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. 2020 Nov 16;9:e59157. doi: 10.7554/eLife.59157

Figure 3. CRMs with activity in the tail epidermis midlines for Ciinte.Klf1/2/4, Ciinte.Nkxtun3, Ciinte.Tox, and Ciinte.Dlx.c.

Representatives examples of X-gal stained embryos at tailbud stages following C. intestinalis embryos electroporation of C. intestinalis genomic regions for Klf1/2/4 (A, B), Nkxtun3 (C, D), Tox (E, F) and Dlx.c (G, H). For each gene, an example for the largest and the smallest regions with robust VDML activity are shown (the size of the region is shown between parentheses after the CRM's name). Embryos are shown in lateral view with dorsal to the top and anterior to the left. Scale bar: 100 μm.

Figure 3.

Figure 3—figure supplement 1. CRMs controlling Ciinte.Klf1/2/4 expression in VDML.

Figure 3—figure supplement 1.

(Top) Snapshot of the Ciinte.Klf1/2/4 locus depicting ATAC-seq profile at mid-neurula stages, tested genomic regions, transcript models and conservation between C. robusta and C. savignyi (from https://www.aniseed.cnrs.fr/ and Dardaillon et al., 2020; Madgwick et al., 2019). Schematic representation of the various constructs and their activity at tailbud stages in DML (blue) and VML (purple) (n indicates the total number of embryos examined, N indicates the number of independent experiments). (Bottom) Identification of putative TFBS for candidate upstream factors in Ciinte.Klf1/2/4-up7 (604 bp). Note that all predicted sites for Msx (6 sites) (Schwartz et al., 2003), Ascl.b (2 sites) (Davidson and Erwin, 2006), and Klf1/2/4 (2 sites) are depicted since this region does not align with the C. savignyi genome (see top panel). The abundance of sites for Msx makes it a likely activator. Up11 is a smaller derivative of up7 (235 bp fragment long) that behaves as a minimal VDML enhancer (Figure 3B) and is deleted in the up8 region (inactive). It is located 2.4 kb upstream of Klf1/2/4, corresponds to an ATAC-seq enrichment detected from late gastrula stages (Supplementary file 2) and contains the 2 Klf1/2/4 sites, suggesting it might be involved in autoregulation/maintenance. Note that the size of the highlighted site for of a given TF may vary depending on the matrix used.
Figure 3—figure supplement 2. CRMs controlling Ciinte.Nkxtun3 expression in VDML.

Figure 3—figure supplement 2.

(Top) Snapshot of the Ciinte.Nkxtun3 locus depicting ATAC-seq profile at mid-neurula stages, tested genomic regions, transcript models and conservation between C. robusta and C. savignyi (from https://www.aniseed.cnrs.fr/ and Dardaillon et al., 2020; Madgwick et al., 2019). Schematic representation of the various constructs and their activity at tailbud stages in DML (blue) and VML (purple) (n indicates the total number of embryos examined, N indicates the number of independent experiments). (Bottom) Identification of putative TFBS for candidate upstream factors in Ciinte.Nkxtun3-up7 aligned with its counterpart from C. savignyi. All putative sites for Msx, Ascl.b, Klf1/2/4 and Nkxtun3 have been mapped, but only conserved sites are shown: 3 Msx, 1 Ascl.b, 2 Klf1/2/4, and 5 Nkxtun3 sites. Deletions on either end of up7 that abolish activity remove 1 Nkxtun3 site, and 1 Msx, 1 Klf1/2/4, and 1 Nkxtun3 sites, respectively. This is suggestive of activation by Msx and Klf1/2/4, and autoregulation/maintenance by Nkxtun3. Note that the size of the highlighted site for of a given TF may vary depending on the matrix used.
Figure 3—figure supplement 3. CRMs controlling Ciinte.Tox expression in VDML.

Figure 3—figure supplement 3.

(Top) Snapshot of the Ciinte.Tox locus depicting ATAC-seq profile at mid-neurula stages, tested genomic regions, transcript models and conservation between C. robusta and C. savignyi (from https://www.aniseed.cnrs.fr/ and Dardaillon et al., 2020; Madgwick et al., 2019). Schematic representation of the various constructs and their activity at tailbud stages in DML (blue) and VML (purple) (n indicates the total number of embryos examined, N indicates the number of independent experiments). (Bottom) Identification of putative TFBS for candidate upstream factors in Ciinte.Tox-int1.1 aligned with its counterpart from C. savignyi. All putative sites for Msx, Ascl.b, Klf1/2/4 and Nkxtun3 have been mapped, but only conserved sites are shown: 1 Msx, 4 Ascl.b, 4 Klf1/2/4, and 3 Nkxtun3 sites. The smaller region Ciinte.Tox-int1.2 contains 3 Ascl.b and 2 Klf1/2/4 suggesting activation by Ascl.b and Klf1/2/4. Note that the size of the highlighted site for of a given TF may vary depending on the matrix used.
Figure 3—figure supplement 4. CRMs controlling Ciinte.Dlx.c expression in VDML.

Figure 3—figure supplement 4.

(Top) Snapshot of the Ciinte.Dlx.c locus depicting ATAC-seq profile at mid-neurula stages, tested genomic regions, transcript models and conservation between C. robusta and C. savignyi (from https://www.aniseed.cnrs.fr/ and Dardaillon et al., 2020; Madgwick et al., 2019). Schematic representation of the various constructs and their activity at tailbud stages in DML (blue) and VML (purple) (n indicates the total number of embryos examined, N indicates the number of independent experiments). (Bottom) Identification of putative TFBS for candidate upstream factors in Ciinte.Dlx.c-up4 aligned with its counterpart from C. savignyi. All putative sites for Msx, Ascl.b, Klf1/2/4, Nkxtun3, and Dlx.c have been mapped, but only conserved sites are shown: 2 Msx, 1 Klf1/2/4, 3 Nkxtun3, and 2 Dlx.c sites. However, these sites are absent from the essential region deleted in up1. Consequently, unidentified factors are likely to regulate Ciinte.Dlx.c-up4 activity and Dlx.c expression. Note that the size of the highlighted site for of a given TF may vary depending on the matrix used.