InteractiveFly: GeneBrief

sequoia : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. The sequoia mutant was identified from a screen for dendrite mutants. Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia is required for specification of a subset of neurons such as the es neurons in the embryo; however, sequoia also functions in regulating dendrite and axon development of every neuronal type examined, including es and other PNS neurons, photoreceptors, and motoneurons in the CNS. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates (Brenman, 2001).

The Drosophila peripheral nervous system (PNS) is a good assay system for the genetic study of dendrite development because it is small enough, containing only 44 neurons per hemisegment, yet most multiple dendritic (md) neurons exhibit complex dendritic branching patterns. Having developed a system using green fluorescent protein (GFP) to visualize dendrite outgrowth in real time in living embryos (Gao, 2000), it was found that dendrite development follows a stereotyped spatial and temporal pattern. Dendritic outgrowth begins after an initial 'bud' site(s) has been chosen. Following primary dorsal dendrite extension, additional lateral dendritic processes elaborate and retract with only a subset becoming stabilized to form secondary dendritic branches (Brenman, 2001).

By monitoring the morphogenesis of these PNS neurons, attempts were made to isolate mutants that would help to dissect the developmental processes in dendrite formation. Some of the questions that might be addressed include: (1) What controls the distinct dendrite morphology of neurons within the same lineage? Several models have been proposed to describe possible lineages that give rise to md neurons. Despite differences in the exact relationship between external sensory (es) and md neurons, these models predict that some of the md and es neurons are derived from a common precursor cell. The dendritic morphologies of the two neuronal types produced by the same precursor are drastically different. The es neuron possesses a single unbranched dendrite, while most md neurons have multiple, highly branched dendritic processes. What causes these neuronal descendents of a common precursor cell to adopt distinct dendrite morphology is unknown. (2) How is the dendritic field of any given neuron specified? Are there 'universal' regulators involved in controlling the morphology of most and possibly all neurons? In the screen, a mutant, sequoia, was identified that provides insight to both of these questions. sequoia alters both the degree of branching and the resulting dendritic field. Dendrites from sequoia mutant embryos display an excessive outgrowth phenotype that is caused by precocious dendrite development as well as an inability to stop dendrite elongation at the appropriate time during embryonic development. sequoia mutants exhibit severe axon breaks in both the PNS and CNS. sequoia also plays a role in cell fate decisions since mutant embryos exhibit excess md neuron production at the expense of es neurons. Thus, similar to prospero, sequoia belongs to an emerging class of genes that affect the cell fate of a subset of neurons and regulate neuronal process formation in a much larger population of neurons. sequoia has more widespread effects on neuronal morphology than other pan-neural genes studied to date. As a likely transcription regulator, sequoia appears to preferentially regulate genes that control morphogenesis rather than genes known for directing cell fates in the nervous system (Brenman, 2001).

The screen utilized a Gal4 enhancer trap line driving GFP expression in md but not es neurons in the dorsal cluster of the embryonic PNS. In sequoia mutant embryos, a range of excessive dorsal dendrite outgrowth was observed for GFP-positive md neurons that fall into two groups. Neurons in one group occupy the same position as that in wild-type embryos. Neurons in the other group are located more dorsally at sites normally occupied by es neurons. The second group of neurons tend to fasciculate their dendritic processes together and extend fewer lateral branches. In either group of neurons, dendrites are abnormally long at late embryonic stages so that dendrites from dorsal clusters in the two hemisegments cross the dorsal midline and intermingle with each other. This is in stark contrast to dendrites from wild-type embryos that are significantly shorter and never reach the dorsal midline in newly hatched larvae (Brenman, 2001).

The excessively long dendrites observed in the mutant could arise from premature growth initiation, faster dendritic growth, and/or an inability to halt growth at the appropriate time. To examine these possibilities, multiple staging criteria were used as well as time-lapse analysis to determine when dendrite growth started and stopped, as well as the rate of growth. Immediately before the formation of cuticle that is impermeable to antibody (stage 16), dendrites labeled with 22C10 antibody were longer in sequoia mutants than those in wild-type embryos. Time-lapse analysis reveals that mutant embryos did not extend dendrites at a faster rate than that in wild-type embryos. Rather, mutant embryos initiate dendritic outgrowth precociously. Immunostaining of embryos at 11-12 hr after egg laying (AEL) revealed significant md dendrite extension in >85% of sequoia mutants but only in <10% of wild-type embryos. At this stage, es dendrites can be seen in wild-type embryos. The es dendrite is morphologically distinct from md dendrites; it has a characteristic dendrite 'squiggle' morphology and a dendritic cap that innervates a cuticular structure of the es organ and exhibits intense staining with the 22C10 antibody. Such es dendrites were not readily discerned in mutant embryos. In addition to cuticle formation, a second independent staging criterion was used to demonstrate precocious md dendrite extension in sequoia mutants. In sequoia mutants, md dendrites were detected as early as stage 14, when the midgut undergoes characteristic cell movements and constrictions. However, wild-type embryos that had progressed beyond this stage of gut development were typically devoid of md dendrites, in contrast to the precocious dendritic outgrowth in sequoia mutants (Brenman, 2001).

Not only did sequoia mutants initiate dorsal dendrite outgrowth abnormally early, they also failed to halt dorsally oriented outgrowth later in embryonic development. Time-lapse analysis revealed that in wild-type embryos, once the dorsal dendrites reach a certain length (at 16-17 hr AEL), dorsally oriented dendritic growth stops and only lateral branches increase in length (Gao, 2000). In sequoia mutants, the dorsally oriented dendritic tips continue growing toward the dorsal midline and eventually approach the dendritic tip from the contralateral homologous cluster. Failure to stop dorsal dendrite elongation is evident for all neurons in the mutant dorsal clusters. For both the md neurons with lateral branches and the extra md neurons that fasciculate their dendrites together and occupy positions of es neurons, their dorsal dendrites extend into the dorsal most zone normally devoid of dendrites at this developmental stage (Brenman, 2001).

Is the extensive overgrowth of dendrites in md neurons associated with any axonal defects? Characterization of the PNS stained with 22C10 antibody reveals that, instead of having normal axon fascicles that have entered the CNS, axons emerging from the dorsal cluster of sequoia mutants were fewer in number and frequently misrouted or stopped altogether in their path of outgrowth toward the CNS. Axons terminating prematurely were observed not only for md neurons but also for all other PNS neurons, resulting in complete axon breaks between PNS clusters. es neurons with the characteristic 'squiggle' morphology were frequently missing in sequoia mutants. Compared to wild-type embryos of the same developmental stage, shortened axons were routinely detected in sequoia mutant exhibiting abnormally elongated dendrites from PNS dorsal cluster md neurons. Immunostaining of sequoia mutants with monoclonal antibody 1D4 shows an axonal phenotype in the CNS similar to that in the PNS. Large fascicles of longitudinal axon tracts evident in the CNS of wild-type embryos are absent in mutant embryos. In addition, motoneuron projections to the periphery are also greatly reduced (Brenman, 2001).

A priori, one could imagine two extreme scenarios to generate the numerous subtypes of neurons, glia, and support cells. At one extreme, every specialized cell type has its own 'master regulator' molecule necessary and sufficient to generate a particular cellular identity. In this case, any cell fate transformation must result in morphological changes, and morphological changes must always be accompanied with cell fate changes. However, it is known from the examples of prospero and Notch mutants that this is not always the case. Cell fate phenotypes can often be separated from morphological phenotypes. This dichotomy clearly illustrates multiple functions for a single molecule. More likely, evolution hones genes and pathways to execute a particular task that will be used repeatedly in multiple developmental processes, including both cell fate specification and morphogenesis. As a consequence, there need not always be a link between them. For instance, a combination of Notch alleles and UAS-Notch transgenes has been used to produce cell fate transformation without axon defects or axon defects without cell fate transformation (Brenman, 2001).

sequoia is similar to the prospero locus in many respects. Both molecules are required to achieve specific cell fates in a small subset of the Drosophila nervous system. However, mutations of both genes affect many other cells besides those that exhibit clear fate changes. Both prospero and sequoia mutants display axon and dendrite phenotypes in addition to cell fate alterations. Clearly, genes involved in regulating neuronal morphology can have independent effects on cell fate and morphology. The prospero mutations affect cell fate in the CNS but not PNS and alter neuronal morphology in both. sequoia thus appears to be similar to prospero but has even broader roles in regulating neuronal morphogenesis (Brenman, 2001).

In sequoia mutant embryos, extra md neurons are generated at the expense of es neurons in the PNS dorsal cluster. This is highly reminiscent of Notch mutant embryos where extra neurons in the PNS are generated, and all of them appear to be md neurons by both enhancer trapping and morphological criteria. Moreover, these extra md neurons in Notch mutant embryos provide an example of generating md neurons at the expense of es neurons. Taken together, these observations support the notion that Sequoia could function in the Notch pathway. Not only are there phenotypic similarities, but Sequoia shares sequence homology (~55%) to the DNA binding zinc finger domains of Tramtrack. Tramtrack has been shown to function downstream of Notch in specifying cell fate in the nervous system. Unlike tramtrack, however, sequoia can alter dendrite morphology without changing cell fate. Whereas the es neurons normally have a single straight dendrite, extra dendritic 'arbors' of es neurons are occasionally observed in adult loss-of-function sequoia mutant clones even though they are clearly associated with external sensory structures typical of es organs. This sets sequoia apart from tramtrack and other mutants that affect cell fate in the nervous system without producing aberrant neuronal morphologies. Another example of this latter group is cut. cut mutants have es organs transformed into chordotonal organs, yet these transformed organs still display dendrites characteristic of their altered identity and axons that enter the CNS. In summary, sequoia resembles prospero and Notch as a molecule that can regulate cell fate independent of morphogenesis (Brenman, 2001).

During Drosophila embryogenesis, dorsal dendrite outgrowth of md neurons stops several hours before the larvae hatches, leaving a large uninnervated region flanking the dorsal midline. This area is eventually covered with dendrites by the second instar larval stage. Studies of the formation of dorsal dendritic territories (Gao, 2000) suggest that both intrinsic and extrinsic factors participate in defining dendritic fields. Several mutants identified in a screen (including sequoia described here) display a dorsal dendrite overextension phenotype, wherein the dorsal dendrites from the two hemisegments meet and/or intermingle at the dorsal midline in the embryo. In theory, this phenotype could be due to earlier dendrite outgrowth, faster dendrite outgrowth, and/or failure to halt dorsally oriented dendrite outgrowth. Mutants such as flamingo fail to stop dorsally oriented outgrowth (Gao, 2000), while sequoia mutants not only start dendrite outgrowth abnormally early but also fail to stop outgrowth later in embryogenesis. The nature of the dorsal dendrite outgrowth 'stop' signal in embryos remains unknown but is unlikely to be governed by competition from homologous neuronal dendrites, since these dendrites do not normally meet during embryogenesis. One could imagine different ways in which developing dendrites recognize 'stop' signals. In one scenario, an inherent 'ruler' predetermines the final dendrite length. This inherent ruler is either absent or reset to a longer length in sequoia mutant embryos. In another scenario, developing dendrites detect a 'stop' signal (e.g., a ligand present at the dorsal midline), and sequoia is required to generate the components necessary to respond to and transduce this 'stop' signal (Brenman, 2001).

If cytoskeletal or other components are required for assembly of both axons and dendrites, could it be that mutants that result in dendrite 'rich' structures may come at the expense of axonal structures and vice versa? Several of the mutants isolated in prior mutant screens display 'extra' dendritic branching or dorsally oriented dendrite outgrowth. These include tumbleweed, flamingo, kakapo, and sequoia. Interestingly, all of these mutants exhibit axonal aberrations. Specifically, some of the axons from these mutants fail to extend fully to reach their targets. Conversely, mutants such as shrub and shrinking violet that display a decrease in dendritic structure do not exhibit shortened axons. It remains to be seen whether these correlations between dendrites and axons reflect a true competition for resources between them or simply the involvement of the same genes in both axon and dendritic outgrowth. Alternatively, the presence of different molecular machinery for the elaboration of axons and dendrites could explain why the same gene would lead to very different effects on axon versus dendrite outgrowth (Brenman, 2001).

sequoia appears to be a universal regulator of morphology in Drosophila neurons. No another pan-neural gene, when removed, causes as widespread and profound defects in neuronal morphogenesis. Both phenotypic and microarray analyses indicate that sequoia regulates multiple aspects of neuronal morphogenesis, including dendrite arborization, length of neurites, and axon guidance. Preliminary microarray analysis further indicates that sequoia regulates, directly or indirectly, the expression of several genes involved in axon guidance as well as novel genes potentially important for neurite outgrowth, without affecting transcript levels for the vast majority of genes examined. This includes genes that specify cell fates and many genes expected to be required by all neurons. Further mechanistic insight may be gleaned from future studies of the downstream genes of sequoia at the cellular level for their potential roles in controlling neuronal morphology (Brenman, 2001).

sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system

Neural progenitors typically divide asymmetrically to renew themselves, while producing daughters with more limited potential. In the Drosophila embryonic ventral nerve cord, neuroblasts initially produce daughters that divide once to generate two neurons/glia (type I proliferation mode). Subsequently, many neuroblasts switch to generating daughters that differentiate directly (type 0). This programmed type I>0 switch is controlled by Notch signaling, triggered at a distinct point of lineage progression in each neuroblast. However, how Notch signaling onset is gated was unclear. Sequoia (Seq), a C2H2 zinc finger transcription factor with homology to Drosophila Tramtrack and the positive regulatory domain (PRDM) family, has been identified as important for lineage progression. This study found that seq mutants fail to execute the type I>0 daughter proliferation switch, and also display increased neuroblast proliferation. Genetic interaction studies reveal that seq interacts with the Notch pathway, and seq furthermore affects expression of a Notch pathway reporter. These findings suggest that seq may act as a context-dependent regulator of Notch signaling, and underscore the growing connection between Seq, Ttk, the PRDM family and Notch signaling (Gunnar, 2016).

In seq mutants, an increase was found in the number of cells in the NB5-6T and NB3-3A lineages, as well as aberrant daughter divisions in both lineages. This effect is mirrored globally, with elevated daughter divisions in both the thorax and abdomen. From these results it is concluded that seq plays a key role in promoting the type I>0 daughter proliferation switch. Surprisingly, it was found that seq overexpression also triggers aberrant type I>0 switches. These results could indicate that Seq expression levels are instructive, with high levels promoting the type I proliferation mode and lower levels promoting type 0. On that note, there is precedence for transcription factors switching between repressor and activator function in a concentration-dependent manner. Elevated NB proliferation was also observed in seq mutants and with seq overexpression, in NB3-3A and globally, indicating that the precise NB cell cycle exit at the end of lineage progression is affected by seq function and levels. It was found that seq represses CycE and E2f1, both in thoracic NBs in general and specifically in NB5-6T. Dap expression was weakly increased globally, but weakly reduced in NB5-6T, suggesting an aspect of context dependency with respect to seq regulation of Dap (Gunnar, 2016).

Notch signaling also plays a key role in triggering the type I>0 switch, and Notch reporter [E(spl)HLHm8-GFP] expression is turned on in NBs during the latter stages of lineage progression (Ulvklo, 2012; Bivik, 2016). This indicates that the timing of Notch signaling onset is important for the precision of the type I>0 switch. Notch activation results in the formation of a tripartite protein complex comprising the Notch intracellular domain (NICD), the DNA-binding factor Su(H) and the co-factor Mastermind (Mam). Molecular and genetic in-depth analysis of how Notch signaling controls the type I>0 switch points to a multi-level model whereby NICD/Su(H)/Mam activates E(spl)HLH and dap, and E(spl)HLH subsequently represses CycE, E2f1 and stg (Bivik, 2016) (Gunnar, 2016).

The findings point to a complex balancing interplay between seq, the Notch pathway and the cell cycle. Premature and elevated Notch reporter [E(spl)HLHm8-GFP] expression was observed in seq mutants, indicating that seq represses Notch signaling in NBs. However, seq mutants phenocopy Notch pathway perturbation, and both result in a failure to execute the type I>0 switch. Moreover, seq and E(spl)HLH interact strongly genetically in transheterozygotes, as is evident by increased Ap cell numbers. Based on these findings, it is proposed that Seq acts at several steps of the Notch type I>0 cascade by repressing not only E(spl)HLH but also CycE and E2f1, and that the balance in regulation of these different targets is sensitive to the levels of Seq. Specifically, it is proposed that Seq is a stronger repressor of CycE and E2f1 than of E(spl)HLH. Combined with the repressive role of E(spl)HLH on CycE and E2f1, this model might also help to explain why seq mutants phenocopy seq overexpression (Gunnar, 2016).

Notch signaling plays a key role during development of the external sensory organs (ESOs) in Drosophila. Notch acts at multiple steps of ESO development: in the process of lateral inhibition, to select the sensory organ precursor (SOP), and during subsequent asymmetric cell division events in the SOP lineage. Previous studies of seq in the developing ESOs revealed defects both in the external lineage, with a conversion of shaft cells into socket cells, and the internal lineage, with loss of both neuron and glia specification. These phenotypes could to some extent be explained by the loss of expression of three Notch target genes: D-Pax2 (shaven - FlyBase), pros and hamlet (ham) . Interestingly, of these three seq targets, only ham was affected in the embryonic PNS. Comparing the role of Notch signaling with that of seq during ESO development, in spite of sharing several target genes seq only partly phenocopies Notch pathway mutants: Notch clones show supernumerary SOP cells and conversion of the entire SOP lineage into neurons, whereas seq clones do not show extra SOP cells but instead shaft-to-socket conversion and neuron/glia specification defects (Gunnar, 2016).

Similar to the lack of SOP selection effects in ESOs in seq mutants, no effects were observed upon NB selection in the embryonic neuroectoderm, a process also controlled by Notch-mediated lateral inhibition. Instead, seq acts at a later stage to modulate expression of the Notch targets E(spl)HLH and CycE, during the subsequent type I>0 daughter proliferation switch. These studies reveal that the interplay between seq and the Notch pathway is highly context dependent, and seq appears to act on different Notch subroutines in different settings. Elevated NB divisions were also observed in seq mutants, in NB3-3A and globally. This is in contrast to Notch signaling, which does not appear to affect NB cell cycle exit in the VNC. Hence, seq can also play roles that are independent of Notch signaling during nervous system development (Gunnar, 2016).

The C2H2 zinc fingers of Seq are highly homologous to the zinc fingers in Ttk. Seq and Ttk furthermore share homology in their zinc fingers with members of the PRDM family. In addition to related zinc fingers, Seq, Ttk and the PRDM family also share an involvement in Notch pathway signaling. ttk has been identified as a Notch pathway target gene and effector, both during oogenesis and ESO development. The PRDM family plays important roles during development and is also intimately linked to Notch signaling. The Drosophila ham gene, a PRDM family member, was found to control ESO development, chiefly by modulating Notch signaling. Similarly, in olfactory sensory lineages, ham also modifies Notch signaling, intriguingly by repressing E(spl)HLH gene expression via direct binding of Ham to this complex. In the developing mammalian CNS, Prdm8 and Prdm16 were found to be regulated by the bHLH HES genes Hes1, Hes3 and Hes5 (Gunnar, 2016).

Dynamic protein expression is another common denominator. Seq, Ttk and Ham were all found to be dynamically expressed in the developing SOPs In addition, dynamic Seq protein expression levels govern photoreceptor axon targeting to the optic lobe (Gunnar, 2016).

In summary, Seq, Ttk and the PRDM family have in common their intimate connection to the Notch pathway, acting to regulate the pathway, and/or being regulated by it, and/or regulating Notch downstream targets. They also share the property of controlling cell fate and proliferation in the developing nervous system, in some cases acting on the same targets (e.g., CycE). Finally, they are highly dynamic in their expression, and the expression levels can act in instructive and temporal manners. These results for seq support the role of the extended PRDM family as context-dependent, temporally controlled and level-sensitive modifiers of Notch signaling during nervous system development (Gunnar, 2016).

A switch in tissue stem cell identity causes neuroendocrine tumors in Drosophila gut

Intestinal stem cells (ISCs) are able to generate gut-specific enterocytes, as well as neural-like enteroendocrine cells. It is unclear how the tissue identity of the ISC lineage is regulated to confer cell-lineage fidelity. This study shows that, in adult Drosophila midgut, loss of the transcriptional repressor Tramtrack in ISCs causes a self-renewal program switch to neural stem cell (NSC)-like, and that switch drives neuroendocrine tumor development. In Tramtrack-depleted ISCs, the ectopically expressed Deadpan acts as a major self-renewal factor for cell propagation, and Sequoia acts as a differentiation factor for the neuroendocrine phenotype. In addition, the expression of Sequoia renders NSC-specific self-renewal genes responsive to Notch in ISCs, thus inverting the differentiation-promoting function of Notch into a self-renewal role as in normal NSCs. These results suggest an active maintenance mechanism for the gut identity of ISCs, whose disruption may lead to an improper acquisition of NSC-like traits and tumorigenesis (Li, 2020).

In addition to the nervous system, neuroendocrine (NE) cells are found in many non-neural tissues and can develop neoplasias that are known as NE tumors (NETs). The NE cells in non-neural tissues display characteristics that are typical of neurons, such as membrane excitability and hormone secretion, yet many of these NEs are generated from adult stem cells of endodermal origin. It is, thus, intriguing how the tissue identity of stem cells is regulated and controlled to safeguard cell-lineage fidelity (Li, 2020).

The intestinal epithelium in adult Drosophila midgut is maintained by intestinal stem cells (ISCs)-the multipotent cells that are capable of generating both absorptive enterocytes (ECs) and secretary enteroendocrine cells (EEs). EEs are neural-like cells and are able to secrete sets of hormone peptides that are similar to those secreted by NE cells in the Drosophila brain. While the initiation of EC generation is driven by Delta (Dl)/Notch-mediated lateral inhibition between the two immediate stem cell daughters, the initiation of EE generation occurs at the stem cell level, with a transient expression of the proneural gene Scute (Sc) that induces ISCs to self-renew and to generate an EE progenitor cell (EEP). Each EEP then divides one more time before terminal differentiation to yield a pair of EEs. Sc encodes a basic helix-loop-helix (bHLH) transcription factor and belongs to the achaete-scute gene complex (AS-C), a Drosophila proneural gene cluster that is expressed in neural progenitor cells and is important for the development of the embryonic central nervous system and sensory organs of both larva and adult. Thus, it appears that there is a transient activation of neural-like programs in ISCs that directs EE generation from ISCs (Li, 2020).

Tramtrack (Ttk, or Ttk69 isoform), which encodes a BTB-domain-containing transcriptional repressor, acts as a master repressor of the differentiation of EEs from ISCs. Depletion of ttk in ISCs causes derepression of AS-C genes including Sc and Asense (Ase). The continuous expression of Sc and Ase directs ISCs to continuously generate EEs, leading to the formation of EE-like tumors, or NETs. One intriguing observation from the ttk-depleted ISCs is that continuous derepression of the differentiation-promoting factors does not compromise ISC maintenance, but continuous overexpression of Sc in normal ISCs will cause regional ISC loss over time. This study characterized the ttk-depleted ISCs and, surprisingly, found that the original self-renewal program of ISCs had switched to a neuroblast-like self-renewal program that is responsible for NET tumorigenesis (Li, 2020).

The results reveal an ISC-to-NB switch in the tissue stem cell self-renewal program that drives NET development from ttk- depleted ISCs. Loss of ttk causes the derepression of NB-specific transcription factors, including dpn and seq, and the concomitant loss of ISC-specific factors. The ectopically expressed Dpn acts as a major self-renewal factor for the self-duplication of tumor cells, while Seq has a 'selector' function in selecting Notch target genes by recruiting Su(H) to the enhancer regions of NB-specific genes, thereby rendering these genes responsive to Notch in the tumor cells. In addition, Seq also has a role in NE differentiation by inducing AS-C gene expression. The cooperative function of Dpn and Seq leads to the activation of a NB-like self-renewal program as well as a NE differentiation program and the continuous activation of these two programs leading to NET development from ISCs (Li, 2020).

There are two types of NBs in the central brain of Drosophila larva: type I and type II. Dpn is specifically expressed in the type II but not the type I NBs. Interestingly, Notch appears to be more important in type II NBs than in type I NBs. Thus, it appears that the ectopically activated self-renewal program in ISCs described in this study is more similar to that used in the type II NBs. Compared to the type I NB lineage, the type II NB lineage goes through one extra type of transient amplification progenitors before terminal cell fate specification, indicating that this type II-like self-renewal program, if hijacked by tumor cells, could potentially be more potent to initiate tumorigenesis (Li, 2020).

As the loss of a single factor, Ttk, in ISCs is sufficient for the switch of tissue stem cell program and for the subsequent development of NETs in the midgut, Ttk could be viewed as a specific class of stem cell factors, which is proposed in this study as a tissue identity maintenance (TIM) factor; such factors function to safeguard the tissue identity of stem cells. ISCs not only give rise to ECs that function to digest and absorb nutrients but also give rise to neural-like EE cells. Conceivably, ISCs may need to use a basal or transient neural-like program in order to endow their capacity to generate EEs, as the proneural factor Sc is transiently expressed in ISCs, and this transient expression initiates EE generation. In this context, a stem cell identity factor like Ttk may be necessary to enable ISCs to maintain a gut identity and thereby prevent excessive acquisition of NSC-like traits (Li, 2020).

The Ttk protein is characterized by having a BTB domain in addition to a zinc-finger DNA-binding domain, and although there is no direct protein ortholog of Ttk in mammals, BTB-domain-containing transcription factors are found in all eukaryotes, including mammals. Moreover, alteration of Notch activity as well as increased expression of proneural genes are also known to occur in mammalian models of NETs and in human NETs. Thus, it is possible that there are TIM factors that function in stem cells in other tissues and organisms and that 'stem cell identity switch' could be a common mechanism underlying NET formation and, possibly, other stem-cell-mediated tumorigenesis (Li, 2020).

Regulation of expression of autophagy genes by Atg8a-interacting partners Sequoia, YL-1, and Sir2 in Drosophila

Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is mammalian LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. This study shows that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and it is acetylated in nutrient-rich conditions. Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. These results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2 (Jacomin, 2020).

Autophagy is a fundamental, evolutionary conserved process in which cytoplasmic material is degraded through the lysosomal pathway. It is a cellular response during nutrient starvation; yet, it is also responsible in basal conditions for the removal of aggregated proteins and damaged organelles and therefore plays an important role in the maintenance of cellular homeostasis. There are three main types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy, referred to as autophagy, is the best-described type of autophagy. During macroautophagy, cytoplasmic material is isolated into double-membrane vesicles called autophagosomes. Autophagosomes eventually fuse with lysosomes, allowing for the degradation of cargoes by lysosomal hydrolases. The products of degradation are transported back into the cytoplasm through lysosomal membrane permeases and can be reused by the cell (Jacomin, 2020).

One of the most important and well-studied autophagy-related proteins is LC3 (microtubule-associated protein 1 light chain 3, called Atg8 in yeast and Drosophila), which participates in autophagosome formation. LC3 interacts with LIR (LC3-interacting region) motifs also known as AIM (Atg8-interacting motifs) on selective autophagy receptors that carry cargo for degradation, and is one of the most widely used markers of autophagy. Despite growing evidence that LC3 is enriched in the nucleus, little is known about the mechanisms involved in targeting LC3 to the nucleus and the nuclear components with which it interacts (Jacomin, 2020).

This study shows that Drosophila Atg8a protein, homologous to mammalian LC3 and yeast Atg8, interacts with the transcription factor Sequoia in a LIR motif-dependent manner that is not responsible for the degradation of Sequoia. This study shows that Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. Atg8a was found to be acetylated and interacts with YL-1, a component of the NuA4/Tip60 nuclear acetyltransferase complex. This study show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. These results suggest a novel mechanism of regulation of autophagy gene expression by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2 (Jacomin, 2020).

Atg8 family proteins have been extensively described for their implications in autophagosome formation and cargo selection in the cytoplasm. Although Atg8 family proteins also localize in the nucleus, their role in this compartment remains largely unexplored. This study has uncovered a nuclear role for Drosophila Atg8a in the regulation of autophagy gene expression and induction of autophagy via a LIR motif-dependent mechanism, regulated by Atg8a acetylation. The transcription factor Sequoia interacts with Atg8a in the nucleus to control the transcriptional activation of autophagy genes. It is suggestd that the acetylation status of Atg8a at position K48 contributes to the modulation of the interaction between Sequoia and Atg8a in the nucleus. This study also identified that YL-1, a component of a nuclear acetyltransferase complex, and deacetylase Sir2 interact with Atg8a, and that they act as regulators of Atg8a acetylation (Jacomin, 2020).

A working model is proposed in which in fed conditions, histone-interacting protein YL-1 contributes to the acetylation of Atg8a, while Sequoia resides at the promoter regions of autophagy genes to repress their expression. In such conditions, the interaction between Sequoia and Atg8a contributes to the sequestration of Atg8a in the nucleus. Atg8a cannot therefore translocate to the cytoplasm to take part in the formation of autophagosomes. This hypothesis is supported by the observation that mutation of the Sequoia LIR motif results in an increased accumulation of autophagosomes and autolysosomes in the fed condition. This is observed alongside a reduction in the enrichment of Sequoia at the promoter region of autophagy genes, resulting in their increased expression. Hence, in the absence of an interaction between Atg8a and Sequoia, the subsequent translocation of Atg8a to the cytoplasm may also play a key role in relieving the repressive abilities of Sequoia at the promoter regions of autophagy genes. Upon starvation, Sir2 interacts with and deacetylates Atg8a. Deacetylated Atg8a interacts more strongly with Sequoia, which cannot be maintained at the promoter regions of autophagy genes, leading to the activation of their transcription. Deacetylated Atg8a is then able to translocate to the cytoplasm and contribute to the formation of autophagosomes. It is proposed that Atg8a plays an essential role in relieving Sequoia from the promoter regions of autophagy genes specifically during starvation-induced autophagy as Atg8a loss of function results in the repression of the expression of autophagy genes (Jacomin, 2020).

The results support previous findings about the yeast and mammalian homologs of Sequoia, Rph1, and KDM4A, respectively, which have been shown to negatively regulate the transcription of autophagy genes (Bernard, 2015). The current study elucidates how the LIR-dependent interaction between Sequoia and Atg8a is involved in modulating the expression of autophagy genes during starvation. Mammalian KDM4A also directly interacts with GABARAP-L1; however, the interaction does not require a functional LIR motif. This may be related to the loss of the functionality of the LIR motif during evolution, as it has been shown for Kenny, another LIR-motif containing protein in Drosophila, and its mammalian homolog inhibitor of nuclear factor κB kinase (NF-κB) subunit γ/NF-κB essential modulator (IKKγ/NEMO). The current study also supports previous reports about the role of acetylation and deacetylation of LC3 in mammals and the regulation of autophagy by acetylation (Jacomin, 2020).

Higher eukaryotes express YL-1, a highly conserved Swc2 homolog, which has specific H2A.Z-binding properties. Drosophila YL-1 has been shown to have a H2A.Z-binding domain that binds H2A.Z-H2B dimer (Liang, 2016). The current study reports a novel role for YL-1 in the regulation of acetylation of non-histone proteins and the regulation of autophagy induction (Jacomin, 2020).

In conclusion, these results unveil a novel nuclear role for Atg8a in the regulation of autophagy gene expression in Drosophila, which is linked to its acetylation status and its ability to interact with transcription factor Sequoia. This study highlights the physiological importance of the non-degradative role of LIR motif-dependent interactions of Atg8a with a transcription factor and provide novel mechanistic insights on an unanticipated nuclear role of a protein that controls cytoplasmic cellular self-eating (Jacomin, 2020).

REGULATION

Targets of Activity

The wide-ranging defects in dendrites and axons indicate that sequoia functions to regulate axonal and dendritic morphogenesis in most neurons. Alternatively, it is conceivable that sequoia regulates the expression of genes generally required for neuronal differentiation. To gain mechanistic insight into sequoia function, the transcript profiles in wild-type and sequoia mutant embryos were compared based on microarray analyses of over 3,000 genes or ESTs, corresponding to about 25% of the Drosophila genome. The vast majority of these genes show comparable expression levels, including genes for cytoskeletal elements, genes that specify neuronal cell fates, and genes generally required for neurite outgrowth such as cdc42. Interestingly, a small fraction of the genes/ESTs analyzed showed clearly distinct expression ratios in sequoia mutants. Of these, 93 (3.1%) different transcripts were reduced by at least one-third of the wild-type level, and 34 (1.1%) different transcripts were increased by at least 75% of the wild-type level. A number of genes that appear to be regulated by sequoia, directly or indirectly, correspond to genes implicated in the control of axon morphogenesis rather than neuronal fate. These include known genes such as connectin, frazzled, roundabout 2, and longitudinals lacking, in addition to novel molecules with homology to axon guidance molecules including slit/kekkon-1 and neuropilin-2. It is noteworthy that two of the genes showing increased transcript ratios, roundabout 2 and CG1435, a novel calcium binding protein, were both also identified in a gain-of-function screen affecting motor axon guidance and synaptogenesis (Kraut, 2001). In addition to genes that have clearly been implicated in axon development based on previous studies or sequence similarity, microarray data reveal that other genes potentially regulated by sequoia include peptidases, lipases, and transporters, as well as novel zinc finger proteins. It should be noted that transcripts that are broadly expressed and increased or decreased in sequoia mutants may actually be altered to a greater extent within neurons, because sequoia likely functions cell autonomously and is only expressed in the nervous system (Brenman, 2001).

Sequoia establishes tip-cell number in Drosophila trachea by regulating FGF levels

Competition and determination of leading and trailing cells during collective cell migration is a widespread phenomenon in development, wound healing and tumour invasion. This issue was analyzed during in vivo ganglionic branch cell migration in the Drosophila tracheal system. Sequoia (Seq) was identified as a negative transcriptional regulator of Branchless (Bnl), a Drosophila FGF homologue, and it was observed that modulation of Bnl levels determines how many cells will lead this migrating cluster, regardless of Notch lateral inhibition. These results show that becoming a tip cell does not prevent others in the branch taking the same position, suggesting that leader choice does not depend only on sensing relative amounts of FGF receptor activity (Araujo, 2011).

Although high levels of FGF can induce the terminal cell fate in all tracheal cells, small variations in FGF levels can also establish how many cells in the migrating cluster behave as leading cells irrespective of Notch inhibition. This argues for different mechanism being responsible for the branching morphogenesis of ganglionic branches (GBs) and dorsal branches (DBs). In the case of the DBs, it has been proposed that tip cell choice requires Notch-driven selection of a leader cell. This study reports that GB tip-cell selection is not dependent on a Notch lateral inhibitory mechanism. DB migration is in many ways very different from GB migration. For most of their migration, DBs maintain two leading cells, the one that will become the terminal cell and the one that will take the fusion cell fate. Notch lateral inhibition plays a crucial role in singling out the fusion cell. Thus, Notch-mediated effects in DB migration might be associated with this fate choice rather than with tip-cell selection. Fusion cells are not present at the tip of GBs, and therefore their migration is not affected autonomously by Notch. In addition, Notch signalling has a non-autonomous effect in tracheal development by negatively regulating bnl expression, which might mask its real autonomous effects in DB migration. In this scenario, it is proposed that the Notch-independent mechanism, observed for GB migration, is likely to be the norm for most tracheal clusters and other migratory cell groups where fate choices are not an issue (Araujo, 2011).

In conclusion, FGF signals received by tracheal cells, associated with FGF receptor activation, can induce more than one tip cell, irrespective of the migratory behaviour of the neighbouring tracheal cells. These results show that one cell becoming the tip cell does not inhibit others in the migrating cluster taking up the same position. This suggests that the distinction between leading and trailing cells could depend not only on a competition mechanism sensing the relative amounts of FGFR activity, but also on a level of FRGR activity above a critical threshold, induced by a variation in levels of Bnl (Araujo, 2011).

DEVELOPMENTAL BIOLOGY

Embryonic

Abnormal dendrite and axon outgrowth could originate from defects in the surrounding tissue, defects within the neurons themselves, or defects in both tissues. However, in sequoia mutant embryos, muscle differentiation, cuticle formation, epidermal structure, and trachea formation all appear normal (Gao, 2000). The sequoia mRNA is expressed exclusively in the developing and mature nervous system, consistent with the observed phenotype within the nervous system but no detectable phenotype outside it. sequoia RNA expression begins early (Stage 3/4) but without obvious maternal contribution. sequoia mRNA is expressed in the procephalic neurogenic head region, as well as neuroblasts and their progenitors. Two different Sequoia antisera generated a similar immunostaining pattern that reproduced the mRNA expression pattern. Sequoia is clearly localized to the nuclei in the developing nervous system. It is detected in nuclei of neurons and sheath cells but not the outer support cells in the dorsal cluster. By late stage 16, Sequoia is most abundant in neurons but not detectable in sheath cells (Brenman, 2001).

EFFECTS OF MUTATION

Given the homology to tramtrack, a gene shown to function in regulating cell fate in the developing nervous system, it was of interest to see whether there might be alterations in cell identity in sequoia mutants. The original Gal4 enhancer trap line, 109(2)80, is expressed in all eight md neurons, including the single bipolar dendrite neuron, the single tracheal innervating neuron, and six dendritic arborization (da) neurons, but not es neurons in the dorsal cluster. Hence, this Gal4 line would label eight neurons in each dorsal cluster in the wild-type embryo. The dorsal cluster in sequoia²² mutants contains 9.5 ± 0.5 GFP-positive neurons. Typically, 9-10 GFP-positive neurons are seen, but it is not unusual to find all 12 neurons in the dorsal cluster labeled with GFP. To further verify the generation of extra md neurons, a different lacZ enhancer trap, E7-2-36, was used that labels all six da neurons and the single bd neuron in the dorsal cluster. Similar results were observed with E7-2-36 as observed with Gal4 line109(2)80 -- extra ß-galactosidase immunostaining neurons at the expense of other es neurons in the cluster (Brenman, 2001).

Since tramtrack has been implicated in the Notch signaling pathway, it was of interest to see whether alteration of Notch activity would change the expression of sequoia. Given that sequoia mRNA expression preceded the formation of neurons, a priori it was hard to predict the relationship between Sequoia protein expression and Notch activity. Antibody staining of Sequoia in Notch mutant embryos revealed that all extra Elav-positive cells produced in the Notch neurogenic phenotype indeed expressed Sequoia. Conversely, overexpressing UAS-Notch with Hairy Gal4 resulted in decreased Elav-positive cells, which were the only cells with detectable Sequoia expression. Thus, the number of Sequoia expressing neurons depends on Notch activity (Brenman, 2001).

Is the sequoia gene product required for normal dendritic morphogenesis in the adult? sequoia mutants are embryonic lethal. To circumvent this problem, sequoia function was examined in the adult by making mitotic clones doubly mutant for sequoia and yellow. On both the notum and scutellum, external microchaetae and macrochaetae were found with yellow hair and socket in these mitotic sequoia loss-of-function mutant clones. There was no cell fate transformation of these external cells, in contrast to tramtrack clones that show loss of hair and socket. Remarkably, in these sequoia mutant clones marked with yellow bristles, the accompanying sensory neurons often show a range of morphological defects. The wild-type es neuron projects a single unbranched dendritic process that innervates the external hair. In sequoia mutant clones, however, some neurons have thick dendrites and laterally protruding processes and other neurons fail to extend dendrites to innervate the accompanying hair (Brenman, 2001).

Sequoia is present in a pan-neural nuclear pattern in the adult head, including photoreceptors. To determine whether sequoia is also required for proper axon morphogenesis in the visual system, the effects of loss of sequoia function in ommatidia were examined. The axon projections from photoreceptor retinal neurons to the brain optic lobe are well characterized; it is easy to identify aberrant axonal projections. Clones of sequoia mutant cells were generated. sequoia mutant ommatidia appear largely indistinguishable from wild-type when viewed by examining the R1-R7 cell numbers and arrangement in toluidine blue-stained tangential sections. This indicates that the photoreceptor cell fate specification is normal. In order to look at the projection of photoreceptor axons, cryostat sections of fly heads homozygous for either the parental the chromosome or sequoia mutant chromosome were stained with mAb24B10, which marks the axons of all photoreceptor cells, R1-R8. The Drosophila photoreceptor neurons project their axons to the optic lobe of the brain, producing a stereotyped retinotopic map. The R1-R6 growth cones terminate in the lamina, forming a dense layer of immunoreactivity, the lamina plexus. The R7 and R8 neurons project their axons through the lamina and instead terminate in the next layer, the medulla. Axons from the various photoreceptors interweave and bundle together, forming cartridges that are particularly evident in the lamina and medulla, where alternating columns of stained axon bundles and unstained areas intersperse. These cartridges in the lamina and medulla are roughly parallel to each other. In sequoia mutant heads, the regular cartridge appearance is either malformed or completely absent, indicative of either failure to extend axons fully or failure to project them appropriately (Brenman, 2001).

Insect dendritic arborization (da) neurons (one of three classes of multiple dendritic neurons) provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. The 15 da neurons in hemisegments A2-A6 are arranged in four clusters (ventral, ventral', lateral and dorsal). The nomenclature for the da neurons identifies their position within one of these four clusters with the prefix v, v', l or d, their status as a da neuron and an alphabetic suffix that orders the cells from ventral to dorsal within each cluster. Three neurons (vpda, v'ada and v'pda) do not conform to this naming scheme. In this study, each neuron has been named according to its typical position within a cluster; however, the primary criterion for identifying each cell is its peripheral dendritic morphology. Four neurons were identified grouped together in a ventral cluster (vdaA-D), one lone ventral neuron (vpda), the previously identified v'ada and v'pda neurons, the two lateral neurons ldaA and ldaB and six dorsal da neurons (ddaA-F) (Grueber, 2002).

The morphologies of Drosophila da neurons have been examined by using the MARCM (mosaic analysis with a repressible cell marker) system. Each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. These neurons were placed into four distinct morphological classes (termed class I, II, III and IV neurons) distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. The data indicate that cells within two morphological classes partition the body wall into distinct, non-overlapping territorial domains and thus are organized as separate tiled sensory systems (for a more complete treatment of the meaning of the word tiling, see The Tilings Around Us). In contrast, the dendritic domains of cells in different classes can overlap extensively. The cell-autonomous roles of starry night (stan) (also known as flamingo (fmi)) and sequoia (seq) in tiling were examined. Neurons with these genes mutated generally terminate their dendritic fields at normal locations at the lateral margin and segment border, where they meet or approach the like dendrites of adjacent neurons. However, stan mutant neurons occasionally send sparsely branched processes beyond these territories that could potentially mix with adjacent like dendrites. Together, these data suggest that widespread tiling of the larval body wall involves interactions between growing dendritic processes and as yet unidentified signals that allow avoidance by like dendrites (Grueber, 2002).

Morphological characterization of Drosophila da neurons indicates that they are similar to the da neurons of the moth Manduca sexta, of which there are at least three distinct morphological classes. In Manduca, the alpha, beta and gamma da neurons show morphological similarities to the class I/II, IV and III da neurons of Drosophila, respectively. Manduca alpha neurons appear to function as proprioceptors, whereas gamma neurons probably function as touch receptors. Whether the Drosophila da neurons are functionally similar to Manduca da neurons remains to be determined. At least two lines of evidence, however, suggest that the morphological classes that have been identified in Drosophila represent functionally distinct types of neurons. (1) pickpocket (ppk), a degenerin/epithelial sodium channel subunit, appears to be expressed only in the class IV neurons ddaC, v'ada and vdaB. Since ppk may have a physiological role in mechanotransduction, its expression in class IV neurons could underlie a functional specialization of these cells. (2) Drosophila da neurons have dichotomous axonal projections, which probably reflect their functional distinctions. Most da neurons project into the ventral neuropil, a characteristic of tactile projections. vpda and an unidentified neuron in the dorsal cluster, by contrast, have more dorsal projections, which is similar to proprioceptive neurons. The ventral-projecting neurons appear to correspond to the class II, III and IV neurons, whereas the dorsal projections belong to at least a subset of the class I neurons (Grueber, 2002).

An important issue arising from this characterization of the Drosophila da system is how the morphological properties of each neuron relate to their genetic specification. Previous studies have shown that the Drosophila da system consists of genetically distinct subgroups of neurons. Most da neurons require proneural genes in the achaete-scute complex (ASC) arise as components of external bristle lineages, and express the Cut protein. The only da neurons that do not share these characteristics are vpda and two dorsal neurons. vpda remains in ASC-mutant embryos but is lost in animals mutant for the proneural gene, atonal. One dorsal da neuron requires a third proneural gene, amos. Finally, vpda and two dorsal da neurons fail to express Cut (Grueber, 2002 and references therein).

Reconciling these accumulated data with a cell-by-cell characterization of the da system, it appears that the class II, III and IV neurons are those that require ASC genes and express Cut. In contrast, Class I neurons do not express detectable levels of Cut and could correspond to the da neurons that require atonal and/or amos (although only one ASC-independent neuron has been described in the dorsal cluster). Consistent with these assignments, during mosaic analysis class III and class IV neurons were often observed co-labeled with es neurons, suggesting that these cells could have arisen from a common precursor. The class II neurons are likely also es related because all four neurons in the ventral cluster are lineally related to es organs and two class II neurons reside here. Understanding how the genes required for early da specification are linked to the activation of distinct programs of dendritic morphogenesis is an important goal for future studies (Grueber, 2002).

Tiling is a principle of dendrite organization in which functionally similar neurons completely fill available receptive territories with little or no redundancy. Two independent tilings of the Drosophila epidermis by da neurons bearing similar morphologies have been identifed. A similar type of tiling has recently been identified in Manduca (Grueber, 2001), suggesting that this is an evolutionarily conserved plan for organizing the da sensory system. In Drosophila, tiling occurs between class III da neurons and between class IV da neurons, which each partitions the body wall into a collection of non-overlapping territories. Furthermore, class III and IV neurons each provide a nearly complete segmental coverage. Dendrites with distinct morphologies, by contrast, can cross extensively (Grueber, 2002).

The tiling of the Drosophila epidermis by class III and IV neurons appears analogous to the tiling among physiologically alike vertebrate retinal ganglion cells. In this system, ON-center and OFF-center, four ON-OFF direction-selective classes and several other cell types, including amacrine cells, provide independent tilings of the retina. Such an arrangement ensures that each region of the visual field is 'viewed' by each physiological type of ganglion cell. Additionally, as visual information is distributed to the appropriate centers of the brain, the location of its origin is unambiguous, thereby maintaining a coherent representation of sensory space. The same rules of organization applied to the insect da system would likewise be advantageous. Mechanical and thermal stimuli often necessitate rapid and finely directed behavioral responses, particularly when they could lead to damage to the cuticle. If future studies show that the da neurons comprising each tiling class are united by their physiology, as is suspected, then these modalities would have the capacity to provide accurate spatial resolution of stimuli landing anywhere on the body wall (Grueber, 2002).

Although the cellular mechanisms that control dendritic tiling are not yet understood, several developmental scenarios can be envisioned. Individual dendrites could repel like dendrites where they meet. Alternatively, neurons could be endowed with a limited capacity for dendritic growth (depending, for example, on cell size) and form their territories without influence from neighboring neurons. Finally, in the case of the da neurons, interactions with the epidermis or surrounding tissue, such as muscle, could provide permissive or restrictive growth signals. These mechanisms are not mutually exclusive and could conceivably act in concert. In the da system, however, dendritic boundaries could not always be correlated with physical boundaries, such as muscle insertion sites, and terminal dendrites typically turned abruptly where they met like dendrites. Thus, these data do not provide strong support for the latter two mechanisms (limited growth capacity and physical boundaries) but do suggest that branch interactions could contribute to tiling. Experimental studies of the effects of adding neurons to, and removing neurons from, the da system will provide essential tests of the importance of these mechanisms. Additionally, because the data are taken from mature da neurons, a crucial question still to be addressed is how dendrites of like neurons behave during development as their territories are established. Dendrites could show exclusion throughout their development or, alternatively, refine their boundaries as a maturational step (Grueber, 2002).

Regulation of tiling by dendritic branch interactions is a likely scenario in the vertebrate retina, where contact-mediated avoidance signals appear to operate in a cell-type-specific manner. Furthermore, morphological data from mammalian retinal neurons show that dendro-dendritic contacts are made between like neurons but not between unlike neurons. Such contacts could provide an opportunity for these neurons to signal to each other by their activity or cell surface composition. Similarly, typically single apparent dendritic contacts are observed between tiling class IV neurons. Whether these contacts are important for exclusion among the remaining branches remains to be determined (Grueber, 2002).

The molecular mechanisms of dendritic tiling in the vertebrate retina have not been established. Mutant screens of the second chromosome in Drosophila might be informative in this regard, since several candidate loci have been identified that cause early overextension of dendrites. Alleles of two of these genes, stan and seq, have been tested for possible roles in tiling. In seq²² and stan⁷² mutant embryos, dendrites show an overextension phenotype and exhibit abnormal crossing of the dorsal midline. MARCM analysis using the seq²² and stan^E59 alleles suggests that such overextension might not reflect a widespread defect in dendritic exclusion, because a majority of the dendrites terminate or turn where contact with an adjacent like neuron occurs or would be expected. Importantly, however, one or two sparsely branched processes were seen extending beyond the normal boundary of the cell in 18% of the class IV stan-mutant neurons (Grueber, 2002).

If branch recognition and exclusion are required for tiling, which appears to be the case for the class IV neurons, one interpretation of the stan phenotype is that the dendrite is overextending because it does not receive or transduce a repulsive signal that requires Stan function. Alternatively, because exclusion occurs among terminal dendritic branches in wild-type neurons, a lack of exclusion in stan-mutant neurons could arise if the overextended processes are equivalent to primary trunks and thus lack the machinery for tiling. However, without information about the fields of surrounding like neurons, the possibility remains that exclusion is intact in stan-mutant neurons. By extending earlier, or more rapidly, than the rest of the dendritic field, these single processes could have successfully invaded an uninnervated region of the body wall. This latter scenario seems to provide a reasonable explanation for why a dorsal branch from ddaC is observed overextending along the dorsal midline (one of the last regions of the body wall to become innervated). Whether a similar 'invasion' scenario could account for the overextended processes from vdaB might ultimately depend on the timing and pattern of outgrowth of its class IV neighbors (i.e. how far can a dendrite of a stan^- vdaB neuron extend before encountering like dendrites?). Because MARCM experiments suggest that stan acts cell autonomously in the dendritic arborization neurons, future studies might be conducted using cell-type specific markers of the class IV neurons in a stan (and seq) mutant background. Such markers would allow the visualization of all neurons together and, as a result, provide a better indication of the relationship between early dendritic overextension phenotypes and tiling (Grueber, 2002).

In addition to the dendritic exclusion that occurs between like neurons, exclusion between dendrites that belong to the same neuron is frequently observed. Such 'self-avoidance' has been identified in Manduca sensory neurons (Grueber, 2001) and characterized experimentally in leech sensory axons; however the underlying mechanisms are not understood. In theory, self-avoidance and tiling might not require distinct signals or signaling pathways (among like neurons) because isoneuronal dendrites could be developmentally identical to 'like' heteroneuronal dendrites. It will therefore be of special interest to compare the mechanisms of exclusion by isoneuronal and heteroneuronal branches during development. Ultimately, an understanding of the distinction between these two processes will require the elucidation of their molecular underpinnings (Grueber, 2002).

Temporal identity in axonal target layer recognition

The segregation of axon and dendrite projections into distinct synaptic layers is a fundamental principle of nervous system organization and the structural basis for information processing in the brain. Layer-specific recognition molecules that allow projecting neurons to stabilize transient contacts and initiate synaptogenesis have been identified. However, most of the neuronal cell-surface molecules critical for layer organization are expressed broadly in the developing nervous system, raising the question of how these so-called permissive adhesion molecules support synaptic specificity. This study showed that the temporal expression dynamics of the zinc-finger protein Sequoia is the major determinant of Drosophila photoreceptor connectivity into distinct synaptic layers. Neighbouring R8 and R7 photoreceptors show consecutive peaks of elevated sequoia expression, which correspond to their sequential target-layer innervation. Loss of sequoia in R7 leads to a projection switch into the R8 recipient layer, whereas a prolonged expression in R8 induces a redirection of their axons into the R7 layer. The sequoia-induced axon targeting is mediated through the ubiquitously expressed Cadherin-N cell adhesion molecule. The data support a model in which recognition specificity during synaptic layer formation is generated through a temporally restricted axonal competence to respond to broadly expressed adhesion molecules. Because developing neurons innervating the same target area often project in a distinct, birth-order-dependent sequence, temporal identity seems to contain crucial information in generating not only cell type diversity during neuronal division but also connection diversity of projecting neurons (Petrovic, 2008).

In the compound eye, each ommatidium contains eight photoreceptor neurons (R1-R8) that form synapses in distinct optic lobe ganglia, the peripheral lamina and the deeper medulla. Axons of the colour-sensitive R8 and R7 cells project into the medulla and segregate into two out of ten synaptic layers, M3 and M6 respectively. In a mosaic screen for genes that control R8/R7 target layer selection, mutants of sequoia (seq⁵ and seq⁶) were identified, with a frequent loss of R-cell innervation in M3 and M6. Using the MARCM (mosaic analysis with a repressible cell marker) technique for the selective labelling of homozygous mutant R8 and R7 cell axons, it was found that more than 90% of seq mutant R7 cells terminate their axon projections in the outer M1-M3 layers, correlating with an innervation gap in the M6 layer of the same medulla column. Similarly, seq mutant R8 axons frequently (87%) stop above their prospective M3 target layer. The analysis of different R8/R7 genetic mosaics showed that wild-type R8/R7 axons are not influenced by a seq mutant neighbour axon of the same medulla column, suggesting independent target recognition by these two R cells (Petrovic, 2008).

sequoia encodes a nuclear protein with two putative zinc-finger domains related to the DNA-binding domain of Tramtrack. In the visual system, the four independently isolated sequoia alleles seq⁵, seq⁶, seq²² and seq^SH1898 lead to indistinguishable R8/R7 axon-targeting phenotypes. No expression could be detected in homozygous seq⁵ and seq⁶ mutant R cells with an anti-sequoia monoclonal antibody and the R7 targeting defects could be rescued after the expression of a sequoia transgene in the mutant cells. To determine whether sequoia functions cell-autonomously in R cells to control axon target selection, R7 cell-type-specific MARCM clones were induced. Almost all of the single homozygous mutant R7 terminals surrounded by heterozygous R8 and R7 axons stop prematurely in M3. Because the loss of sequoia does not lead to changes in early R8 and R7 specification or their subsequent projection towards their optic lobe ganglion, it is concluded that sequoia is required for R8/R7 axons to connect to their synaptic target layer (Petrovic, 2008).

The sequoia targeting phenotype becomes obvious during early pupal development when the R8 and R7 axons terminate sequentially into two separate medulla layers. In contrast to the adult brain, the medulla target field during initial R8/R7 ingrowth appears more homogeneous and distinct layer organization becomes visible in the subsequent segregation of lamina and medulla neuron processes. seq mutant R8 and R7 axons reach the medulla target area at the appropriate time and in the correct topographic order, but gaps in the initial R7 layer can be detected; this is due to the termination of R7 axons in the R8 layer. Similarly to the R7 projection defect in the adult visual system, the mis-targeting of R7 into the R8 layer during the pupal stage seems to be due to the disruption of seq function directly in R7 and is not influenced by defects in R8 (Petrovic, 2008).

The expression pattern of sequoia is highly dynamic in early differentiating R8/R7 cells during the phase of axonal growth and medulla innervation. The onset of sequoia expression in R cells reflects their sequential birth and differentiation order. As a result of the temporal gap in neuronal differentiation between R8 and R7 from the same ommatidium, together with a rapid downregulation of sequoia, the periods of elevated sequoia expression in these two R cells are non-overlapping. By the time the R7 cell initiates axonal projection accompanied by a high expression level of sequoia, the axon of the neighbouring R8 cell has already reached the medulla, and sequoia expression has been turned off. Therefore a tight correlation is observed between the R-cell-specific sequoia expression profile and the sequential R8/R7 medulla target layer innervation (Petrovic, 2008).

The fact that sequoia is expressed in both R8 and R7, but the loss of sequoia leads to specific R8/R7 axon-targeting defects, raises the possibility that connection specificity is mediated through the difference in the temporal pattern of sequoia expression. To test the importance of precise sequoia expression timing, synchronized the R8/R7 sequoia expression profiles were syncronized. Similarly to the seq loss of function situation, constitutive seq expression in all R cells does not lead to changes in the cell fate of R8 or R7 or their initial axonal projection towards the medulla. However, whereas in wild-type individuals R8/R7 growth cones transform their morphology over subsequent hours from an 'expanded' to a 'condensed' appearance, growth cones of R8/R7 cells with a prolonged sequoia expression remain in the 'expanded' state and R8 axons extend towards and terminate precisely in the R7 layer. The co-innervation of R8 and R7 axons is maintained during the subsequent steps of medulla reorganization, because a single R8/R7 layer in M6 can be detected in the medulla of adult flies. These data indicate that the downregulation of sequoia expression in R8 cells is critical for their growth cones to become stabilized in their initial target layer. Prolonged sequoia expression specifically in R8 or R7 and the sequoia overexpression in R8 in an R7 deficient-background support the model in which the endogenous level of sequoia determines the competence of ingrowing R-cell axons to connect to their appropriate target layer (Petrovic, 2008).

To obtain insights into the molecular mechanisms through which R8/R7 axon targeting competence is mediated, whether the loss of sequoia leads to changes in the expression of neuronal cell adhesion/receptor molecules was tested. A significant decrease in the expression levels of the receptor protein tyrosine phosphatase Lar (leukocyte-antigen-related-like) and the homophilic cell adhesion molecule Cadherin-N (CadN) was found in sequoia mutant R cells. Although CadN is the only factor known so far to be essential for the initial R8/R7 axon targeting, its widespread expression in R cells and in the target region has made it unlikely that homophilic CadN interactions alone are sufficient to specify R8/R7 target choice. Therefore, whether the sequoia targeting mechanism would allow CadN to provide spatio-temporal specificity in initial axon-target adhesion was examined. Loss of CadN and sequoia leads to an identical early termination of R7 axons in the outer medulla layer and later co-innervation of R7 and R8 in the adult M3 layer. After the removal of CadN from sequoia-overexpressing R8/R7 cells, a complete suppression of the M3-->M6 layer switch can be observed because all brains show the characteristic M6-->M3 layer switch of CadN mutant R7 axons. This result indicates that the sequoia-mediated axon targeting functions through CadN. However, the sequoia-induced M3-->M6 layer switch is not caused simply by an increase in CadN adhesion, because overexpression of CadN does not affect the R8/R7 projections into the medulla and the overexpression of sequoia does not lead to a significant increase in the level of CadN in R cells. From these data it is conclude that homophilic CadN interactions are sequentially used in R8 and R7 to mediate the sequoia-regulated axon-target interaction (Petrovic, 2008).

On the basis of these results it is proposed that initial afferent lamination in the developing visual system of Drosophila is not controlled by the layer-specific expression of recognition molecules but is mediated through temporally restricted competence of ingrowing axons to interact with the target region. Here, permissive cell adhesion molecules provide spatio-temporal recognition specificity in axon-target interaction. Interestingly, Cadherin-N has been shown to function in subsequent steps of medulla innervation by lamina neurons, supporting the idea that the same type of adhesion molecule could be used in a repetitive fashion to support sequential layer formation. A critical aspect of the specificity of temporal recognition is the rapid downregulation of the targeting competence after the initial axon-target contact to prevent the reactivation of early projecting axons during the subsequent steps of innervation. The sequoia-mediated axon targeting mechanism controls an essential step in the coordinated development between sensory neurons and their central nervous system (CNS) target field in the Drosophila visual system. Starting with afferent-derived signals inducing early target neuron differentiation, the sequoia-controlled patterning of initial axon innervation ensures the correct R8/R7 positioning for subsequent afferent-induced target field organization and layer-specific molecule expression to stabilize the initial innervation (Petrovic, 2008).

Neural progenitors often generate distinct subtypes of neurons in an invariant temporal sequence during development. Similarly to its role in generating cell type diversity during neuronal division, temporal identity is used subsequently to generate connection diversity in projecting neurons. Mis-expression of sequoia in more mature R cells during the later steps of differentiation has no effect on axonal projection, indicating that R cells lose their competence to respond to the sequoia-induced targeting identity. A narrow developmental window of competence has also been described for the temporal identity factors in the embryonic and postembryonic nervous system. Similarly, dynamic expression of sequoia can be observed throughout the development of the nervous system in Drosophila, and seq mutants show a severe disruption of the connectivity pattern in various brain regions. In vertebrates, retina development is also characterized by the sequential generation of distinct cell types followed by their assembly into a highly laminated CNS structure. Recent in vivo imaging studies in zebrafish have shown that axon-dendrite interactions occur in a sequential manner and illustrate a transient requirement for some of the cell types during the assembly of laminated retinal circuits. Thus, a sequoia-related control mechanism might be more broadly applicable to the development of circuit specificity in the CNS (Petrovic, 2008).

An allele of sequoia dominantly enhances a trio mutant phenotype to influence Drosophila larval behavior

The transition of Drosophila third instar larvae from feeding, photo-phobic foragers to non-feeding, photo-neutral wanderers is a classic behavioral switch that precedes pupariation. The neuronal network responsible for this behavior has recently begun to be defined. Previous genetic analyses have identified signaling components for food and light sensory inputs and neuropeptide hormonal outputs as being critical for the forager to wanderer transition. Trio is a Rho-Guanine Nucleotide Exchange Factor integrated into a variety of signaling networks including those governing axon pathfinding in early development. Sequoia is a pan-neuronally expressed zinc-finger transcription factor that governs dendrite and axon outgrowth. Using pre-pupal lethality as an endpoint, a screened was performed for dominant second-site enhancers of a weakly lethal trio mutant background. In these screens, an allele of sequoia has been identified. While these mutants have no obvious disruption of embryonic central nervous system architecture and survive to third instar larvae similar to controls, they retain forager behavior and thus fail to pupariate at high frequency (Dean, 2013).

REFERENCES

Search PubMed for articles about Drosophila sequoia

Araujo, S. J. and Casanova, J. (2011). Sequoia establishes tip-cell number in Drosophila trachea by regulating FGF levels. J. Cell Sci. 124(Pt 14): 2335-40. PubMed Citation: 21693579

Bernard, A., Jin, M., Gonzalez-Rodriguez, P., Fullgrabe, J., Delorme-Axford, E., Backues, S. K., Joseph, B. and Klionsky, D. J. (2015). Rph1/KDM4 mediates nutrient-limitation signaling that leads to the transcriptional induction of autophagy. Curr Biol 25(5): 546-555. PubMed ID: 25660547

Bivik C, MacDonald RB, Gunnar E, Mazouni K, Schweisguth F, Thor S. (2016). Control of neural daughter cell proliferation by multi-level Notch/Su(H)/E(spl)-HLH signaling. PLoS Genet. 12(4):e1005984. PubMed ID: 27070787

Brenman, J. E., Gao, F.-B., Jan, L. Y. and Jan, Y. N. (2001). Sequoia, a Tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila. Dev. Cell 1: 667-677. 11709187

Dean, K. E., Fields, A., Geer, M. J., King, E. C., Lynch, B. T., Manohar, R. R., McCall, J. R., Palozola, K. C., Zhang, Y. and Liebl, E. C. (2013). An allele of sequoia dominantly enhances a trio mutant phenotype to influence Drosophila larval behavior. PLoS One 8: e84149. PubMed ID: 24376789

Gao F.B., Kohwi M., Brenman J.E., Jan L.Y. and Jan Y.N. (2000). Control of dendritic field formation in Drosophila: The roles of flamingo and competition between homologous neurons. Neuron, 28: 91-101. 11086986

Grueber, W. B., Graubard, K. and Truman, J. W. (2001). Tiling of the body wall by multidendritic sensory neurons in Manduca sexta. J. Comp. Neurol. 440: 271-283. 11745623

Grueber, W. B., Jan, L. Y. and Jan, Y. N. (2002). Tiling of the Drosophila epidermis by multidendritic sensory neurons. Development 129: 2867-2878. 12050135

Gunnar, E., Bivik, C., Starkenberg, A. and Thor, S. (2016). sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system. Development143(20):3774-3784. PubMed ID: 27578794

Jacomin, A. C., Petridi, S., Di Monaco, M., Bhujabal, Z., Jain, A., Mulakkal, N. C., Palara, A., Powell, E. L., Chung, B., Zampronio, C., Jones, A., Cameron, A., Johansen, T. and Nezis, I. P. (2020). Regulation of expression of autophagy genes by Atg8a-interacting partners Sequoia, YL-1, and Sir2 in Drosophila. Cell Rep 31(8): 107695. PubMed ID: 32460019

Kraut, R., Menon, K. and Zinn, K. (2001). A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila. Curr. Biol. 11: 417-430. 11301252

Li, Z., Guo, X., Huang, H., Wang, C., Yang, F., Zhang, Y., Wang, J., Han, L., Jin, Z., Cai, T. and Xi, R. (2020). A switch in tissue stem cell identity causes neuroendocrine tumors in Drosophila gut. Cell Rep 30(6): 1724-1734. PubMed ID: 32049006

Liang, X., Shan, S., Pan, L., Zhao, J., Ranjan, A., Wang, F., Zhang, Z., Huang, Y., Feng, H., Wei, D., Huang, L., Liu, X., Zhong, Q., Lou, J., Li, G., Wu, C. and Zhou, Z. (2016). Structural basis of H2A.Z recognition by SRCAP chromatin-remodeling subunit YL1. Nat Struct Mol Biol 23(4): 317-323. PubMed ID: 26974124

Marchetti, G. and Tavosanis, G. (2019). Modulators of hormonal response regulate temporal fate specification in the Drosophila brain. PLoS Genet 15(12): e1008491. PubMed ID: 31809495

Petrovic, M. and Hummel, T. (2008). Temporal identity in axonal target layer recognition. Nature 456(7223): 800-3. PubMed Citation: 18978776

Ulvklo C1, MacDonald R, Bivik C, Baumgardt M, Karlsson D, Thor S. (2012). Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression. Development. 139(4):678-89. PubMed ID: 22241838

date revised: 17 December 2020

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