Figure 4. Molecular dissection and mutation of element G.

(A) Summary of GFP expression regulated by element G truncations using EpGFPII reporter constructs. Serial truncation of element G was performed based on boundaries defined by chromatin accessibility and the kirrelL 5′-UTR. Criteria for strong and weak primary mesenchyme cell (PMC) expression are defined in Figure 2. Ectopic expression is defined as majority of injected embryos exhibiting GFP expression in cells other than PMCs. (B) Summary of GFP expression driven by G.ATAC element mutants using EpGFPII reporter constructs. (C) Analysis of element enhancer activity in modified EpGFPII reporter constructs containing the endogenous Sp-kirrelL promoter elements.

Figure 4—figure supplement 1. Element G truncation and mutational analysis.

(A) Spatial expression patterns of GFP reporter constructs containing element G truncations in S. purpuratus embryos at 48 hpf. (B) Spatial expression patterns of GFP reporter constructs containing element G truncations in L. variegatus embryos at 28 hpf. Representative images of primary mesenchyme cell (PMC)-specific and ectopic GFP expression are shown for some constructs. Top rows: GFP fluorescence. Bottom rows: GFP fluorescence overlayed onto differential interference contrast (DIC) images. Scale bar: 50 μm.

Figure 4—figure supplement 2. Mutational analysis of G.ATAC element.

(A) Clustal alignment of Sp-kirrelL and Lv-kirrelL G.ATAC sequences. Violet shading indicates conserved sequences. Red boxes highlight putative transcription factor-binding sites. Mutations generated in binding sites are indicated in red letters below the aligned sequences. (B) Spatial expression pattern of GFP reporter constructs containing different G.ATAC element mutants. Top row: GFP fluorescence. Bottom row: GFP fluorescence overlayed onto differential interference contrast (DIC) images. Scale bar: 50 μm.

Figure 4—figure supplement 3. Interactions between Sp-kirrelL cis-regulatory elements (CREs) and the endogenous Sp-kirrelL promoter.

(A) Diagram showing the backbone of a modified, EpGFPII reporter construct containing the endogenous Sp-kirrelL promoter upstream of the Sp-endo16 promoter. (B) Spatial expression patterns of the modified GFP reporter constructs containing the various Sp-kirrelL elements indicated (elements B, C, E, F, H, and I). (C) Analysis of C element enhancer activity in modified EpGFPII reporter constructs. (D) Analysis of C.ChIP element enhancer activity in constructs containing wild-type (WT) or shuffled C element spacer sequences. Criteria for strong and weak primary mesenchyme cell (PMC) expression are defined in Figure 2. (E) Spatial expression patterns of the GFP reporter constructs. Top rows: GFP fluorescence. Bottom rows: GFP fluorescence overlayed onto differential interference contrast (DIC) images. Scale bar: 50 μm.

Figure 4—figure supplement 4. Stacked bar plots showing summary of GFP expression patterns of injected embryos scored at 48 hpf.

Each spatial expression category is expressed as a percentage of total injected embryos. (A) GFP spatial expression patterns of transgenic embryos injected with element G and truncation of element G mutant reporter constructs. (B) GFP spatial expression patterns of embryos injected with reporter constructs containing G.ATAC and G.ATAC with different transcription factor-binding site mutations. (C) GFP spatial expression patterns of transgenic L. variegatus embryos injected with Sp-kirrelL reporter constructs at 24 hpf. (D) GFP spatial expression patterns of embryos injected with constructs comprising of elements A–G in a modified EpGFPII vector containing the endogenous Sp-kirrelL promoter. (E) GFP spatial expression patterns of embryos injected with reporter constructs containing C.ChIP and wild-type (WT) or shuffled spacer sequences.