Name: live imaging of mouse secondary palate fusion
Uploaded: 2017-07-27T14:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

我们感谢道格拉斯·本森先生关于二次上颚成像的初步对话。我们还承认David Castaneda-Castellanos（Leica）和Chris Rieken（蔡司）帮助调整共聚焦显微镜中的成像条件。感谢Lynsey Hamilton（Bitplane）对使用Imaris软件进行定量图像分析的有用建议。这项工作由NIH / NIDCR R01 DE025887资助。

所有动物实验均按照加利福尼亚大学旧金山机构动物护理和使用委员会的协议进行。 活动影像制作准备<ol><li> 液体培养基的制备 <ol><li>向Dulbecco&#39;s Modified Eagle培养基（DMEM）/ F12中加入20％胎牛血清（FBS），2mM L-谷氨酰胺，100U / mL青霉素，100μg/ mL链霉素，200μg/ mL L-抗坏血酸，15mM HEPES媒体。 注意：在成像之前，实时成像介质是新鲜的。这种介质含有酚红，?...

<ol>
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使用3D器官外植体培养的组织形态发生的活体成像可以提供关于固定组织切片的常规染色分析中不能显示的细胞过程的详细信息。使用小鼠胚胎中性腭裂的体外外植体培养，我们观察到几个有趣的细胞行为，导致我们提出了一种涉及上皮收敛和细胞挤出的腭裂融合的新机制。 在这种研究中的一个常见挑战是通过长时间的连续激光激发进行光漂白。在我们的研究中，使用...

组织融合是多器官发育的重要一步。主要的人类出生缺陷如唇腭裂，脊柱裂和心脏畸形可能会导致从在组织融合1缺陷。小鼠二级腭融合已被广泛研究，以确定细胞和分子机制发展2，3，4控制组织融合。在小鼠中，二尖瓣发育开始于E11.5附近，其中每个双侧上颌过程均产生二级腭骨架。腭架的初始生长沿着舌头垂直发生，直到大约E14.0，此时腭架在舌头上方水平上升。中度指导的生长导致两个腭搁架的上皮的物理接触，形成中线上皮（MES）在E14.5。必须从中间腭架之间移除插入的MES，以允许间充质融合，并通过E15.5 3开发完整的，完全融合的中腭。 如何在两个单独的腭架之间形成共享的上皮MES细胞层，然后移除以实现间充质融合，一直是腭发育的中心问题。基于小鼠组织学和电子显微镜（EM）研究，外植体培养研究和功能小鼠遗传学实验，几个基本细胞行为已经涉及到这个过程。从每个货架腭的内侧边缘上皮MEE丝状伪足状突起便于初始接触5， 图6，随后将这些上皮细胞的嵌入到共享单个MES 6,7。删除生成的共享MES已被提议通过三个非排他性机制进行。采用组织学观察和离体谱系用活体染料示踪的早期研究表明，MES可能通过上皮被除去以MES细胞的间充质转换（EMT）8,9，虽然最近，上皮细胞的遗传谱系追踪已升高的不确定性，以上皮细胞的长期贡献腭间质架10，11，12。凋亡细胞的数量显著，并在他们的人数在某些突变体不能进行适当的味觉融合的减少，导致了想法，凋亡可能是MES溶解2,3的主要驱动力。最后，最初基于涉及上皮标记和进行时间点静态观察的研究MES细胞提出在口鼻及前后尺寸11，13迁移，但这样的动态细胞行为最初未经证实的由于无法观察他们在活组织腭。最近，我们能够通过开发一种新的活体成像方法直接观察这些行为，该方法结合了荧光标记的小鼠遗传方法与外植腭架的共聚焦活检。 首先，腭融合过程中可视化腭上皮细胞动态细胞行为，我们通过杂交ROSA26-mTmG FLOX小鼠Keratin14-CRE小鼠14,15生成的上皮特异性报道小鼠。所产生的胚胎的腭外植体培养物的共聚焦活体成像证实了一些先前提出的细胞行为，并在融合过程中鉴定了新的事件6 </SUP&gt;。上皮细胞膜突起先于初始细胞 - 细胞接触，然后通过细胞插入和鼻孔细胞置换上皮收敛。值得注意的是，我们还发现，电池挤出，报道在上皮内环境稳定中发挥重要作用的过程，是一个重大的机构驱动鼠标继发腭融合6,16。该成像方法可与其他记者行一起使用;我们利用Lifeact-mRFPruby转基因小鼠17，18在融合过程来检查肌动蛋白细胞骨架的动态。其他记者也可以用来观察腭部融合的其他具体方面，这种方法可以适用于激光扫描共焦显微镜或旋转盘共焦显微镜，这取决于成像需要和显微镜可用性。现场影像越来越成为发育生物学中的基石方法。班驳轻微地，颅面形态发生是复杂的，影响面部的人类出生缺陷是常见的。这种共焦现场成像方法将有助于更好地了解潜在的基本发育机制以及人类颅面异常的起源。

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			<td height="20" style="height:20px;width:348px;">Reagents</td>
			<td style="width:360px;"></td>
			<td style="width:225px;"></td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM/F12</td>
			<td>Life technology</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal Bovine Serum</td>
			<td>Life technology</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-glutamine</td>
			<td>Life technology</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544-100G</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pennicillin/Streptomycin</td>
			<td>Life technology</td>
			<td>15140-122</td>
			<td></td>
		</tr>
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			<td height="19" style="height:19px;">Low melting agarose</td>
			<td>BioExpress</td>
			<td>E-3111-125</td>
			<td></td>
		</tr>
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			<td height="19" style="height:19px;">35mm glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-10-C</td>
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			<td height="19" style="height:19px;">Petrolieum Jelly (Vaseline)</td>
			<td>Sigma</td>
			<td>16415-1Kg</td>
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			<td height="20" style="height:20px;">Mice</td>
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			<td height="19" style="height:19px;">Keratin14-cre</td>
			<td></td>
			<td>MGI: J:65294</td>
			<td>Allele = Tg(KRT14-cre)1Amc</td>
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			<td height="22" style="height:22px;">ROSA26mTmG</td>
			<td></td>
			<td>MGI: J:124702</td>
			<td>Allele = Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo</td>
		</tr>
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			<td height="19" style="height:19px;">Lifeact-mRFPruby</td>
			<td></td>
			<td>MGI: J:164274</td>
			<td>Allele = Tg(CAG-mRuby)#Rows</td>
		</tr>
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			<td height="20" style="height:20px;">Microscope</td>
			<td></td>
			<td></td>
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			<td height="19" style="height:19px;">White Light SP5 confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
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			<td height="19" style="height:19px;">Cell Observer spinning disk confocal microscope</td>
			<td>Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of mouse secondary palate fusion

二级腭架融合形成完整的二级腭是哺乳动物发育的关键过程，其破坏可导致腭裂性腭裂，这是人类常见的先天性异常现象。二次腭融合已被广泛研究，导致几个可能介导该过程的细胞机制。然而，这些研究主要在开发期间的进行时间点或在静态时间点分析的固定外植体培养物中对固定的胚胎组织进行。静态分析限于动态形态发生过程的分析，如腭裂融合，以及什么类型的动态细胞行为介导腭融合不完全了解。这里我们描述了一种用于小鼠胚胎体外二次腭融合的活体成像方案。为了检查腭裂融合的细胞行为，上皮特异性角蛋白14 -cre用于标记ROS中的腭上皮细胞A26-mTmG flox记录胚胎。为了可视化丝状肌动蛋白，使用Lifeact-mRFPruby报道小鼠。通过解剖最近粘附的胚胎期（E）14.5期胚胎的次级腭架并在玻璃底盘上的含琼脂糖的培养基中培养以使用倒置的共聚焦显微镜成像来进行中度腭融合的活体成像。使用这种方法，我们检测到在二次腭融合期间的各种新型细胞行为。对不同细胞行为在空间和时间上的协调的欣赏大大有助于我们对这种动态形态发生过程的理解。该方案可应用于突变小鼠系，或用药理学抑制剂处理的培养物，以进一步提高对二次腭融合如何控制的理解。

腭外植体活体成像显示多种细胞过程介导腭裂融合6 。两个上皮细胞之间的初始接触由膜突起（ 图 2B-2E ）制成。当两个上皮层相遇时，它们形成多细胞分层MES，然后插入以通过上皮收敛形成整合的单细胞层MES（ 图 2A-2E和图 2K-2O ）。较深Z水平的上皮...

Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

Live Imaging of Mouse Secondary Palate Fusion

在这里，我们提出了使用共聚焦显微镜的小鼠二次腭融合的活体成像方案。该方案可以与各种荧光报告小鼠系列结合使用，并可与途径抑制剂一起用于机械性的洞察。该协议可适用于其他开发系统中的实时成像。

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

The overall goal of this confocal live imaging method is to directly observe the cellular processes, the drive's secondary palate fusion during cranial facial development. Begin by separating the head from the body of the embryo under a dissecting microscope in a petri dish containing fresh, room temperature PBS. Grasp the head with one pair of fine number five forceps and use a second pair of forceps to remove the upper brain, cutting away any extra tissue outside of the palatal shelves with a second pair of forceps.Remove the mandible carefully without damaging the palatal shelves. When the mandible has been detached, carefully remove the tongue and confirm the presence of a robust reporter signal in the midline epithelia seam. It's important not to damage it, palatal explained during dissection to keep the midline epithelia seam intact.Next, remove the hind brain, brain stem and both maxilli with the adhered palatal shelves. Cut the anterior upper jaw tissue, keeping both the primary and secondary palates intact. Remove the nasal septum on the nasal side of the explant to expose the midline epithelia seam and confirm that the epithelia layer between the tissue is intact.Now, add live imaging medium to a 35 millimeter glass bottom dish and place the dissected palatal shelves, oral side down in the dish as close to the glass bottom as possible. Then, place dry ice pellets on a conductive surface near the culture dish to expedite the solidification of the agarose within the imaging medium. When the agarose is semi-solid, mount the culture dish in an inverted confocal microscope equipped with a 37 degree celsius incubation chamber and use a syringe to apply petroleum jelly between the culture dish and the culture cover to prevent evaporation of the imaging medium.Select a low magnification objective to locate the region of interest. Then, select a high magnification objective and scan multiple z stacks with five micron steps for 16 to 24 hours, using the appropriate laser excitation. Live imaging of palate explant culture reveals multiple cellular processes that mediate palate fusion, with the initial contacts between the two epithelia cells being made by membrane protrusions.When the two epithelia layers meet, they form a multi cell layered midline epithelia seam, followed by intercalation to generate an integrated single cell layer, midline epithelia seam through epithelia convergence. The epithelia cells in deeper z levels are also progressively displaced to the oral side of the midline epithelia seam, contributing to the epithelia convergence process. Posterior directed cell migration is observed in middle palate midline epithelia seam.With epithelia cell extrusion events observed during palate fusion, suggesting that the epithelia cells in the midline epithelia seam can be removed by this active process. This integrated epithelia seam will ultimately break down to achieve confluency. Further, filamentis actin becomes enriched at the border between the epithelia and mazenkemo areas, forming a cable like structure along the anterior posterior axis.Software can be used to track specific cellular behaviors of interest, during palate fusion with the best method for cell tracking depending on the reporter signal used in the experiment.

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

In Vitro Differentiation of Mature Myofibers for Live Imaging

In Vitro Differentiation of Mature Myofibers for Live Imaging

在成熟的肌纤维诱导分化实时成像

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

Live Confocal Imaging of Developing Arabidopsis Flowers

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Long-term Live Imaging of Drosophila Eye Disc

Long-term Live Imaging of Drosophila Eye Disc

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Cell Imaging of Chromosome Segregation During Mitosis

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...