Integration of Drosophila and Human Genetics to Understand Notch Signaling Related Diseases

Abstract

Notch signaling research dates back to more than one hundred years, beginning with the identification of the Notch mutant in the fruit fly Drosophila melanogaster. Since then, research on Notch and related genes in flies has laid the foundation of what we now know as the Notch signaling pathway. In the 1990s, basic biological and biochemical studies of Notch signaling components in mammalian systems, as well as identification of rare mutations in Notch signaling pathway genes in human patients with rare Mendelian diseases or cancer, increased the significance of this pathway in human biology and medicine. In the 21^st century, Drosophila and other genetic model organisms continue to play a leading role in understanding basic Notch biology. Furthermore, these model organisms can be used in a translational manner to study underlying mechanisms of Notch-related human diseases and to investigate the function of novel disease associated genes and variants. In this chapter, we first briefly review the major contributions of Drosophila to Notch signaling research, discussing the similarities and differences between the fly and human pathways. Next, we introduce several biological contexts in Drosophila in which Notch signaling has been extensively characterized. Finally, we discuss a number of genetic diseases caused by mutations in genes in the Notch signaling pathway in humans and we expand on how Drosophila can be used to study rare genetic variants associated with these and novel disorders. By combining modern genomics and state-of-the art technologies, Drosophila research is continuing to reveal exciting biology that sheds light onto mechanisms of disease.

Keywords: Drosophila; Functional genomics; Mendelian diseases; Notch signaling; Translational research.

Figure 1:. Schematic diagram of the Drosophila Notch signaling pathway.

Canonical Notch signaling takes place between two juxtaposed cells (left: signal sending cell, right: signal receiving cell). Different steps of signal activation and functions of molecules depicted here are described in detail in the main text. Note that some players depicted here only regulate Notch signaling in specific contexts. Abbreviations for proteins shown are based on FlyBase gene symbol nomenclature (also see Table 1).

Figure 2:. Notch signaling is required for lateral inhibition and lineage decisions during mechanosensory organ development.

A) Photograph of the fly notum. Large (macrochaetae) and small (microchaetae) are organized in a stereotypical fashion. B) Schematic diagram of a single mechanosensory organ (bristle). C) Schematic diagram representing the development of a single bristle. “N” indicates cells that activate Notch signaling. D-D’) Schematic diagrams of lateral inhibition during the selection of a sensory organ precursor (SOP) cell. In the beginning both cells have the potential to become an SOP. As development progresses, two cells acquire distinct fates through amplification of small differences through transcriptional feedback loops built into the stem. Cells that become the net signal sending cell becomes the SOP (labeled in red), and the net signal receiving cell(s) takes the epithelial cell fate. Panels B and C were adapted and mofidied from [108].

Figure 3:. Phenotypic consequences of Notch signaling loss during mechanosensory organ development.

A) Notch signaling mediates the lateral inhibition to specify an SOP from a proneural cluster. Cells that receive high Notch signaling becomes epidermal cells. B) Upon loss of Notch signaling during lateral inhibition, all cells takes the SOP cell fate. Photographs show SOPs marked by Senseless expression (Red). C) Reiterative Notch signaling is required to specify the four cell fates of the mechanosensory organ. The cells that receives the highest amount of Notch signaling becomes the Socket cells while the cells that receive the least becomes the neuron. D) Upon loss of Notch signaling during lineage decisions all cells take on the neuronal fate. Photographs show neuronal nuclei and membrane, labeled by antibodies against Elav (Red) and Hrp (Green). Panels A and B were adapted and mofidied from [108].

Figure 4:. Notch signaling is required for inductive signaling during wing margin development.

A) Photograph of a DAPI-stained wing imaginal disc that is pseudocolored for the dorsal domain (green) and the future wing margin (red). B) Schematic diagram of a transverse section of a part of the fly thorax. The dorsal wing imaginal disc (green) gives rise to the notum (dorsal thorax) and the dorsal wing blade. The ventral wing imaginal disc forms the ventral wing blade. The boundary between the dorsal and ventral compartment becomes the wing margin (red). C) Notch mediated inductive signaling specifies the future wing margin during imaginal disc development. Serrate (Ser) signals from the dorsal to the ventral compartment (red arrows) whereas Delta (Dl) signals from the ventral to the dorsal compartment (yellow). D) Upon Notch signaling activation at the dorsal-ventral boundary, genes such as Wingless (red) and Cut (not shown) are expressed, specifying the wing margin. E) Upon loss of Notch signaling during inductive signaling, these genes fail to be expressed and the wings exhibit a “notching” phenotype. Abbreviation of axes in panels A-B: D (Dorsal), V (Ventral), A (Anterior), P(Posterior), M (Medial), L (Lateral). Panel C was adapted and mofidied from [108].

Figure 5:. Strategies to “humanize” Drosophila genes in vivo.

A) For genes that have coding introns (introns flanking two coding exons), one can insert a T2A-GAL4 cassette via CRISPR and HDR (homology directed repair). When the gene of interest is translated, the splice acceptor (SA) forces the splicing machinery to include the T2A-GAL4 cassette. The transcriptional termination site (polyA) stops the transcription, preventing the rest of the gene to be transcribed. When the transcript (mRNA) is translated, N-terminal of the fly protein is made but is prematurely truncated due to the T2A (2A) ribosomal skipping sequence, leading to generation of nonfunctional proteins in most cases. T2A sequence further allows the GAL4 protein to be translated, which in turn translocates to the nucleus to activate the expression of human cDNAs (wild-type/reference or mutant/variant) under the control of UAS elements. B) For genes that do not have a coding intron, one can knock-in a GAL4 in the fly gene of interest. GAL4 will be transcribed and translated in the same temporal and special manner as the fly gene, allowing one to express the human cDNA in a mutant background. Grey boxes: 5’ and 3’ untranslated regions. Orange box: Fly coding sequence (CDS).