Lecture D111L Week 05 S13

D111L D111L Developmental and Cell Biology LabDevelopmental and Cell Biology Lab

Week 5Week 5

Schedule for Today

• Learning Goals• Group Time to prepare for

Week 4 summaries• Week 4 summary• Lab Report• C.elegans mutant screen• Sparklix• Time for Groups to discuss

experimental setup

Lab Experiments Lab Report

Week

Lecture Theme

Pollen Tube

Germination

C.elegans Sea UrchinsFibroblast

3T3-L1Chick

5Genetic Screening

Student Designed Experiment

F2 Genetic Screen

6 Fertilization

Maintance of stock

Fertilization Draft of Lab Report in Criterion

7Sea Urchin Development

Whole Mounts & DAPI Staining

Check Development of Sea Urchins

Text Entry to CPR (Revised Draft)

8 Cell CultureMaintance of stock

Addition of Diet Supplement

Peer Review of Lab Reports

9Memorial Day

Whole Mounts & DAPI Staining

Image JRevised Lab Report to TA

10Chick Development

Chick Development

Learning Goals we are focusing on

• Understand the requirements of working with a research organism– C.elegans

• Maintain a lab notebook• Present the results of your experiment

– Lab Report

Questions about Questions about Last Week?Last Week?

Papaver hyridum ‘Lavender Breadseed”

Group TimeGroup TimeWeek 4Week 4

Successes and ChallengesSuccesses and ChallengesTalk with another group Talk with another group

from a different dayfrom a different day

Last Fall ClassLast Fall ClassWednesday’s Morning Wednesday’s Morning

Bench 3Bench 3Distictis buccinatoriaDistictis buccinatoria

Week 5 Lab Session

• Should be light (screening worms)

• All the Pollen germination materials will still be in the lab– You may (should) continue your Week 4

experiment

Lab ReportLab Report

Draft corrected in Criterion 6 points Week 6 CPR Results 10 points Week 7 & Week 8Final Lab Report (graded by TA) 40 points Week 9

Lab Report Grading

Content Points• Title• Introduction• Materials & Methods• Results• Discussion Points deducted for

Inaccurate citationInaccuracy of statementsWordiness or use of unscientific phrasesGrammatical and Spelling Errors

TA Rubric Mastery GoodSome

EvidenceLittle

Evidence

CPR Rubric Mastery GoodSome

Evidence

Loss of points due to

• Content Score minus deductions

• Late• Inaccurate citation• Inaccuracy of statements• Wordiness or use of unscientific phrases• Grammatical and Spelling Errors

General Objectives• An informative and focused title

Introduction• Organized in a logical manner- - Inverse pyramid format• Summarized the important background information relevant to

the study.• Explained the rational for the experiment in a logical fashion

citing primary literature• Balanced correctly the amount of general background and

focused information• Linked the rational from prior studies to the hypothesis

statement.• Stated a testable hypothesis• 3 references from primary articles

Materials and Methods• Included all pertinent information and details• Avoided step by step instructional style• Included a description of data analysis• Written in the past tense and first person active voice avoided

General Objectives

General Objectives

Results• Summarized the data.• No interpretation• Important trends or patterns emphasized• Graphs and tables have the correct units identified• Only necessary Illustration/s (graphs, tables) present• Graphs or tables referred to correctly in the text• Figure legends are complete and in the correct place• Assumptions made are stated

Discussion• Interpreted the results relative to the hypothesis. • Backed up ALL interpretation statements with data from results.• Chose only the most logical explanation not every possible

explanation.• Discussed other relevant primary literature in relationship to the

results and interpretation.• Used 3 primary articles• Discussed how confident they were of the results and where

there might be error that is relevant to the discussion.• Discussed how the results and interpretation fits with the bigger

picture of the topic.

General Objectives

Specific Details of Report Related to Objectives• An informative and focused title• Introduction• Organized in a logical manner- - Inverse pyramid format• Summarized the important background information relevant to the study.• Explained the rational for the experiment in a logical fashion citing primary literature• Balanced correctly the amount of general background and focused information• Linked the rational from prior studies to the hypothesis statement.• Stated a testable hypothesis• 3 references from primary articles• Materials and Methods• Included all pertinent information and details• Avoided step by step instructional style• Included a description of data analysis• Written in the past tense and first person active voice avoided• Results• Summarized the data.• No interpretation• Important trends or patterns emphasized• Graphs and tables have the correct units identified• Only necessary Illustration/s (graphs, tables) present• Graphs or tables referred to correctly in the text• Figure legends are complete and in the correct place• Assumptions made are stated• Discussion• Interpreted the results relative to the hypothesis. • Backed up ALL interpretation statements with data from results.• Chose only the most logical explanation not every possible explanation.• Discussed other relevant primary literature in relationship to the results and interpretation.• Used 3 primary articles• Discussed how confident they were of the results and where there might be error that is relevant to the discussion.• Discussed how the results and interpretation fits with the bigger picture of the topic.

Overall Objectives• Easy to read and understand• The approach is insightful• Student conveys a clear understanding of the material • The ideas are clearly communicated with focus and

specificity• Reader is effortlessly guided through progression of

ideas• Sources used to develop writer’s purpose• Avoided unnecessary information

Criterion Draft• Due Wednesday, Week 6 PM• One week later- CPR text Entry

Caenorhabditis elegans

Post Lab Questions• How did your worm plate differ from the plate you used in

Week 4? • Carefully describe the phenotype of one of your mutants

(If you have do not find a mutant, describe one of the adult worms you see).

• Include in your description the following information. – Why you thought the worm had a mutation. – Compare and contrast the morphology of the mutant. – Compare and contrast the behavior (movement) of the

mutant. – Do you think your mutation is dominant or recessive and

why.

How did your P0 worm plate differ from what you expected?

A. More worms than I saw on my wild type transfer

B. Less worms than I saw on my wild type transfer

C. Worms were not a variety of stagesD. Number of worms looked just like I expectedE. Was not sure what I really expected to see

http://www.wormatlas.org/ver1/handbook/anatomyintro/anatomyintro.htm

Jorgensen, E.M., Mango, E.M. 2002 The Art and Design of Genetic Screens: C. elegans. Nat. Rev. Genet. 3: 356-69.

Sparklix NotebookSparklix NotebookRubricRubric

Rubric for Week 5: Notebook CheckContent Topic Full Credit Half Credit No Credit

Descriptions of Procedure

The notebook describes and explains all the critical procedural steps. The notebook can not have missing or inaccurate steps.

The notebook contains the basic steps but does not include additional explanations. The notebook can not have missing or inaccurate steps.

Notebook does not have any of the required elements or a reader can not understand the text.

Goals or Anticipated Outcomes Notebook contains explanations of the goals of the experiment or any predicted outcomes.

Notebook contains explanations of the goals of the experiment but not the predicted outcomes.


Experimental Design The notebook identifies the independent, and dependent variables.

The notebook identifies the variables involved but does not identify whether they are independent or dependent variables.


MaterialsThe notebook contains relevant detailed information about the materials used in the experiment.

The notebook contains some information about the materials being used in the experiment


ControlsThe notebook identifies any positive or negative controls. Controlled and uncontrolled variables are described

The notebook identifies any positive or negative controls. Controlled and uncontrolled variables are not describe.


Organization of Data

The data is clearly organized in a manner that would be understandable to someone that has not done the experiment.

The data is somewhat organized but a reader would need to be familiar with the experiment to understand the data.


Units and Calculations All measurements have the correct number of significant figures and units. Calculations are described.

Some measurements may not have the correct number of significant units. The description of calculations is incomplete.


Observations and/or Interpretations

The notebook contains all required images or drawings. The notebook has additional information or observations. Comments about what may be occurring in the experiment are included.

The notebook contains all required images. The notebook has some additional information or observations.


Reflections

The notebook contains a discussion and assessment of errors. Details about possible changes in the procedure is included in the notebook.

The notebook contains a discussion and assessment of errors.


Overall Quality Above expectations Meets expectations Below expectations

Observations and/or Interpretations• The notebook contains all required images or

drawings. The notebook has additional information or observations. Comments about what may be occurring in the experiment are included.

• The notebook contains all required images. The notebook has some additional information or observations.

• Notebook does not have any of the required elements or a reader can not understand the text.

ObservationsTake This Fish and Look at It

• "In the Laboratory With Agassiz," – by Samuel H. Scudder

• “Entymologist Samuel H. Scudder (1837-1911) wrote this account of his first learning encounter with the renowned ichthyologist Dr. Louis Agassiz (1807-1873), founder of Harvard University’s Lawrence School of Science. The story took place around 1859, was first published (anonymously) in 1873, and has since become a classic lesson in the value of close observation for analysis.”

Samuel L. Scudder, “Take This Fish and Look at It,” in Jo Ray McCuen and Abthony C. Winkler, Reading for Writers, 54th ed. (New York, San Diego, Chicago: Harcourt, Brace, and Jovanovich, Inc., 1983, 1980, 1977, 1974), pp.82-85.

The Student, the Fish, and Agassiz; by the Student

It was more than fifteen years ago that I entered the laboratory of Professor Agassiz, and told him I had enrolled my name in the Scientific School as a student of natural history. He asked me a few questions about my object in coming, my antecedents generally, the mode in which I afterwards proposed to use the knowledge I might acquire, and, finally, whether I wished to study any special branch. To the latter I replied that, while I wished to be well grounded in all departments of zoology, I purposed to devote myself specially to insects.

"When do you wish to begin?" he asked.

"Now," I replied.

This seemed to please him, and with an energetic "Very well!" he reached from a shelf a huge jar of specimens in yellow alcohol. "Take this fish," he said, "and look at it; we call it a haemulon; by and by I will ask what you have seen."

With that he left me, but in a moment returned with explicit instructions as to the care of the object entrusted to me.

"No man is fit to be a naturalist," said he, "who does not know how to take care of specimens."I was to keep the fish before me in a tin tray, and occasionally moisten the surface with alcohol from the jar, always taking care to replace the stopper tightly. Those were not the days of ground-glass stoppers and elegantly shaped exhibition jars; all the old students will recall the huge neckless glass bottles with their leaky, wax-besmeared corks, half eaten by insects, and begrimed with cellar dust. Entomology was a cleaner science than ichthyology, but the example of the Professor, who had unhesitatingly plunged to the bottom of the jar to produce the fish, was infectious; and though this alcohol had a "very ancient and fishlike smell," I really dared not show any aversion within these sacred precincts, and treated the alcohol as though it were pure water. Still I was conscious of a passing feeling of disappointment, for gazing at a fish did not commend itself to an ardent entomologist. My friends at home, too, were annoyed when they discovered that no amount of eau-de-Cologne would drown the perfume, which haunted me like a shadow.

In ten minutes I had seen all that could be seen in that fish, and started in search of the Professor—who had, however, left the Museum; and when I returned, after lingering over some of the odd animals stored in the upper apartment, my specimen was dry all over. I dashed the fluid over the fish as if to resuscitate the beast from a fainting fit, and looked with anxiety for a return of the normal sloppy appearance. This little excitement over, nothing was to be done but to return to a steadfast gaze at my mute companion. Half an hour passes—an hour—another hour; the fish began to look loathsome. I turned it over and around; looked it in the face—ghastly; from behind, beneath, above, sideways, at a three-quarters' view—just as ghastly. I was in despair; at an early hour I concluded that lunch was necessary; so, with infinite relief, the fish was carefully replaced in the jar, and for an hour I was free.

On my return, I learned that Professor Agassiz had been at the Museum, but had gone, and would not return for several hours. My fellow-students were too busy to be disturbed by continued conversation. Slowly I drew forth that hideous fish, and with a feeling of desperation again looked at it. I might not use a magnifying-glass; instruments of all kinds were interdicted. My two hands, my two eyes, and the fish: it seemed a most limited field. I pushed my finger down its throat to feel how sharp the teeth were. I began to count the scales in the different rows, until I was convinced that that was nonsense. At last a happy thought struck me—I would draw the fish; and now with surprise I began to discover new features in the creature. Just then the Professor returned.

"That is right," said he; "a pencil is one of the best of eyes. I am glad to notice, too, that you keep your specimen wet, and your bottle corked."

With these encouraging words, he added, "Well, what is it like?"

He listened attentively to my brief rehearsal of the structure of parts whose names were still unknowns to me: the fringed gill-arches and movable operculum; the pores of the head, fleshy lips and lidless eyes; the lateral line, the spinous fins and forked tail; the compressed and arched body. When I finished, he waited as if expecting more, and then, with an air of disappointment, "You have not looked very carefully; why," he continued more earnestly, "you haven't even seen one of the most conspicuous features of the animal, which is a plainly before your eyes as the fish itself; look again, look again!" and he left me to my misery.

I was piqued; I was mortified. Still more of that wretched fish! But now I set myself to my tasks with a will, and discovered one new thing after another, until I saw how just the Professor's criticism had been. The afternoon passed quickly; and when, towards its close, the Professor inquired, "Do you see it yet?"

"No," I replied, "I am certain I do not, but I see how little I saw before."

"That is next best," said he, earnestly, "but I won't hear you now; put away your fish and go home; perhaps you will be ready with a better answer in the morning. I will examine you before you look at the fish."

This was disconcerting. Not only must I think of my fish all night, studying, without the object before me, what this unknown but most visible feature might be; but also, without reviewing my discoveries, I must give an exact account of them the next day. I had a bad memory; so I walked home by Charles River in a distracted state, with my two perplexities. The cordial greeting from the Professor the next morning was reassuring; here was a man who seemed to be quite as anxious as I that I should see for myself what he saw. "Do you perhaps mean," I asked, "that the fish has symmetrical sides with paired organs?"His thoroughly pleased "Of course! of course!" repaid the wakeful hours of the previous night. After he had discoursed most happily and enthusiastically—as he always did-—upon the importance of this point, I ventured to ask what I should do next.

"Oh, look at your fish!" he said, and left me again to my own devices. In a little more than an hour he returned, and heard my new catalogue.

"That is good, that is good!" he repeated; "but that is not all; go on"; and so for three long days he placed that fish before my eyes, forbidding me to look at anything else, or to use any artificial aid. "Look, look, look," was his repeated injunction.

This was the best entomological lesson I ever had—a lesson whose influence has extended to the details of every subsequent study; a legacy the Professor had left to me, as he has left it to many others, of inestimable value, which we could not buy, with which we cannot part.

A year afterward, some of us were amusing ourselves with chalking outlandish beasts on the Museum blackboard. We drew prancing starfishes; frogs in mortal combat; hydra-headed worms; stately crawfishes, standing on their tails, bearing aloft umbrellas; and grotesque fishes with gaping mouths and staring eyes. The Professor came in shortly after, and was as amused as any at our experiments. He looked at the fishes. "Haemulons, every one of them," he said; "Mr. ---- drew them."

True; and to this day, if I attempt a fish, I can draw nothing but haemulons.

The fourth day, a second fish of the same group was placed beside the first, and I was bidden to point out the resemblances and differences between the two; another and another followed, until the entire family lay before me, and a whole legion of jars covered the table and surrounding shelves; the odor had become a pleasant perfume; and even now, the sight of an old, six-inch worm-eaten cork brings fragrant memories.

The whole group of haemulon was thus brought in review; and, whether engaged upon the dissection of the internal organs, the preparation and examination of the bony framework, or the description of the various parts, Agassiz’s training in the methods of observing facts and their orderly arrangement was ever accompanied by the urgent exhortation not to be content with them.

“Facts are stupid things,” he would say, “until brought into connection with some general law.”

At the end of eight months, it was almost with reluctance that I left these friends and turned to insects; but what I had gained by this outside experience has been of greater value than years of later investigations in my favorite groups.

Take this Wormand Look at It

Procedure

• Observe your worms in your F1 and Po plates• Transfer any worm that appears to be

different – Try and only transfer a single worm

• Label the transferred worm plate– Lab section: Date: Bench #: Worm ID #– Worm ID # Your initials followed by 01-03

Worm Tracts

Types of Common Mutants

• Broad Categories

• Morphology• Behavioral

– Defects in movement

Uncoordinated mutants

Wild type movement• Sine wave on agar

– Dorsal-ventral bends– Both forward and reverse

• Head can move in any directionMutants• Wide variety of phenotypes

unc mutants• Neuron defects

– Early and late– Conserved sequences

• Navigation problems– Neurons grow in the wrong directions

• Receptor defects– Acetylcholine receptors

• Muscle defects

http://www.hhmi.org/news/caldwell2.html

Movies at http://www.bama.ua.edu/~gcaldwel/video.html



Roller Mutants

• Rotates around its body length as it moves– Moves in circles

• Form craters• Usually seen in adults

• Video• http://www.youtube.com/watch?v=V8p62xeI2Ec

http://www.youtube.com/watch?feature=endscreen&NR=1&v=mwlEWqz_5yY

Published on Jun 12, 2012 by wormbehavior C.elegans experiment - gene: del-1;unc-8, strain:AQ2937, allele:ok150;e15lb145, chromosome:X;IV, genotype:unc-8(e15lb145)IV; del-1(ok150)X. This experiment is part of a collection of experiments that are analyzed in c.elegans behavioural database - http://wormbehavior.mrc-lmb.cam.ac.uk

Future Classes

Morphology/ Size Mutants

• Shorter than wild type– Normal head size

• Dumpy-small diameter• Small- thinner• Long- longer and thinner

From Brenner, S. 1973. The Genetics of Caenorhabditid elegans. Genetics. 77: 71-94

Dumpy

• Connective tissue mutationCollagen (cuticle)• Cuticle requires at least 32 different collagens• dpy-13: dumpy phenotype• sqt-1: dumpy, long, roller phenotypes• rol-6: right roller

Figure 3. Cuticle collagen mutants and phenotypes. Panel A depicts Normaski images of a wild type animal, a dpy-7(qm63) and a dpy-13(e458);dpy-7(qm63) double adult stages. The dpy-13(e458) mutant is comparably Dpy to the dpy-7(qm63) mutant (not shown) whereas the dpy-13(e458);dpy-7(qm63) double displays a more severe dumpiness than either single mutant. Panel B shows scanning electron micrographs of the surface of adult stage wild type and mutant animals. The annuli of the mutant dpy-13(e458) are narrower than those of the wild type and are completely absent from dpy-7(qm63) mutants.

http://www.wormbook.org/chapters/www_cuticle/cuticle.html

Breeding

• Sterility• Do not bred true• Mixed offspring

• Dominant– Often lethal when homozygous

Group WorkDiscussion Activity

• Talk with your group about changes you may need to do for this week’s Pollen Experiment

• Hand in a brief summary of last week’s results and what you plan to do Week 5

Lecture D111L Week 05 S13

Documents

Transcript of Lecture D111L Week 05 S13