L 1.Figure 2. In Vivo NMS-E973 In Vitro Birthdating Evaluation of Trigeminal Sensory

L 1.Figure 2. In Vivo NMS-E973 In Vitro Birthdating Evaluation of Trigeminal Sensory Neurons Using BAPTIEmbryos carrying the huc:kaede transgene had been analyzed making use of the Birthdating Analysis by Photoconverted fluorescent SPI-1005 Protocol protein Tracing In vivo strategy (BAPTI). (A) As schematized, the trigeminal sensory neurons initially seem 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 seem red. Following 48 hrs incubation, neurons born prior to 24 hpf retain huc:kaedered and express de novo huc:kaedegreen even though neurons born just after 24 hpf express only huc:kaedegreen. Earlyborn neurons seem red and green although lateborn neurons appear green only. (B) Converted embryos were imaged at 72 hpf. Earlyborn neurons are identifiable by their red and green signals (arrow) even though lateborn neurons are identifiable by their green only signal (arrowhead). Neurons with weak (prime arrow) or sturdy red signals (bottom arrow) had been counted as earlyborn neurons. The entire trigeminal sensory ganglion was imaged by confocal microscopy. Note that at this plane of confocal section only 1 lateborn neuron is present (arrowhead). Side view, anterior towards the left; scale bar represents 10 m.Caron et al.PageNIHPA Author ManuscriptFigure 3. Simultaneous In Vivo Evaluation of Trigeminal Sensory Neuron Birthdate and Fate Using BAPTISMEmbryos carrying the huc:kaede transgene together using a subpopulation:egfp transgene have been analyzed applying the Birthdating Analysis by Photoconverted fluorescent protein Tracing In vivo approach combined having a Subpopulation Marker (BAPTISM). (A) As schematized, the trigeminal sensory neurons initially appear all green (huc:kaedegreen or huc:kaedegreen subpopulation:egfpgreen). Following a 1st conversion at 24 hpf, earlyborn neurons are labeled red and those neurons that express the subpopulation marker seem red and green (huc:kaedered subpopulation:egfpgreen) whereas neurons that don’t express the subpopulation marker appear red only (huc:kaedered). Following a 48 hr incubation, earlyborn neurons retain the converted, redfluorescent Kaede but also express de novo unconverted greenfluorescent Kaede (huc:kaedered huc:kaedegreen or huc:kaedered huc:kaedegreen subpopulation:egfpgreen). Lateborn neurons express nonconverted green Kaede (huc:kaedegreen or huc:kaedegreen subpopulation:egfpgreen). Following a second conversion at 72 hpf, each earlyborn and lateborn neurons contain redfluorescent, converted Kaede (huc:kaedered) and only those neurons that also express the subpopulation transgene retain green fluorescence (huc:kaedered subpopulation:egfpgreen). (B) Comparison on the signals in single neurons prior to and after the second conversion reveal regardless of whether a provided subpopulation marker is expressed in an earlyborn and/or a lateborn neuron. Early born neurons seem yellow prior to the second conversion even though lateborn neurons seem green only. Trigeminal neurons that express the subpopulation marker (subpopulation ) appear yellow immediately after the second conversion although the ones that usually do not express it appear red only (subpopulation ).NIHPA Author Manuscript NIHPA Author ManuscriptDevelopment. Author manuscript; offered in PMC 2009 April 1.Caron et al.PageNIHPA Author Manuscript NIHPA Author Manuscript NIHPA Author ManuscriptFigure 4. LateBorn but not EarlyBorn Neurons Are Restricted in their FateEmbryos carrying the huc:kaede transgene with each other with either the p2x3b:egfp or.