Information

How to watch a Zebrafish embryo in detail?

How to watch a Zebrafish embryo in detail?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

How much microscope zoom would I need to watch the development of a Zebrafish embryo in certain detail? Thanks guys!


From personal experience, it should be sufficient to observe zebrafish at 10-20x magnification for broad structural changes during development. If your microscope has a 10x eyepiece, then that would be 1-2x zoom. If you are observing sub-cellular changes, you would benefit from higher magnification.


Zebrafish

Abstract

Zebrafish is recognized as an ideal model organism for investigating the cellular and molecular mechanisms underlying heart formation and regeneration. Zebrafish has a closed cardiovascular system and a cardiac cycle that is highly reminiscent of human cardiovascular physiology. Zebrafish can be genetically manipulated, and their genome has been fully sequenced. Genetic studies in zebrafish have discovered novel genes and pathways in cardiovascular development and function, with close correlation with cardiovascular diseases. Zebrafish heart regeneration occurs following injury through the dedifferentiation and proliferation of mature cardiomyocytes. Embryo-based small-molecule screens have discovered chemical probes and drug candidates for enhancing cardiovascular development, function, and regeneration.


Zebrafish, the Living Looking Glass

In the basement of the Life Sciences Building, around 1,500 fish tanks, ranging in size from briefcases to small crates, are systematically laid out in rows on metal shelves. From fertilized egg to adult, the roughly 20,000 fish represent the entire zebrafish lifecycle, providing Bruce Draper with a comprehensive view of their growth.

“If you’re looking at the process of development—so going from a fertilized egg to a swimming, feeding organism—all that process in mammals is happening in utero, so you actually have to sacrifice the mom to get the embryos out to study them,” says Draper. “With zebrafish, it’s all external fertilization.”

Part of Draper’s research focuses on problems of reproductive development. Zebrafish (Danio rerio) are well-suited for this research as their embryos are clear, providing a window into the biological machinery behind their formation. As the fish age, they develop stripes and lose their transparency.

Researchers bypass this problem by genetically modifying zebrafish with a gonad—the organ responsible for producing sperm and eggs—that glows under ultraviolet light. This allows continuous monitoring of gonad development as the fish grows, providing clues about reproductive development diseases like ovarian cancer.

Previously, Draper and his colleagues identified the gene fgf24 as important for gonad development in zebrafish. Mutant zebrafish developed defective gonads and had limited reproductive abilities. While this specific gene signaling isn’t known to be involved in mammalian gonad development, many aggressive ovarian cancers correlate with an overactive signaling pathway related to this gene. Overall, about 84 percent of the genes associated with human disease have counterparts in zebrafish.

Associate Professor Bruce Draper uses zebrafish to study gonad development. Designed by Steve Dana/UC Davis

Draper and his colleagues are investigating how single-cell RNA sequencing could help advance their research. The technique allows a high-resolution view of individual cells and the genes they express.

“We’re now identifying on a much more refined level what genes are expressed in particular cells,” he says, noting that the most aggressive forms of ovarian cancer typically occur in the organ’s cell linings. “We’re very interested in trying to identify those epithelial cells in our dataset so that we can start asking what other genes are expressed in there.”

Draper’s techniques for this project are being informed by Celina Juliano, whose office is just a few doors down from his.


Zebrafish Help Unlock Clues to Human Disease

May 2018—From floor to ceiling, in row after row of small, bubbling tanks, 30,000 tiny tropical fish are unlocking the secrets to diseases that have vexed generations of researchers and physicians.

The humble zebrafish, an inch-and-a-half-long freshwater member of the minnow family, is native to the streams, ponds and puddles of the Himalayan region. It doesn’t look much like a zebra, though it does have five horizontal stripes adorning either side.

One of the most interesting things about zebrafish is that when they lose something — an eye, a fin, a tail, even individual cell types — they grow a new one.

Because their genetic profile is remarkably similar to our own, zebrafish are also found in the Miller Research Building on the medical campus of The Johns Hopkins University, where they provide scientists with genetic clues to medical mysteries. The fish have helped investigators at Johns Hopkins make important breakthroughs toward regenerating eye tissue, understanding thyroid cancer and making sense of the DNA tangles that regulate cell activity.

The Center for Functional Investigation in Zebrafish — or the FINZ center — is a research core facility of the McKusick-Nathans Institute of Genetic Medicine. Thirty different cores from departments across The Johns Hopkins University offer more than 500 different research-related services. The centers allow investigators to share valuable resources and expertise, saving both time and dollars.

FINZ center co-director Jeff Mumm, of the Wilmer Eye Institute and the McKusick-Nathans Institute, says that, in addition to three Johns Hopkins researchers currently maintaining tanks of fish there, the center collaborates with other scientists.

For a fee, FINZ offers numerous genetic services to colleagues throughout the institution, including modifying the genome to produce fish with traits that researchers want to study. For instance, a researcher wanting to study how the liver develops and functions can ask the FINZ center to produce zebrafish with glowing livers. Taking a gene from jellyfish that enables their incandescent glow, the FINZ team introduces that gene into a zebrafish, lighting up the liver and allowing researchers to observe its growth and function.

“That’s the kind of work we do all the time,” says Mumm. “It’s a cheap and efficient process to work with us to create genetically modified zebrafish for specific research needs.”

In 2004, geneticist Andy McCallion, along with former colleagues Shannon Fisher and Steven Leach, proposed that Johns Hopkins build its own zebrafish facility. McCallion, who co-directs the FINZ center, and Fisher, now a faculty member at Boston University, convinced medical school leadership that zebrafish would offer a more efficient, less expensive way to do genetics research.

McCallion and his lab team dig deep into genetic codes that control when and where genes are switched on/off, combining cutting-edge genomic tools and computational artificial intelligence to find the abnormalities that can influence diseases such as Parkinson’s. He says that, while that work remains arduous, early in his career it was far more difficult.

“We could see sequences that stood out as highly similar among different species, but we didn’t have a large-scale, genomewide way to test our hypotheses,” McCallion recalls. “We needed a way to test hundreds and hundreds of these things. For a lot of reasons, studying the fish gives us that ability.”

In addition to their genetic similarity to humans, zebrafish have other attributes that make them attractive to scientists who need to research large numbers of the same organism. The fish reproduce and mature quickly, they’re easy to maintain and their eggs are fertilized outside their bodies, allowing researchers to harvest newly fertilized embryos. To produce the desired trait in a fish, they can introduce new genes or remove genes from the harvested embryos. And since zebrafish embryos are translucent, scientists can observe their development in real time, watching organs and whole systems grow from stem cells.

Mumm says that, while science has focused on mice as models to study disease since the creation of the first genetically altered mouse in 1980, zebrafish have, in recent years, emerged as an important model species as well.

Zebrafish Research | Behind the Scenes of the Johns Hopkins Zebrafish Facility

Zebrafish play an important role in research at Johns Hopkins. Frazer Matthews and Hannah Edelman take us on a tour of the facility where 30,000 fish help researchers unlock clues to disease.

For many years, fruit flies were the species of choice for genetics researchers, sharing better than 61 percent of genes with humans. And while the insects still play important roles, key physiological differences make fish, as fellow vertebrates, attractive.

While zebrafish share about 71 percent of the human genome, in some key cases, fish genes are near-perfect matches for people. According to a 2013 study by British researchers, 82 percent of genes associated with human diseases and disorders have a zebrafish counterpart.

Mice and humans are a closer genetic match, at about 85 percent. But Mumm says, “zebrafish provide a fresh perspective, affording unique opportunities beyond what is possible in mice.” The Mumm Lab specializes in “high-throughput biology,” where automated equipment processes large numbers of samples to enable large-scale chemical and genetic screening.

This approach is difficult and expensive to apply to mice and thus has largely been limited to cell culture work. However, due to their small size, zebrafish have emerged as a living disease model platform for high-throughput drug discovery. The Mumm Lab has developed a zebrafish-based screening approach that evaluates drug effects faster than ever, processing tens of thousands of fish per day.

Because their genes can be modified so inexpensively and in great numbers, Mumm says zebrafish allow scientists to pursue large-scale genetic research initiatives as well, interrogating the function of larger numbers of genes in shorter amounts of time.

Johns Hopkins surgeon Jason Prescott maintains numerous zebrafish tanks in the FINZ center. Three days a week, he operates on patients who have thyroid cancer. But he also has a research lab aimed at, one day, reversing the disease’s progress and eliminating the need for surgery. Zebrafish help Prescott better understand how genetic mutations can cause healthy thyroids to turn cancerous.

“The goal is to put myself out of the surgery business,” he says.

Prescott and his lab team study the defective gene coding in patients who come to him for thyroid cancer surgery. When they isolate a patient’s faulty gene, they introduce a similar gene in zebrafish. Then they observe the fish to learn the cancerous cells’ origins and how they replicate.

“The fish have thyroid glands that are very similar to ours,” Prescott says. “Anatomically, they’re in just about the same place and they perform a similar function. And because they’re living, we can actually see the biology in real time, as it happens.”

The fish also allow for fast and efficient testing of drugs to combat disease.
“Bypassing more primitive screening techniques, we’re able to save time and money by testing drugs on hundreds of fish at once,” he says. In one example of Prescott’s research, individual fish, genetically modified to have cancerous thyroids, are placed in shallow dishes of water. The fish have been engineered so that their thyroids glow when a particular drug causes a particular reaction. The researchers introduce various drug compounds to the water and study the results.

Zebrafish have played a key role in Prescott’s pursuit of pharmaceutical breakthroughs to combat thyroid cancer.

“The gold standard for preclinical drug trials is to work with a living organism,” he says. “It allows us a much more realistic environment than working with cells growing on a plate.”

The Capacity for Self-Repair

Medical student Hannah Edelman is six years into her M.D./Ph.D. program, researching human genetics and pediatric diabetes. She spends many hours in the FINZ center studying how zebrafish never get the disease.

In type 1 diabetes, a patient’s immune system attacks and destroys the cells in the pancreas that produce insulin, the hormone that regulates blood sugar. While humans cannot regenerate these cells, zebrafish can.

“They can regrow pancreatic beta cells,” she says, referring to the cells that store and release insulin. “I want to know what’s so special that allows them to do this. We’re trying to find a way for humans to be able to regenerate these cells. That would mean a lot in the treatment of type 1 diabetes.”

In Mumm’s work studying degenerative eye disease, he takes advantage of the fact that humans and zebrafish share a trait — a specific cell type that can revert to being a stem cell — that allows them to produce new cells in response to injury or disease in the retina. Thus both species have the capacity to produce new cells to heal the eye.

“Somewhere along the way in their evolutionary selection, zebrafish developed the capacity for self-repair,” says Mumm.

The difference, Mumm says, is that, while the new cells lead to a brand new retina in a zebrafish, humans are not so lucky. “For us, the new cells become scar tissue. It actually does humans more harm than good.” In cell culture, however, these injury-responsive human retinal stem cells have the ability to produce new neurons.

He hopes that by learning how the zebrafish’s robust regenerative abilities are controlled, we can harness dormant regenerative capacities in patients with degenerative eye diseases. He says the fish are speeding up the process by helping eliminate the scientific dead ends faster than ever before.

Combining these studies with the high-throughput screening techniques in his lab, Mumm says, “We can find out what doesn’t work at step one rather than at step 52. That amounts to a whole lot of time and money saved.”


Photoreceptor progenitor dynamics in the zebrafish embryo retina and its modulation by primary cilia and N-cadherin

Background Photoreceptors of the vertebrate neural retina are originated from the neuroepithelium, and like other neurons, must undergo cell body translocation and polarity transitions to acquire their final functional morphology, which includes features of neuronal and epithelial cells.

Methods We analyzed this process in detail on zebrafish embryos using in vivo confocal microscopy and electron microscopy. Photoreceptor progenitors were labeled by the transgenic expression of EGFP under the regulation of the photoreceptor-specific promoter crx, and genes of interest were knocked-down using morpholino oligomers.

Results Photoreceptor progenitors detached from the basal retina at pre-mitotic stages, rapidly retracting a short basal process as the cell body translocated apically. They remained at an apical position indefinitely to form the outer nuclear layer (ONL), initially extending and retracting highly dynamic neurite-like processes, tangential to the apical surface. Many photoreceptor progenitors presented a short apical primary cilium. The number and length of these cilia was gradually reduced until nearly disappearing around 60 hpf. Their disruption by knocking-down IFT88 and Elipsa caused a notorious defect on basal process retraction. Time-lapse analysis of N-cadherin knock-down, a treatment known to cause a severe disruption of the ONL, showed that the ectopic photoreceptor progenitors initially migrated in an apparent random manner, profusely extending cell processes, until they encountered other cells to establish cell rosettes in which they stayed acquiring the photoreceptor-like polarity.

Conclusion Altogether, our observations indicate a complex regulation of photoreceptor progenitor dynamics to form the retinal ONL, previous to the post-mitotic maturation stages.


Biomedical research III: communication between the brain and other organs

Human puberty is a dynamic process that initiates the complex interactions of the hypothalamic-pituitary-gonadal axis (HPG axis), which refers to single endocrine glands as individual entities. The HPG axis plays a critical role in developing and regulating many of the body’s systems, particularly reproduction 39 . Gonadotropin-releasing hormone (GnRH), secreted by the hypothalamus in the brain, circulates through the anterior portion of the pituitary hypophyseal portal system and binds to receptors on the secretory cells of the adenohypophysis 40 . In response to GnRH stimulation, these cells produce luteinizing hormone and follicle-stimulating hormone, which circulate in the bloodstream 41 . Therefore, an adolescent develops into a mature adult with a body capable of sexual reproduction 42 . Kallmann syndrome (KS) is a genetic disorder known to prevent a person from starting or fully completing puberty. In a study showing that the WDR11 gene mutation is involved in KS pathogenicity, the zebrafish wdr11 gene was demonstrated to be expressed in the brain region, indicating a potential role for WDR11-EMX1 protein interaction 43 .

Additionally, acute inflammation is known to initiate regenerative response after traumatic injury in the adult zebrafish brain. The cysteinyl leukotriene receptor 1 (cysltr1)–leukotriene C4 (LTC4) pathway is required and sufficient for enhanced proliferation and neurogenesis 44 . LTC4, one of the ligands for CysLT1, binds to its receptor Cysltr1 expressed on radial glial cells in the zebrafish brain 44 . In a study by Kyritsis et al., cysltr1 was increasingly expressed on radial glial cells after traumatic brain injury, suggesting cross talk between components of the inflammatory response and the central nervous system during traumatic brain injury 44 .

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family is involved in the production of reactive oxygen species in response to various extracellular signals. The NOX family member dual oxidase (DUOX) was identified as thyroid NADPH oxidase. In humans, DUOX2 mutations were identified among children diagnosed with congenital hypothyroidism. Recently, it was demonstrated that, in addition to goitrous thyroid glands and growth retardation, defects in anxiety response and social interaction were found in duox-knockout zebrafish 45 . These results suggest that duox-knockout zebrafish could serve as an effective animal model for studies in thyroid development and related neurological diseases, including intellectual disability and autism.

A large percentage of children with ASD are known to have gastrointestinal problems, such as constipation, diarrhea, and abdominal pain. Recent studies on the brain-gut axis have also shown that interactions with host-associated microbial communities, either directly by microbial metabolites or indirectly via immune, metabolic or endocrine systems, can act as sources of environmental cues. Molecular signals from the gut provide environmental cues for communication between the gut and the brain during episodes related to anxiety, depression, cognition or autism spectrum disorder (ASD) 46 . Moreover, modulation of intrinsic signaling pathways and extrinsic cues in resident intestinal bacteria enhances the stability of β-catenin in intestinal epithelial cells, promoting cell proliferation 47 .


The protein secretory pathway

The secretory pathway generates, trafficks and processes proteins destined for the extracellular space or the plasma membrane. It comprises the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), the Golgi complex and the vesicles that carry cargo between them (Fig. 2). Protein synthesis and glycosylation occur in the ER and Golgi, respectively. The coat protein II (COPII) complex facilitates cargo selection, vesicle formation and anterograde trafficking from the ER to the Golgi, whereas retrograde transport occurs in COPI vesicles (Fig. 1F Fig. 2). Diverse protein complexes function at each step of this pathway to recruit Rab GTPases and SNARE proteins, which direct and tether vesicles to target organelles and facilitate membrane fusion.

Secretory pathway components studied in zebrafish and implicated in human diseases. Secretory pathway proteins implicated in human disease are shown in black with corresponding zebrafish tools to study these in red. NTD, neural tube defects CLSD, cranio-lenticular-sutural dysplasia CDAII: congenital dyserythropoietic anemia II CMRD, chylomicron retention disease LGMD, limb girdle muscular dystrophy SEDL, X-linked spondyloepiphyseal dysplasia tarda ID, intellectual disability.

Secretory pathway components studied in zebrafish and implicated in human diseases. Secretory pathway proteins implicated in human disease are shown in black with corresponding zebrafish tools to study these in red. NTD, neural tube defects CLSD, cranio-lenticular-sutural dysplasia CDAII: congenital dyserythropoietic anemia II CMRD, chylomicron retention disease LGMD, limb girdle muscular dystrophy SEDL, X-linked spondyloepiphyseal dysplasia tarda ID, intellectual disability.

Work in unicellular organisms and cultured cells created an assumption that protein secretion is uniformly regulated across cell types. Studies in human, zebrafish and other vertebrates, however, revealed that this pathway is regulated in a spatio-temporal and paralog-specific manner (Melville and Knapik, 2011 Unlu et al., 2013). Although all cells secrete proteins, some – including B-cells, chondrocytes, hepatocytes and the endocrine and exocrine pancreatic cells – are considered ‘professional’ secretory cells and these cells were assumed to be particularly sensitive to secretory pathway disruption. Zebrafish mutants in secretory pathway genes both supported and refuted this hypothesis: some mutants show phenotypes in most highly secretory cells, whereas others have phenotypes that are restricted to only a subset of cells.

The emerging view is that the secretory machinery is integral to morphogenesis and organ function in a cell-specific fashion. The availability of zebrafish genetic mutants and fluorescent in vivo reporters provides novel insight into organismal functions of the secretory pathway. The effects of secretory pathway disruption relevant to development and disease are discussed below.

Developmental consequences of secretory pathway disruption

The COPII complex comprises the Sar1 GTPase, Sec23–Sec24 dimers of the inner coat, and Sec13–Sec31 heterotetramers of the outer coat (Fig. 2 Kaiser and Schekman, 1990 Novick et al., 1980). Zebrafish mutants of sec23a (crusher) and sec24d (bulldog) develop craniofacial dysmorphology, kinked pectoral fins and short body length (Lang et al., 2006 Sarmah et al., 2010). These are attributed to a failure of extracellular matrix (ECM) secretion during chondrocyte differentiation. Sec23A- and Sec24D-deficient animals fail to export collagen and other N-glycosylated proteins from the chondrocyte ER, arresting differentiation and ultimately causing cell death (Lang et al., 2006 Sarmah et al., 2010 Unlu et al., 2013), whereas collagen secretion and skeletal development are intact upon depletion of the close paralog Sec24C (Sarmah et al., 2010). Sec23B mutations in humans and zebrafish disrupt erythropoesis (Bianchi et al., 2009 Schwarz et al., 2009), a different phenotype from the chondrocyte defects observed in crusher and bulldog mutants. These COPII phenotypes are distinct from the dwarf mutants sneezy, happy and dopey, which disrupt genes that encode the α, β and β′ subunits of the COPI complex, respectively (Fig. 2), which are characterized by defects in notochord and melanosome formation (Coutinho et al., 2004). These data suggest that although COPI and COPII are required for efficient secretion and membrane recycling in all cells, loss of specific members of each complex have profound and disparate effects on a subset of cells. Mutations in individual COPII components cause an array of phenotypes in highly secretory cell types in organs such as cartilage, notochord, eye and gut (Niu et al., 2012 Schmidt et al., 2013 Townley et al., 2008 Townley et al., 2012), and in erythrocytes (Bianchi et al., 2009 Schwarz et al., 2009 Unlu et al., 2013), whereas cells that depend on vacuole formation are most sensitive to COPI depletion.

So how do transcriptional regulatory mechanisms direct the secretory pathway to assure a timely availability of cargo-specific coats? A large-scale screen in zebrafish identified the feelgood mutant, which carries a missense variant in the creb3L2 gene (Driever et al., 1996 Knapik, 2000 Neuhauss et al., 1996) – the first known transcription factor that regulates availability of the COPII components sec24d and sec23a, but not sec24c (Melville et al., 2011). Similarities between feelgood, crusher and bulldog mutant phenotypes suggest that a ‘secretory module’ consisting of Creb3L2–Sec23A–Sec24D specializes in procollagen secretion. Given that zebrafish depleted of Sec24C do not manifest skeletal dysmorphology and the gene is not a target of Creb3L2, it is likely that other cargo-specific secretory modules regulate sec24c and other genes in this pathway. Future studies in zebrafish and other animal models will be needed to crack the code of physiologically relevant, cargo-specific secretory networks.

Diseases caused by secretory pathway disruption in zebrafish and humans

Several human syndromes are associated with secretory pathway defects (De Matteis and Luini, 2011), some of which are recapitulated in mutations in orthologous zebrafish genes (Fig. 2). The concurrent identification of crusher/sec23a mutants in zebrafish and patients with SEC23A/cranio-lenticulo-sutural dysplasia (CLSD) variants (Boyadjiev et al., 2006 Lang et al., 2006) provides an excellent example of convergence between human genetics and zebrafish developmental biology to uncover physiological ramifications caused by disrupting basic cell biological processes. Both crusher mutants and CLSD patients present with craniofacial dysmorphology and axial skeleton defects attributed to backlog of ECM proteins in the ER (Boyadjiev et al., 2006 Lang et al., 2006). The closely related SEC23B gene is mutated in congenital dyserythropoietic anemia type II patients who have multinucleated erythroblasts in bone marrow, a phenotype recapitulated in zebrafish sec23b morphants (Bianchi et al., 2009 Schwarz et al., 2009).

It is unclear why mutations in SEC23A and SEC23B paralogs, which differ only by an 18 amino acid stretch, cause such different phenotypes. One possibility is that spatio-temporal differences in expression confer cell-specific functions of some COPII complex genes. However, as sar1a and sar1b are ubiquitously expressed early in development and become enriched in distinct tissues later on (E.W.K., unpublished observations), it is unlikely that their gene expression pattern is solely responsible for the divergent phenotypes observed in these mutants.

The craniofacial phenotypes of COPII mutants suggest that chondrocytes are highly sensitive to ECM secretory defects. This predicts that other manipulations that block the secretory pathway would also cause craniofacial dysmorphology. This, however, is not supported by data from zebrafish or humans when factors functioning at other steps in the secretory pathway are depleted. The transport protein particle (TRAPP) complex tethers ER-derived vesicles to the cis-Golgi membrane (Fig. 2 and Sacher et al., 2008). Fibroblasts from patients with TRAPPC11 or TRAPPC2 mutations (Bögershausen et al., 2013 Scrivens et al., 2009), and cultured cells depleted of TRAPPC11 (Scrivens et al., 2011 Wendler et al., 2010) display Golgi fragmentation and secretory protein retention. However, when these proteins are depleted in whole organisms their cell-specific roles are uncovered: patients with TRAPPC2 mutation develop spondyloepiphyseal dysplasia tarda (SEDT) distinguished by skeletal defects, short stature and microcephaly (Gedeon et al., 1999 Huson et al., 1993). This is recapitulated in trappc2 zebrafish morphants, which have a short trunk and microcephaly (A. M. Vacaru and K. C. Sadler, unpublished). The short stature and/or trunk phenotype might reflect a defect in chondrocyte formation or in ECM deposition, as in COPII mutants, but patients and zebrafish with TRAPPC11 mutation present very differently. TRAPPC11 mutation in humans causes myopathy, intellectual impairment and hyperkinetic movements (Bögershausen et al., 2013). Interstingly, zebrafish foie gras (foigr) mutants carrying a mutagenic viral insertion in the trappc11 gene display a different phenotype from other models of TRAPP complex disruption: they develop fatty liver, hepatomegaly, smaller gut and jaw, and fin defects (Cinaroglu et al., 2011 Sadler et al., 2005). The foigr/trappc11 fatty liver phenotype is partially attributed to activation of the unfolded protein response (Cinaroglu et al., 2011) however, neither mammalian cultured cells that are depleted of TRAPPC11 nor trappc2 morphants induce this response (A.M.V. and K.C.S., unpublished observations), highlighting the utility of in vivo cell biology studies in vertebrates.

The unique phenotypes that differentiate TRAPP complex disruption from COPII or COPI mutations indicate that a global block in protein secretion is not the only mechanism that underlies their associated phenotypes. Moreover, although depleting individual TRAPP or COP complex factors has similar effects in isolated cells, the physiological consequences could not be predicted without use of whole animals. These findings point to cell- and developmental-specific roles for each gene involved in protein secretion and underscore the need for comparative whole animal models to decipher the cellular and physiological functions of this pathway.


Chapter 9 - Analysis of cilia structure and function in zebrafish

Cilia are microtubule-based protrusions on the surface of most eukaryotic cells. They are found in most, if not all, vertebrate organs. Prominent cilia form in sensory structures, the eye, the ear, and the nose, where they are crucial for the detection of environmental stimuli, such as light and odors. Cilia are also involved in developmental processes, including left–right asymmetry formation, limb morphogenesis, and the patterning of neurons in the neural tube. Some cilia, such as those found in nephric ducts, are thought to have mechanosensory roles. Zebrafish proved very useful in genetic analysis and imaging of cilia-related processes, and in the modeling of mechanisms behind human cilia abnormalities, known as ciliopathies. A number of zebrafish defects resemble those seen in human ciliopathies. Forward and reverse genetic strategies generated a wide range of cilia mutants in zebrafish, which can be studied using sophisticated genetic and imaging approaches. In this chapter, we provide a set of protocols to examine cilia morphology, motility, and cilia-related defects in a variety of organs, focusing on the embryo and early postembryonic development.


Glossary

Digital scanned laser light sheet fluorescent microscopy (DSLM): An improved version of SPIM, which uses a thin laser beam rather than a full light sheet, thus reducing damage to both specimen and fluorescent dye.

Gastrulation: The phase in early embryonic development during which the three germ layers are formed: ectoderm, mesoderm and endoderm. The timing and molecular mechanism of gastrulation differ between organisms.

Genetic strain: A genetically uniform group of animals, used in laboratory experiments. A genetic strain can be developed by inbreeding, mutation or genetic engineering.

Single-plane illumination microscopy (SPIM): This method allows 3D observation of processes in living organisms, even in deep tissue layers. It detects fluorescence at an angle of 90° relative to the axis of illumination with a sheet of laser light, permitting optical cutting. The specimen is not positioned on a microscope slide but in a liquid-filled chamber which is rotated during observation.


Math Model Helps Show How Zebrafish Get Their Stripes

A mathematical model developed by Brown University researchers, including doctoral student in Applied Mathematics Alexandria Volkening, is shedding new light on how zebrafish get their iconic stripes. The model helps to demonstrate how two dynamic processes—the movement of pigment cells across the skin, and the birth and death of cells as the fish grows—combine to keep zebrafish stripes in line.

Zebrafish have become quite a popular model organism for biology researchers over the past few decades. The small freshwater fish begin life as transparent embryos and develop in just a few months to full size, giving scientists the chance to watch their development in detail. The emergence of their namesake stripes of dark blue and bright yellow has been the subject of much research. The stripes have been shown to be the result of interplay between three types of pigment cells: black melanophores, yellow xanthophores, and silvery iridophores.

“The stripe pattern forms dynamically as the fish develops,” said Volkening, who is the lead author on the new paper. “It’s not like these pigment cells are filling out some kind of prepattern that’s already there. It’s the interactions of the cells over time that causes the patterns to form. We wanted to build a model that simulates this based as much as possible on what’s known about the biology.”


Watch the video: Zebrafish egg development over 24 hours (November 2022).