Understanding metamorphosis of the amphibian olfactory system at the morphological, molecular, and functional level
Final Report Abstract
In contrast to the single sensory surface present in teleost fishes, the mammalian olfactory system is defined by segregated subsystems with distinct molecular and functional characteristics, chief among them the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Most amphibians also have a segregated MOE and VNO. Xenopus laevis, a Pipidae frog, already has a MOE located in the so-called principal cavity (PC) and a VNO during larval life. In contrast to most other amphibians, during metamorphosis of Xenopus, a second sensory cavity, the middle cavity (MC), develops. Simultaneously a reorganization of the PC takes place. After metamorphosis, Xenopus has a tripartite olfactory organ including the MC (‘water nose’), the PC (‘air nose’), and the VNO. Interestingly, the distribution of vomeronasal receptors (V1Rs and V2Rs) among the epithelia of larval Xenopus is drastically different from any other tetrapod species investigated so far. Only late-derived V2Rs are found in the VNO, but early-derived V2Rs and V1Rs are restricted to the MOE. The distribution of early-derived V2Rs closely correlates with the distribution of amino acid responses in larval MOE. The greater goal of our research was to obtain a thorough understanding of morphological, molecular, and functional changes during metamorphosis of the Xenopus olfactory system. Specifically, we tried to answer if the adult water nose forms de novo, or, alternatively, is derived from a segregated segment of the larval MOE. Next, we investigated if amino acid responses and the expression of V2Rs in the larval MOE persist during metamorphosis, and if so, in which of the two adult MOEs (PC or MC) it takes place. Finally, we set out to directly prove whether MOE-associated V2Rs indeed constitute receptors for amino acids. We undertook the first stage-by-stage survey of the anatomical changes of the Xenopus olfactory organ during metamorphosis. We visualized and quantified the degree of cell death within the epithelium of the PC, MC, and VNO and investigated the origin of the receptor neurons and supporting cells of the newly formed MC. Our results show that during MC formation some supporting cells, but not receptor neurons, are relocated from the PC to the MC and that they are eventually eliminated during metamorphosis. These findings illustrate the structural and cellular changes of the olfactory organ during metamorphosis and indicate that supporting cells, but not receptor neurons, transiently support the formation of the sensory epithelium of the MC during metamorphosis. Next, we showed that the MOE- associated V2Rs are gradually lost from the PC as it transforms into the adult air nose. Concomitantly, these V2Rs begin to be expressed in the MC (adult water nose). We observed the same transition for responses to amino acid odorants. These findings further support the hypothesis that amino acid responses may be mediated by V2Rs. Together, our results indicate that the epithelium of the postmetamorphic MC might be an anatomical, morphological and functional replica of the the larval PC. Finally, we set out to investigate if V2R receptors indeed are amino acid sensors. As in vitro approach we planned heterologous expression of V2Rs. However, here we ran into technical problems, as the homology cloning even of V2RC was not successful. Our fallback to obtain the X. laevis sequences from transcriptomes was not realizable in time for this grant, due to both delay of the (finally successful) generation of transcriptomes and the lack of V2R annotation for the X. laevis genome, which necessitates a different approach. As in vivo approach we planned to generate V2RC gene knockout, the likely co-receptor of early-derived V2Rs. However, already in our proof-of-principle knockout of the zebrafish ortholog, homozygous mutants were not viable (we could generate the knockout, and raise heterozygous mutants). We successfully finished five of the seven experiments (A-G) which we proposed in our grant application. We have extensive initial results for the other two experiments. Here, several complications have delayed our work. However, we will continue to work together on these questions.
Publications
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Metamorphic remodeling of the olfactory organ of the African clawed frog, Xenopus laevis. J Comp Neurol. 2016, 524(5):986-98
Dittrich K, Kuttler J, Hassenklöver T, Manzini I
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Coordinated shift of olfactory amino acid responses and V2R expression to an amphibian water nose during metamorphosis. Cell Mol Life Sci. 2017, 74(9):1711-1719
Syed AS, Sansone A, Hassenklöver T, Manzini I, Korsching SI
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Functional reintegration of sensory neurons and Transitional dendritic reduction of mitral/tufted cells during injury-induced recovery of the larval Xenopus olfactory circuit. Front Cell Neurosci. 2017, 11:380
Hawkins SJ, Weiss L, Offner T, Dittrich K, Hassenklöver T, Manzini I
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Dye electroporation and imaging of calcium signaling in Xenopus nervous system. Methods Mol Biol. 2018, 1865:217-231
Weiss L, Offner T, Hassenklöver T, Manzini I
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Whole-brain calcium imaging in larval Xenopus. Cold Spring Harb Protoc. 2020
Offner T, Daume D Weiss L, Hassenklöver T, Manzini I
