## 4 Incoherent FFLs
### Incoherent FFLs in Eukaryotes  
Biochemical interactions that determine gene expression and lead to the formation of network motifs are well-known in unicellular organisms, whereas there is more uncertainty about higher eukaryotes [53; 54]. Nevertheless, some studies have pointed out how  
Figure 6: Incoherent FFLs used in _E. coli_ consortia. (**A**) A canonical II-FFL gives a pulse in fluorescence in the presence of its input, AHL. (**B**) Non-canonical incoherent FFL adopted to achieve pattern formation on solid media. incoherent FFLs are present, among multicellular organisms, in gene networks that are involved in complex tasks such as neuron signal regulation and adaptation. Mammalian skin is known to be innervated with C fibers that transmit itch, pain, heat, and other noxious stimuli. Sensory neurons, which are associated with C-fibers, include VGLUT3+ low-threshold c-mechanoreceptors (CLTMs), MrgprD+ polymodal nociceptors, MrgprA3+ pruriceptors, and MrgprB4+ c-mechanoreceptors. Incoherent FFLs have been reported to control both the segregation and the genesis of mammalian sensory neuron sub-types [55,56]. These results are based on the phenotypic changes observed in knock-out mice. Runx1 (X, activator) and Zfp521 (Y, repressor) form an I1-FFL that controls the molecular identities that belong to the MrgprD+ polymodal nociceptors (Z). Moreover, Runx1 forms (with an unknown factor) an I3-FFLs to regulate the expression of MrgprA3 and Mrgpb4. The occurrence of several types of incoherent helps understand how sensory neuron subtypes are formed. Furthermore, it points out that incoherent FFLs have a critical impact in sensory neuron evolution and sensory decoding. In a different study concerning neuron expression, [PERSON] et al. [57] suggested that intermediate neural progenitors (INPs), in the brain of _Drosophila melanogaster_, make use of an I1-FFL to express different neurons in a precise chronological order (Dichaete, Grainyhead, Eyeless). The genes _Osa_, odd-paired (_Opa_), and Dichaete (_D_) should be linked via an I1-FFLs where Osa activates directly both D and its repressor Opa. However, the slower activation kinetics of Opa permits the accumulation of D that turn on the downstream cascade prior to its repression by Opa. Thus, the balanced expression of Opa and D seems to regulate the transition between successive temporal identities, though this network topology is not fully understood yet (see Figure 7). Any inconsistencies with this scheme, e.g., the overexpression of Osa, would result in incorrect temporal patterning and immature differentiation. Adaptation in eukaryotic chemotaxis (i.e., the movement along a gradient of a certain substance) has been studied in the social amoeba _Dictyostelium discoideum_[58]. Adaptation occurs when the output of a system returns to a fixed baseline after a change in the input signal. In the case of amoeba chemotaxis, the concentration of a chemo-attractant (the input) underwent a uniform change that triggered [30] a response from the chemotaxis pathway downstream of a G protein-coupled receptor (GPCR) [59]. In particular, Ras-GTP, i.e., the activated Ras--the protein that regulates signal transduction--showed almost perfect adaptation. This is achieved via a non-canonical incoherent feedforward loop, where the chemoattractant binds GPCR that leads to the activation of both RasGEF and RasGAP. The former activates Ras, the latter represses it. Thus, the simultaneous activation of a Ras activator and a Ras inhibitor is the key for adaptation. Coherent feedforward loops, in contrast, do not appear to be suitable for adaptation, as reported in [PERSON] et al. [60]. Previously, we have seen how an I1-FFL is adopted in _E. coli_ to direct the usage of arabinose or galactose under glucose starvation. [PERSON] et al. [30], in contrast,  
Figure 7: Drosophila neural stem cell neurogenesis lies, potentially, on an I1-FFLs.