INQUIRY & An Authentic Research Experience INVESTIGATION for Undergraduates in the Developmental Biology and Physiology Using the Chick Embryonic Heart

• JACQUELINE S. McLAUGHLIN, MIT A. PATEL

ABSTRACT 2014; Lopatto, 2003; Seago, 1992). A four-step pedagogical frame- The lab presented in this paper utilizes a proven four-step pedagogical work that includes all essential elements of authentic research has framework (McLaughlin & Coyle, 2016) to redesign a classic Association of been developed that can serve to simplify and streamline the develop- Biology Laboratory Education (ABLE) undergraduate lab (McLaughlin & ment and implementation process in an introductory biology labora- McCain, 1999) into an authentic research experience on vertebrate four- tory setting (McLaughlin & Coyle, 2016). This framework has been chambered heart development and physiology. The model system is the shown to improve student perceptions of science and research, skills chicken embryo. Through their research, students are also exposed to the and knowledge levels in scientific research practices set forth by the embryonic anatomy and physiology of the vertebrate heart, the electrical National Research Council (2000), and confidence to effectively circuitry of the developing heart, and the effects of pharmacological drugs on heart rate and contractility. Classical embryological micro-techniques, engage in scientific research practices including the ability to write explantation of the embryo, surgical removal of the beating heart, isolation strong scientific lab reports and read scientific papers (McLaughlin of the heart chambers, and more advanced tissue culture methods are also et al., 2017). These results are consistent with other studies which conducted. In this laboratory paradigm, students work in pairs to ask their measured students’ perceptions and attitudes (Brownell et al., 2012; own questions concerning the effects of two human cardiovascular drugs, Harrison et al., 2011; Caruso et al., 2009; Weaver et al., 2008; ™ ™ denopamine and acebutolol on both in vivo and in vitro chicken Howard & Miskowski, 2005), ability to design experiments and inter- embryonic heart rate and contractility, develop testable hypotheses based on pret data (Myers & Burgess, 2003), and ability to develop information information gathered from relevant scientific literature, devise and carry out fluency (Gehring & Eastman, 2008) in laboratory courses that inte- a controlled experiment, and present the data in a professional scientific manner pertaining to a topic of clinical significance. grated authentic research-based experiences into the curriculum. Course-based The chicken embryo is a classic model Key Words: authentic research; heart development; organism used to illustrate the principles of chicken embryo; developmental biology laboratory; undergraduate physiology laboratory; undergraduate research. basic vertebrate embryology primarily because research experiences it is easily accessible, inexpensive, and direct observation of a living embryo is possible even Introduction (CUREs) provide a at the early stages of embryogenesis. With the aid of a dissecting , the key stages Course-based undergraduate research experi- way to increase and of heart development and the circulatory pat- ences (CUREs) provide a way to increase broaden terns can be observed in vivo, even when the and broaden participation of students in chicken embryo is still attached to the yolk. authentic research (Auchincloss et al., 2014; participation of Another advantage to using the live chicken Bangera & Brownell, 2014), and train them embryo to demonstrate the principles of heart in the essential elements of authentic students in authentic development is that the embryo can be research, which include reading scientific lit- research. removed from the shell and maintained in vitro erature, generating questions (that don’t cur- in a temperature controlled environment for rently have answers), forming hypotheses, designing experiments, several hours while the beating of the heart, flow of blood, and collecting and analyzing data, working toward significant findings, the developmental stages accompanying the formation of the four and presenting results in both oral and written forms (Spell et al., chambers are even more clearly visible. In addition, the chicken

The American Biology Teacher, Vol. 79, No 8, pages. 645–654, ISSN 0002-7685, electronic ISSN 1938-4211. © 2017 National Association of Biology Teachers. All rights reserved. Please direct all requests for permission to photocopy or reproduce article content through the University of California Press’s Reprints and Permissions web page, www.ucpress.edu/journals.php?p=reprints. DOI: https://doi.org/10.1525/abt.2017.79.8.645.

THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 645 embryonic heart is large enough for properly trained students to surgically isolate it from the embryo. Thus, students who work with this particular embryo in their development and/or physiology lab- oratories not only learn principles of vertebrate mor- phogenesis, organogenesis, and cardiac physiology, but are also exposed to simple, yet useful micro- manipulative techniques. These particular dexterous manipulations allow many of those who aspire to become physicians to try their hands (no pun intended) at surgical technique. We thoroughly redesigned a classic cookbook Figure 1. Illustration of human heart organogenesis from (A) a “tubular Association of Biology Laboratory Education (ABLE) heart” to (B) an S-shaped tube with the prominent ventricle bulging to the undergraduate lab on vertebrate heart development right (Gray, 1918/2000). © 2000 copyright Bartleby.com, Inc. (McLaughlin and McCain, 1999) to incorporate the above highlighted four-step laboratory pedagogical framework. This allows students to carry-out a higher level inquiry-based, authentic research experience (McLaughlin & Coyle, 2016; Goedhart & McLaugh- lin, 2016; McLaughlin et al., 2017) using present-day cardiovascular used as a model system for human heart development. The hearts drugs and updated tissue culture techniques and equipment. Stu- of the human and chicken embryos develop from the fusion of dents also master more advanced developmental biology, cardio- paired precardiac mesodermal tubes (aka “endocardial tubes”) vascular physiology, and pharmacology concepts. located on either side of the developing foregut, on the ventral sur- face. These paired tubes begin to fuse at the anterior end (head) and continue to fuse posteriorly to form one continuous tube Lab Protocol known as the tubular heart (Figure 1a). Once formed, the tubular heart is completely ventral to the foregut, and it has five distinct Developmental and Physiological Aspects of the regions that can be identified from anterior to posterior: truncus Chicken Embryonic Heart: Chronotropic and arteriosus, bulbus cordis, primitive ventricle, primitive atrium, and Inotropic Effects of Select Drugs sinus venosus (Figure 1b). Blood flows anteriorly, from the sinus venosus to the truncus arteriosus (future ascending aorta and pul- monary trunk). The heart begins to beat just after the paired heart Objectives tubes begin to fuse. It is the sinus venosus that becomes the future embryonic pacemaker. The key objectives in this lab are three-fold: (1) to think critically The tubular heart then elongates on the right side, looping and about, and understand, how the four-chambered vertebrate heart bending to form an S shape, with the prominent ventricle bulging develops using the chicken embryo as a model system; (2) to think to the right (Figures 1b and 2). The heart continues to fold upon critically about specific pharmacological agent(s) and their modes itself, moving the sinus venosus and atrium to a position anterior of action(s) at both the cellular and organ (heart) levels; and (3) to carry out a scientific research experiment. To accomplish these objectives, you will have eight weeks to carry out a four-step pro- cess wherein you will design and execute an authentic research project. This experimental framework is consistent with the man- ner in which professional research scientists design, execute, inter- pret, and communicate their experimental results. The over-arching experimental question that you will be attempting to answer through your research is the following: How do the cardiovascular drugs, denopamine™ and acebutolol™, affect the in vitro heart rate (chronotropic effect) and force of contraction (inotropic effect) of the developing isolated chicken heart?

Background Knowledge The development of the four-chambered vertebrate heart involves a series of cellular migrations, fusions, and specific differentiations— that is, a multitude of morphogenetic events. Although gestation of Figure 2. Head and S-shaped heart of a developing chick the human and chick differ, their four-chambered hearts develop in embryo at 1.5 days (~36 hours), viewed from the ventral surface a very similar fashion, and because of this the chicken embryo is (Gray, 1918/2000). © 2000 copyright Bartleby.com, Inc.

646 THE AMERICAN BIOLOGY TEACHER VOLUME. 79, NO. 8, OCTOBER 2017 Figure 3. The developmental stages of a human heart beginning with the fusion of two endocardial tubes up to specific compartmentalization. Illustration of development of heart by OpenStax is licensed under CC by 3.0. and dorsal to the ventricle and the bulbus cordis (Figure 3). The ven- Step 2. Design an Experiment tricle is now U-shaped and in the medial ventral position. The blood You will work with your lab partner to research, select, and read flows posteriorly and then makes a sharp turn to flow anteriorly. primary literature articles (at least three) related to the above The atrium then expands to the left in preparation for its divi- guided question in order to devise a more specific, self-directed sion into the right and left atria (Figure 3). Although the heart still research question. You will then formulate a hypothesis directed has two chambers at this time, communication between the sinus toward your specific question that is substantiated by the reference venosus and the atrium is via the right side of the atrium. This is literature you select. Your next step is to design an experiment that the first step toward the sinus venosus becoming part of the future attempts to test your hypotheses and incorporates the knowledge right atrium (SA node). The bulbus cordis will eventually give rise you have gained on your selected drug(s), using an appropriate to parts of the ventricles, while the truncus arteriosus will become control. Your experimental design must be written up as a tradi- the future ascending aorta and pulmonary trunk. Eventually, when tional research protocol, including detailed procedures and data the atrium and ventricle have each divided into a pair of chambers, a typical four-chambered heart is present (Figure 3). interpretation. You should consider the following when writing your protocol: (a) how you will administer the drug to an isolated chicken heart; (b) what concentrations you will use (you must have Laboratory Assignment Below is a description of the four steps you will be following to carry out authentic research and to address the question at hand (Figure 4). Each step will require at least two weeks.

Step 1. Learn Essential Lab Techniques During step one, you will learn essential and relevant in vivo and in vitro experimental techniques associated with both chicken embryo and heart manipulation via instruc- tor(s) demonstrations and hands-on train- ing. This step will include a small-scale and instructor-led exercise (use Appendix 1 Figure 4. The four-step pedagogical framework for authentic scientific research worksheet). (McLaughlin & Coyle, 2016).

THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 647 at least three different concentrations); (c) what will be your con- • Beaker for egg waste (400 mL) trol; (d) what kind of data you will collect; (e) how you will collect • DI water squirt for rinsing off embryological tools the data; and (f) how you will represent these data in their final • Scotch Magic tape form. Your instructor(s) must review your protocol to ensure that • it is adequately written, relevant, referenced, and executable within stopwatch (digital or cell phone) the allotted time frame before you are allowed to proceed with your • biohazard waste disposal experiment. Notes for Instructors Step 3. Conduct the Experiment *Fertilized eggs at specific days of development can be ordered and Conduct this experiment using learned techniques while gathering shipped in-state or out-of-state through Moyer’s Chick Farm, Qua- data in your notebook. This will require coordinated work with kertown, PA (215-536-3155). † your lab partner, and you will be expected to make good use of Any cardiovascular drugs with agonist/antagonist effect can be open laboratory time, in addition to scheduled class time. Your used in this experiment. ‡ instructor(s) will be available for assistance or questions, but the Contact Bioptechs, Butler, PA (420-050-4212) directly, if you expectation is that your work will be self-directed and paced would like to try an EDU system using chick embryos in your appropriately. classroom and to discuss pricing. §See Appendix 2 for detailed instructions for making micro-knives, Step 4. Interpret Data & Communicate Results embryo spoons, and doughnuts. Review your results and interpret your data. If your data are puz- I. Preparation of Chicken Egg and Bioptechs™ Equipment zling or unclear, your instructor(s) can help you make sense of it. Once you are able to draw the conclusions of your experiment, 1. Order eggs (in advance) from a chicken farm to receive fer- you will present it to your instructor(s) in the form of a formal tilized eggs of the appropriate day(s) (i.e., ages). Following research paper or an oral presentation. pick-up, store eggs in a chicken egg at 41°C until use. 2. Place CMRL media in a 45°C water bath two hours prior to Techniques use. (CMRL media has been shown to be the optimal media for in vitro chicken heart experimentation in our lab.) Materials and Equipment (per student pairs) 3. Place all the glassware in incubator at 41°C two hours prior • chicken eggs: 3, 4, or 5 days old (half dozen eggs per week)* to use. • chicken egg humidified incubator 4. Set up Bioptechs EDU Culture Dish Control System on one • CMRL (Connaught Medical Research ) media 1066 of the two dissecting scopes. Verify that EDU dish is [Ward’s Science] inserted correctly and system is attached to the power supply. • dish (110 mm diameter) lined with cotton (2) 5. Add a small amount of warm CMRL media to the Delta T • stock solution of R(–) denopamine™ [0.1mg/ml] Sigma Aldrich † culture dish. Allow the culture dish about two minutes to D7815 reach selected 41°C (avian) temperature. Close the lid of • stock solution of acebutolol HCl™ [0.1mg/ml] Sigma Aldrich † the dish. A3669 II. Windowing an Egg (modified from Cruz, 1993) • Falcon centrifuge tubes, 15 mL (6) 1. Obtain an egg (day 3, 4, or 5) from the incubator and place • water bath horizontally on a glass dish lined with cotton (under a • incubator for glass dishes and drugs gooseneck lamp). • dissecting microscope (illumination from above and below the 2. Use Scotch Magic tape to tape along the center of the long specimen) (2) axis of the egg, covering most of the top surface of the • gooseneck lamp with 100 W bulb (2) egg. Place two more pieces of tape on either side of the cen- ter piece (Figure 5a). • Syracuse culture dishes, 65 mm (2) ‡ 3. Puncture the rounded end of the egg using a 20 G needle. • Delta T-EDU Culture Dish Control System [Bioptechs] Insert the needle pointing down into the egg. Withdraw • 20 G needle attached to a syringe 1–2 mL of albumen. This allows the embryo to move away • iris fine scissors (2) from the upper surface of the egg, where you will be cutting • iris micro-dissecting scissors out the window. Discard the albumen and rinse syringe with water. • fine forceps (2) 4. CAREFULLY puncture the tape-covered surface of the egg • micro-knife (hatchet)§ with the tip of your fine scissors. The location of the punc- • § embryo spoon ture should be about half an inch off-center (Figure 5a). • § filter paper doughnuts 5. Cut out an oval opening while pulling up with your scissors • transfer (beral) , 1.5 mL (5) to keep them as far away as possible from the embryo and

648 THE AMERICAN BIOLOGY TEACHER VOLUME. 79, NO. 8, OCTOBER 2017 Figure 5. The isolation of heart from a 5-day-old chicken embryo illustrated in a series of six steps: (A) preparing to “window an egg” using three vertical strips of tape placed across the long axis of an egg; (B) cutting an oval opening using fine scissors to expose the embryo; (C) “explantation of the embryo” within its amniotic membrane using embryo spoon; (D) removal of amniotic and allantois membranes with fine forceps and/or micro-dissecting scissors in Bioptechs equipment; (E) surgical “isolation of the heart” from the embryo using fine forceps and micro-dissecting scissors/micro-knife; and, (F) “administration of drug” to the isolated heart using plastic transfer .

THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 649 vitelline membrane (membrane around yolk). The size of bpm in your lab notebook. How does this heart rate com- the opening depends on the size of the egg, but it should pare with the in vivo heart rate? – be about 1.5 2.0 inches in diameter. Remove the shell cap 9. Use Atlas of Descriptive Embryology to identify the exact stage of with forceps, exposing the window (Figure 5b). development of your embryo. Record this in your laboratory 6. If you are going to observe the embryo for more than a notebook. couple minutes while it is still in the egg, add several drops IV. Isolation of the Heart (modified from McLaughlin & McCain, of CMRL media to the surface of the embryo to prevent 1999) dehydration. Do not add any CMRL media if you are going 1. The explanted chicken embryo should be oriented ventral to immediately explant the embryo. This will prevent the side up; this allows you to access the beating heart. filter paper doughnut from adhering to the vitelline 2. Surgically remove the beating heart by cutting it above the membrane. bulbus cordis and below the sinus venosus of the atrium in vivo embryo 7. Determine the heart rate of the three times (Figure 5e and 1b). Take a picture of the isolated heart. at 15-second intervals. Record the heart rate in beats per 3. Determine the in vitro heart rate of the isolated heart three minute (bpm) in your lab notebook. [It is recommended times at 15-second intervals. Record the heart rate in bpm that one student record the time using a stop-watch while in your lab notebook. another student counts heart beats of the embryo looking † V. Administration of Cardiovascular Drugs through the microscope.] 1. Prepare the serial dilutions of the drug(s) that you chose to III. Explantation of a Chicken Embryo (modified from Cruz, 1993) work with. Make each dilution in a separate Falcon centri- 1. Explantation refers to removal of an embryo from its nor- fuge tube using the warm CMRL media as your solvent. mal in vivo environment and placement in a new location; Store your labeled tubes in the rack at 45°C water bath or in this case the embryo is transferred to a Bioptechs EDU incubator. Culture Dish. This is a useful method because it is much 2. Design table(s) similar to the ones in Appendix 1, wherein easier to manipulate or operate on an embryo when it is you adequately record all of your heart rate data per your in a dish, rather than in an egg. To begin this process, experimental design. Remember to include both in vivo add about quarter inch of warm CMRL media into a and in vitro embryonic heart rates (control baseline heart pre-warmed Syracuse dish. Then, place the dish on the rates) before you exogenously expose your embryos to any stage of your first dissecting microscope and angle the drugs. gooseneck lamp as close to the dish as possible in order 3. Remove as much CMRL media bathing the isolated heart as to keep the media warm. This is a critical step since the possible using transfer pipette, and add 1 mL of the lowest ’ chicken embryo s normal body temperature needs to remain concentration of the drug to the isolated heart in the dish (Fig- at 41°C. ure 5e). Wait at least 30 seconds for it to equilibrate, then 2. *Using forceps, gently place a filter paper doughnut around record the heart rate (three times at 15-second intervals), not- the embryo such that it frames the embryo. The filter paper ing any arrhythmias (such as tachycardia, bradycardia, atrial will stick to the vitelline membrane, and the embryo will be flutter, fibrillation, etc.) or changes in heart contractility (vari- exposed through the hole. ation in force of contraction in any or all chambers). Remove 3. Using surgical scissors, cut along the edges of the filter the drug as best as you can from the Bioptechs EDU dish using paper (vitelline membrane) off the surface of the egg while a transfer pipette, then immediately add the next higher dilu- holding it with forceps. As you release the filter paper, the tion of the drug. Repeat the procedures in this step to obtain embryo will remain attached to it. in vitro heart rate for all your drug dilutions. 4. Quickly transfer the filter paper doughnut along with 4. It is imperative that you work up at least five embryos per the attached embryo into a Syracuse culture dish already placed age group(s) selected. For example, if you chose to observe the ™ on the microscope. effect of acebutolol on the 4- and 5-day old isolated chicken hearts, you will need to work up five 4-day-old eggs and five 5. If the embryo doesn’t stay attached to the filter paper, use 5-day-old eggs. Work up your embryos one at a time, repeat- your embryo spoon to collect it from the underlying yolk. ing Steps II–VI and recording the control heart rates at all Remember to replace the yolk that transferred over from this stages. process with fresh CMRL media several times (Figure 5c). 5. Carry out your experiment! 6. Remove the embryo from all attached extra-embryonic mem- branes and extraneous yolk using fine forceps (Figure 5d). 7. Then, carefully transfer the embryo from the Syracuse dish Notes for Students into the Bioptechs EDU dish placed on your second dissect- *Use a filter paper donut on 3- and 4-day-old embryos; use an ing microscope using fine forceps. Check to make sure the embryo spoon only on 5-day-old embryos. † temperature is set to 41°C (avian). Add warm CMRL media, This step is preformed following the approval of your lab protocol if needed. by your instructor(s). 8. Determine the in vitro heart rate of the explanted embryo Important: Discard egg shells, embryonic waste and embryos in three times at 15-second intervals. Record the heart rate in appropriate biohazard containers.

650 THE AMERICAN BIOLOGY TEACHER VOLUME. 79, NO. 8, OCTOBER 2017 Conclusion Additionally, the four steps outlined herein should scaffold the sci- entific process, allowing students who are novices to the scientific The simplicity and flexibility involved in the four-step pedagogical process to progressively gain familiarity and comfort with the laboratory framework allows it to be easily adopted for use within essential elements of scientific inquiry. Lastly, it is recommended the unique infrastructure and resourceful environments at a variety that instructor(s) read how this framework was used by other of institutions and at different levels of biological study, effectively instructors to rework the semester with regard to time and work- increasing student access to authentic scientific research. For exam- load (McLaughlin & Coyle, 2016; Goedhart & McLaughlin, 2016). ple, this pedagogical framework was used to successfully transform a sophomore-level, introductory, cell biology laboratory for majors at a 4-year college branch campus (McLaughlin & Coyle, 2016) and an honors, introductory biology laboratory for non-majors at Acknowledgement a 2-year college (Goedhart & McLaughlin, 2016) into authentic We would like to extend our gratitude to Michelle Lynn for sketch- research experiences. For the former, students investigated various ing a realistic illustrations of the chicken heart isolation procedure. factors affecting the growth and viability of a mammalian cell cul- We would also thank Dr. Elizabeth McCain, who worked tirelessly ture line, Vero cells, and related their findings to a current issue with Dr. Jacqueline McLaughlin on publishing the original version in the field of cell biology, nutritional and/or physical stress of cells. (ABLE) of this lab. For the latter, students selected and investigated factors affecting microalgae cell growth and related their findings to a real-life appli- cation of social significance. A short video documenting the use of the pedagogical framework from both instructor and student pers- References pectives, From Cookbook to Critical Thinking (https://vimeo.com/ 118326855), was created by Citrus College and published on Vimeo, Auchincloss, L. C., Laursen, S. L., Branchaw, J. L., Eagan, K., Graham, M., … Inc. In both laboratory paradigms, students worked within groups to Hanauer, D. I., Dolan, E. L. (2014). Assessment of course-based undergraduate research experiences: A meeting report. CBE-Life learn modern cellular biology techniques, ask their own questions, Sciences Education, 13,29–40. develop testable hypotheses based on information gathered from rele- Bangera, G., & Brownell, S. E. (2014). Course-based undergraduate research vant scientific literature, devise and carry out a controlled experiment, experiences can make scientific research more inclusive. CBE-Life and present the data in a professional scientific manner. Sciences Education, 13, 602–606. The lab presented in this paper also utilizes the four-step ped- Brownell, S. E., Kloser, M. J., Fukami, T., & Shavelson, R. (2012). Undergraduate agogical framework, but this time to transform a sophomore-level, biology lab courses: Comparing the impact of traditionally based introductory, developmental biology laboratory for biology majors. “Cookbook” and authentic research-based courses on student lab Students investigate the development of the vertebrate four- experiences. Journal of College Science Teaching, 41(4), 36–45. chambered heart using the chicken embryo as a model system, Caruso, E. M., Mead, N. L., & Balcetis, E. (2009). Political partisanship while simultaneously being exposed to the embryonic anatomy influences perception of biracial candidates’ skin tone. Proceedings of and physiology of vertebrate heart, the electrical circuitry of the the National Academy of Sciences of the United States of America, 106 – developing heart, and the effects of pharmacological drugs on heart (48), 20168 20173. rate and contractility. Classical embryological micro-techniques and Cruz, Y. P. (1993). Laboratory exercises in developmental biology. San Diego, CA: Academic Press. procedures are conducted as well. Student, working in pairs, devise Gehring, K. M., & Eastman, D. A. (2008). Information fluency for undergraduate a hypothesis concerning the effects of two human cardiovascular ™ ™ biology majors: Applications of inquiry-based learning in a drugs, denopamine and acebutolol ,onin vitro heart rate and developmental biology course. CBE-Life Sciences Education, 7(1), 54–63. heart contractility. These drugs were selected for their known ago- Goedhart, C. M., & McLaughlin, J. S. (2016). Student scientists: Transforming nist or antagonist effects on the human heart rate. Other drugs that the undergraduate research laboratory into a research environment. have been successfully used in this lab to substitute these drugs American Biology Teacher, 78(6), 502–508. include dobutamine, verapamil, ractopamine, epinephrine, nico- Gray, H. (1918/2000). Anatomy of the Human Body. Philadelphia: Lea & tine, and pseudoephedrine. Indeed, the list of drug substitutions Febiger; Bartleby.com, 2000. is endless, and as such, allows the instructor(s) semester-to-semester Harrison, M., Dunbar, D., Ratmansky, L., Boyd, K., & Lopatto, D. (2011). versatility while maintaining the authentic research nature and, as Classroom-based science research at the introductory level: Changes in such, enhanced performance and engagement of students (lack of career choices and attitude. CBE-Life Science Education, 10, 279–286. plagiarism). To best exemplify the authentic research nature and Howard, D. R., & Miskowski, J. A. (2005). Using a module-based laboratory enhanced performance of students who have experienced the to incorporate inquiry into a large cell biology course. Cell Biology Education, 4, 249–260. chicken lab in particular is this reality: Many students have pre- sented their work at peer-reviewed conferences or have published Lopatto, D. (2003). The essential features of undergraduate research. Council on Undergraduate Research Quarterly, 24, 139–142. their research (e.g., Gonzalez et al., 2015). McLaughlin, J. S., & Coyle, M. S. (2016). Increasing authenticity and inquiry It is important to point out the following when utilizing the in the cell and molecular biology laboratory. American Biology Teacher, framework: Instructors should act as research chaperones, guiding 78(6), 492–501. student scientists through each step, providing constant feedback McLaughlin, J. S., Favre, D. E., Weinstein, S., & Goedhart, C. M. (2017). The and environments that allow time for student self-reflection, mis- impact of a four-step laboratory pedagogical framework on biology takes, and dialogue over assignments before their final submission students’ perceptions of laboratory skills, knowledge and interest in for grading (i.e., protocol, notebook, scientific paper, poster, etc.). research. Journal of College Science Teaching, 47(1), 83–91.

THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 651 McLaughlin, J. S., & McCain, E. R. (1999). Developmental and physiological Tyler, M. S. (1994). A guide for experimental study. Sunderland, MA: aspects of the chicken embryonic heart. In C. A. Goldman (Ed.), Tested Developmental Biology. Studies for Laboratory Teaching. Proceedings of the 20th Workshop/ Weaver, G. C., Russell, C. B., & Wink, D. J. (2008). Inquiry-based and Conference of the Association for Biology Laboratory Education (ABLE) (vol. 20, pp. 85–100). research-based laboratory pedagogies in undergraduate science. Myers, M. J., & Burgess, A. B. (2003). Inquiry-based laboratory course Nature Chemical Biology, 4(10), 577–580. improves students’ ability to design experiments and interpret data. Advances in Physiology Education, 27, 2633. National Research Council (NRC). (2000). Inquiry and the national science education standards. Washington, DC: National Academic Press. JACQUELINE S. MCLAUGHLIN ([email protected]) is an Associate Professor Seago, J. L. (1992). The role of research in undergraduate instruction. of Biology in the Department of Biology at the Pennsylvania State American Biology Teacher, 54, 401–405. University—Lehigh Valley, 2809 Saucon Valley Rd., Center Valley, PA Spell, R. M., Guinan, J. A., Miller, K. R., & Beck, C. W. (2014). Redefining authentic 18034. MIT A. PATEL ([email protected]) is a Postbac IRTA at National research experiences in introductory biology laboratories and barriers to Institute of Health in the Department of National Institute of Allergy and their implementation. CBE-Life Sciences Education, 13(1), 102–110. Infectious Diseases, 9000 Rockville Pike, Bethesda, MD 20892.

APPENDIX 1 Examining the Heart Rate of the Embryonic Heart Table 1. Summary of in vivo and in vitro heart rates of embryo and isolated heart. In vivo In vitro

Embryo Explanted Embryo Isolated heart HR (15 sec) HR (bpm) HR (15 sec) HR (bpm) HR (15 sec) HR (bpm) Trial 1 Trial 2 Trial 3 Average

Table 2. Summary of in vitro heart rates in each separated region of heart. In vitro

Bulbus Cordis Sinus Venosus Atrium Ventricle HR HR HR HR HR HR HR (15 sec) (bpm) (15 sec) (bpm) (15 sec) HR (bpm) (15 sec) (bpm) Trial 1 Trial 2 Trial 3 Average

1. In your lab notebook, using an embryological atlas like Atlas of Descriptive Embryology, diagram and thoroughly label 3-day-old and 4-day-old chicken hearts (whole mounts). 2. Window a 3- or 4-day-old egg, then determine and record the in vivo heart rate three times at 15-second intervals in Table 1. 3. Explant the embryo out of the egg, then determine and record the in vitro heart rate of the embryo three times at 15-second intervals in Table 1. • How does this heart rate compare to that of in vivo heart rate? 4. Surgically isolate the heart. Observe the beating of the isolated heart, then determine and record the in vitro heart rate of the heart three times at 15-second intervals in Table 1. • Where does the beating begin and end? Be precise. • Is the isolated heart rate similar or different from the heart beat of the explanted, in vitro embryo? • Draw a picture of the isolated heart and label each region.

652 THE AMERICAN BIOLOGY TEACHER VOLUME. 79, NO. 8, OCTOBER 2017 5. Use the micro-knife or iris micro-dissecting scissors to separate the four regions of the isolated heart (bulbus cordis, sinus venosus, atrium, and ventricle). Observe each isolated region of the heart, then determine and record the in vitro heart rate of each region three times at 15-second intervals in Table 2. • Does each region have an intrinsic heart beat? If so, are they synchronous? • Does each region beat at the same rate as the in vivo or in vitro explanted heart? • Draw a picture of the isolated regions of heart and appropriate labels. APPENDIX 2 Directions for Making Embryological Tools Micro-knives (modified from Tyler, 1994) 1. Break a single-edge razor blade into small fragments with the pliers or tin snips, making sure that each fragment has the cutting edge on it. Avoid damaging this edge. The fragment should be between 5 and 10 mm long. 2. Soak one end of the wooden applicator stick (2 mm diameter; 100–150 mm long) in water for about 5 minutes to soften the wood. Make a 5-mm split down the center of the moistened end of the stick with a fresh razor blade. Do not splay out the two sides of the split; just create a crack in the stick large enough for the razor blade fragment to fit in. 3. Carefully insert the rough edge of the razor blade fragment into the split at 45° angle to the axis of the stick. You can’t use the pliers to do this because they will damage the cutting edge. You must use your fingers—be careful! The cutting edge of the blade should not be embedded in the wood at all, and you should be able to comfortably hold the stick like a pencil and have the razor’s edge almost flat with a desk surface. 4. Let the wood completely dry. Paint the split edges with super glue to firm the placement of the razor blade fragment. 5. Store the micro-knives such that their edges are protected. One method is to stick Styrofoam peanuts on the razor edge. Note: Micro-knives can only be used for one lab period; they dull quickly.

Embryo Spoon (modified from Tyler, 1994) 1. Heat an insect or dissecting pin over a flame and puncture 8–10 holes in the bowl area of the plastic spoon (ice cream spoon), as shown in the picture. 2. Use the sandpaper (220 or 400 grit) to remove the sharp plastic edges.

Filter Paper Doughnuts 1. Fold a piece of filter paper (Whatman #1, 3.2 cm diameter) in half, then cut out a semi-circle in center of the filter paper, keeping 0.5 cm distance between two circles. 2. The end result should be an oval-shaped doughnut.

THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 653