins, via heparan sulfate proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.

Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness.

Owing to its phylogenetic position at the base of the chordates, the cephalochordate amphioxus is an emerging model system carrying immense significance for understanding the evolution of vertebrate development. One important shortcoming of amphioxus as a model organism has been the unavailability of animal husbandry protocols to maintain amphioxus adults away from the field. Here, we present the first report of successful maintenance and spawning of Branchiostoma lanceolatum adults in a facility run on artificial seawater. B. lanceolatum has been chosen for this study because it is the only amphioxus species that can be induced to spawn. We provide a step-by-step guide for the assembly of such a facility and discuss the day-to-day operations required for successful animal husbandry of B. lanceolatum adults. This work also includes a detailed description of the B. lanceolatum spawning behavior in captivity. Our analysis shows that the induced spawning efficiency is not sex biased, but increases as the natural spawning season progresses. We find that a minor fraction of the animals undergo phases of spontaneous spawning in the tanks and that this behavior is not affected by the treatment used to induce spawning. Moreover, the induced spawning efficiency is not discernibly correlated with spontaneous spawning in the facility. Last, we describe a protocol for long-term cryopreservation of B. lanceolatum sperm. Taken together, this work represents an important step toward further establishing amphioxus as a laboratory animal making it more amenable to experimental research, and hence assists the coming of age of this emerging model.

Amphioxus spawning behavior in an artificial seawater facility.

Posterior body elongation is a widespread mechanism propelling the generation of the metazoan body plan. The posterior growth model predicts that a posterior growth zone generates sufficient tissue volume to elongate the posterior body. However, there are energy supply-related differences between vertebrates in the degree to which growth occurs concomitantly with embryogenesis. By applying a multi-scalar morphometric analysis in zebrafish embryos, we show that posterior body elongation is generated by an influx of cells from lateral regions, by convergence-extension of cells as they exit the tailbud, and finally by a late volumetric growth in the spinal cord and notochord. Importantly, the unsegmented region does not generate additional tissue volume. Fibroblast growth factor inhibition blocks tissue convergence rather than volumetric growth, showing that a conserved molecular mechanism can control convergent morphogenesis through different cell behaviours. Finally, via a comparative morphometric analysis in lamprey, dogfish, zebrafish and mouse, we propose that elongation via posterior volumetric growth is linked to increased energy supply and is associated with an overall increase in volumetric growth and elongation.

Estelle Hirsinger

Département de Biologie du Développement et Cellules Souches,
Department of Developmental and Stem Cell Biology

Institut Pasteur

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Estelle Hirsinger

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Département de Biologie du Développement et Cellules Souches,Department of Developmental and Stem Cell Biology

Institut Pasteur

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Département de Biologie du Développement et Cellules Souches,
Department of Developmental and Stem Cell Biology