Biology 306DEVELOPMENTAL BIOLOGYLABORATORY MANUAL FALL 2000

This laboratory manual was written and compiledby Dr. Frances E. Weaver

Note: In this second edition of the Biology 306 manual, exercises came from a variety of sources and therefore are in a variety of formats.

Instructions for Laboratory Assignments

Laboratory Notebooks: Each student will keep a detailed, dated laboratory notebook of all activities in the lab, which will include, but are not limited to, all methodologies and observations (many of which will take place outside of the scheduled lab period). Accurate drawings, raw and analyzed data, answers to questions, discussions and suggestions for future experiments or improvements to the labs must also be included.

Lab notebooks will be collected and graded twice during the "standard" laboratories, and returned to each student the following week (or sooner as necessary). I expect your lab note book to improve each time it is collected. A GOOD LAB NOTEBOOK IS PRECISE, DETAILED AND LEGIBLE!!!!!

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GENES AND DEVELOPMENT MINI PROJECT

Literature review and project report:

General: This paper and the subsequent presentation are, because of budget constraints, somewhat of a hodgepodge of lab report and research proposal. The literature review should refer only to the "unknown" gene you have been assigned, although you are going to work with knowns in the laboratory.

Reports must be typewritten, including tables, table and figure titles and figure legends. Graphs may be hand drawn but must be done neatly. Begin each section with the title of the section i.e. Results:

Reports must follow the guidelines below. Points will be taken off if the report does not adhere to these standards. Plagiarism, collaborative reports (unless authorized) and other forms of academic fraud are not permitted and will be grounds for FAILURE in the course. All sources must be referenced.

Your paper must read like a scientific paper published in a referred journal, by this time you should be very familiar with such articles.

Title:Will include the title of the study and the your name. Titles must be descriptive of the

work. Titles such as Lab Report or Gene Report are not acceptable.

Literature review: The literature review should refer only to the "unknown" gene you have been assigned, although you are going to work with knowns in the laboratory. Here we propose that the gene you have been assigned exists and functions in the embryonic development of the horseshoe crab.

Clearly you must learn something about how horseshoe crabs develop. I will provide the initial references for you, as some of these are difficult to obtain quickly, but you should do some research on your own. Relate horseshoe crabs to other species, why is this approach valid? Which species are the best sources of information that might relate closely to the way horseshoe crabs do things? What is there in the scientific literature about the function of the gene you have been assigned in these other species? Using the articles you are able to obtain and the textbook write a brief, review of the role of this gene in embryonic development. Limit this section to about five pages.

Your final literature review must include at least five research (that is, experimental articles) papers and at least one literature review article relating directly to the gene you have been assigned, as well as additional references about horseshoe crabs in particular or developmental pathways in general (as in what you glean from the text book). I will mark the paper down if you don't follow these rules.

The preliminary review will likely be sketchy, as you may not have or yet understand the articles you need. The second draft of the literature review will a complete draft and must

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include either more information or more depth than the first draft. The final draft, obviously, include either more information or more depth than the second draft. I will mark the paper down if all you do from one draft to another is correct typos.

You must include a bibliography in any draft you turn in. Please remember that a responsible scientist works from full text articles, not abstracts* and that plagiarism is unacceptable. If you are not sure how properly reference your sources, please see me.

Methods and Results (the lab report part)Clearly write out in your own words what we did in the laboratory using the primers to

known genes. Write this section in the past tense. Use a paragraph format, and remember that someone must be able to repeat your work from this section. Illustrating your methods section with tables or flow charts often helps to clarify what was done, but flow charts do not substitute for the text of the methods section. This section should include a straightforward, factual presentation of the outcome of laboratory efforts. Do include gel pictures as figures if we are able to get some!

Discussion of the laboratory work : Discuss the relative success or failure of our laboratory work with the known genes. What could you do to improve our success? If we have been sucessful, in what direction might we know go?

Proposed future experiments with your gene: Now use what we did in the laboratory to design an approach that you would use for a new set of primers to your assigned gene. Then, assume that you will be successful in obtaining a cDNA to your gene, and propose additional studies to ascertain the function of this gene in development. This requires a good understanding of the experiments you have read about and some creativity on your part. Explain exactly what you propose to do, giving hypotheses and methodology. Then dissect possible outcomes, for example, you propose to study gene expression using in situ hybridization, what do you conclude if signal is detected in the limb buds, or in the gut or not at all? Note: transgenic horseshoe crabs are not possible, so limit your experiments to what can be done directly with embryos.

Bibiliography or References You must list any sources you used in preparing this paper, including your lab. manual or

handout, and textbook. References must be properly cited in the text, as well. DO NOT USE FOOTNOTES. Consult a style manual (reference section of the library) for appropriate usage of citations and how to prepare a reference list. MLA format is best, although many scientific journals have a preferred format.

This is a citation in the body of the text:

Our results disagree with those obtained by Morris and Weaver (1996) by nearly ten percent.

This is the (fictional) reference list that follows:

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Dewy, T. H. and Howe, D. N. (1956) Mechanisms of olfaction in insects. J. Entomological Soc. 23:1-4

Morris, R. W. and Weaver, F. E. (1996) Positive taxis of Sarcophaga larvae in response to fat substitutes. J. Entomological Soc. 86:1-4

More help:

I keep copies of several source books on scientific writing in my office, don't hesitate to borrow one. Feel free to consult me, multiple times if necessary, before you hand anything in for a grade.

If your report is all marked up for grammatical, spelling or composition errors, you may wish to contact the Writing Center (X 4332)

*Why? Because the abstract is the authors' assessment of what was done and what it means. A sensible scientist examines the data first and then decides if the authors are justified in making these claims.

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Biology 306 Laboratory IV: Journal Club

Practicing scientists frequently share newly published findings with their immediate colleagues in a forum called a journal club. We will conduct one journal club this semester.

The procedure for preparation to give a journal club talk is:

1. Choose one article on the gene you have been assigned topic for presentation. It must be a research paper, not a review article, news and views article or something from the popular press. Choose one that will help you do the best job of explaining the function of the gene you have been assigned.

2. Obtain a full text version of the article and look it over carefully. Please give me a copy at least three days before the presentation. If you are able to understand most of the article using only your textbook and your personal store of knowledge as references, its presentable, if you'll need more than one other background source, it is not.

4. Review your choice with me.

5. Prepare a 15 minute presentation on the research described in the article. WE GOTTA HAVE VISUALS!! Flow charts of procedures really help.

Journal club presentations should consist of the following:

Background: in which you place this article in context. Assume we know very little about the system(s) that you are about to describe.

Methods and Results: Present the experiments that were conducted in the study one at a time* by answering these questions for each:

What was the experiment designed to do? (you can say this experiment attempts to show….)

How was it done? Your audience will appreciate diagrams of complex procedures.What were the data (show and explain tables or figures)? What new questions arose from these results?

Discussion: Did the experiments conducted support or refute the hypothesis(es) of the study? Why or why not? Where there any flaws in the study? What new questions do you have?

*Not all experiments of a lengthy paper can be covered in 15 minutes, if it seems that you must cut something please consult me before you prepare the talk.

Remember to: Organize your information into a clear and concise sequence and make smooth transitions from section to section. PRACTICE YOUR TALK BEFORE A LIVE AUDIENCE (preferably of humans), practice more than once, practice more than twice, practice more than

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three times (you get the idea). Speak clearly and look at your audience (even if they are not looking at you). POINT THINGS OUT ON YOUR VISUALS.

No one is expecting you to have this stuff memorized, but the only way to avoid the pitfall of reading the paper with your head down is to practice! Nervousness is normal! Take a deep breath, consult your notes, and resume! Finish your talk with the words: “ Thank you”. Your audience will appreciate it!

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GENES AND DEVELOPMENT MINI PROJECT

Power point or web based presentation

Prepare a visually intense presentation that will introduce us to the gene you are working on, how it operates in other species, what it might do in horseshoe crabs and how you propose to test that (those) hypothesis. Assume we do not know much about this gene or the species that have been studied, but don’t bore us with a laundry list of details that we can't grasp in 10 or 15 minutes; hit us with the essential information.

The presentation should consist of an introduction, a review of the role of this gene in embryonic development in other species, a proposed role for this in horseshoe crab development, how you might detect this gene in the crabs and what experiments you propose to do if it was indeed found.

Use figures scanned in from any source, I'll help, to illuminate the role(s) of this gene in development. Make extensive use of bulleted lists, diagrams and flow charts to illustrate your points, which must be presented in a logical fashion. Please don't waste time making fancy transitions from slide to slide, concentrate on the information you are presenting.

Review my advice on journal club presentations before standing up with your presentation.

Peer and instructor review of presentationsEach student and the instructor will review in written form each presentation according to

a rubric that we will agree on prior to the date of the presentations. It is my feeling that you will best be able to perform to and evaluate with guidelines that you have participated in developing.

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Biology 306 Laboratory IDifferentiation

In this laboratory you will examine an organism that is capable of regeneration, and a single celled organism, Dictyostelium, that is capable of assembly and differentiation. We will initiate the observations in this lab, but you must return to your cultures several times in the next week to complete the exercise.

Cactus spine needles:

A cactus spine needle is useful for bisecting such organisms as Hydra, Lumbriculus or Planaria, and for microsurgery on chick embryos.

With a razorblade slit and slightly sharpen the end of an applicator stick. Obtain a cactus spine; insert the blunt end into the slit and seal with nail polish. Make several needles using different spines and different angles of insertion. Store your needles in the instrument cups provided.

Regeneration in Lumbriculus

Lumbriculus variegatus, the California blackworm or mud worm is a segmented, hermaphroditic annelid that prefers extremely wet to aquatic environments. The body is composed of about 120 to 250 segments. Both sexual reproduction, which involves embryonic development from a cocoon and asexual reproduction by self-fragmentation occur. It is possible to culture these organisms in the laboratory under very simple conditions by taking advantage of their ability to reproduce asexually.

Capture and handle worms and worm fragments while they are in spring water using transfer pipettes. Do not use forceps or needles, as the worms will self fragment (automize). When a worm is separated into fragments, the pieces regenerate over a period of about two weeks. A regeneration blastema, a region of dedifferentiated cells will form at the cut surfaces. The regenerated segments are smaller and lighter in color than the original segments, and the original segments may have undergone a developmental reorganization (or re-specification, if you will) to perform new functions.

Obtain a specimen of Lumbriculus in a dish of spring water, and observe its behavior under the dissecting microscope. Locate the head and “tail” ends and observe locomotion.

You should fragment the worm using a razor blade or cactus spine needle into pieces small enough to fit in eppendorf tubes. Don’t make the pieces too small or you will have far too many to record over the next two weeks.

Consult your partner: How will you determine that regeneration has indeed taken place? Count segments? Measure lengths?

Record the initial length or segment number of each fragment you create.

Fill each eppendorf tube with ~1 ml of spring water, and label the tube to indicate the specific pieces that you will transfer into it. Transfer each segment to the tube as you cut it. Cap the

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tubes tightly and store them in the laboratory until next week’s laboratory when you will observe and document the progress of regeneration in these segments.

Differentiation in Dictyostelium discoideum

The “cellular slime mold”, which is not a mold is an excellent system in which to study differentiation. Single celled myxamoebae feed on bacteria, aggregate when starved, and the multicellular aggregate called a grex or slug migrates in a coordinated fashion, seeking further food. If none is encountered, the grex differentiates into a sporocarp to weather out the bad times. New myxamoebae are produced when the sporocarp “germinates”. The life cycle of Dictyostelium is diagrammed below, see Chapter 2 of your textbook for a review of some of the interesting work that has been done of Dictyostelium.

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Culturing Dictyostelium

Dictyostelium and E. coli or Enterobacter stock cultures will be provided.

Wash hands and disinfect surfaces. Mark bottom of two lactose agar plates with name, date and compass directions (N, S, E, W)

Using a flamed metal loop (or sterile plastic one), aseptically inoculate a dish of lactose agar with E. coli using a North –South streak.

Using a flamed metal loop (or sterile plastic one), scrape up enough Dictyosteliumgrowth, about the size of a pea, for inoculation.

Inoculate the same lactose agar plate with the Dictyostelium, streaking now in the East –West direction, and being sure to leave bacteria free areas where the Dictyostelium can migrate and

differentiate. Repeat with second plate.

Dispose of all contaminated materials in the biohazard bags provided.

Disinfect the work area, and wash your hands.

Incubate the plate at room temperature in sunlight for 7 days, checking on the culture every two days (approx.). In the past, cultures have grown well in Room 423 on the windowsill. It is best to leave the cultures upside down so that condensation on the lids does not spread bacteria over the entire plate.

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EW

N

S

Record your observations, which should be made every day, in your laboratory notebook.

References:

Cactus spine needles: England, M.A. (1981) The application of the scanning electron microscope to the analysis of morphogenetic events. J. Micros. 123:133-146

Dictyostelium culture and life cycle: Carolina Biological Supply handout CB161389602, 1996Dictyostelium life cycle diagram dowloaded from http://biodidac.bio.uottawa.ca/Thumbnails/MYXO002B-GIF.htm on 8/11/00

Lumbriculus: Carolina Tips, 59 (3) 1996

Thanks to Leland Johnson for ideas presented at the Developmental Biology Laboratory Teaching workshop, 1997

Frances E. Weaver 1/24/1999; 8/11/00

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Biology 306 Laboratory II

Meiosis and fertilizationChlamydomonas OedogoniumSea Urchins (if available)

Gametes and gametogenesis: slide studyAscarisFrogsChicksMammals

Ongoing: Differentiation in DictyosteliumRegeneration in Lumbriculus

Isogamous fertilization in Chlamydomonas ( see Gilbert, Developmental Biology 6 th ed. Fig 2.8; p. 33)

These haploid cells exist as two mating types, + and - both participate equally in contributing cytoplasm to the zygote. Recognition is between adhesion molecules on the flagella membranes. The plus cell extends a fertilization tube, contacting a receptor site on the minus individual. In practice, cells tend to aggregate in clumps. Once two cells have mated, cell walls are dissolved at the anterior poles, the cytoplasms fuse, and the nuclei unite to form a zygote (diploid). For a brief period a quadriflagellated zygospores may be visible, these loose their flagellae when they become zygotes. After a period of dormancy a zygote “matures” undergoing meiosis (just like other diploid cells) and then germinates, producing 2 + and 2 - haploid organisms. This organism thus displays both sex and reproduction.

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Both sexual and asexual reproduction are depicted here.

Four hours before lab the + and - strains were placed under a lighted desk lamp at a distance of 18 to 25 cm. At t = 2hr the equal amounts of the mating solution (provided by Carolina Biological Supply) was added to the cultures, and they were gently agitated and replaced under the lamp. These cells should now be ready to mate.

Mix a drop of + with a drop of - strain on a slide, preferably in an observation chamber constructed using a cover slip and Carolina observation gel. Work quickly!! Use just enough fluid to keep the cells moist, and seal the edges of the coverslip with the gel or nail polish. Place the slide on the compound microscope, observe the cells, and continue observations at convenient times throughout the lab period. In a healthy culture the entire life cycle will be complete in 3 hours.

Mating in Oedogonium

In the lab we will have a representative of these oogamous filamentous green algae which are often found growing epiphytically on aquatic plants such as water lilies. Oedogonium may reproduce asexually via budding of zoospores from vegetative cells. Zoospores posses about 120 flagellae!

In the sexual phase, the large egg is produced in an oogonium. Single filaments are either "male" or "female" or both depending on the species. An oogonial pore opens on one side when the egg is ready for fertilization. Sperm are multiply flagellated and produced in shortened box- like structures called antheridia.

In those species that have both “male and female” on the same filament, mature eggs produce a substance chemotaxtic for sperm (sound familiar?) this compound is one of a group of substances called erotactins. A diagram of Oedogonium:

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antheridia

oogonium

Obtain a specimen of the Oedogonium mating culture on a microscope slide and observe under the compound microscope. You are looking for oogonia and antheridia. Depending upon the length of time since the initiation of mating, you may also see zoospores (use a higher power objective and adjust the lighting). Young germlings might be found at the edge of the mating dish. Sketch and label what you see. This organism can also be used for student projects, but be aware that 48-72 hours must elapse between the initiation of mating and the appearance of large quantities of oogonia and antheridia.

Fertilization and early cleavage in sea urchins:

Sea urchins are deuterostome organisms that have radial symmetry as adults, but bilateral symmetry as larvae. The eggs are variously colored, but the developing embryos become progressively less opaque. A jelly coat, which releases peptides that attract sperm, very useful when the adults are meters apart in the ocean, surrounds the each egg. This egg jelly may or may not be visible. Some echinoderm species invest their jelly with pigment granules. Fertilization is external, and the entering sperm stimulates the exocytosis of cortical granules to the egg surface, resulting in the elevation and eventual hardening of a fertilization envelope. The fertilization envelope represents a slow block to polyspermy, and also serves to protect the embryo until it reaches the blastula stage. Blastulae, which hatch from the fertilization envelope, are ciliated and capable of swimming.

For a summary of early embryonic development in sea urchins, view the appropriate sections of the video "A Dozen Eggs"

The gonads lie close to the under side of the aboral surface. The sexes look alike, so the only way to tell them apart is by getting them to release their gametes. Eggs and sperm are obtained by intra-coelomic injection of 0.5 M KCl. The female is placed over a beaker of seawater to collect the eggs. Sperm are collected dry onto a plastic petri dish.

Observe gamete collectionObtain a sample of eggs on a microscope slide, and observe. The dot in the eggs is the

egg nucleus or germinal vesicle. Add a drop of "dry" sperm to the edge of the coverslip and watch the eggs carefully as

the sperm make contact. Can you see the fertilization envelope as it rises? If thing are too murky, try diluting the sperm with seawater.

Sketch your results, refer to chapter 2 of your atlas.We will batch fertilize some eggs and check for early cleavage throughout the period. If

we are successful, we will keep our cultures and observe them over the next week.

Gametogenesis: Study of Prepared slidesBring Mathews and Schoenwolf Atlas of Descriptive Embryology 5th edition with you to lab

Using your compound microscope, your textbook (see Chapter 19 and study, compare and sketch the prepared slides of:

Frog ovary and testis; see chapter 5 of your atlas.Chick ovary and testis (lower power lenses are best for the ovary);refer to chapter 6 of your atlas.Small mammal ovary and testis

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Human sperm smear (use oil immersion lens)Human or cat ovary and testicle

One process that you are observing is meiosis. Meiosis takes place in the gonad of all these species. Following or accompanying meiosis are the differentiation steps. The end products; oocytes, eggs or sperm are very different from each other.

Once you have completed your sketches answer the following thought and discussion questions for each of the species observed: How do the differences in the morphology of the eggs and sperm arise during gametogenesis. How do these visible differences relate to the differing functions of the gametes in fertilization and early development? Are there cellular or acellular materials associated with the egg once it is ovulated, such as follicle cells? How do the differences in structure of the eggs from the various species relate the environment in which the embryos will develop?

Nuclear events of fertilization: Prepared slides of Acaris

The order and extent of the nuclear events of fertilization vary with species, but these usually include sperm entry, decondensation of sperm DNA, swelling and union of sperm and egg pronuclei. In most mammals meiosis II resumes and completes at the time of fertilization in order to create the female pronucleus, so there are a number of things happening at once.

Examine prepared (use 45X and 100X oil immersion lenses) of uteri of the parasitic roundworm Ascaris megalocephala also known as Parascaris aequorum. Sperm are amoeboid and crawl into the uterus where there are many ova in various stages of maturation. The sperm are darkly staining, almost triangular bodies, maturation (decondensation) and fusion of the male and female pronuclei are easy to see, as is the beginning of mitosis. There are only 2 chromosomes per haploid cell (4 per diploid), so you should be able to spot the condensed chromosomes. Remember that you are looking at the whole worm on most slides so your first job is to locate the uterus.

Make detailed, labeled sketches of the stages of fertilization you are able to locate referring to chapter 2 of your atlas. When might the DNA synthesis needed for cleavage occur?

If we are successful with the sea urchins, you may have to complete your slide studies in the next laboratory period.

ReferencesCarolina Biological Supply Company "Unialgal Mating Kits" handout #cb160617810

Information on the life cycle of Oedogonium was taken from Morphology of Plants, 3 ed. by Harold C. Bold, Harper and Row, New York, 1973.

Oedogonium life cycle diagram downloaded from the site below on 8/11/00http://biodidac.bio.uottawa.ca/Thumbnails/filedet.htm?File_name=CHLO013B&File_type=GIFThanks to Leland Johnson for ideas presented at the Developmental Biology Laboratory Teaching workshop, 1997

Frances E. Weaver 1/27/1999; 8/11/00

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Biology 306 Laboratory III Lab notebooks due on the Monday that follows this lab.

Early Frog Development: Exercise taken from S. L. Biroc, Developmental Biology. A Laboratory Course with Readings, Macmillan, New York, 1986, attached.

Early development of Zebra fishhttp://zfish.uoregon.edu/zf_info/zfbook/stages/stages.html

Ongoing: Differentiation in DictyosteliumRegeneration in Lumbriculus Sea Urchin development (if available)Slide study, if incomplete from last week.

Bring Mathews and Schoenwolf Atlas of Descriptive Embryology 5th ed. and your text book with you to lab.

We must work on frog and zebra fish embryos at the same time, so be prepared to go back and forth.

Early development of Zebra fish

Zebra fish development is considerably more rapid than frog. Depending on the time of embryo collection this morning, our embryos may be well into cleavage by lab time.

Obtain a dish of zebra fish embryos in aged tap water, observe and sketch the resulting stages of development approximately every half-hour. Refer to the on-line manual of developmental stages available at http://zfish.uoregon.edu/zf_info/zfbook/stages/stages.html (which we should be able to access during the laboratory period) and your text book. At the end of the lab period, transfer your embryos to a clean dish of tap water and count them, discarding any obviously defective ones. Record the number of embryos in your lab book. Return to the lab to observe them on Friday and Monday, record the numbers and developmental stages each time. What percentage of your embryos matured to larvae?

Early Frog Development: Exercise taken from S. L. Biroc, Developmental Biology. A Laboratory Course with Readings, Macmillan, New York, 1986, attached.

Dictyostelium: complete observations if you have not already done so.

Lumbriculus: Record in a table (% length change/piece or % increase in segment number) a numerical assesment of ; and comment on the extent of regeneration in Lumbriculus. What factors such as size of piece or position of the cut influence regeneration? Did your middle pieces show signs of developing both heads and tails? Are the regenerating segments smaller or larger than the "old" segments?

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Biology 306 Laboratory IV-VI Genes and Development Mini-Project

Detailed laboratory instructions will be provided prior to the start of week IV. Project instructions and guidelines for journal club appear in this booklet.

We will be attempting to prepare and clone cDNAs of developmental interest from RNA isolated from horseshoe crab embryos.

Outline of Activities:

Week IVEach student will be assigned a gene of interest and a pair of degenerate oligonucleotides to sequences derived from homologs of that gene in vertebrate and invertebrate species.We will go over the requirements for this research project, journal club and presentations.

We will perform RT-PCR using these oligonucleotide primers on total RNA isolated from horseshoe crab embryos.

It will be your responsibility to research the gene you have been assigned for your paper and presentation. We will make use of on-line data bases and search engines to locate, order and/or print out the first few references during this period. The remainder of the literature based research will be done on your own time.

Week V

We will determine our success, or lack of it in the RT-PCR reactions by gel electrophoresis. If successful we will attempt to ligate our cDNAs into plasmid vectors. Otherwise, we will "trouble shoot" the RT-PCR

Week VI

We will transform our successful ligations into E. coli and select for transformants orWe determine our success, or lack of it, in the repeated RT-PCR reactions by gel electrophoresis. If successful we will attempt to ligate our cDNAs into plasmid vectors. Otherwise, we will continue to "trouble shoot" the RT-PCR.

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Biology 306 Laboratory VII

Drosophila Development and Imaginal Disk DissectionSee exercise attached from Mary S. Tyler, Developmental Biology, A Guide for Experimental Study Sinauer Associates, Sunderland MA 1994

Eversion of Imaginal Disks

Ongoing:Genes and development mini project, if needed.

Premature Eversion of Imaginal Disks

In Drosophila, the mouthparts, antennae, eyes, legs, wings, halteres and genitalia of the adult develop during pupation from imaginal disks. These disks represent tissue set aside from the larval structures. The original disk invaginates from the body wall (see figure 17.1 from Cruz, below) and becomes, effectively, a collapsed pouch. The pouch everts at pupation, in a motion that is rather like the extension of a telescoping structure such as a pointer or a car antenna (see figure 19.16 from Gilbert, below). Molecular analysis has shown that the anterior posterior axis as well as the specific regions of each structure is determined prior to eversion and that natural eversion is primarily the result of shape changes of the disk cells. Although disk extension during pupation is normally under hormonal control (ecdysone), we can prematurely evert disks by treating them with trypsin.

Obtain several third instar larvae and place them in a drop of Insect Ringers in a small petri dish.

Anesthetize the larvae by placing the dish on ice for a few minutes.

Place an anesthetized larva on a microscope slide

Using the naked eye or the dissecting microscope, firmly grasp a larva by its anterior end with one pair of fine forceps and about 1/3 of the way down with another pair.

Pull the larva apart, the digestive tube should be expelled from the preparation.Most of the disks are associated with the esophagus and the anterior end of the digestive tract. They are clearer than fat bodies or digestive tract and recognizable by the concentric marks of the imaginal tissue.

Put a little more Insect Ringers on the slide and dissect the disks away from the rest of the larva using catcus spine needles. Take care not to let the disks dry out.

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Using the compound microscope, sketch and label the disks using the figures below and in the previous exercise as guides.

Push your dissected disks to one side of the slide, and remove any other debris.

Remove as much Ringer's as possible, and cover the disks with 0.1% Trypsin in Insect Ringers, pH 7.0.

Set the slide aside and make observations at half-hour intervals. Eversion make take up to an hour (or more) to complete.

When eversion appears to be completed, sketch the results of the trypsin treatment, being sure to use the same magnification as the disks sketched before eversion. Did eversion occur? Speculate as to why trypsinization leads to eversion.

References

Yolana P. Cruz Laboratory Exercises in Developmental Biology Academic Press, San Diego 1993

Scott F. Gilbert Developmental Biology 5th edition, Sinauer Associates, Sunderland MA, 1997

Mary S. Tyler, Developmental Biology, A Guide for Experimental Study Sinauer Associates, Sunderland MA 1994

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Observation of Chick Development: Exercise taken from Johnson and Volpe, Patterns and Experiments in Developmental Biology; Wm. C. Brown, Dubuque IA 1973, attached Explantation of chick embryos and cardia bifida Exercise from S. L. Biroc, Developmental Biology. A Laboratory Course with Readings, Macmillan, New York, 1986, attached.

Bring Mathews and Schoenwolf Atlas of Descriptive Embryology 5th edition with you to lab.

A word of caution: Eggs may b e contaminated with Salmonella bacteria. Typically the contamination is on the outside of the shell, but occasionally bacteria are found internally. We are handling a great number of raw eggs, please exercise great caution. Wash the eggshells gently and be certain to wash your hands thoroughly before leaving the lab.

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Biology 306 Laboratory X

Explantation of chick embryos, staining for cartilage

Bring Mathews and Schoenwolf Atlas of Descriptive Embryology 5th edition with you to labOther references on chick development will be available in the laboratory.

Ongoing: Repeat of cardia bifida

In the development of the appendages cartilaginous skeletal elements precede the formation of bone proper. This process is known as endochondral ossification. Here we will explant and stain chick embryos at relatively early stages for the formation of cartilage. Our expectation is that the developing spinal vertebrae and the bones of the developing appendages will be intensely stained. In earlier embryos we may see more diffuse staining, if so we will attempt to correlate these patches with our understanding of chondrogenesis. The procedure is simple, but does require that you return to the lab to change solutions.

The fixative and staining solutions are either caustic or toxic or both. Take note of the position of the eye wash station. Wear a lab coat and take care not to spill or spatter your fixative and staining solution. Report any spills to the lab instructor.

Read the procedure carefully. Some steps must be done 1 and 2 days after lab. Devise a plan for sharing the labor before starting the explantations.

Chicken eggs that have been incubated between 3 and 10 days will be available.

Crack incubated eggs into saline and explant embryos from at least two different stages into a jar of 5% TCA (trichloroacetic acid). Note the developmental stage of each embryo you explant, and in particular the status of the limb buds, by referring to the available atlases and sketching the embryos in your notebook. The acid treatment will fix the tissues in place.

Fix the embryos for 3hr or longer in the 5% TCA. During this time, explant 24hr embryos and repeat cardia bifida experiment if last week's was unsuccessful.

Use just enough of the solutions to cover the embryos.

Pour the TCA off into a waste bottle and wash the embryos once in distilled water (use forceps to transfer embryos or hold them in the jar).

Cover the embryos with Alcian Green stain (0.1% in acid alcohol) Acid alcohol is 1% HCl in 70% ethanol. Leave several hours or over night. Stain will bind tightly to cartilage and loosely to other tissues so the embryos will be very green after staining.

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Pour off the stain and transfer the embryos to acid alcohol (destain), incubate overnight.

Replace the acid alcohol with absolute ethanol, two changes of one hour each. This is a dehydration step to remove excess water.

Transfer the embryos to methyl salicylate to clear, clearing may take several hours.Caution - methyl salicylate is toxic! Handle with care, and clean up any spills.

Observe the stained embryos in methyl salicylate under the dissecting microscope. Sketch and label what you see, paying particular attention to the limb buds. Use your textbook or a comparative vertebrate anatomy book as a reference for the bones of the chick wing and leg.

Reference: Tickle, C. (1993) Chick Limb Buds in Essential Developmental Biology A Practical Approach, C. D. Stern and P. W. H. Holland ed., IRL Press at Oxford University Press, Oxford UK pp. 119-125

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Developmental Biology Laboratory XI

Effect of retinoic acid on zebrafish developmenthttp://zfish.uoregon.edu/zf_info/zfbook/stages/stages.html

Obtain several zebrafish embryos and sort them by developmental stage. Be sure to have both control and experimental embryos free of debris Design an experiment to test the effects of retinoic acid on embryonic development. The RA concentration can vary from 0.1 M to 1 M. Depending on the number of embryos we get, you may be able to use only one or several concentrations.

We will have:aged tap watersmall glass petri dishes transfer pipets A stock solution of retinoic acid in aged tap water, the concentration of which will dictate how much or how many dilute solutions we can make. microscopes

Your experiment could include prolonged exposure or be a pulse chase (washout) experiment. You must plan at least one time outside of this period to view the embryos (larvae), which can include Friday at 10 a.m. (our class time). Make groups a manageable size (5-10 if we have that many) and be sure to reserve some untreated embryos as a control. Keep accurate logs of your treatment (s) and their results in your lab book.

Ongoing: Observation of chick embryos stained for cartilage

Caution - methyl salicylate is toxic! Wear gloves and tip the embryos out (or transfer them with forceps) into a glass petri dish under the hood. Pour excess methyl salicylate back into your staining bottle, leaving just enough to cover the embryo. Handle stained embryos with care, and clean up any spills.

Observe the stained embryos in methyl salicylate under the dissecting microscope. Sketch and label what you see, paying particular attention to the limb buds. Use your textbook or a comparative vertebrate anatomy book as a reference for the bones of the chick wing and leg.

Biology 306 Laboratory

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Developmental Biology Laboratory XII

Comparative study of stages of mammalian embryonic development.

See typewritten exercise by Dr. Weaver, attached.

Bring Mathews and Schoenwolf Atlas of Descriptive Embryology 5th edition with you to labOther references on rat development will be available in the laboratory.

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Biology 306 Laboratory XIVPresentations and Peer Reviews

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