Introduction to the zebrafish Materials for Sunday and Monday – to help introduce the zebrafish, stages of embryonic developmental and mutants (Based on Chuck Kimmel’s notes with modifications by Lila Solnica-Krezel, Diane Sepich and Yinzi Liu)

I. Sunday evening introductory lecture. “Zebrafish as a model of vertebrate development”

II. Monday morning lab introduction. “Watching embryos develop – a primer for the first lab”. III. Monday Lab notes “Exploring the zebrafish embryo: stages, mutants and cytoskeleton” We separately provide copies for you of the 1995 Kimmel et al. paper “Stages of embryonic development of the zebrafish”, which will be useful in the lab. A PDF version is on the lab’s Mac and also PDF files of the following five introductory papers: 1. Ho, R. and Kane, D. (1990) Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors. Nature 348, 728-730. 2. Hafter et al., (1996) The identification of genes with unique and essential functiona in the development of the zebrafish, Danio rerio. Development, 123, 1-36. 3. Solnica-Krezel et al., (1996) Mutations affecting cell fates and cellular rearrangements during gastrulation in zebrafish. Development, 123, 67-80. 4. Langdon, Y. G, Mullins, M. C. (2011) Maternal and zygotic control of zebrafish dorsoventral axial patterning. Annu Rev Genetics. 45, 357-377. 5. Solnica-Krezel, L. & Sepich, D. (2012) Vertebrate gastrulation: making and shaping germ layers. Annu Rev Cell & Developmental Biology

I. Sunday evening Status of the zebrafish as a model for vertebrate genetics and development Some features of the zebrafish and its embryo make it an attractive system for learning about development and how genes control development. 1. The zebrafish as a genetic model:

• George Streisinger: Founder of the 'modern era' of studies: External fertilization. Clutch size. Generation time.

2. Mutants! Useful tools to understand how axis is induced the nervous system is wired, and many other problems.

• First developmental mutants in zebrafish identified by such screens. Chuck Kimmel and Charline Walker find spadetail, cyclops.

• Janni Nusslein-Volhard (Tubingen) & Wolfgang Driever (Boston): The large scale genetic screens. Now many mutations, identifying hundreds of developmentally acting genes are available, and are being characterized and mapped.

• Mary Mullins and Francisco Pelegri maternal-effect screens. • Nancy Hopkins and retroviral insertion mutants. • TILLING & ILLING mutants • Zinc Finger Nucleases

• TALE Nucleases 3. Transgenics. 4. Axis specification. 5. Germ layers specification and patterning. 6. Gastrulation movements.

• Epiboly • Internalization • Convergence & Extension

II. Monday morning Watching embryos develop – a primer for the first lab In the Stages paper (see ZFIN for better images), embryonic development is divided into a set of developmental periods (e.g. the period of gastrulation), and further subdivided into an openended series of stages. We use time-lapse movies in this class meeting to introduce what is happening during the early developmental periods:

• 1-cell (or zygote period) (0-0.7 hours post fertilization; hpf) Newly fertilized egg. The non-yolky blastomere segregates towards the animal pole. You can watch the cytoplasmic segregation, the formation of the first blastomere and then its division.

• Cleavage (1-2.2 hpf) Rapid cell divisions, without cell growth, occur within the blastodisc. Cell number tells you the stage. Meroblastic cleavage. Stereotyped but not invariant division patterns. Lineage tracing. Metasynchronous timing. Deep cells and the enveloping layer (EVL).

• Blastula (2.2-5.2 hpf). Cell size and shape of the blastodisc tells you the stage. Yolk syncytial layer (YSL) formation at the midblastula transition (3hpf), and the likely function of the dorsal YSL as a Nieuwkoop organizing center. Midblastula transition and the origin of zygotic gene expression. Early ‘random’ (?) cell movement. Morphogenesis begins in the late blastula. Epiboly by radial intercalation -- scatter of clonally-related cells. The blastoderm margin as zone of lower cell-scattering.

• Gastrulation (5.2-10 hpf). Epiboly continues and conveniently indicates stage. Beneath the EVL the blastoderm begins marginal ingression to develops two layers. The outer epiblast feeds the inner hypoblast (or mesendoderm). Both layers undergo convergence and extension. The first organ, the notochord, becomes delineated by establishment of the axial/paraxial boundaries in the mesoderm (eventually the notochord/somite boundary).

III. Monday afternoon In the Lab: Exploring the embryo – developmental stages, mutants, and the cytoskeleton Before you start: Young embryos are very delicate! 1. Young embryos cannot tolerate the pressure of tearing a chorion roughly around

them. 2. Passing through the air/water interface will rupture dechorionated embryos, so keep

them submerged. 3. Embryos in their chorions are surprisingly robust. They can tolerate being placed in a

dish and having the surrounding buffer removed with a pipette tip. This is handy when the buffer is being changed or the embryos transferred into fix or when they are dropped onto the counter.

4. The working stock for most applications with dechorionated embryos is 0.3x or 30% Danieau (1.8mM Ca2+).

5. For performing microsurgery or dechorionating very young embryos with pronase, use 1x Danieau Buffer (100%; 5.4mM Ca2+).

6. 2% Methylcellulose is very dense and will hold embryos reasonably well for time lapse, but it will allow you to turn the embryo if needed. Lower percentage Methylcellulose can be used with a trade off in terms of stability for ease of movement.

WHAT WE DO: We do three ‘experiments’ to answer the questions below. Our purpose is to begin to become familiar with the early developmental pattern, normal and abnormal, of our zebrafish Danio rerio, and ask about the role of microtubule cytoskeleton in the early inductive and morphogenetic processes. Experiment 1. Using Methyl Cellulose to Mount Embryos for Time-lapse Recording of early cleavages. Questions: How would you characterize the division pattern of the early cleavages? Is the cleavage pattern correlated with the dorsal-ventral embryonic polarity? 1. Dechorionate embryos in 0.3x Danieau Buffer. Take your time. Young embryos

cannot tolerate pressure, so tear a hole in the chorion with a pair of watchmaker forceps, adjust your forceps and tear again until you have a hole large enough for the embryo to fall through. Then turn the chorion so the embryo can fall out.

2. Put a drop (or several drops) of Methyl Cellulose solution to fill the circle in the center

(approx 500 µL) on the glass bottom dish or (about 250 µL) on a 24x60 mm bridged coverslip. Both configurations are intended for inverted microscopes.

3. Take embryo and buffer in a large bore Pasteur pipette. 4. Place the tip of the pipette deeply into the bottom of the Methyl Cellulose and allow

the embryo to fall down the pipette to the tip and out into the methylcellulose. * Passing through the air/water interface will rupture embryos, so keep them submerged;

* The embryo should fall down from the tip of the pipette into the methylcellulose slowly by itself. But if not, give it a little bit pressure without introducing too much buffer; * If the embryo is dropped near the surface instead of the bottom of the methylcellulose, it may take a long time to sink to the bottom. Stirring the surrounding methylcellulose would help them get to the bottom in a few minutes. But never touch the embryo anywhere as it would possibly cause damage.

5. To adjust the position of the embryo stir the methylcellulose near the embryo with

your forceps. If you want to place additional embryos on the dish, put them far enough away so your stirring will not inadvertently disturb the position of the others.

6. Gently add buffer to the glass bottom dish or place a coverslip on the bridged

coverslip. Experiment 2. Disrupt movement of dorsal determinants with Nocodazole Questions: What is the organization of microtubule cytoskeleton during early zebrafish development? What are the roles of microtubules during the cleavage stages? What is the organization of microtubule cytoskeleton during gastrulation? What are the roles of microtubules in gastrulation movements? All solutions and tips should be collected as hazardous waste as we are required to do. Wear gloves. Intraperitoneal (Mouse) LD50: 39.53 mg/kg (LD50 for 50 kg mouse is 1977mg or 1.98 g IP). 1. Prepare 100 ml of 0.3x Danieau (1.8 mM Ca2+) solution, and warm solution to 28.5 C. 2. Coat the bottom of 5 wells of a 24-well plate with 0.25 ml of 1% agarose in Danieau

buffer. Place dish in a secondary container, like a tray (for safety purposes). Distribute 1 mL solution to 7 wells. This will make 0 mg/ml control, 3 nocodazole treatments, and 3 recovery wells.

3. Make two intermediate dilutions: - 1 mg/mL by putting 2 µL of 10 mg/mL stock solution in 20 µL of 100% DMSO. - 100 µg/mL by putting 2 µL of stock 10 mg/mL nocodazole solution in 200 µL of 100%

DMSO. 4. Make solutions to treat embryos. - To make 10 µg/mL nocodazole solution--add 1 µL of 10 mg/mL nocodazole solution in

100% DMSO to one of the wells, pipette up and down to mix. - To make 1 µg/mL --- add 1 µL of the 1 mg/mL stock, pipette up and down to mix. - To make 0.1 µg/mL --- add 1 µL of the 100ug/mL stock, pipette up and down to mix. - To be precise, you can add 1 µL of DMSO to the control well to reach 0.1% DMSO in

the final solution. But it is not necessary since the difference is negligible. 5. Only now allow the fish to breed. Collect embryos at 3-minute intervals. Dechorionate

embryos with watchmaker forceps and drop 1-cell stage embryos into nocodazole solutions, letting them fall into solution from the pipette.

6. Incubate at 28.5 C for 2 minutes then remove to new well. Examine the progress of

development over time. Alternative experiments 1. Treat cleavage stage embryos (16 cell stage, 1.5 hpf) with 10 µg/mL nocodazole to

block cell division. 2. Treat early epiboly stage (sphere, 4 hpf) embryos with 10 µg/mL nocodazole to

disrupt microtubules. 3. Test the effectiveness of nocodazole on embryos inside chorion versus those

dechorionated. 4. Test the speed and effectiveness of the nocodazole effect on microtubules by adding

a solution to a Tg microtubule-GFP embryo that is already being observed on the fluorescent microscope.

Experiment 3. Treat Cleavage or Epiboly stage embryos with Taxol Questions: What is the effect of stabilization of microtubules on early cleavage stages and gastrulation movements? All solutions and tips should be collected as hazardous waste as we are required to do. Wear gloves. LD50 (Mouse) =12 mg/kg. (LD50 for 50 kg mouse is 600mg or 0.6 g). 1. Prepare 100 mL of 0.3x Danieau (1.8 mM Ca2+) solution with 2.7% DMSO, and warm

solution to 28.5 C. 2. Prepare 25 mL of 1% agarose in 0.3x Danieau (1.8 mM Ca2+) solution. Distribute

0.25 mL to wells of 24 well plate, which is in a secondary container, like a tray. Allow to set.

3. Distribute 0.75 mL of 0.3x Danieau (1.8 mM Ca2+) solution with 2.7% DMSO to each.

This becomes 2% DMSO when equilibrated with the agarose. 4. Add 1µL 10mM Taxol stock, pipette up and down very gently to mix (final working

concentration will be 10µM). 5. Dechorionate early embryo and drop in 1-cell stage or 2-cell stage embryos, letting

them fall into solution from the pipette. 6. Incubate at 28.5 C for 30 minutes then examine the progress of development. Alternative experiments 1. Treat Early Epiboly stage embryos with 10 µM Taxol to inhibit epiboly. 2. Test the effectiveness of taxol on embryos inside chorion versus those

dechorionated.

Solutions Danieau Buffer 0.3 x Danieau medium: 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM Ca(NO3)2 1.5 mM HEPES, pH 7.6 which forms a pH of 7.6 without adjustment *I love this name, but the numbering is confusing (0.3x is 30%). To be clear, we will indicate Ca 2+ molarity. The working stock for most applications is 0.3x or 30% Danieau (1.8mM Ca2+); *For performing surgery or dechorionating very young embryos with pronase, use 1x which is 100% (5.4mM Ca2+) Danieau Buffer; *A concentrated stock of 10x version so 3x Danieau (18mM Ca2+) can be autoclaved for sterile use and longer storage. 10 mg/mL Taxol solution 1 mg taxol 117 µL 100% DMSO * Taxol has a molecular weight of 853.9 and is poorly soluble in water. 10 mg/mL nocodazole solution 2mg of nocodazole 200 µL of 100% DMSO  2% MethylCellulose 2 g Methylcellulose 100 mL of 0.3x Danieau Buffer (i.e., 1.8mM Ca2+). *Don't heat Methylcellulose as it will become MORE dense (think molecular gastronomy's hot ice cream); *Expect to rock this material for 4 days at 4C. Inspect and shake several times; *Even after it looks dissolved uniformly, there may still be a few undissolved particles which will be visible under microscope and can be disturbing in your images or movies. Centrifugation of the aliquots in 1.5mL Eppendorf tubes at high speed would help get rid of most of them; *2% Methylcellulose is very dense and will hold embryos reasonably well for timelapse, but it will allow you to turn the embryo if needed; Low melting point agarose (Seaplaque cat 50100) can be used to mount embryos instead.