Name: targeted laser ablation in the embryo of saccharina latissima
Uploaded: 2022-03-11T15:00:00
Description: Watch this Scientific Journal Video about Targeted Laser Ablation in the Embryo of Saccharina latissima at JoVE.com

SB'nin doktora hibesi, Bretagne Bölgesi (ARED hibe numarası COH20020) ve Sorbonne Université tarafından finanse edilmektedir. I.T.is doktora hibesi, Bretagne Bölgesi (ARED hibe numarası COH18020) ve Norvegian NMBU Üniversitesi tarafından finanse edilmektedir. Bu proje, MITI disiplinlerarası programları aracılığıyla CNRS'den finansal destek almıştır. MRic, Fransız Ulusal Araştırma Ajansı (ANR-10-INBS-04) tarafından desteklenen ulusal altyapı France-BioImaging üyesidir.

1. Saccharina latissima gametophytes üretimi
<ol><li>S. latissima'nın olgun sporofitlerini daha önce tarif edildiği gibi vahşi doğadan toplayın20,21. Seçilen sporofitlerin epifitlerden (bıçağın yüzeyinde görülebilen küçük organizmalar) veya iç parazitlerden (ağartılmış alanlarda veya bıçaktaki lekelerde bulunur) bulunmadığından emin olun. 
</li>
	<li>Bir neşter kullanarak, bıçağın orta...

<ol>
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G., Charrier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+zygotes+and+embryos+of+the+kelp+saccharina+latissima+for+cell+biology+approaches.">Preparation of zygotes and embryos of the kelp saccharina latissima for cell biology approaches.</a> Bio-protocol. 101, 4132 (2021).</li><li>L&#252;ning, K., Dring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction+induced+by+blue+light+in+female+gametophytes+of+Laminaria+saccharina.">Reproduction induced by blue light in female gametophytes of Laminaria saccharina.</a> Planta. 104 (3), 252-256 (1972).</li><li>de Medeiros, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+manipulation+with+ultrashort+infrared+laser+pulses+in+light-sheet+microscopy.">Cell and tissue manipulation with ultrashort infrared laser pulses in light-sheet microscopy.</a> Scientific Reports. 10 (1), 1942 (2020).</li><li>Liang, X., Michael, M., Gomez, G. 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Lokal hücresel lazer ablasyon, yüksek hassasiyetle zamansal ve mekansal ablasyona izin verir. Bununla birlikte, etkinliği hedef hücrelerin erişilememesi nedeniyle engellenebilir; örneğin, tüm hücreler üç boyutlu bir embriyoya aittir. Bu protokol, tüm hücrelerin bir lazer ışını ile ayrı ayrı kolayca ayırt edilebileceği ve yok edilebileceği tek katmanlı bir lamina geliştiren alg Saccharina latissima'nın embriyosu üzerinde geliştirilmiştir.
Lazer güc?...

Lazer ablasyon, embriyo gelişimini incelemek için onlarca yıldır kullanılmaktadır. Embriyo hücrelerinin bir lazer ışını ile ışınlanması, embriyogenez sırasında rejeneratif potansiyeli ve hücre soyunun modifikasyonunu izlemeyi ve hedeflenen ablasyonun hücre bölünmesi ve hücre kaderi üzerindeki etkisini araştırmayı mümkün kılar. Lazer ablasyon yöntemlerinde kullanılan model organizmalar tipik olarak böcekler1,2, nematodlar3,4, omurgalılar5,6 ve bazen debitkiler 7,8 gibi hayvanlardır. Ek olarak, 1994 ve 1998 yıllarında kahverengi alg Fucus üzerinde bir lazer mikro-ablasyon yaklaşımı, hücre duvarının erken embriyonun fotopolarizasyonundaki rolünü göstermek için kullanılmıştır 9,10.
Kahverengi algler, 1.6 milyar yıl önce ökaryotik ağacın kökünde ayrılan Stramenopiles grubuna aittir. Sonuç olarak, hayvanlar ve bitkiler gibi diğer çok hücreli organizmalardan filogenetik olarak bağımsızdırlar11. Saccharina latissima, daha yaygın olarak yosun olarak bilinen Laminariales takımına aittir ve dünyadaki en büyük organizmalar arasındadır, 30 m'nin üzerindeki boyutlara ulaşırlar. Saccharina sp., gıda ve yem gibi birçok uygulama için kullanılan büyük bir deniz yosunudur ve polisakkaritleri dünya çapında tarım, farmakolojik ve kozmetik endüstrilerinde kullanılmak üzere ekstrakte edilir12, 13. Özellikle Asya'da ve daha yakın zamanda Avrupa'da yetiştirilmesi, yavruları açık denizde serbest bırakmadan önce kuluçkahanelerde embriyoların hazırlanmasını gerektirir. Tüm kelpler gibi, bir haploid gametofitin büyüdüğü ve döllenme için gametler ürettiği mikroskobik bir gametofitik fazdan oluşan bifazik bir yaşam döngüsüne ve büyük bir düzlemsel bıçağın deniz tabanına veya kayalara tutturulmuş tutucusundan geliştiği diploid makroskopik bir sporofitik faza sahiptir. Sporofit, olgunlukta haploid sporları serbest bırakır, böylece yaşam döngüsü14,15,16'yı tamamlar.
S. latissima bazı ilginç morfolojik özellikler sunar17. Embriyosu, farklı doku tiplerinin ortaya çıkmasıyla çakışan çok katmanlı bir yapı elde etmeden önce tek katmanlı bir düzlemsel tabaka15,18,19 olarak gelişir. Ek olarak, Laminariales, embriyoları maternal gametofitik dokularına bağlı kalan kahverengi alglerin tek taksonlarından biridir (Desmarestiales ve Sporochnales de15). Bu özellik, maternal dokunun bu gelişimsel süreçteki rolünü inceleme ve kahverengi alglerdeki maternal kontrol mekanizmalarını hayvanlardaki ve bitkilerdeki mekanizmalarla karşılaştırma fırsatı sunar.
Bu makalede, erken bir kelp embriyosunda lazer ablasyon için ilk tam protokol sunulmaktadır. UV ns-pulsed tekniğini içeren bu protokol, embriyogenez sırasında kendi rollerini incelemek için bireysel embriyo hücrelerinin spesifik yıkımına neden olur. Prosedür, Laminariales'te embriyogenez sırasında hücre etkileşimlerini ve hücre kaderini araştırmak için güvenilir bir yaklaşım sunar.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25 mm glass bottom petri dish</td>
			<td>NEST</td>
			<td>801001</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved sea water</td>
			<td>-</td>
			<td></td>
			<td>Collected offshore near the Astan buoy (48&#176;44.934 N 003&#176;57.702 W) close to Roscoff, France, at a depth of 20 m.</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>MED 2</td>
			<td>83.3951</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#181;m</td>
			<td>Corning / Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture cabinets</td>
			<td>Snijders Scientific Plant Growth Cabinet ECD01</td>
			<td></td>
			<td>Any other brand is suitable provided that the light intensity, the photoperiod and the temperature can be controlled.</td>
		</tr>
		<tr>
			<td>LSM 880 Zeiss confocal microscope</td>
			<td>Carl Zeiss microscopy, Jena, Germany</td>
			<td></td>
			<td>Ablation and imaging were performed using a 40x/1.2 water objective</td>
		</tr>
		<tr>
			<td>Pellet pestles</td>
			<td>Sigma Aldrich</td>
			<td>Z359947</td>
			<td>Blue polypropylene (autoclavable)</td>
		</tr>
		<tr>
			<td>Provasoli supplement</td>
			<td>-</td>
			<td></td>
			<td>Recipe is available here: http://www.sb-roscoff.fr/sites/www.sb-roscoff.fr/files/documents/station-biologique-roscoff-preparation-du-provasoli-2040.pdf</td>
		</tr>
		<tr>
			<td>Pulsed 355 laser (UGA-42 Caliburn 355/25)</td>
			<td>Rapp OptoElectronic, Wedel, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Paramount</td>
			<td>PDSS 11</td>
			<td></td>
		</tr>
		<tr>
			<td>SysCon software</td>
			<td>Rapp OptoElectronic, Wedel, Germany</td>
			<td></td>
			<td>Laser-driver software</td>
		</tr>
		<tr>
			<td>ZEN software</td>
			<td>Carl Zeiss microscopy, Jena, Germany</td>
			<td></td>
			<td>Imaging software, used together with the SysCon software; Black 2.3 version</td>
		</tr>
	</tbody>
</table>

targeted laser ablation in the embryo of saccharina latissima

Saccharina latissima'da embriyo, lamina veya bıçak adı verilen tek katmanlı bir hücre tabakası olarak gelişir. Her embriyo hücresinin gözlemlenmesi kolaydır, komşularından kolayca ayırt edilebilir ve ayrı ayrı hedeflenebilir. Onlarca yıldır, lazer ablasyon embriyo gelişimini incelemek için kullanılmıştır. Burada, kahverengi alg S. latissima'nın erken embriyoları için hücreye özgü lazer ablasyon için bir protokol geliştirilmiştir. Sunulan çalışma şunları içerir: (1) kültür koşulları da dahil olmak üzere kritik parametrelerin bir açıklaması ile Saccharina embriyolarının hazırlanması, (2) lazer ablasyon ayarları ve (3) ışınlanmış embriyonun sonraki büyümesinin hızlandırılmış mikroskopi kullanılarak izlenmesi. Ek olarak, embriyoların görüntüleme platformundan laboratuvara geri taşınması için en uygun koşullar hakkında ayrıntılar sağlanır ve bu da sonraki embriyo gelişimini derinden etkileyebilir. Laminariales takımına ait algler, Saccharina'ya benzer embriyogenez kalıpları gösterir; Bu protokol, bu taksondaki diğer türlere kolayca aktarılabilir.

S. latissima'nın gametofitleri yetiştirildi ve gametogenez, zigot ve embriyo üretmek için indüklendi. Gametogenezin indüksiyonundan on iki gün sonra, embriyolara lazer ablasyon uygulandı. Burada deney, S. latissima embriyolarının genel gelişiminde spesifik hücrelerin rolünü değerlendirmeyi amaçladı. En apikal hücre, en bazal hücre ve medyan hücreler hedef alındı. Karo taramasından sonra, ilgilenilen bir embriyo olan Petri kabının tamamı (Şekil 2A)...

Watch this Scientific Journal Video about Targeted Laser Ablation in the Embryo of Saccharina latissima at JoVE.com

Targeted Laser Ablation in the Embryo of Saccharina latissima

Embriyodaki spesifik hücrelerin yok edilmesi, hücre kaderinde yer alan hücresel etkileşimleri incelemek için güçlü bir araçtır. Mevcut protokol, kahverengi alg Saccharina latissima'nın erken embriyosundaki hedeflenen hücrelerin lazer ablasyonu için teknikleri açıklamaktadır.

Saccharina latissima Embriyosunda Hedefe Yönelik Lazer Ablasyonu

In Saccharina latissima, the embryo&#160;develops as a monolayered cell sheet called the lamina or the blade. Each embryo cell is easy to observe, readily ...

Bu protokol erken kelp embriyosundaki ilk tam lazer ablasyon protokolüdür. Prosedür, kahverengi alglerde embriyogenez sırasında hücre kaderini ve etkileşimini araştırmak için güvenilir bir yaklaşım sunmaktadır. Lokal hücresel lazer ablasyon, hücreler üzerinde yüksek hassasiyet ve doku seviyesi ile temporal ve özel ablasyona izin verir.Bu yaklaşım, kahverengi alglerin erken embriyosundaki hücre kaderlerini ve etkileşimini incelemek için umut verici bir yöntemdir. Birkaç değişiklikle diğer alg sistemlerine uygulanabilir. Ablasyon adımı sırasında seçilecek embriyoları bulmak ve seçilen embriyoların sonraki gelişimini izlemek için tüm Petri kabını görüntüleyerek başlayın.Kutucuk taramasını başlatın. Petri kabının dört kardinal noktasının konumunu kaydedin. Metin yazısında tanımlanan parametreleri girin ve tüm Petri kabının iletilen veya floresan görüntülerini düşük çözünürlükte elde edin.Döşeme tarama görüntüsünü kaydedin ve görüntü alma yazılımı penceresinde açık tutun. Petri kabını çıkarmadan hedefi değiştirin. Taramada ilgilendiğiniz embriyoları bulun.Lazeri kalibre etmek için, lazer sürücüsü ve görüntü alma yazılım paketini açın. Ardından lazer yolunu başlatın. Lazer sürücüsü yazılım paketinde edinimi başlat'a tıklayarak her iki yazılım paketini de senkronize edin.Lazer ablasyon parametrelerini ayarlayın ve canlı üzerine tıklayın. Çanak üzerinde boş bir alan seçin ve cam tabana odaklanmak için sahne seviyesini Petri kabının üst yüzeyinin 20 mikrometre altına indirin. AOI seç düğmesine tıklayarak ve UV lazer sürücüsü yazılım paketindeki görüntünün kenarlarına tıklayarak ilgi çekici bir alan bulmak için.Kalibrasyonu başlat'a tıklayın ve ablasyon lazeri ve görüntüleme lazer yörüngelerini ayarlamak için manuel kalibrasyonu seçin. Tüm kepenklerin açık olduğundan emin olun. Canlı görüntünün ortasında, cam kapak kaymasındaki deliğe karşılık gelen siyah bir nokta görecek kadar yüksek bir lazer gücü seçin.Fare imleci ile merkezi siyah noktaya tıklayın ve hizalama prosedürünü tamamlamak için yazılım tarafından önerilen 18 ek noktayı seçin. Tıklama ve ateş moduna tıklayın. Aynı kapak kapağındaki kalibrasyonu kontrol edin ve lazer etkisinin görülebildiği cam tabakaya tıklayın.Fayans taramasında ilgilendiğiniz bir embriyoyu seçin ve üzerine tıklayarak sahneyi hareket ettirin. Zaman serisini başlatın. Zaman atlamalı parametreleri canlı olarak test edin ve gerekirse ayarlayın.Görüntü alma yazılımında maksimum hızda hızlandırılmış bir kayıt başlatın. İlgi alanına odaklanmak ve tüm embriyoyu görselleştirmek için yakınlaştırmayı ayarlayın. Ablasyonun kaydedilmesi sırasında embriyoyu takip etmek için, düğmeye tıklayın, en son görüntüyü elde edin.Parametreleri, maksimum 40 mikrowatt ve bir milisaniyelik darbe süresi süresine karşılık gelen% 45 lazer iletimine ayarlayın. Lazer sürücü yazılımı tıklama ve ateş fonksiyonunu kullanarak ilgili embriyo hücrelerine zararlı ışınlamayı uygulayın. 688 nanometrenin altında, sitoplazmadan otofloresan kloroplastların atılmasını izleyin.Hücre içeriği hücrede kalırsa, hücredeki ihlalin boyutunu artırmak için tıklatma ve tetikleme işlevini bir kez daha kullanın. Hücre içeriğinin çoğu serbest bırakılana kadar bu işlemi tekrarlayın ve çekim sayısını minimumda tutun. Embriyo stabilize olduktan sonra hızlandırılmış kaydı durdurun ve daha fazla hücreler arası hareket tespit edilemez.Kutucuk tarama görüntüsündeki ek açıklamayı güncelleştirin, varolan kutucuk taramasının üzerine yazın ve görüntüyü kaydedin. Lazer ablasyondan sonra gelişen embriyo sayısını izleyerek hayatta kalma oranını belirleyin ve bunları ölenlerle karşılaştırın. Her gün lazer atışı embriyolarının uzunluğunu ölçerek ve sağlam embriyolarla karşılaştırarak büyüme gecikmesini belirleyin.Ablatlanmış hücrelere bitişik hücrelerin reaksiyonunu izleyerek bitişik hasarı bulun. İlgilenilen hedeflenen hücreler en apikal hücre, en bazal hücre ve medyan hücrelerdi. Tüm Petri kabının karo taramasından sonra, ilgilenilen bir embriyo lazer çekimi için uygun bir aday olarak tanımlandı.Hücre,% 45 güçte bir darbeli UV lazer ışını ile vurulduğunda içeriğini serbest bırakırken, ışınlanmış hücrelere bitişik bir hücrenin hücreler arası boşluğa genişlemesi. Işınlanan embriyoların pozisyonu 10 gün boyunca her 24 saatte bir kaydedildi. Işınlanmış embriyoların çoğu gelişmeye devam etti, ancak büyüme değişikliği gösterdi.Buna karşılık, diğer lazer parametreleriyle test edilen embriyolar, hücre ağartması, solma veya şekil değişiklikleri gibi ciddi stres belirtilerini hızla gösterdi. Bu şekilde vurulan embriyoların neredeyse tamamı deneyden sonraki beş gün içinde öldü. Görüntü formatı veya yakınlaştırma gibi bazı parametreler kalibrasyondan sonra değiştirilmemelidir.Lazer ablasyon sırasında ve sonrasında izleme, neler olduğuna dair fikir sahibi olmak için gereklidir. Lazer ablasyon, kahverengi alglerin gelişimsel çalışmasında yeni keşiflere ve farklı metabolitlerin veya hücrelerinin bileşenlerinin etkileşimlerinin daha derin bir şekilde anlaşılmasına yol açmaktadır.

targeted-laser-ablation-in-the-embryo-of-saccharina-latissima

Research

JoVE Journal

Developmental Biology

Targeted Laser Ablation in the Embryo of Saccharina latissima

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Zebra balığı Embriyo microbead İmplantasyonu

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Gelişim Biyolojisi

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Yaşayan Zebra balığı Embriyo Hücre alt yapıları görüntüleme

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

Kordalı embriyo Mikroenjeksiyon ve Elektroporasyon<em&gt; Ciona intestinalis</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

Cerrahi Ablasyon Tahlili Planaryalar Göz Rejenerasyon incelenmesi için

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

AFM ve Zebra balığı embriyo sarısı hücre Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

Hava sıyırma iki Salmonid balık, etkinliğini gökkuşağı alabalığı (Oncorhynchus Mykiss) ve kahverengi alabalık (Salmo Trutta morfin fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

En düşük Invaziv embriyo transferi ve tavşan modelinde optimum embriyo aşamasında embriyo vitrifikasyon

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Embriyonik Cardiomyocytes kuş embriyo implantasyonu Vivo için Mikroenjeksiyon tabanlı sistem

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

İzole Caenorhabditis elegans Embriyo Blastomerler ve Yapıştırıcı Polistiren Boncuklar ile Mekansal Hücre Temas Desenleri In Vitro Reconstitution

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Zebrabalığı İnternöromast Hücrelerinde Konfokal Mikroskop Tabanlı Lazer Ablasyon ve Rejenerasyon Tonu

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...