FIGURE SUMMARY
Title

Hoxa1 targets signaling pathways during neural differentiation of ES cells and mouse embryogenesis

Authors
De Kumar, B., Parker, H.J., Paulson, A., Parrish, M.E., Zeitlinger, J., Krumlauf, R.
Source
Full text @ Dev. Biol.

Hoxa1-bound regions function as enhancers. (A, B) Browser shots of two Hoxa1 bound regions (A, Dok5) and (B, Wls1) along with occupancy of TALE cofactors (Pbx and Meis), co-activator p300, modified histone marks characteristic of enhancers (H3K4me1 and H3K27Ac) and open chromatin states (ATAC-seq) are shown. ChIP-nexus data reveals the presence of multiple binding motifs (bottom) for Hoxa1 and its cofactors in the protected regions. (C, D) Regulatory analysis of the (A, Dok5) and (B, Wls1) Hoxa1-bound regions along with 250 bp flanking sequences in mouse and zebrafish embryos using LacZ and GFP transient transgenic reporter assays, respectively. In C, zebrafish embryos with mCherry inserted in the endogenous egr2b locus (krox20), mark r3 and r5 with reporter expression (Distel et al., 2009). The bound region from each gene mediates neural-specific expression in mouse and zebrafish embryos, indicating conservation of activity. OV, otic vesicle, m/h, mid/hindbrain boundary and r, >rhombomere.

Evolutionary conservation of Hoxa1 targets. (A) Ectopic expression of Hoxa1 in zebrafish. In control embryos, Red, mCherry reporter expression in rhombomeres 3 and 5 (r3&r5) and green,GFP reporter expression under control of a Hoxb1-ARE enhancer mediates spatially-restricted expression in r4 and the rostral brain. Injection with Hoxa1 or epitope-tagged Hoxa1 RNAs repressed mCherry-specific expression in r3 and anteriorizes r4 expression of GFP (brackets). (B, C) >MA plots showing differentially expressed (DE) genes (as red and green dots) in zebrafish after injection with mouse Hoxa1_3flag_myc (B) or mouse Hoxa1 (C) mRNA. (D) A scatterplot of gene expression values (log2-fold-change) from the two assays show minimal differences (R = 0.96). DE genes are shown in color. (E) Pi chart showing fraction of mouse orthologous genes modulated by Hoxa1 in zebrafish. Nearly half of the orthologous genes have a nearby Hoxa1 binding site, suggesting evolutionary conservation of Hoxa1 targets between mouse and zebrafish. (F) Transcriptional profiling of zebrafish embryos comparing control and mouse Hoxa1 injected embryos (20 hpf). Differential gene expression at FDR ≤ 0.05 are shown as a heatmap with values capped at±3 for visibility. At the left are examples of up and downregulated genes.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image.

Reprinted from Developmental Biology, 432(1), De Kumar, B., Parker, H.J., Paulson, A., Parrish, M.E., Zeitlinger, J., Krumlauf, R., Hoxa1 targets signaling pathways during neural differentiation of ES cells and mouse embryogenesis, 151-164, Copyright (2017) with permission from Elsevier. Full text @ Dev. Biol.