Abstract
The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression.
Highlights
During embryogenesis, the Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) is dynamically expressed predominantly in the primordia of the visceral mesoderm (VM), the developing CNS, the amnioserosa (AS) and in a restricted manner in the epidermis [1]
Using a robotic protein-DNA interaction assay, we identified the Zic family transcription factor Odd-paired as a factor that binds to regulatory elements in the Alk locus
We have identified additional enhancer regions regulated by the Bin and Bap transcription factors (TFs), likely together with additional TFs, that work with the Opa binding cis-regulatory module (CRM) to regulate Alk expression in the VM in a combinatorial manner
Summary
The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) is dynamically expressed predominantly in the primordia of the visceral mesoderm (VM), the developing CNS, the amnioserosa (AS) and in a restricted manner in the epidermis [1]. Alk plays a critical role during VM development, where it is activated in response to the secreted ligand Jelly Belly (Jeb) driving the Ras/MAPK/ERK pathway [2,3,4,5]. This leads to expression of founder cell (FC) specific transcription factors (TFs) such as Hand [6], optomotor-blind related-1 (org-1) [4] and factors important in the muscle cell fusion process like dumbfounded/kin of irre (duf/kirre) [3,4,5]. Signaling regulated by Jeb/Alk is critical for embryonic FC-specification and the subsequent fusion with FCMs to form a functional larval midgut muscle [2,3,4,5]
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