Figure 4.

Overview of the results from the in vivo transgenic enhancer assays in Xenopus

Representative images of EGFP reporter expression in living, transgenic Xenopus tadpoles, driven by SED vector reporter constructs containing non-coding regions of interest. The respective Xenopus species injected and the Nieuwkoop and Faber (NF) stage shown in the pictures are indicated on top of the images, while the injected construct and the stage of injection are indicated left of the images. For each stage, the number of tadpoles displaying the depicted EGFP reporter expression pattern over the total number of analyzed transgenic tadpoles is given.

(A and B) Dorsal view of transgenic Xenopus tropicalis embryos upon unilateral injection of the reporter construct containing IRX1_cCRE7. EGFP reporter expression was visible at the neural plate and tube (NP) on the injected side, indicated by the arrow, while no expression was observed on the non-injected side.

(C and D) Ventral view of transgenic tadpoles, displaying EGFP reporter expression in the craniofacial cartilage, a derivative of the neural crest (NC), and the eye (E) on the injected side.

(E and F) At the same stages, DsRed (positive control) expression was apparent in the myotomes (M) of the tadpoles.

(G and H) Detailed view of the eye at NF stage 55 indicates that EGFP reporter expression was maintained on the injected (left) side, while no expression was observed in the non-injected (right) side.

(I and J) Lateral and dorsal view of transgenic albino Xenopus laevis tadpoles upon unilateral injection of the reporter construct containing IRX1_cCRE7 in the two-cell stage demonstrated EGFP reporter expression in the eye and brain (B).

(K and L) Lateral and dorsal view of transgenic tadpoles injected with the same construct, but in two of the dorsal blastomeres at the four-cell stage, also displayed EGFP reporter expression in the eye and brain.

(M and N) Lateral and dorsal view of transgenic tadpoles introduced with the IRX1_cCRE10 reporter construct demonstrated low EGFP reporter expression in the eye and brain.

(O–Q) DsRed (positive control) was expressed in the myotomes of the corresponding tadpoles.

(R and S) Lateral and dorsal view of transgenic albino Xenopus laevis tadpoles upon injection of the reporter construct containing the PRDM13_cCRE1 region demonstrated EGFP reporter expression in the eye and brain.

(T) Lateral view of transgenic tadpoles injected with mutational hotspot-1 (PRDM13_cCRE3) showed no EGFP reporter expression in the eye or brain.

(U and V) Lateral and dorsal view of transgenic tadpoles injected with mutational hotspot-2 (PRDM13_cCRE5) demonstrated EGFP reporter expression in the eye and brain.

(W) For the five cCREs analyzed using in vivo enhancer detection assays, DNase-seq profiles generated in human embryonic retinal tissue at five different developmental stages are given. In case of IRX1_cCRE7, IRX1_cCRE10, PRDM13_cCRE1, and PRDM13_cCRE7, open chromatin is observed at/until day 103 of development, while PRDM13_cCRE1 is closed exclusively at this period.