Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

Quantification of liver size is crucial in studies aimed at understanding liver development, regeneration, and oncogenesis. The protocol described here is a relatively quick, easy, and cheap technique for liver size quantification in larval zebrafish. Exercising appropriate caution while performing certain aspects of the protocol can aid in increased accuracy of results and decreased frustration.

Proper fixation of the larvae is crucial towards getting well-preserved biological samples and preventing their disintegration. Dilution of the 4% PFA solution can occur when PBS is not removed completely before the addition of PFA to the rinsed larvae. Using well-made PFA solutions and pipetting out all or most of the PBS solution prior to PFA addition is helpful to address this issue.

Although fast and easy to perform after much practice, the dissection technique requires substantial manual dexterity. While dissecting, it is crucial to remove the skin and yolk completely off from above the liver such that the whole liver is exposed. Failure to do this can result in images where the view of the liver boundary is obscured (Figure 2A,B). Unskilled and forceful movements while dissecting can lead to pinching off of parts of the liver (Figure 2C) or loosening of liver attachments, resulting in the liver being displaced (Figure 2D) or missing entirely (Figure 2E). Users should put in adequate numbers of hours towards honing their dissecting skills on practice samples before moving on to experimental samples.

During mounting, the skin above the liver has been removed, increasing the probability of the liver falling out during subsequent steps. To avoid that possibility, gentle pipetting movements should be employed during this process.

During image procurement using the brightfield microscope, it is crucial that good quality images are taken. Blurry, out-of-focus images will make it difficult to assess the true boundary of the liver (Figure 2F). As this method involves measuring the surface area of the left lobe of the liver, it is crucial that the larva is oriented well on its side and not tilted (Figure 2G). Make sure that both eyes of the larva are aligned (one eye covering the view of the other). While measuring surface area using image processing software, it is important to draw the boundary as close as possible to the real outline of the liver so as to avoid measurement discrepancies. Exclude any images where the liver cannot be accurately measured (Figure 2A–G). However, keep in mind that excluding livers can skew the data, as bigger livers are more likely to be disrupted than smaller livers.

One of the limitations of this protocol is that it applies only to fixed larvae. Alternative methods such as fluorescence microscopy can be used to measure liver size in live larvae expressing hepatocyte-specific fluorescent reporters^5,7,10. These alternative methods enable sequential measurements to be made on the same animal, and they are also quicker, since they do not require fixation or dissection of the tissues overlying the liver. The advantages of this protocol compared to fluorescence microscopy in live animals are: 1) more flexibility with respect to when livers are measured, as zebrafish can be kept in fixative for weeks or months before photographing them; 2) no requirement for incorporating a fluorescent reporter, which can be cumbersome when dealing with homozygous mutants; and 3) applicability of steps 1 and 2 for other experiments, including immunofluorescence staining or in situ hybridization studies. We use both methods, depending on the particular application. For example, we typically use live imaging and hepatocyte-specific reporters for high-throughput screening³, and follow up on potential hit compounds using the protocol described here³.

This protocol takes only the surface area into account for quantification of liver size, so it does not detect changes in cell metabolism or morphology, nor does it differentiate between increases in cell number and increases in cell size. In order to address this limitation, complementary assays to assess steatosis¹⁶, histology⁶, cell number^8,11, cell size^3,17, proliferation^3,18, and/or apoptosis¹⁹ can be performed.

Another limitation of this protocol is that it assumes that increases or decreases in the surface area of the left liver lobe are reflective of the changes in surface area and volume of liver as a whole. This assumption may not apply when liver growth is non-uniform. To examine liver shape and check for non-symmetric increases in liver growth, we routinely do light sheet fluorescence microscopy⁸ or confocal microscopy³ on our transgenic models. Light sheet fluorescence microscopy can be used to directly quantify larval liver volume⁸. In transgenic zebrafish expressing hepatocyte-specific Yap1, liver area and liver volume were similarly increased compared to non-transgenic control siblings⁸.

The dissection technique described here can be combined with immunofluorescence staining, cell-specific fluorescent reporter lines, and/or other labeling techniques to study other aspects of liver development besides liver size^3,19,20. As this dissection protocol also exposes the gut and pancreas, it may be helpful for studies of other visceral organs as well.