The Notch signaling pathway

The Notch signaling pathway is one of the "six major and universal signal trans- duction devices in the cell" [61]. Notch is present in all metazoans and it has been conserved through evolution. This universal device influences cell fate speci- fication, cell proliferation and apoptosis in almost all vertebrate and invertebrate tissues and organs [62, 63]. Indeed, Notch pathway is involved in different as- pects of the developing embryo at different stages. This encompasses a wide variety of processes, ranging from the creation of the somites along the body in vertebrates [14], angiogenesis, which is the creation of red blood vessels [64], neu- rogenesis [54, 65], boundary determination in tissues [66] and so on. Moreover, such pathway is directly related to different important diseases, like cancer [67]. The firsts studies involving the Notch pathway were performed on inverte- brates, being first in Drosophila, and then in the worm C. elegans. Later on, a homolog was found in the Xenopus frog, and finally it was also found in humans. The Notch term comes from a story that happened around 1913 1914. On March 1913, a Drosophila mutant exhibited several serrations or notches on its

1.3. Our focus: salt-and-pepper patterns due to the Notch signaling pathway ligand Notch receptor NICD Proneural genes Neural differentiation

Figure 1.2: Simplified cartoon of the Notch signaling pathway. A Notch ligand (e.g. Delta) (left cell) binds to the Notch receptor in a neighboring cell (right cell). Then, a series of proteolytic cleavages (not shown) releases the Notch intracellular domain (NICD) towards the cell that is harboring the receptor. NICD ultimately inhibits the proneural genes expression, which drive the expression of further ligand and neural differentiation. Note that in this cartoon we omit the Hes/Hey Notch targets and therefore its direct regulation on the Notch ligands.

wings [68]. Such phenotype was firstly reported on 1914 [69] and it was shown to be a dominant effect of a sex-linked mutation in the X-chromosome [68, 70]. In 1937 Poulson showed that deficiencies in the X-chromosome in Drosophila lead to altered embryos with a prominent nervous system [71], what would correspond to a massive production of neurons, a feature that is named neurogenic pheno- type [62]. Almost 50 years later, the group of Artavanis-Tsakonas discovered the locus of the Notch gene and cloned it, revealing that it was related to a surface membrane receptor [63]. Later on, in 1990, Fehon et al. showed that cells expressing the Delta ligand formed aggregates with Notch expressing cells, indicating a specific interaction for Notch with Delta anchored in neighboring membranes [72]. Afterwards, many works have shown this interaction and have focused on its downstream events (see for instance reviews [73, 74]).

Nowadays, the Notch pathway is a very active field of research, and new elements of this pathway are still being revealed [63].

anchored on the membrane of neighboring cells (Fig. 1.2) [72]. These intercellular or ’trans-interactions’ result in a complex that undergoes a series of proteolytic cleavages, leading to the release of the Notch intracellular domain (NICD) towards the cell that is harboring the receptor. NICD translocates to the cell nucleus, where it forms a complex with the so-called CLS transcription factor and a co- activator called Mastermind/Lag-3. Then, such ternary complex binds to specific DNA regulatory sequences, activating the transcription of different Notch target genes (see reviews [73, 74] for a detailed explanation of this process). Among these genes there are the family of Hes/Hey genes [75, 76]. Hes/Hey genes are transcriptional inhibitors of the Notch ligands genes itself and a group of genes named proneural genes, which are essential for cell differentiation into the neu- ral and other cellular fates. Proneural genes expression also inhibits the Notch ligands genes [77]. For understanding salt-and-pepper patterning, the fact that Notch receptor, upon binding with its ligand on a neighboring cell, inhibits the production of ligand is crucial, as will be described below.

Notch receptor has different canonical ligands. In vertebrates these ligands are Delta and Jagged (in Drosophila, the Jagged homologous ligand is Serrate). In worms, the Notch homolog receptors are called GLP and LIN, and its ligands LAG and APX. In vertebrates, receptor and ligand types can present different subtypes (paralogs). Specifically, in vertebrates four Notch receptor subtypes have been characterized (Notch1-4) and six ligands, four belonging to Delta family (Delta- like 1-4, homologous to Delta) and two to Jagged family (Jagged 1-2, homologous to Serrate). In Drosophila there is just one Notch receptor and two different ligands, Delta and Serrate. Moreover, there are numerous non-canonical factors that can modulate Notch pathway. For instance, the non-canonical Notch ligands Delta-like homologs Dlk1/2, which are similar to the canonical ligands but lack the DSL domain contained by the canonical ligands [78, 79].

There are additional aspects of the Notch pathway that appear to be pivotal for its regulation at transcriptional and post-transcriptional levels [80]. Some of the most studied are ligand endocytosis [81], ligand regulated degradation [82] and receptor modifications through Fringe glycosiltransferases [83, 84]. More- over, Notch pathway coexists with other signaling pathways in the cell. Though, these pathways do not act independently, but there is crosstalk among them [63]. For instance, the Notch pathway shares some elements with the Wingless/Int1

1.3. Our focus: salt-and-pepper patterns due to the Notch signaling pathway (Wnt) [61] and the Hedgehog (Hh) [63] pathways, being both of them two other important pathways in metazoan development. Hence, Notch pathway is very complex, and the context in which is acting will drive to very distinct phenom- ena.

Notch pathway has been reported to function in two different operating modes: lateral inhibition and lateral induction (Fig. 1.3). In lateral inhibition, a cell heading for a particular fate inhibit its neighbors to adopt such fate. Thus, a cell that is expressing high ligand amount will inhibit its neighboring cells to express such ligand. This kind of behavior will be essential for the creation of salt-and- pepper patterns in a wide variety of sensory and non-sensory tissues, like in the vertebrate and invertebrate eyes, the vertebrate inner ear, and the epidermis of zebrafish. Lateral induction acts in the opposite way, driving nearby cells to adopt the same fate. Lateral induction has been proposed to occur in lens formation, in pancreas development, in angiogenesis and in inner ear development, among others [18].

Notch signaling can also undergo synchronous and asynchronous oscillations in the tissue [85]. One of the most studied phenomena where genetic oscillations have been observed is in somitogenesis, where synchronous oscillations and delays are of paramount importance [14, 86–88].

In this Thesis we mainly focus on lateral inhibition, since we are mostly in- terested in salt-and-pepper patterns and how different elements in the Notch pathway modify them. Moreover, lateral inhibition has been a subject of study by biologists in the last thirty years, so there is enough literature regarding this patterning mechanism for doing a theoretical work with a strong motivation in previous experimental observations. In Chap. 6 we also deal with lateral induc- tion, which is a less studied phenomenon.

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Figure 1.3: Notch can act in two different operating modes. (A) Lateral induction, in which a cell induce its neighboring cells to adopt the same fate and (B) lateral inhibition, in which a cell inhibits its neighboring cells to adopt the same fate. We illustrate this phenomenon in terms of ligand expression. (C,D) Possible dynamical outputs for (C) lateral induction and (D) lateral inhibition. Red intensity is related to the amount of ligand concentration: high ligand cells are red, while cells without ligand are white. Arrows in the last panel of (A,C) and (B,D) indicate activation and inhibition, respectively.