Investigating the effects of altered thyroid hormone levels on neural stem cell proliferation in the larval zebrafish hypothalamus.

Priyanjali Ghosh, PhD-University of Massachusetts-Amherst
Rolf O. Karlstrom, PhD- University of Massachusetts-Amherst
Location: Massachusetts

Abstract

The central nervous system of most vertebrate species consists of zones of neural stem cell (NSC) proliferation which retain the ability to undergo neuro/gliogenesis well into adulthood [1]. The two primary regions of adult neurogenesis in mammals are the ventricular and subventricular zones (V-SVZ) of the lateral ventricles and the subgranular zone (SGZ) of hippocampus [2–7]. Additionally, adult neurogenesis in the mammalian hypothalamus has also been reported [8–11]. Unlike mammals, neurogenesis is more abundant in reptiles, amphibians and fish [3, 12]. In fact, studies have identified 16 different regions of proliferation and neurogenesis in the adult zebrafish brain, and unlike mammalian species, neurogenesis occurs in all of these subdivisions in the zebrafish brain [2, 13, 14]. This makes the zebrafish a fantastic model organism for studying NSC proliferation and neuro/gliogenesis. Recent studies show that there are striking similarities and differences across all vertebrate species in the factors and mechanisms that regulate NSC proliferation and neuro/gliogenesis [1]. Thus, understanding these mechanisms is critical to understanding regenerative neurogenesis and to developing treatments for neurodegenerative diseases. One such interesting factor known to regulate NSC behavior throughout vertebrate life is thyroid hormone (TH). Appropriate amounts of TH are necessary for proper brain development in all vertebrates and studies have shown that TH plays an important role in maintaining NSC proliferation and fate determination in the central nervous system [15]. However, studies performed in rats and mice to understand the effects of TH on NSC proliferation reveal contradictory results. For example, low levels of TH are shown to decrease the proliferative rates of NSC in SVZ of mice [16] whereas the opposite effect is observed in the rat SVZ [17]. Not only does this suggest that the effect of TH may vary between species, it encourages us to explore the role of TH in the zebrafish brain. Specifically, for this CURE course, we are interested in studying the role of TH on NSC proliferation in the zebrafish hypothalamus. Why the hypothalamus? For one, the hypothalamus is the most ancient and evolutionarily conserved part of the vertebrate brain [18]. Second, life-long hypothalamic neurogenesis has been documented in rodents, zebrafish, and likely humans [5, 11, 19]. Lastly, very little is known about the role of TH in regulating the NSC proliferation in the hypothalamus (including that of the zebrafish), making the goal of thus CURE course novel.

Student Goals

  1. Students will generate hypotheses and predictions prior to their experiments.
  2. Students will learn how generate, troubleshoot and analyze data.
  3. Students will provide biological justifications for their experimental observations and generate a "new" set of hypotheses and predictions for future experiments.

Research Goals

  1. Students will investigate the effects of hypothyroidism on neural stem cell proliferation in the zebrafish hypothalamus.
  2. Students will investigate the effects of hyperthyroidism on neural stem cell proliferation in the zebrafish hypothalamus.

Context

This CURE course was developed to provide UMass-Amherst Biology Majors with a "real-world" research experience in the areas of physiology and neurobiology. The zebrafish model system will facilitate an exploration of neural stem cell proliferation in the vertebrate brain and allow students to generate and test the hypotheses pertaining to changes in hypothalamic proliferation rates in response to altered physiological conditions.

At UMass-Amherst, a Biology Major is required to take at least two laboratory based upper-level courses. This course, BIO397N, is one such upper level lab course. Most of the upper-level labs (including previous renditions of BIO397N) are comprised of experiments with known outcomes along with some form of an inquiry and/or discovery-based experimental component, usually in the form of an independent project (students design and implement towards the end of the semester). The current version of BIO397N has been specifically designed to meet the distinctive educational goals of a CURE—which includes providing students the opportunity to experience "real-world-research" , where the experimental outcome is unknown and the results from their work may be of importance to outside stakeholders.

Target Audience: Juniors and Seniors within the Biology Major
Number of students per section: 18
Number of sections per semester: 2 (Fall) and 4 (Spring)
CURE Duration: 13-week semester
Pre-requisites: Physiology and Genetics in addition to the standard series of courses a Biology Major takes in their Freshman and Sophomore years.

Target Audience:Major, Upper Division
CURE Duration:A full term

CURE Design

As described in the Abstract, the goal of this CURE is to manipulate thyroid hormone levels in larval zebrafish and quantify the number of proliferating neural stem cells in the hypothalamus. Due to the 13-week structure of the semester, the first 7 weeks of the course focus on three main components:
1.Developing the technical skills to perform the experiments--which include:
a. Staging and sorting zebrafish embryos.
b.Treating larval zebrafish with Propylthiouracil (PTU) (to mimic hypothyroidism), L-thyroxine (to mimic hyperthyroidism), and EdU (5-ethynyl-2´-deoxyuridine: a thymidine analog that incorporates into dividing cells).
c. Dissecting larval zebrafish brains.
d. Performing the EdU reaction.
e. Imaging brains.

Note: The Instructor, undergraduate and graduate TA's are experienced in zebrafish biology and having 1:6 ratio (instructor/TA: Student) allows each student to get a significant amount of personal help and guidance.

2.Understanding the background information pertinent to the research topic--which includes:
a. Zebrafish Brain Anatomy.
b. Hypothalamus-Pituitary-Thyroid Axis.
c. Neural Stem Cells, with a focus on understanding the difference between proliferation and differentiation.
d. Cell signaling pathways like the Notch Pathway.

Note: In order to cater to the different learning styles, each "topic" is presented in multiple ways. For example, the instructor often lectures on the white board and supplements the "lecture" with online diagrams, papers, website articles and videos. All these resources are easily accessible through the internet and Moodle. The classroom is also equipped with 8 computers and one printer allowing students to easily access the material.

3.Generating Hypotheses and Predictions:
a. Read pre-selected review articles.
b. Use the background information and information from review article to generate hypothesis and predictions.

4.Developing teamwork skills:
In this course, students will be working in pairs throughout the 13-week semester. They will be performing the experiments, analysis, and all the assignments as a group. This creates and environment where they can develop their "teamwork" skills including (but not limited to) inter-personal communication, adhering deadlines, problem-solving, decision-making and conflict resolution.

At the end of the first 7-weeks, the students present their preliminary results in the form of a PowerPoint Presentation. This "half-way" point in the semester is usually when students finally appreciate the full process of the experimental steps, come up with explanations for successes and failures and most importantly, they get a chance to see if their initial data supports or rejects their hypothesis and predictions.

During the second half of the semester, the students repeat the experiments implementing appropriate changes (as deemed from Trial 1) and focus on rectifying previously made experimental errors and increasing the number of samples analyzed. Additionally, students spend the second half of the semester reading primary literature papers (short-listed by instructors) to construct biological arguments to justify their experimental observations and also to generate a new hypothesis and predictions for future/ "next-step" experiments.

Core competencies: Generating hypotheses and predictions, learning how to follow and execute experimental protocols, imaging techniques and use of software like ImageJ, troubleshooting experiments, analysis of data, science communication, teamwork.

Nature of research: Zebrafish, Neurobiology, Physiology.

Results from this study will shed light on how thyroid hormones may potentially regulate neural stem cell proliferation in the hypothalamus. To date, this question has not been explored in great details and results from this study may be of interest to research groups (including the Karlstrom Lab at UMass-Amherst) studying the behavior of neural stem cells in the context of the developing and aging brain. Interested parties will have access to student presentations and papers.

Core Competencies:Analyzing and interpreting data
Nature of Research: Basic Research, Wet Lab/Bench Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Students will investigate the effects of hypothyroidism on neural stem cell proliferation in the zebrafish hypothalamus.
Research Goal 2: Students will investigate the effects of hyperthyroidism on neural stem cell proliferation in the zebrafish hypothalamus.

 

 


Student Goal 1: Students will generate hypotheses and predictions prior to their experiments.

Students will read a set of (pre-screened) review articles which will enable them to identify the "gaps in the knowledge" in the context of how thyroid hormones affect neural stem cell proliferation. Students will then generate hypotheses and predictions based on their readings. The readings were selected in a such a manner that the students can generate several acceptable hypotheses. This will enable students to learn and practice a key component of the scientific method—a skill that can be used in any field of research.

Students will read a set of (pre-screened) review articles which will enable them to identify the "gaps in the knowledge" in the context of how thyroid hormones affect neural stem cell proliferation. Students will then generate hypotheses and predictions based on their readings. The readings were selected in a such a manner that the students can generate several acceptable hypotheses. This will enable students to learn and practice a key component of the scientific method—a skill that can be used in any field of research.


 


Student Goal 2: Students will learn how generate, troubleshoot and analyze data.

1) Students will learn the following skills that will help them generate data:
a) Treating larval zebrafish with Propylthiouracil (PTU) (to mimic hypothyroidism), L-thyroxine (to mimic hyperthyroidism), and EdU (5-ethynyl-2´-deoxyuridine: a thymidine analog that incorporates into dividing cells)
b) Dissecting larval zebrafish brains
c) Performing the EdU "Click-it" reaction which allow students to fluorescently tag cells that are undergoing cell division.
d) Imaging brains

2) Students will learn how to troubleshoot their experiments by discussing the importance and purpose of each experimental step and how a mistake in any one of those steps may lead to an experimental failure. 

3) Students will learn the following to analyze data:
a) Students will learn how to use ImageJ software to create "z-stacks" and count cells.
b) Students will participate in a "class-wide" cell count "practice-sessions" to encourage consistency in how a cell is "defined" and "counted" from a fluorescent image.
c) Students will learn how to calculate standard deviations using Excel.
d) Students will receive guidance on how to organize their data in Excel so that they can generate clear, concise and informative graphs.

While some of these skills are very specific to "zebrafish biology", this entire experience will teach students the importance of understanding each step of an experimental procedure, the importance of being careful and thorough, and how communicate scientific data.

1) Students will learn the following skills that will help them generate data:
a) Treating larval zebrafish with Propylthiouracil (PTU) (to mimic hypothyroidism), L-thyroxine (to mimic hyperthyroidism), and EdU (5-ethynyl-2´-deoxyuridine: a thymidine analog that incorporates into dividing cells)
b) Dissecting larval zebrafish brains
c) Performing the EdU "Click-it" reaction which allow students to fluorescently tag cells that are undergoing cell division.
d) Imaging brains

2) Students will learn how to troubleshoot their experiments by discussing the importance and purpose of each experimental step and how a mistake in any one of those steps may lead to an experimental failure. 

3) Students will learn the following to analyze data:
a) Students will learn how to use ImageJ software to create "z-stacks" and count cells.
b) Students will participate in a "class-wide" cell count "practice-sessions" to encourage consistency in how a cell is "defined" and "counted" from a fluorescent image.
c) Students will learn how to calculate standard deviations using Excel.
d) Students will receive guidance on how to organize their data in Excel so that they can generate clear, concise and informative graphs.

While some of these skills are very specific to "zebrafish biology", this entire experience will teach students the importance of understanding each step of an experimental procedure, the importance of being careful and thorough, and how communicate scientific data.


 


Student Goal 3: Students will provide biological justifications for their experimental observations and generate a "new" set of hypotheses and predictions for future experiments.

After the first trial, students will determine whether their results support or reject their hypothesis. Then they will read additional primary literature papers to discuss and justify their biological observations. This will be the "discussion" section of the final paper that they will have to write. Additionally, students will also be asked to design "future experiments" and generate corresponding hypotheses and predictions for these new experiments. This exercise will allow students to practice their "critical thinking" skills and appreciate the fact that research is a cycle of observations and experiments that lead to the genesis of more questions.

After the first trial, students will determine whether their results support or reject their hypothesis. Then they will read additional primary literature papers to discuss and justify their biological observations. This will be the "discussion" section of the final paper that they will have to write. Additionally, students will also be asked to design "future experiments" and generate corresponding hypotheses and predictions for these new experiments. This exercise will allow students to practice their "critical thinking" skills and appreciate the fact that research is a cycle of observations and experiments that lead to the genesis of more questions.


Instructional Materials

EdU reaction protocol (Microsoft Word 2007 (.docx) 203kB Jan8 20)


 

Assessment

Research Paper (Microsoft Word 2007 (.docx) 126kB Jan8 20)

 

Instructional Staffing

Rolf O. Karlstrom, PhD: The Karlstrom lab has a long history of studying forebrain patterning and pituitary development in the zebrafish. The lab has published several papers studying the effects of thyroid hormones on thyrotropes. Recently, the lab has also expanded its interests to understanding the regulation and maintenance of neural stem cells in the zebrafish. Thus, linking the two topics and trying to understand how thyroid hormones may potentially regulate neural stem cell proliferation was of interest to the lab. Dr Karlstrom's expertise in the field and his zebrafish facility have been a great source of support for this course.

Katherine Dorfman, PhD and Kurt Schellenberg: As the department's laboratory coordinators, they have played in integral role in ordering and preparing reagents. They are also helpful in teaching students how to make solutions and use the microscopes in the classroom.

Wayne Barnaby: As a Graduate TA, Wayne's primary role is supporting the instructor (Dr. Ghosh) in teaching the content, clarifying core concepts, and grading assignments.

Grace Lawson and Anna Aristarkhova: As Undergraduate TA's and research students from the Karlstrom lab, Grace and Anna provide additional technical support during and outside the class. They play a key role in zebrafish husbandry, help students with experimental procedures, and they clarify basic concepts.


 

Advice for Implementation

The content of this lab is very interesting, and students love working with a model organism and doing "real-science". With that said, there are several factors to consider while implementing this course.

  1. It is extremely helpful to have access to a fish facility. Zebrafish husbandry is straightforward, but like most model organisms, you are limited by the number of embryos you can collect per crossing/mating event. This can affect the number of variables and trials that can be conducted for the experiments, not to mention the "game-plan" for the semester. Furthermore, having access to a fish facility allows one to show students around the facility. While the course can be taught by ordering embryos from ZIRC (Zebrafish International Resource Center), it does require a lot of planning and can potentially get very expensive.
  2. The EdU reaction Click-it kit is commercially available, but it is very expensive. The kit is also designed for cells plated on coverslips which introduces experimental variability when used in larval zebrafish brains.
  3. It is critical to have access to fluorescence microscopes. Ideally you would want a confocal microscope so as to collect serial optical sections of the specimen. However, our teaching labs have a TE2000 Inverted Compound Fluorescent Microscope, which allows us to take fluorescence images and students have to manually change the focal plane to get multiple images throughout the specimen—which they later merge into a single z-stack image using ImageJ. This process works well as long as the students are careful in capturing all the focal planes of the tissue. Nevertheless, there remains a concern as to whether the entirety of tissue was properly imaged.
  4. Teaching the anatomy of the zebrafish brain is always challenging. Repeated practice and using published virtual models often help students orient themselves.

Iteration

As mentioned before, the EdU Click-it reaction was designed for cell lines plated on coverslips. Using the same protocol on larval zebrafish brains introduces experimental variability. However, it proves to be a great teaching moment. Students can go through every step of the experimental procedure, starting from the treatments with PTU/T4 all the way to imaging and then discuss why they see the variability. It gives them the opportunity to critically think about and understand the importance of each step of the experiment and postulate why they are seeing variabilities in their results.

Resources

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