Evolutionary Changes in Left-Right Visceral Asymmetry in Astyanax Cavefish

Li Ma, Mandy Ng, Janet Shi, Aniket V. Gore, Daniel Castranova, Brant M. Weinstein, William R. Jeffery

Preprint posted on 16 May 2020

Sinister creatures: Li Ma et al. explore the mechanisms behind unusual changes in left-right asymmetry in blind Mexican cavefish.

Selected by Sophia Friesen

Background and context:

When looking at another human, or at most other vertebrates, one might be forgiven for assuming that we are bilaterally symmetric. Apart from subtle differences such as handedness, our left and right sides usually appear identical. Beneath the surface, though, our internal organs display profound and functionally critical asymmetries: the heart is on the left side of the body in all but 0.01% of cases [1], and disruption of left-right patterning can lead to severe heart defects [2]. The sidedness of asymmetric organs is highly conserved within most vertebrate species; for instance, in the Mexican tetra, a freshwater bony fish, the heart consistently forms a loop to the right and the liver forms on the left.

However, as the authors of this preprint discovered, some cave-dwelling subspecies of the Mexican tetra show unusual left-right asymmetry, with higher rates of “mirror-reversed” organs. These fish thus provided a great opportunity to study naturally evolved changes in left-right asymmetry. To figure out the mechanism behind these changes in asymmetry, the authors determined the extent and frequency of altered asymmetry, identified changes in the expression of a gene that helps establish the left-right axis, and used hybridization experiments between cave and surface fish to observe the pattern of inheritance of altered asymmetry.

Key findings:

  1. Two populations of cavefish have unusual left-right patterning

To quantify how often cavefish showed altered asymmetry, the authors examined left-right asymmetry in the developing heart – the first visibly asymmetric organ – in fish from the Pachón and Tinaja cave systems, as well as surface fish. Both populations of cavefish had much higher rates of altered asymmetry compared to the surface fish: while more than 95% of surface fish embryos had normal left-right patterning, only 80% of Pachón embryos and 87% of Tinaja embryos appeared normal, with the remainder showing either mirror-reversed hearts (13% of Pachón and 4% of Tinaja fish) or completely symmetric hearts. Embryos with mirror-reversed asymmetry survived normally to adulthood, but, as in humans, loss of asymmetry was usually lethal.

Sidedness of another notably asymmetric organ, the liver, was also changed in the Pachón cavefish, with 11% showing mirror-reversed livers. Intriguingly, liver sidedness was unchanged in the Tinaja population, despite that population’s high rate of altered heart asymmetry.

  1. Changes in the expression of Lefty2, a conserved regulator of left-right patterning, may be involved in the unusual asymmetry of cavefish

As in other bony fish, left-right asymmetry in the Mexican tetra involves asymmetric expression of the signaling molecule Southpaw (Nodal) and the transcription factor Pitx2 [3]. To investigate whether disruptions in these genes might underlie the altered asymmetry seen in cavefish, the authors measured the embryonic expression patterns of Pitx2 and Southpaw. In surface fish, expression of Southpaw and Pitx2 becomes restricted to the left side, but in Pachón cavefish, Southpaw and Pitx2 expression is often mirror-reversed (20% of fish) or, less frequently, remains totally symmetric (5%). This suggested that altered left-right asymmetry was set up at or before the level of these expression changes.

The unusual expression of Southpaw prompted the authors to investigate expression of two of its negative regulators, Lefty1 and Lefty2. While Lefty1 expression appeared normal, Lefty2 expression was almost entirely absent from its normal location on the left side of the embryo. Sequencing of the coding region of cavefish Lefty2 showed no differences from surface fish Lefty2, indicating that the change in expression of this gene was due to regulatory changes.

  1. Heart left-right asymmetry is dependent on maternal factors

One of the great advantages of using cavefish to study evolved changes, such as heart asymmetry, is that they’re genetically similar enough to their surface-dwelling relatives to hybridize. The authors took advantage of the close relationship between cave and surface fish to study the pattern of inheritance of cavefish asymmetry, by crossing cavefish to surface fish and observing the asymmetry of the offspring. They found that, when a female surface fish is crossed to a male cavefish, the offspring have normal asymmetry, but when a female cavefish is crossed to a male surface fish, the offspring show the same ratios of altered asymmetry as pure cavefish. This showed that the unusual left-right asymmetry of cavefish is determined by maternal factors.

Why I liked this paper:

For me, one of the most fascinating aspects of development is the establishment and refinement of progressively more complex body patterns. Despite our outward appearance of bilateral symmetry, the breaking of left-right symmetry is absolutely critical to vertebrate development. Given this, I was surprised to see differences in asymmetry between two such closely related populations, and without widespread changes in viability. I think that the presence of a high proportion of “mirror-reversed”, apparently healthy cavefish is a testament to the robustness of developmental patterning.

I also really enjoyed this preprint because apart from the inherent charisma of studying cave-dwelling fish, I love how powerful they are as a model of evolution. They only branched off from their surface-dwelling cousins a few million years ago [4], an evolutionary eyeblink, but the different selective pressures in a deep cave versus a surface lake have led to dramatic morphological changes – not just changes in left-right asymmetry, but eyelessness, loss of pigmentation, and metabolic changes [5][6]. Despite their many differences, they’re closely related enough to surface fish to hybridize, a fact which the authors used to great effect to study inheritance of asymmetry.

Questions for the authors:

  1. I find it interesting that heart asymmetry and liver asymmetry don’t appear to be correlated, particularly in the Tinaja population. Would you expect increased lethality in embryos that have some organs “switched” and others not, as compared to completely mirror-reversed embryos?
  2. What prompted you to investigate LR asymmetry in cavefish in the first place? Did you have reason to suspect that asymmetry would be different in cave-dwelling subspecies, or was this a chance discovery?
  3. The changes you observed included loss of asymmetry and resulting embryonic lethality in a notable minority of cases. Could you speculate on why this apparently harmful variation is present at such a high frequency?


  1. Jain VV, Gupta OP, Jain J. A rare case of situs inversus with dextrocardia, Lutembacher syndrome, and pericardial effusion. Heart Views 12(3): 107-111 (2011).
  2. Desgrange A, Le Garrec J-F, Meilhac SM. Left-right asymmetry in heart development and disease: forming the right loop. Development 145 (2018). doi: 10.1242/dev.162776
  3. Essner JJ, Amack JD, Nyholm MK, Harris EB, Yost HJ. Kupffer’s vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut. Development 132: 1247-1260. doi:10.1242/dev.01663
  4. Gross JB. The complex origin of Astyanax BMC Evol Bio 12:105 (2012). doi: 10.1186/1471-2148-12-105
  5. Jefferey WR. Regressive evolution in Astyanax Ann Rev Genetics 43:25-47 (2009).
  6. Moran D, Softley R, Warrant EJ. Eyeless Mexican cavefish save energy by eliminating the circadian rhythm in metabolism. PLoS One 9(9): e107877 (2014).

Tags: astyanax, cavefish, evolution, left-right patterning

Posted on: 5 June 2020 , updated on: 7 June 2020


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Author's response

William R. Jeffery shared

Hi Sophia:

We are glad you enjoyed the preprint.  Your summary was excellent!

Your questions were also very good.  Here are some answers.

Lack of heart and liver symmetry correlation in Pachon and Tinaja. We would expect lethality in embryos that have some individual organs switched based on information from zebrafish and mouse mutants and humans with heterotaxia.  However, despite the differences in reversals between Tinaja and Pachon, their heart and liver changes could still be correlated. We will have to find a way to double assay heart looping and liver positioning to find out for sure. It is also possible that we are simply observing different levels of cave-related trait evolution in Pachon compared to Tinaja, which has been demonstrated for eye and pigment regression, or less advanced cave related traits in Tinaja due to later colonization of this cave by surface fish. To investigate the latter possibilities, we will have to determine organ asymmetry in additional cavefish populations.  Luckily, there are more that 30 different cavefish populations recorded in the wild, and we will need to get more of them into the laboratory.

What prompted you to investigate LR asymmetry in cavefish in the first place? It was a chance discovery by one of the co-authors, but it was to some extent expected because it is known that degenerating cavefish eyes sometimes show asymmetry in size, and asymmetry of bone growth is seen adult cavefish craniofacial features, including skulls skewed from the midline. Perhaps the embryonic changes we report are predecessors to the later changes.

Loss of asymmetry and embryonic lethality in a notable minority of cases. Cavefish spawn hundreds of embryos and only a about 50% normally survive to adulthood in the lab (and possibility less in the wild). So, the frequency of loss of asymmetry is actually not high and much less than the frequency of asymmetric larvae that do not make it through the larval stages. We can also imagine that the rate of asymmetry may be an artifact of the time of assay, which is very early during heart morphogenesis.  It might be that the hearts scored as non-asymmetric at 2-3 dpf are actually slow developers and become normally asymmetric later in development, and thus survive.  Note that some larvae lacking heart asymmetry do survive in our study, although most do not.

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