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Metamorphic remodeling of the olfactory organ of the African clawed frog, Xenopus laevis.
Dittrich K
,
Kuttler J
,
Hassenklöver T
,
Manzini I
.
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The amphibian olfactory system undergoes massive remodeling during metamorphosis. The transition from aquatic olfaction in larvae to semiaquatic or airborne olfaction in adults requires anatomical, cellular, and molecular modifications. These changes are particularly pronounced in Pipidae, whose adults have secondarily adapted to an aquatic life style. In the fully aquatic larvae of Xenopus laevis, the main olfactory epithelium specialized for sensing water-borne odorous substances lines the principal olfactory cavity (PC), whereas a separate olfactory epithelium lies in the vomeronasal organ (VNO). During metamorphosis, the epithelium of the PC is rearranged into the adult "air nose," whereas a new olfactory epithelium, the adult "water nose," forms in the emerging middle cavity (MC). Here we performed a stage-by-stage investigation of the anatomical changes of the Xenopus olfactory organ during metamorphosis. We quantified cell death in all olfactory epithelia and found massive cell death in the PC and the VNO, suggesting that the majority of larval sensory neurons is replaced during metamorphosis in both sensory epithelia. The moderate cell death in the MC shows that during the formation of this epithelium some cells are sorted out. Our results show that during MC formation some supporting cells, but not sensory neurons, are relocated from the PC to the MC and that they are eventually eliminated during metamorphosis. Together our findings illustrate the structural and cellular changes of the Xenopus olfactory organ during metamorphosis.
Figure 1. Three-dimensional visualization of the spatial organization
of sensory epithelia in the whole olfactory organ. The images
represent dorsolateral views of the olfactory organ at selected
developmental stages. Initially, tadpoles possess sensory epithelia
in the principal cavity (PC, green) and vomeronasal organ (VNO,
gray). During premetamorphosis, the middle cavity (MC, magenta)
is newly formed at the anterodorsal boundaries of the PC. During
prometamorphosis, the MC grows in size and localizes dorsally to
the VNO. Planes in the volumetric reconstructions represent 5
mm thickness. A, anterior; P, posterior; M, medial; L, lateral; D,
dorsal; V, ventral. Scale bar = 100 mm.
Figure 2. Development of the principal cavity (PC) and the vomeronasal organ (VNO) and formation of the middle cavity (MC). Images
show slices of olfactory organs with the whole population of olfactory sensory neurons labeled with biocytin-streptavidin via olfactory nerve
tracing. Depicted are maximum intensity projections of image stacks. Schematic representations of the depicted stages illustrate the size
and localization of the three epithelia during larval development (right panels). A dashed square indicates the approximate location of the
field of view shown in the microscopic images. The arrangement in three different sensory epithelia (PC, VNO, MC) is depicted over larval
development. During premetamorphosis, the MC emerges and grows in size until metamorphosis. Metamorphosis reshapes the larval PC
into a thinner, more extensive sensory surface thought to be specialized for airborne odorant detection. Also, the VNO undergoes spatial
expansion during development. A narrow duct (asterisk) connects PC and MC at higher stages. Numbers indicate the developmental stage.
A, anterior; P, posterior; M, medial; L, lateral. Scale bars5100 mm.
Figure 3. Active caspase-3-positive cells in the sensory epithelia of principal cavity (PC), vomeronasal organ (VNO), and middle cavity
(MC). A: Maximum intensity projection of a sliced larval olfactory organ and connected olfactory nerve (ON) labeled for active caspase-3
at stage 58. Note that the MC is not visible in the depicted horizontal plane. B: Higher magnification of the PC (boxed area in A). Among
labeled apoptotic cells, sensory neurons with apical dendrite and/or basal axon can be identified (open arrowhead). C: Higher magnification
of the VNO/PC (boxed area in A). D: Maximum intensity projection of a sliced olfactory organ illustrating active caspase-3-like immunofluorescence
in the MC at stage 58/59. The number of apoptotic cells is much lower than in the PC and VNO. E: Average number of
active caspase-3-positive cells per section in the PC (green), VNO (gray), and MC (magenta) over the course of larval development. Statistical
significance was determined by using an unpaired t-test (*P<0.05, **P<0.01). A, anterior; P, posterior; M, medial; L, lateral. Scale
bars5200 mm in A; 50 mm in B–D.
Figure 4. Active caspase-3-positive axons in the olfactory nerve. A: Maximum intensity projection of a slice of the anterior telencephalon
illustrating active caspase-3-like immunofluorescence in olfactory sensory neurons. Axons of apoptotic olfactory sensory neurons (green)
extend into the olfactory nerve (ON) and olfactory bulb (OB). B: Representative immunostainings of whole intact olfactory nerves of different
developmental stages (46–/57) are depicted as maximum intensity projections. C: A scatter diagram shows the average total number
of active caspase-3-positive axons in the unsectioned olfactory nerve of each developmental stage (number of olfactory nerve samples per
surveyed stage: 46/3, 47/3, 48/2, 49/11, 50/4, 51/7, 52/11, 53/9, 54/4, 55/4, 56/6, 57/9, 58/7). Statistical significance was
determined by using an unpaired t-test (*P<0.05, **P<0.01). Numbers indicate the developmental stage. Scale bars550 mm in A; 20
mm in B.
Figure 5. Formation of the middle cavity (MC) and indistinct localization of sensory neurons on the boundaries to the principal cavity (PC).
A–F: Maximum projections of sliced olfactory organs depicting representative examples of the early stages of MC formation. Images highlight
the unclear position of sensory neurons at the boundaries of PC and MC (open arrowheads), labeled via olfactory nerve tracing. The
right panel shows the PC/MC border at higher magnification. Some individual sensory neurons cannot be clearly attributed to an individual
sensory epithelium (solid arrowheads). It is possible that these PC neurons are incorporated into the emerging MC. Numbers indicate the
developmental stage. A, anterior; P, posterior; M, medial; L, lateral. Scale bars5100 mm in A,B,D (left), E (right); 200 mm in C,E (left); 50
mm in F (left), 50 mm in A–C (right); 20 mm in D,F (right).
Figure 6. Incorporation of supporting cells, but not sensory neurons, into the forming middle cavity (MC). A: Larval olfactory organs were
electroporated with dextran (magenta) before the MC was apparent (at stages 47–/50). Successful electroporation was verified by in vivo
two-photon microscopy. Images depict maximum projections of an image stack. The whole epithelium of the principal cavity (PC) and the
olfactory nerve (ON) showed widespread and uniform dextran labeling in supporting and sensory neuron layers. The higher magnification
view in the right panel highlights that both supporting cells (solid arrowheads) and sensory neurons (arrows) were labeled. B–D: After formation
of the MC (at stages 54–/57), all sensory neurons were traced via the olfactory nerve with biocytin-streptavidin (green). Images
depict maximum intensity projections of slices of olfactory organs. Dextran-labeled cells represent cells that were already present before
MC formation and persisted until the olfactory organ was investigated. The right panel shows the MC at higher magnification. Punctate,
dextran-labeled cell debris and supporting cells (solid arrowheads) can be visualized in the MC (open arrowheads). In the emerging MC,
mature sensory neurons (marked by an asterisk), already extending their axon into the olfactory nerve, can be identified via biocytinstreptavidin
tracing (green). Dextran labeling never revealed a cell with neuronal morphology and/or colabeling with biocytin-streptavidin
in any of the investigated MCs (n515, 30 slices of the MC). The slices depicted in A and B originate from the same animal. Numbers
indicate the developmental stage. PC, principal cavity; A, anterior; P, posterior; M, medial; L, lateral. Scale bars5100 mm in A–D (left), 20
mm in A,B (right); 50 mm in C,D (right).
