Mending a frozen heart: cryoinjury of the zebrafish heart models myocardial infarction in mammals

When several articles are published within a few days that cover the same topic, you know that some corner of science has advanced to a point where certain ideas suddenly emerge as the next logical step. A field that has attracted considerable attention lately is vertebrate heart regeneration. In humans, of course, cardiac muscle cells do not regenerate, which is of great concern for heart attack patients. Myocardial infarction locally disrupts the blood supply to the heart. Deprived of oxygen, heart muscle cells begin to die and are eventually removed by immune cells. What remains is a scar whose main function is to prevent further damage to the heart. However, in the long run the scarred tissue is likely to have negative effects, by changing the fine-tuned interplay of muscle contraction and the hydrodynamics of blood flow through the ventricles. As a result, ensuing heart failure becomes more likely.

Zebrafish, as is well known by now, are champions when it comes to regenerating cardiac muscle that has been lost by amputation. The regenerated myocardium is derived from existing dedifferentiated cardiomyocytes that re-enter the cell cycle and proliferate to replace the missing tissue. Research on teleosts therefore holds promise to identify alternative avenues into overcoming the limited regeneration response in the mammalian heart. Unfortunately, ventricular amputation is not the best model to understand the healing processes, because an infarcted heart experiences a different kind of damage, in which debris of dead cells is left behind that needs to be cleared before healing processes can occur. Now, a number of research groups have found an alternative way of injury that overcomes this limitation.

The laboratories of Anna Jazwinska (Fribourg, Switzerland), ¹ Nadia Mercader (CNIC, Spain), ² and Gilbert Weidinger (Dresden, Germany) ³ have found that cryocauterization, in which a cooled piece of metal wire is inserted through a hole in the pericardial sac to kill a portion of ventricular muscle by extreme frostbite, induces massive cardiac necrosis in up to 25% of the ventricular wall in a manner similar to myocardial infarction in mammals. After cryoinjury, three phases can be distinguished on the way to recovery. First, the infarcted area is invaded by leukocytes that mount an inflammatory response to clear cellular debris from the epicardium, myocardium, and endocardium, all of which are affected by necrosis and apoptosis, within a day.

Subsequently, a fibrin layer is formed that seals the wound and fibroblasts accumulate to produce a collagen scaffold and initiate the reparative phase. At this stage significant differences to heart amputation become apparent. While amputation usually results in fibrin-rich deposits, but little collagenous ones, two of the groups reported extensive fibrotic scar formation after cryocauterization. These scars consisted of a network of connective collagenous tissue in the interior of the wound that took several months to be cleared.

The epicardium, a layer that surrounds the myocardium, also seems to play a role in regeneration. Early after the injury, genes that played an earlier role in heart development are re-expressed. At the same time, proliferation was massively increased in the epicardium surrounding the injured site, such that the single-layered sheet grew to several layers that cover the wound like a cap and trapped the blood clot underneath. Finally, within 3 weeks after injury, a new myocardium invades the infarcted lesion from the boundaries and replaces the fibrin-collagen-based matrix. Endocardial cells also proliferate in the regions surrounding the lesion. Within 3–7 weeks, coronary vessels invade the injured area and completely reconstitute the blood supply.

The prospects for transferring insights from the teleost model to research on the human heart seem favorable. One group found the electrocardiogram (ECG) recordings to be remarkably similar to human ECGs, even though the fish heart has only two chambers. At the time of scar formation the collagenous insulation resulted in longer intervals before the ventricle repolarized, which in human patients is a risk factor for sudden death. Further, an infarct in mammals can lead to long-term ventricular remodeling, a situation also observed in cryoinjured zebrafish hearts. In this sense, even in fish the functional recovery of the heart is incomplete.

What is driving research into zebrafish heart regeneration is the long-term goal to repair human hearts. Adult mammalian cardiomyocytes do not proliferate in response to injury. But thanks to the zebrafish model it might one day be feasible to activate resident progenitor cells identified within the adult mammalian heart. These cells are endowed with cardiomyocyte potential but do not seem to be activated to replace injured tissue. If research in zebrafish should identify avenues into altering appropriate signaling pathways to activate their intrinsic regenerative potential, then the experimental potential offered by the cryoinjury procedure is the next step in this direction.

Variation in limb position: how pelvic fins develop in unusual positions

There is tremendous variation in the shape, size, and position of fins in teleost fish. Pelvic fins are homologous to tetrapod hindlimbs, yet their position along the body axis has experienced dramatic evolutionary shifts: in zebrafish, as in other cypriniform fishes, pelvic fins are in an abdominal position just rostral to the anus. Cichlids have thoracic pelvic fins in proximity to the pectoral fins, whereas in cod the pelvic fins lie in front of the pectoral fins. This remarkable variation begs the question whether pelvic fin position is associated with adaptive advantages. In 1938, John Edward Harris, a biomechanist at Cambridge University, tried to find out by amputating the thoracic pelvic fins of a sunfish. Watching the resulting swimming behavior he learned that pelvic fins are used as stabilizing devices that allow fish to efficiently come to a full stop. The counter‐movement of the pelvic fins is crucial for sun fish to avoid shooting upward with their noses during a sudden full stop. ¹

Unfortunately, the developmental mechanisms of pelvic fin positioning are not fully understood. To find out how the development of abdominal pelvic fins differs from that of thoracic ones, Yumie Murata and Mika Tamura with colleagues in the lab of Mikiko Tanaka (Tokyo Institute of Technology, Yokohama, Japan) compared the process in zebrafish and Nile tilapia (Oreochromis niloticus). ²

Among the usual suspects that most likely confer axial identity to pelvic fin precursor cells in the lateral plate mesoderm (LPM) are the Hox genes. In particular, in tetrapod embryos hindlimb buds arise at the levels of Hox10 paralog expression in the spinal cord, and motorneurons that innervate the hindlimbs originate inside Hox10 expression domains. To examine whether in teleosts the neural Hox10 expression boundary also aligns with pelvic fin precursors, the researchers traced the fates of embryonic cells in the thin layer of LPM at the axial level of hoxc10a expression. LPM cells at the level of somite 16 were marked with lineage tracer molecules early during somitogenesis and observed throughout the next 4 weeks until pelvic fins formed.

The cluster of labeled cells grew in size through cell proliferation and already by 30 h postfertilization its rostral end extended up toward the level of somites 8–9. This is interesting, as the pelvic fin develops at this level a few weeks later. Murata et al. suggest that growth rates in the trunk are higher than in the LPM, so that pelvic fin precursors are rapidly displaced to face more anterior somites. In zebrafish this shift is further enhanced by the radical re‐shaping experienced by the caudal end of the yolk cell, which is gradually constricted to form an elongated tube, the yolk extension, that stretches toward the anus.

In Nile tilapia, the situation is rather different. Pelvic fins develop much earlier, around 9 days after fertilization, and in a thoracic position, adjacent to somites 3–4. Still, pelvic fin precursors originate from axial levels lateral to neural hoxc10a expression and form streams of lineage‐labeled cells that extend between the site of labeling during somitogenesis and the eventual position of the pelvic fin. Thus, evolutionary alterations to the shape of the underlying yolk might have played a role in the emergence of diverse pelvic fin positions. In addition, the streams of labeled cells that form between the place of specification and final fin position are evocative of a role for active pelvic fin precursor cell migrations in the evolution of diverse pelvic fin positions in teleosts.

In tetrapods, inactivation of the growth differentiation factor 11 (Gdf11) gene is known to shift hindlimb position and Hoxc10 expression caudally. To test whether the loss of zebrafish gdf11 affects pelvic fin position, the authors injected gdf11‐specific morpholinos into embryos and found that pelvic fins were shifted backward by 1 somite. Cell tracking experiments confirmed that in gdf11 morphants hoxc10a expression was shifted caudally. Therefore, pelvic fin precursors, aligning with hoxc10a expression, might have originated from a more caudal position. One of the roles of gdf11 in the presomitic mesoderm is to induce the expression of cyp26a1, coding for an enzyme that maintains a retinoic acid‐free zone caudal to the somites. ³ When Gdf11 is knocked out in the mouse, the domain of activated retinoic acid signaling expands posteriorly toward the presomitic territory. It is therefore possible that the anterior boundaries of Hox genes, such as of Hoxc10, is controlled by the interplay of retinoic acid synthesis in somites and its breakdown in presomitic mesoderm.

One aspect of tetrapod limb development, however, turned out not to be conserved in teleosts. Whereas in tetrapods the identity of motorneurons innervating the hindlimb musculature corresponds to the expression of Hox10 paralogs in the spinal cord, labeling these motorneurons in zebrafish and Nile tilapia showed that in teleost fish hoxc10a is expressed more caudally.

This work is the first to address evolutionary differences in pelvic fin positions in teleosts. It shows that although pelvic fin precursors are likely to be specified early in embryogenesis at comparable axial levels, selection on differences in yolk shape, relative growth rates between trunk and LPM, and most likely also migration of fin precursors has given rise to the diversity of pelvic fin positions found today.

Invasion of the body snatchers: symbiotic algae enter cells of salamander host

Amphibians lay eggs in which the embryo is encapsulated by a gelatinous jelly, and quite often they are attached to the vegetation of vernal ponds. Since 1888 it is known that in the wild the egg jelly of the spotted salamander, Ambystoma maculatum, contains spherical green algae. The algae have been named Oophila amblystomatis, for they are only found together with the eggs of A. maculatum. Spotted salamanders spend most of their adult life underground and only visit vernal pools for spawning. Embryos develop in pools all the way through metamorphosis. When the young adults hatch, the algae are released from inside the egg capsule into the environment, while salamanders return to their buried lifestyle.

The association of unicellular algae and vertebrates so far is unique, but a history of investigations into this curious phenomenon has established that this is an endosymbyotic relationship with mutual facultative benefits for both partners. When algae are removed from fertilized eggs by raising them in the dark, salamander larvae hatched later, had a higher embryonic mortality, and at hatching had not developed as far as larvae with “algal guests.” Conversely, algae do not grow well in the absence of an embryo. For the embryo, the presence of the jelly capsule provides protection from predation but also impedes the exchange of respiratory gases. Algae can offer oxygen derived from photosynthesis and probably benefit from some of the embryo's waste products. How intricate is the endosymbiontic relationship, where do the algae come from, and where do they go once the larvae have hatched? Recent work from Ryan Kerney et al. (Dalhousie University, Halifax, Canada) found what looks like an answer to some of these questions. ¹

Kerney et al. took advantage of the autofluorescence of algal chlorophyll to determine when and where they first appear. Using long‐exposure fluorescent microscopy they found that algae from embryos collected in the wild initially bloom next to the blastopore, the site of cell ingression during gastrulation. At later stages of embryonic development, however, several algae could be seen that had invaded the embryonic host tissue.

The mechanism of host tissue invasion is unknown, but algae‐derived 18S rDNA could not be detected in embryos before the neurula stage, ruling out the possibility that they enter through the blastopore opening during gastrulation. It is more likely that algae can enter the embryo through the caudal end of the alimentary canal, the anus, because a considerable number of algae were found in this region. The diversity of tissues jammed with endogenous algae suggests that algae may also enter the embryo directly. Algal autofluorescence from chlorophyll was found between cells of the head mesenchyme, neural tube, and in somites. Algae in young larval stages could only be identified by in situ hybridization to their 18S rDNA, which added cranial bones, endoderm, and the liver as hiding places. Most algae, however, were found in the yolk and alimentary canal.

Surprisingly, many autofluorescent algal cells were embedded within the cytoplasm of differentiated salamander host cells, and often so in close neighborhood with mitochondria. These would benefit from oxygen and maybe also carbohydrates generated through algal photosynthesis. Photosynthate transfer has not yet been detected in this salamander, but now that algal invasion of host tissue is documented, it appears that the methods employed to detect carbon fixed by photosynthesis in older studies may not have been sensitive enough. The vacuoles of algae growing inside egg capsules contain ammonia waste. Since the vacuoles of intracellular algae were often larger, this could mean that algae penetrate the integument on the trail of a gradient of nitrous waste.

Once the salamander larvae start to feed, algal fluorescence could not be detected anymore. Since free‐living Oophila species are not known and researchers have had difficulties raising algae‐free clutches from the laboratory or the wild, it seems possible that they are transmitted from mother to egg, a process known as vertical transmission. Kerney et al. present new data in support of mother‐to‐child transfer. They found that the reproductive tissues of adult salamanders, mainly oviduct and ovary, sometimes contain algal 18S rDNA, and this marker even occurs in reproductive tissues adult males. Hence, are eggs already preloaded with algae before fertilization? If this really is a possible route of transfer, then it must be rare, because none of the larvae used in this study contained algae in their reproductive tracts.

This article shows that photosynthetic endosymbiosis, previously only known from protists and metazoan invertebrates, goes deeper in the spotted salamander than previously thought. In green salamanders the immune system has not yet fully developed when the algal invasion happens, but for other species the evolution of an adaptive immune system in vertebrates might have blocked the development of endosymbionts. On the other hand, maybe we simply have not looked close enough yet. Thanks to this work we now know what to look out for, and where.