Open-Ended Inquiry into Zebrafish Nerve Development Using Image Analysis

Abstract

Open-ended laboratory projects increase student success and retention in the sciences. However, developing organismal-based research projects is a challenge for students with restricted laboratory access, such as those attending courses remotely. Here I describe the use of image analysis of zebrafish neural development for authentic research projects in an introductory biology laboratory course. Zebrafish are a vertebrate model that produce large numbers of externally and rapidly developing embryos. Because zebrafish larvae are transparent, fluorescent reporters marking nervous system structures can be imaged over time and analyzed by undergraduate scientists. In the pilot of this project, remote first-year college students independently developed biological questions based on an image collection comparing zebrafish mutants and wild-type siblings. Students created and mastered techniques to analyze position, organization, and other morphological features of developing neurons and glia in the images to directly test their biological questions. At the end of the course, students communicated their project results in journal article format and oral presentations. Students were able to hone skills in organismal observation and data collection while studying remotely, and they reported excitement at applying lecture-based knowledge to their own independent questions. This module can be adapted by other instructors for both students on- and off-campus to teach principles of neural development, data collection, data analysis, and scientific communication.

Keywords: Fiji; ImageJ; authentic inquiry; first-year research; image analysis; lateral line; motor nerves; neuroanatomy; neurodevelopment; open-ended laboratory; remote learning; sensory nerves; zebrafish.

Figure 1

Lateral schematic of key nervous system features in a zebrafish larva. The anterior lateral line nerve (ALLn) and posterior lateral line nerve (PLLn) are sensory nerves. Motor nerves are shown emanating from spinal cord. Boundaries of body wall muscle segments (somites) are shown in gray. Compass denotes dorsal (D)-ventral (V) and anterior (A)-posterior (P) axes. (B–E) Patterning of wild-type sibling (B–C) and stl159 mutant (D–E) larvae at 72 hours post-fertilization (hpf). (B, D) Brightfield lateral view shows normal melanocyte patterning in sibling and disrupted in mutant (arrowhead). (C, E) Merged nbt:dsRed; sox10:megfp fluorescence in larvae shown in B and D indicating features scored by students. PLLn position is marked with white arrow, spinal cord is marked with bracket, subset of motor nerves from spinal cord are marked with asterisks. PLLn and motor nerve phenotype are disrupted in stl159 mutants. Merged image created in Fiji; students scored merged or single channel images depending on research question.

Figure 2

dsRed expression in a 72 hpf zebrafish taken at 80x magnification. (A) Raw low-exposure .czi image as it appears in Fiji. The spinal cord (bracket) is prominent, and PLLn (arrow) is visible. Motor nerves are too dim to observe. Histogram (right) is scaled 0–255, with most pixel brightness values to the left. (B) Scaled image shows PLLn (arrow) and motor nerves, including fine branched processes (arrowheads). Spinal cord (bracket) is saturated and should not be analyzed using this image. In the B&C box, the maximum is scaled so the display values correspond to the peaks in the histogram.

Figure 3

Counting and measuring features in Fiji. (A) nbt:dsRed expression in 72 hpf larvae. Spinal cord is indicated with bracket. Five motor nerves are counted with the multi-point tool as indicated. (B) Results window shown for five points on motor nerves. Mean, Min, and Max report fluorescence brightness; because this is a point (with area 0), they are all the same, as there can be only one fluorescence value for a single point. X and Y are the coordinate position for the point in the image. Clicking additional points in the image would number those points and add values to the same Results window. (C) The image shown in A is marked to measure motor nerve length. Three motor nerves (exit points marked with asterisks) were measured, with the third marked with a yellow line. (D) Results window for three motor nerve measurements. Mean, Min, and Max report fluorescence intensity. Length and angle are line-specific features that represent useful data for analysis. The first two results are for lines not represented in the image, as they disappear when new lines are drawn.