Ectopic eyes outside the head in Xenopus tadpoles provide sensory data for light-mediated learning

Abstract

A major roadblock in the biomedical treatment of human sensory disorders, including blindness, has been an incomplete understanding of the nervous system and its ability to adapt to changes in sensory modality. Likewise, fundamental insight into the evolvability of complex functional anatomies requires understanding brain plasticity and the interaction between the nervous system and body architecture. While advances have been made in the generation of artificial and biological replacement components, the brain's ability to interpret sensory information arising from ectopic locations is not well understood. We report the use of eye primordia grafts to create ectopic eyes along the body axis of Xenopus tadpoles. These eyes are morphologically identical to native eyes and can be induced at caudal locations. Cell labeling studies reveal that eyes created in the tail send projections to the stomach and trunk. To assess function we performed light-mediated learning assays using an automated machine vision and environmental control system. The results demonstrate that ectopic eyes in the tail of Xenopus tadpoles could confer vision to the host. Thus ectopic visual organs were functional even when present at posterior locations. These data and protocols demonstrate the ability of vertebrate brains to interpret sensory input from ectopic structures and incorporate them into adaptive behavioral programs. This tractable new model for understanding the robust plasticity of the central nervous system has significant implications for regenerative medicine and sensory augmentation technology.

Fig. 1.

Eye primordia transplants produce ectopic eyes at the site of graft in Xenopus tadpoles. Donor tissue was excised from the eye field of stage 24 embryos (A, red arrow), grafted along the recipient's body, and healed within 30 min (B, white arrow). Ectopic eyes develop at the same rate as native eyes, and wounds heal completely within 24 h post-surgery (C). Eyes transplanted to caudal regions develop within pockets of tissue (D) or tightly along the trunk of the tail (E). Tadpoles receiving grafts could also have their native eyes removed through surgery at stage 46 (F, red arrows in G), leaving only ectopic visual structures.

Fig. 2.

Ectopic eyes transplanted to caudal regions innervate host tissue. When donor eye primordia from a td-Tomato⁺ host was transferred to the trunk of a recipient, two distinct innervation patterns were observed. In the first, neuronal processes proceeded anteriorly though the fin and terminated along the lining of the stomach of the host (Ai,ii). In the second, labeled neuronal processes penetrated the trunk of the recipient and proceeded towards the spine of the host in either a highly arborized branching pattern (Bi,ii) or along a single path (Biii,iv). White arrows indicate the location of ectopic eyes. The red arrow in panel Biv indicates the termination point of ectopic neuronal processes in the trunk (not continuing to the brain).

Fig. 3.

Eyeless tadpoles respond to changes in light intensity. Tadpoles were placed in a behavior analysis system (A) that contains a circular arena with overhead illumination delivered through red and blue LEDs (B). A machine vision camera system located below the dish tracked tadpole movement rates in response to changing light colors and intensities within the experimental arena. The movement rates of wild-type animals (C) and individuals with eyes removed through surgery (D) were compared. When presented with alternating lighting conditions between red and blue light, wild-type tadpoles showed an increase in movement rates when presented with blue light (Ei, and collapsed for each time period in Eii). In addition, eyeless tadpoles also showed a significant increase when presented with blue light, suggesting that they were able to sense blue wavelengths though a mechanism independent from visual sensory organs (Fi, and collapsed for each time period in Fii). Shaded red and blue areas indicate trial periods in which red or blue illumination was present. Error bars indicate ±1 s.e.m.; N=24 for both conditions; different letters represent significantly different columns by repeated-measures ANOVA.

Fig. 4.

Tadpoles can learn associative stimulus avoidance in an automated assay. Tadpoles were placed individually into the behavior apparatus, and the automated software executed a training cycle. Animals were first tested for red or blue light preference in the absence of shock, followed by a training period where animals receive a shock when occupying the red half of the arena. Following training, all animals received a 90 min rest period in blue light before being tested for red and blue color preference in the absence of shock. Training, rest and testing sessions were repeated a total of six times across the trial (A). Prior to training, wild-type tadpoles demonstrated no preference for either red or blue halves of the dish. However, following two training sessions, a significant red light aversion was generated (Bi). In contrast, animals subjected to the same experiment in the absence of shock did not demonstrate red light aversion during testing (Bii). Furthermore, animals with eyes surgically removed at stage 45 were unable to learn red light avoidance in the presence of shock (Biii). During the training periods, when tadpoles are punished for occupying red halves of the arena, wild-type animals spent significantly less time in punishing areas compared with eyeless tadpoles (C). Error bars indicate ±1 s.e.m.; N=33, 36 and 36, for wild type, no shock and no eyes, respectively; *P<0.05, repeated-measures ANOVA (B), Student's t-test (C).

Fig. 5.

Xenopus can learn a light-mediated task using ectopic eyes present in the tail of the tadpole. Tadpoles with their native eyes removed, but containing a single ectopic eye on the tail, were sorted based on the innervation pattern of the ectopic structure and tested for learning in the automated device. Each group was trained twice, with randomized placement between days of training, to avoid positional effects in the device and to reduce the possibility of false positives. Forty percent of wild-type tadpoles demonstrated learning on both days, while none of the eyeless tadpoles met the learning criteria. Tadpoles with eyes in the tail showed no innervation; those with eyes in the tail that innervated the stomach also showed no appreciable learning in the device. However, 20% of tested individuals with an ectopic eye in the tail that innervated the trunk and spine of the animal learned over both days of testing. Error bars indicate 95% confidence intervals.