Fig. 3

TEMPO reveals spatiotemporal cell histories in the zebrafish hindbrain

(A) Schematic representation of atoh1a progenitor domain in orthogonal hindbrain sections during early (ti) or late (tf) developmental stages. Inset in (A): known differences in proliferation and migration between dorsal and ventral atoh1a progenitors (1, 2, first or second cell division, respectively).

(B) Co-injection of Tol1 constructs (UAS:TEMPO-gRNA-1 and her4.1:Cas9-gRNA-2 switch) into Tg[atoh1a-Gal4] positive zebrafish embryos.

(C) Representative images of TEMPO transitions in the atoh1a domain. Upper panels: dorsal view of zebrafish hindbrain. Lower panels: orthogonal z-projections of the insets in upper panels. Arrow: Representative YFP+ clone derived from a late-born progenitor. Arrowhead: CFP+ clone derived from early-born progenitors.

(D) Percentage of atoh1a+ fluorescent cells and color distribution along time.

(E) Medio-lateral distribution of atoh1a derivatives within a given clone. Data in (D) and (E) are shown as percentage ± SEM (n = 5 fish, 3 independent experiments).

(F) Correlation between birthdate and spatial distribution of vsx2 neurons in dorsal (upper panel) and orthogonal (lower panels) views of the zebrafish hindbrain.

(G) TEMPO activation in vsx2 neurons using the transgenic lines Tg[vsx2:Gal4;UAS:TEMPO] and Tg[her4.1:Cas9].

(H) Lateral (left) and dorsal (center) views of a representative fish expressing all transgenes at 6 dpf. (Right) Orthogonal projections of the regions indicated in the left panels (i–iii).

(I) Distribution of TEMPO+ neuronal projections in a representative fish (color-coded arrows point to projections arranged medio-laterally by increasing birthdate). Asterisk: YFP+ neuronal body located medial to the neuronal projections of YFP+ neurons.

(J and K) Dorso-ventral distribution of TEMPO+ vsx2 neurons in rhombomeres R2-5 (J) or R6 (K). Data shown as percent ± SEM. n = 5. Scale bars, 50 μm. hpf, hours post fertilization.