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First published online 28 August 2008
doi: 10.1242/dev.023200

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In vivo birthdating by BAPTISM reveals that trigeminal sensory neuron diversity depends on early neurogenesis

Sophie J. C. Caron^1,2, David Prober^1,*, Margaret Choy^1,2,* and Alexander F. Schier^1,

¹ Department of Molecular and Cellular Biology, Center for Brain Science, Broad Institute, Harvard Stem Cell Institute, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
² Developmental Genetics Program, New York University School of Medicine, New York, NY 10016, USA.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Continuous neurogenesis in the zebrafish trigeminal sensory ganglia. (A) As schematized in a 30 hpf zebrafish embryo, the trigeminal sensory ganglia (green) are located on each side of the head posterior to the eyes. Trigeminal sensory neurons innervate the head and part of the yolk. (B-D) Wild-type embryos were hybridized with a huc antisense probe to count the number of trigeminal sensory neurons per ganglion. The first neurons appear at 11 hpf forming small clusters (B). During subsequent development, the number of neurons per ganglion increases (C). Side view, anterior towards the left; red asterisks label single neurons. Scale bar: 10 µm. The graph represents the average number of neurons per ganglion at different developmental stages ranging from 11 hpf to 24 hpf (D). An individual ganglion contains an average of 14 neurons at 11 hpf and reaches an average size of 30 neurons at 24 hpf. Error bars indicate s.e.m. (n=15). (E-H) To measure the number of neurons added to the trigeminal sensory ganglion after 24 hpf, embryos were injected once with BrdU, fixed at 96 hpf and immunostained for HuC (red) and BrdU (green). Double labeled cells in the trigeminal sensory ganglia of wild-type (E) or neurogenin1 morphant (F) embryos injected with BrdU at 72 hpf are indicated with a white arrowhead. Side view, anterior towards the left; white outline delimits the trigeminal sensory ganglion from the anterior lateral line. Scale bar: 10 µm. The graphs represent the number of neurons per ganglion born after the BrdU injection time point in wild-type (G) and neurogenin1 morphant (H) embryos. Injections were performed at different times ranging from 24 hpf to 92 hpf. Gray dots represent individual samples and red dots represent the average number of BrdU-positive trigeminal sensory neurons found per ganglion at each injection time point (n=30).

View larger version (45K): [in this window] [in a new window]   Fig. 2. In vivo birthdating analysis of trigeminal sensory neurons using BAPTI. Embryos carrying the huc:kaede transgene were analyzed using the birthdating analysis by photoconverted fluorescent protein tracing in vivo method (BAPTI). (A) As schematized, the trigeminal sensory neurons initially appear green. Following illumination of 24 hpf embryos with ultraviolet light, huc:kaedegreen is converted to huc:kaedered and all neurons born prior to 24 hpf appear red. After 48 hours incubation, neurons born before 24 hpf retain huc:kaedered and express de novo huc:kaedegreen, whereas neurons born after 24 hpf express only huc:kaedegreen. Early-born neurons (E) appear red and green (yellow); late-born (L) neurons appear green only. (B) Converted embryos were imaged at 72 hpf. Early-born neurons are identifiable by their red and green signals (arrow), whereas late-born neurons are identifiable by their green only signal (arrowhead). Neurons with weak (top arrow) or strong red signals (bottom arrow) were counted as early-born neurons. The entire trigeminal sensory ganglion was imaged by confocal microscopy. At this plane of confocal section only one late-born neuron is visible (arrowhead). Side view, anterior towards the left. Scale bar: 10 µm.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Simultaneous in vivo analysis of trigeminal sensory neuron birthdate and fate using BAPTISM. Embryos carrying the huc:kaede transgene together with a subpopulation:egfp transgene were analyzed using the birthdating analysis by photoconverted fluorescent protein tracing in vivo method combined with a subpopulation marker (BAPTISM). (A) As schematized, the trigeminal sensory neurons initially appear all green (huc:kaedegreen or huc:kaedegreen + subpopulation:egfpgreen). Following a first conversion at 24 hpf, early-born neurons are labeled red and those neurons that express the subpopulation marker appear red and green (yellow) (huc:kaedered + subpopulation:egfpgreen), whereas neurons that do not express the subpopulation marker appear red only (huc:kaedered). After a 48-hour incubation, early-born neurons retain the converted, red-fluorescent Kaede but also express de novo unconverted green-fluorescent Kaede (huc:kaedered + huc:kaedegreen or huc:kaedered + huc:kaedegreen + subpopulation:egfpgreen). Late-born neurons express non-converted green Kaede (huc:kaedegreen or huc:kaedegreen + subpopulation:egfpgreen). After a second conversion at 72 hpf, both early-born and late-born neurons contain red-fluorescent converted Kaede (huc:kaedered) and only those neurons that also express the subpopulation transgene retain green fluorescence (huc:kaedered + subpopulation:egfpgreen) (yellow). (B) Comparison of the signals in single neurons before and after the second conversion reveal whether a given subpopulation marker is expressed in an early-born and/or a late-born neuron. Early born neurons appear yellow before the second conversion whereas late-born neurons appear green only. Trigeminal neurons that express the subpopulation marker (subpopulation +) appear yellow after the second conversion, whereas the ones that do not express it appear red only (subpopulation-).

View larger version (64K): [in this window] [in a new window]   Fig. 4. Late-born but not early-born neurons are restricted in their fate. Embryos carrying the huc:kaede transgene, together with either the p2x3b:egfp or the trpa1b:egfp transgene were analyzed using BAPTISM. (A-D) huc:kaede was first converted at 24 hpf, imaged at 72 hpf (A,C) and converted a second time (B,D). (A,B) In huc:kaede;p2x3b:egfp embryos, early-born neurons either express the subpopulation marker (green arrow) or do not (white arrow), and late-born neurons either express the subpopulation marker (green arrowhead) or do not (white arrowhead). (C,D) In huc:kaede;trpa1b:egfp embryos, early-born neurons either express the subpopulation marker (green arrow) or do not (white arrow). Late-born neurons do not express the subpopulation marker (white arrowhead). Side view, anterior towards the left. Scale bar: 10 µm. (E) The histogram represents the number of neurons per trigeminal sensory ganglion expressing the p2x3b or trpa1b subpopulation markers derived from either early-born neurons (dark gray) or late-born neurons (light gray). The error bars refer to the s.e.m. (n=7).

View larger version (49K): [in this window] [in a new window]   Fig. 5. Early-born trigeminal sensory neurons contribute to various subpopulations independently of late-born neurons. Subpopulation markers were analyzed in embryos lacking late-born trigeminal sensory neurons. (A-H) Embryos were incubated from 24 hpf to 72 hpf with 2% DMSO alone (A,C,E,G) or 20 mM hydroxyurea and 150 µM aphidicolin (B,D,F,H). Embryos carrying the p2x3b:egfp (A,B) or trpa1b:egfp (C,D) transgene were imaged at 72 hpf. Wild-type embryos were fixed at 72 hpf and in situ hybridization was performed for p2x3b (E,F) and trpa1b (G,H). Side view, anterior towards the left. Scale bars: 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 6. The trigeminal sensory ganglia of neurogenin1 mutant and morphant embryos are solely formed from late-born neurons. Neurogenin1-depleted embryos develop smaller trigeminal sensory ganglia formed from late-born neurons only. (A-D) Embryos carrying the huc:kaede transgene were injected with 6 ng of neurogenin1 antisense morpholino (B,D) or uninjected (A,C). At 24 hpf, the trigeminal sensory ganglia are visible in uninjected embryos by the expression of Kaede (A,C) but no trigeminal sensory neurons are detectable in the neurogenin1 morphants at 24 hpf (B). At 96 hpf, the trigeminal sensory ganglia are visible in neurogenin1 morpholino-injected embryos (D) but contain fewer neurons than uninjected embryos (C). Side view, anterior towards the left. Scale bar: 10 µm. (E-G) The morphology of the neurons of the trigeminal sensory ganglia was analyzed by immunostaining in wild-type (E), neurogenin1 morphant (F) and neurogenin1 mutant (G) embryos with HuC, a pan-neuronal marker, and HNK-1, a marker labeling the cell surface of sensory neurons. White arrowheads indicate the trigeminal sensory ganglia. Side view, anterior towards the left. Scale bar: 100 µm. (H-K) To determine whether the trigeminal sensory ganglia in neurogenin1 morphant embryos are partly formed from early-born neurons, embryos were treated with 2% DMSO alone (H,J) or with 20 mM hydroxyurea and 150 µM aphidicolin (I,K) at 24 hpf. HuC staining (red) labels the trigeminal sensory ganglia. Staining for the mitotic marker phospho-histone H3 (green) was used to monitor the number of proliferating cells in the whole embryos. Proliferation was not affected in mock-treated embryos (H,J) but was significantly reduced in treated embryos (I,K). No trigeminal sensory neurons are detectable in the neurogenin1 morphant embryos treated with the anti-proliferative drugs (K), in contrast to the mock-treated neurogenin1 morphant (I) or wild-type embryos (H,J). Side view, anterior towards the left. Scale bar: 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 7. Cell fate restriction of late-born trigeminal sensory neurons is independent of early-born neurons. Subpopulation markers were analyzed in embryos lacking early-born trigeminal sensory neurons. (A-D) Embryos carrying the p2x3b:egfp (A,B) or trpa1b:egfp (C,D) transgene were injected with 6 ng of neurogenin1 morpholino (B,D) and imaged at 72 hpf. Although both p2x3b-expressing and trpa1b-expressing neurons are visible in uninjected larvae (A,C), only p2x3b-expressing are detectable in neurogenin1 morphant larvae (B). No trpa1b-expressing neurons were detected in these larvae (D). (E-H) Wild-type (E,G) and neurogenin1 morphant (F,H) larvae were stained by in situ hybridization at 96 hpf for p2x3b (E,F) or trpa1b (G,H). No trpa1b-expressing neurons were detected in neurogenin1 morphant larvae (H). Side view, anterior towards the left. Scale bars: 10 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Model for the role of neurogenesis in forming trigeminal sensory ganglia in zebrafish. The trigeminal sensory ganglia in wild-type embryos are formed from early-born neurons (born before 24 hpf) and late-born neurons (born after 24 hpf). In 96 hpf larvae, early-born neurons (blue triangles, squares and circles) are intermingled with late-born neurons (orange squares and circles). Early-born neurons contribute to all identified subpopulations of trigeminal sensory neurons, whereas late-born neurons fail to form the trpa1b-expressing subpopulation (blue triangles). When proliferation is blocked after 24 hpf, embryos lack late-born neurons.

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