Analysis of the wnt1 regulatory chromosomal landscape

Abstract

One of the earliest patterning events in the vertebrate neural plate is the specification of mes/r1, the territory comprising the prospective mesencephalon and the first hindbrain rhombomere. Within mes/r1, an interface of gene expression defines the midbrain-hindbrain boundary (MHB), a lineage restriction that separates the mesencephalon and rhombencephalon. wnt1 is critical to mes/r1 development and functions within the MHB as a component of the MHB gene regulatory network (GRN). Despite its importance to these critical and early steps of vertebrate neurogenesis, little is known about the factors responsible for wnt1 transcriptional regulation. In the zebrafish, wnt1 and its neighboring paralog, wnt10b, are expressed in largely overlapping patterns, suggesting co-regulation. To understand wnt1 and wnt10b transcriptional control, we used a comparative genomics approach to identify relevant enhancers. We show that the wnt1-wnt10b locus contains multiple cis-regulatory elements that likely interact to generate the wnt1 and wnt10b expression patterns. Two of 11 conserved enhancers tested show activity restricted to the midbrain and MHB, an activity that is conserved in the distantly related spotted gar orthologous elements. Three non-conserved elements also play a likely role in wnt1 regulation. The identified enhancers display dynamic modes of chromatin accessibility, suggesting controlled deployment during embryogenesis. Our results suggest that the control of wnt1 and wnt10b expression is under complex regulation involving the interaction of multiple enhancers.

Keywords: ATAC-seq; CAGE-seq; Cis-regulation; Conserved non-coding element; Neural patterning; Spotted gar; Vertebrate; Zebrafish; wnt1; wnt10b.

Figure 1.. Conservation of the wnt1-wnt10b-arf3 genomic interval in vertebrates.

Gene data obtained from the UCSC genome browser, approximate interval size shown is indicated. Genes are indicated with blue arrows that indicate orientation. W1E refers to the conserved wnt1 3’ enhancer identified in mouse and pufferfish. Note the conservation of the interval including W1E through arf3 in all genomes. wnt6 is present between wnt1 and wnt10b in Coelacanth, consistent with a hypothesized ancient Wnt cluster Figure 1. Conservation of the wnt1-wnt10b-arf3 genomic interval in vertebrates. Gene data obtained from the UCSC genome browser, approximate interval size shown is indicated. Genes are indicated with blue arrows that indicate orientation. W1E refers to the conserved wnt1 3’ enhancer identified in mouse and pufferfish. Note the conservation of the interval including W1E through arf3 in all genomes. wnt6 is present between wnt1 and wnt10b in Coelacanth, consistent with a hypothesized ancient Wnt cluster (Nusse, 2001).

Figure 2.. Analysis of sequence conservation in the wnt1-arf3 genomic interval.

(A) Vista plot comparison of zebrafish to stickleback (top bracket) and zebrafish to spotted gar (bottom bracket). Conserved noncoding elements are represented by red peaks. Numbering is according to vista browser output. The absence of element 20 in the zebrafish-gar comparison is attributable to an incomplete gar contig; element 20 is found on a separate contig. (B) Schematic diagram of the reporter construct used to evaluate transcriptional activity of CNEs. Tol2 elements facilitate transposition via the Tol2 system. Constructs include the cfos minimal promoter.

Figure 3.. wnt1 CNEs are transcriptionally active.

All images lateral views, 27 hpf embryos.(A,C,E,G,I,K,M,O,Q) EGFP fluorescence. (B,D,F,H,J,L,N,P,R) In situ hybridizations for EGFP transcripts. Note broad GFP fluorescence not matched by transcription pattern for CNE19 (A,B), 25 (E,F), 26 (G,H), 32 (M,N) and 33 (O,P), indicating earlier expression that terminates prior to 27 hpf. Restricted expression is observed for CNE 20 (C,D), 27 (I,J), 31 (K,L) and 37 (Q,R), but the transcription pattern for CNE 27 indicates the cessation of transcription prior to 27 hpf. In contrast, CNE 20 is transcribed robustly in the MHB and dorsal midbrain at 27 hpf. CNE 31expression in the hatching gland (K, asterisk) is likely a positional effect.

Figure 4.. Spotted gar CNE 20 and 27 are active in zebrafish.

Expression at 27 hpf, lateral views. EGFP fluorescence (A,C) and corresponding in situ hybridizations for EGFP transcripts (B,D). Note relative differences in expression strength at the MHB (arrows), with 27 being more strongly expressed than 20.

Figure 5.. Comparison of zebrafish to gar CNE20 and CNE27 activity.

Lateral views of EGFP fluorescence in 27 hpf zebrafish heads. Note widespread but mottled expression from gar CNE20 (A, arrow) in comparison to the restricted expression of zebrafish CNE20 (B). While the gar 20 element is expressed strongly in the epiphysis (asterisk), zebrafish 20 is expressed at a lower level, but is also observed in the dorsal midbrain (arrowhead). Gar CNE27 is observed strongly in the posterior midbrain and MHB (C, arrow), in comparison to the more diffuse midbrain expression of zebrafish CNE27 (D). This likely reflects different temporal expression dynamics, as zebrafish 27 is not transcribed strongly in the midbrain or MHB at this time (see Fig. 3J).

Figure 6.. ATAC-seq peaks identify conserved and non-conserved cis-regulatory elements.

The window spans rps26 through phf8, with CNEs indicated by their numbers below red box outlines. Putative non-conserved enhancers are indicated by asterisks below red boxes. The embryonic sources for each ATAC-seq line are indicated in the black boxes above each line.

Figure 7.. CAGE-seq identifies transcription start sites for wnt1 and wnt10b.

Refseq transcripts are shown in blue. (A) wnt1 transcripts originate from a ~86 bp window of CNE 26. (B) wnt10b transcripts originate from a more localized focus at or adjacent to the refseq 5’ end.