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Files in this Data Supplement:
Fig. S1. BrdU remains available for 4 hours after injection. To measure the perdurance of BrdU in zebrafish embryos, BrdU was chased with IdU. Embryos were injected with BrdU and/or IdU and were fixed 2 hours after the last injection. Embryos were stained with an antibody that recognized BrdU only (AbD Serotec) and an antibody that recognized both BrdU and IdU (Becton Dickinson). Primary antibodies were detected using an anti-rabbit antibody coupled with rhodamine and an anti-mouse antibody coupled with FITC (Jackson ImmunoLab), respectively. Therefore, cells labeled with BrdU only or with BrdU and IdU have a red and green signal (yellow in merged image), whereas cells that are labeled with IdU only have a green signal. (A-C) Staining of embryos injected with BrdU only (A), IdU only (B), or both (C), revealed that the anti-BrdU antibody specifically recognized BrdU, whereas the anti-BrdU/IdU antibody recognized both nucleotides. (D-H) Embryos were injected first with BrdU at 24 hpf and injected again with IdU 1 hour (D), 2 hours (E), 3 hours (F), 4 hours (G) and 12 hours (H) following the BrdU injection. When IdU was injected 4 hours after the BrdU injection, several green stained cells were visible throughout the embryo, indicating that BrdU is no longer available to be incorporated 4 hours after injection. Side view, anterior towards the left.
Fig. S2. Blocking proliferation eliminates late-born neurons in trigeminal sensory ganglia. To remove late-born neurons from the trigeminal sensory ganglia, proliferation was blocked after 24 hpf by treating embryos with anti-proliferative drugs. (A,B) Wild-type embryos were treated with 2% DMSO alone (A) or 20 mM hydroxyurea and 150 µM aphidicolin (B). Embryos were analyzed using BAPTI at 72 hpf. Late-born neurons contain only green, unconverted, Kaede (white arrowhead), whereas early-born neurons also contain red, converted, Kaede (white arrow). (C) The chart represents the number of late-born neurons per trigeminal sensory ganglion in treated and mock-treated embryos. Late-born neurons are severely reduced or absent upon treatment with the anti-proliferative drugs. The error bar refers to the standard error.
Fig. S3. 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-D) Wild-type embryos were incubated from 24 hpf to 72 hpf with 2% DMSO alone (A,C) or 20 mM hydroxyurea and 150 µM aphidicolin (B,D). Treated embryos were fixed at 72 hpf and stained by in situ hybridization for p2x3a (A,B) and trpv1 (C,D). Side view, anterior towards the left. Scale bar: 10 µm.
Fig. S4. 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) Wild-type (A,C) and neurogenin1 morphant (B,D) embryos were fixed at 72 hpf and stained by in situ hybridization for p2x3a (A,B) or trpv1 (C,D). Side view, anterior towards the left. Scale bar: 10 µm.
Fig. S5. Birthdate affects the sensory modalities of the trigeminal sensory ganglia. (A-F) Wild-type (A,D), neurogenin1 morphant (B,E), and neurogenin1 mutant (C,F) larvae were tested for their response to touch (A-C) and allyl isothiocyanate (D-F). Fish larvae were recorded using a video camera mounted on a dissection scope. Larvae were considered responsive if they instantaneously escaped the source of the stimulus. Touch stimulus was delivered by touching the head of the fish with a glass needle. Wild-type and neurogenin1-depleted 96 hpf larvae are responsive to touch (A-C). Response to allyl isothiocyanate diluted in DMSO (1:100) was tested. Wild-type (D) but neither neurogenin1 morphant (E) nor mutant larvae (F) are responsive to allyl isothiocyanate. For quantification, see Table 1.
Fig. S6. Early-born neurons are located more superficially in the trigeminal sensory ganglia than late-born neurons. Neurons analyzed by BAPTISM (Fig. 4) were subdivided based on their location in the trigeminal sensory ganglion. Each ganglion was imaged by confocal microscopy and single planes were assembled into a stack with the most superficial neurons being at the top of the stack. The stack was then divided into three equivalent sections. Neurons located in the first third of the stack were considered superficial, whereas the ones located in the last third of the stack were considered deep. The chart represents the distribution of neurons in each section (superficial, middle, deep) as a percentage of the total number of neurons. (A) In embryos expressing the p2x3b:egfp transgene, early-born neurons tend to be more superficial (n=99), whereas late-born neurons tend to be located deeper in the stack (n=22). (B) The early-born neurons expressing the trpa1b:egfp transgene are mostly superficial (n=90).
Movie 1. Late-born neurons in a wild-type embryo. Wild-type embryo was injected with BrdU at 72 hpf and immunostained at 96 hpf for HuC (red) and BrdU (green). The entire trigeminal sensory ganglion was imaged on a Pascal confocal inverted microscope using a 40× water-immersion objective. All planes were collected in this movie file. The first plane corresponds to the most superficial cell layer. Planes are separated by 1 µm intervals. Side view, anterior towards the left.
Movie 2. Late-born neurons in a neurogenin1 morphant embryo. Neurogenin1 morphant embryo was injected with BrdU at 72 hpf and immunostained at 96 hpf for HuC (red) and BrdU (green). The entire trigeminal sensory ganglion was imaged on a Pascal confocal inverted microscope using a 40× water-immersion objective. All planes were collected in this movie file. The first plane corresponds to the most superficial cell layer. Planes are separated by 1 µm intervals. Side view, anterior towards the left.
Movie 3. In vivo birthdating analysis of trigeminal sensory neurons using BAPTI. An embryo carrying the huc:kaede transgene was analyzed using the birthdating analysis by photoconverted fluorescent protein tracing in vivo method (BAPTI). The early-born trigeminal sensory neurons of this embryo were converted at 24 hpf with ultraviolet light and imaged at 72 hpf. Early-born neurons are identifiable by their red and green signals, whereas late-born neurons are identifiable by their green-only signal. The entire trigeminal sensory ganglion was imaged on a Pascal confocal inverted microscope using a 40× water-immersion objective. All planes were collected in this movie file. The first plane corresponds to the most superficial cell layer. Planes are separated by 1 µm intervals. Side view, anterior towards the left.
Movie 4. In vivo birthdating analysis of trigeminal sensory neurons expressing p2x3b using BAPTISM before the second conversion. An embryo carrying the huc:kaede;p2x3b:egfp transgene was analyzed using the BAPTI combined with subpopulation markers method (BAPTISM). The early-born trigeminal sensory neurons of this embryo were converted at 24 hpf with ultraviolet light and imaged at 72 hpf. Early-born neurons are identifiable by their red and green signals, whereas late-born neurons are identifiable by their green only signal. The entire trigeminal sensory ganglion was imaged on a Pascal confocal inverted microscope using a 40× water-immersion objective. All planes were collected in this movie file. The first plane corresponds to the most superficial cell layer. Planes are separated by 1 µm intervals. Side view, anterior towards the left.
Movie 5. In vivo birthdating analysis of trigeminal sensory neurons expressing p2x3b using BAPTISM after the second conversion. The embryos shown in Movie 4 was converted and imaged immediately. Neurons expressing p2x3b:egpf retain a green signal. The entire trigeminal sensory ganglion was imaged on a Pascal confocal inverted microscope using a 40× water-immersion objective. All planes were collected in this movie file. The first plane corresponds to the most superficial cell layer. Planes are separated by 1 µm intervals. Side view, anterior towards the left.
Movie 6. In vivo birthdating analysis of trigeminal sensory neurons expressing trpa1b using BAPTISM before the second conversion. An embryo carrying the huc:kaede;trpa1b:egfp transgene was analyzed using the BAPTI combined with subpopulation markers method (BAPTISM). The early-born trigeminal sensory neurons of this embryo were converted at 24 hpf with ultraviolet light and imaged at 72 hpf. Early-born neurons are identifiable by their red and green signals, whereas late-born neurons are identifiable by their green-only signal. The entire trigeminal sensory ganglion was imaged on a Pascal confocal inverted microscope using a 40× water-immersion objective. All planes were collected in this movie file. The first plane corresponds to the most superficial cell layer. Planes are separated by 1 µm intervals. Side view, anterior towards the left.
Movie 7. In vivo birthdating analysis of trigeminal sensory neurons expressing trpa1b using BAPTISM after the second conversion. The embryos shown in Movie 6 were converted and imaged immediately. Neurons expressing trpa1b:egpf retain a green signal. The entire trigeminal sensory ganglion was imaged on a Pascal confocal inverted microscope using a 40× water-immersion objective. All planes were collected in this movie file. The first plane corresponds to the most superficial cell layer. Planes are separated by 1 µm intervals. Side view, anterior towards the left.
Movie 8. Wild-type larvae are responsive to touch and allyl isothiocyanate. Response to touch and allyl isothiocyanate was tested on a 96 hpf wild-type larva. Larval behavior was recorded using a video camera mounted on a dissection microscope. First, a touch stimulus was delivered by touching the head of a larva with a glass needle. Second, allyl isothiocyanate (orange liquid) was applied onto the head of the larva. See Table 2 for quantification.
Movie 9. neurogenin1 morphant larvae are responsive to touch but not to allyl isothiocyanate. Response to touch and allyl isothiocyanate was tested on a 96 hpf neurogenin1 mutant larva. Larval behavior was recorded using a video camera mounted on a dissection microscope. First, allyl isothiocyanate (orange liquid) was applied onto the head of the larva. Second, a touch stimulus was delivered by touching the head of a larva with a glass needle. See Table 2 for quantification.
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