BIOLOGICAL OVERVIEW

Drosophila Tey represses transcription of the repulsive cue Toll and generates neuromuscular target specificity

Little is known about the genetic program that generates synaptic specificity. This study shows that a putative transcription factor, Teyrha-Meyhra (Tey), controls target specificity, in part by repressing the expression of a repulsive cue, Toll. This study focused on two neighboring muscles, M12 and M13, which are innervated by distinct motoneurons in Drosophila. It was found that Toll, which encodes a transmembrane protein with leucine-rich repeats, is preferentially expressed in M13. In Toll mutants, motoneurons that normally innervate M12 (MN12s) form smaller synapses on M12 and instead appear to form ectopic nerve endings on M13. Conversely, ectopic expression of Toll in M12 inhibited synapse formation by MN12s. These results suggest that Toll functions in M13 to prevent synapse formation by MN12s. Tey was identified as a negative regulator of Toll expression in M12. In tey mutants, Toll is strongly upregulated in M12. Accordingly, synapse formation on M12 was inhibited. Conversely, ectopic expression of tey in M13 decreased the amount of Toll expression in M13 and changed the pattern of motor innervation to the one seen in Toll mutants. These results suggest that Tey, which contains no known transcription factor motifs, determines target specificity by repressing the expression of Toll. These results reveal a mechanism for generating synaptic specificity that relies on the negative regulation of a repulsive target cue (Inaki, 2010).

A remarkable feature of the nervous system is the precision of its circuitry. A neural circuit develops through a series of neuronal recognition events. First, neurons find their path, turn at mid-way guideposts, and fasciculate or defasciculate before reaching their final target area. Then, neurons select and form synapses with specific target cells in the target region. The final matching of pre- and post-synapses is thought to be mediated by specific cues expressed on the target cells. However, the regulation and function of such cues remain poorly understood (Inaki, 2010).

The process of neuromuscular targeting in Drosophila features highly stereotypic matchings between 37 motoneurons and 30 target muscle cells, providing a unique model system for the study of neuronal target recognition. Several target cues, including Capricious, Netrin-B and Fasciclin 3, have been identified that are expressed in specific target cells and mediate attractive interactions between the synaptic partners. It has recently been shown that target specificity is also regulated by repulsion from non-target cells. Wnt4, a member of the Wnt family of secreted glycoproteins, is expressed in muscle 13 (M13) and prevents synapse formation by motoneurons targeted to a neighboring muscle, M12. In the absence of Wnt4, motoneurons targeted to M12 form ectopic nerve endings on M13, indicating that Wnt4 repulsion on M13 is required for proper targeting of the motoneurons. In addition to Wnt4, Toll and Semaphorin II (Sema-2a - FlyBase) are known to function as negative regulators of synapse formation in this system. However, whether they have a role in target selection remains unknown (Inaki, 2010).

Another unsolved issue is how the expression of such attractive or repulsive target-recognition molecules is regulated. It is amazing that the expression of these molecules is so precisely regulated that they are present at the right time and place. It is likely that the expression of these molecules is determined as part of the differentiation program of the target cells. However, little is known about the molecules and mechanisms involved. Several transcription factors, such as S59, Krüppel and Vestigial, have been identified as being expressed in subsets of muscle cells. They are expressed from the progenitor stage, and their loss-of-function (LOF) and gain-of-function (GOF) alter the specific characteristics of the individual muscles, such as their size, shape, orientation and attachment sites to the epidermis, indicating that they function as determinants of a particular muscle fate. However, whether these transcription factors regulate the expression of target-recognition molecules and thus determine the innervation pattern is unknown (Inaki, 2010).

A comparative microarray analysis has been conducted of two neighboring target muscles, M12 and M13, that are innervated by distinct motoneurons (Inaki, 2007). By comparing the expression profile of the two muscles, attempts were made to understand the molecular mechanisms that make these muscles distinct targets for the motoneurons. From this screening, ~25 potential target-recognition molecules were identified as preferentially expressed in either muscle cell. Among them was Wnt4, mentioned above. This study reports the functional analyses of two additional genes that were identified in the screening: Toll and teyrha-meyrha (tey). Toll encodes a transmembrane protein with extracellular leucine-rich repeats, and has multiple functions in development. Toll is expressed in subsets of muscles, including 6, 7 and 15-17. Previous studies have shown that Toll inhibits synapse formation by RP3, a motoneuron targeted to muscles 6 and 7. This study shows that Toll is preferentially expressed in M13 over M12 and, like Wnt4, inhibits synapse formation by motoneurons targeted to M12. It was also shown that tey, a previously uncharacterized gene, regulates the expression of Toll in specific muscles. tey is expressed specifically in M12, where it negatively regulates Toll expression. In the absence of tey, Toll is ectopically expressed in M12 and innervation of M12 is inhibited. These results suggest that Tey regulates targeting by downregulation of the repulsive cue Toll specifically in M12. Based on these results, a mechanism is proposed for the generation of synaptic specificity that relies on negative regulation of repulsive target cues (Inaki, 2010).

Toll is preferentially expressed in M13 over M12. The size of M12 terminals was decreased in Toll mutants, with concomitant expansion of M13 terminals. This phenotype is very similar to that of Wnt4 LOF and is likely to be caused by MN12s forming ectopic synapses with M13, although it remains possible that some of the ectopic nerve endings on M13 are formed by other motoneurons. Furthermore, it was observed that the size of M12 terminals is reduced when Toll is misexpressed on the muscle. The LOF and GOF analyses suggest that Toll functions as a repulsive factor in M13 that is important for target selection by MN12s. Thus, Toll provides another example of a repulsive factor that is involved in target selection. How Toll mediates the repulsive signal to motoneurons is currently unknown. A model is that Toll functions as a ligand that is expressed in muscles and signals through receptor(s) expressed in motoneurons. However, no receptor has been identified for Toll. Toll has been shown to function as a receptor, not a ligand, in other systems, such as in dorsoventral patterning and innate immunity. Another possibility is that Toll might mediate the modification or regulation of other targeting molecules, such as Wnt4 (Inaki, 2010).

M13 expresses at least two repulsive cues, Wnt4 and Toll, that are important for the targeting of M12 and M13. It seems that these two molecules contribute to target specificity in a manner that is redundant with yet other molecules because in both single and double mutants of these genes, the connectivity is only partially disrupted. Previously, other potential repulsive cues that are expressed in M13 were identified, including Beat-IIIc and Glutactin (Inaki, 2007). Ectopic expression of these molecules in M12 inhibits synapse formation by MN12s, as observed when Toll and Wnt4 are misexpressed. Although the precise roles of these molecules remain to be verified by LOF analyses, these results suggest the possibility that a single muscle, M13, expresses a number of repulsive cues that are involved in targeting of motoneurons. This is consistent with previous hypotheses that Drosophila neuromuscular connectivity is determined by highly redundant mechanisms. It will be important to determine how the signals from multiple cues are integrated to generate the precise pattern of synaptic connections. It will also be interesting to examine whether other muscles similarly express repulsive cues to prevent inappropriate innervation. The phenotypes of Wnt4 Toll double mutants were of similar severity to those of the single mutants. This might be due to the presence of other targeting molecules, as described above. Toll and Wnt4 might also function in the same signaling pathway. For example, Toll may be involved in the regulation of Wnt4 activity through influencing its secretion, localization or protein modification. Toll and Wnt4 might also act as repellents for distinct MN12s (Inaki, 2010).

This study has shown that a novel nuclear protein, Tey, regulates the expression of Toll and is important for the determination of target specificity. tey regulates the position, orientation and attachment sites of M12. Thus, Tey seems to act as a determinant of several important properties of M12, regulating both the differentiation of the muscle itself and the specificity of nerve innervation. Expression of tey is remarkably specific, being limited within the somatic musculature to a single muscle, M12. Other, known muscle-determinant genes were expressed in broader subsets of muscles (Inaki, 2010).

Tey negatively regulates the expression of Toll in M12. In tey mutants, Toll expression is strongly upregulated in M12. This indicates that tey is required in this muscle to specifically suppress Toll expression. Consistent with this, ectopic expression of tey in M13 partially suppressed Toll expression. Toll is normally expressed in most of the other ventral muscles, including muscles 6, 7, 13-17, but not in M12, suggesting that some positive transcriptional regulator(s) higher up in the hierarchy activate Toll expression in this group of muscles and that negative regulation by Tey is required to suppress Toll expression only in M12. The regulation of Toll by Tey should be at the transcriptional level because the expression of the exogenously introduced Toll enhancer-trap lacZ reporter is affected in tey mutants or when tey is misexpressed. It remains to be determined whether Tey binds directly to the regulatory region of the Toll gene or regulates Toll transcription in an indirect manner (e.g. by regulating other transcription factors). Tey contains no known transcription factor motifs. The expression of another M13-enriched gene, Wnt4, was not affected in tey mutants or when tey was misexpressed. Unlike Toll, Wnt4 is expressed in only two ventral muscles: 13 and 30. Thus, expression of Wnt4 might be regulated in a different manner to Toll, possibly by positive transcription factors that are specifically expressed in these muscles. It will be interesting to determine how the expression of target-recognition molecules is precisely regulated by the combinatorial action of positive and negative transcription factors (Inaki, 2010).

In tey mutants or when tey is misexpressed, neuromuscular connectivity was also altered in a manner consistent with the misregulation of Toll expression. The inappropriate presence of Toll repulsion in tey LOF mutants suppressed synapse formation on M12. Conversely, suppression of Toll expression in M13 in tey GOF mutants led to changes in the innervations of M12 and M13, similar to those observed in Toll mutants. Furthermore, the effects of tey GOF were dramatically reversed when Toll was co-expressed with tey, suggesting that Toll is the major target of tey in causing the GOF phenotypes. These results suggest that Tey regulates neuromuscular connectivity by specifically repressing Toll expression in M12. As noted above, Toll is normally expressed in a number of ventral muscles, but not in M12. Furthermore, Toll is expressed in M12 in the absence of Tey suppression in tey mutants. This suggests that the default state is for Toll to be expressed in all ventral muscles, possibly by the action of positive transcription factor(s) expressed in these muscles. Tey might therefore generate target specificity by suppressing the expression of Toll in one among a group of muscle cells expressing the repulsive cue. The data thus suggest a mechanism of transcriptional control of target specificity, namely, the negative regulation of repulsive cues (Inaki, 2010).

β-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development

β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. This study reports a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. These findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz (Tipping, 2010).

This study demonstrate that the Krz protein is necessary for setting a precise level of activation of two maternal signalling pathways, Torso and Toll. This activity of Krz helps to establish the correct domains of expression of developmental patterning regulators that are under the control of these pathways (Tipping, 2010).

Genetic and protein interaction data suggest a new mechanism by which Krz may limit the activity of Torso. It was observed that Krz preferentially binds and sequesters an inactive form of ERK, thereby making it unavailable for activation by the upstream kinases such as MEK. Such a mechanism of direct inhibition of ERK activation by β-arrestin binding has not been previously reported. This mechanism is consistent with the observed in vivo effects of loss of krz on ERK activity. In krz maternal mutant embryos, ERK is not sequestered and therefore more ERK is available to transduce Torso signals, resulting in hyperactivation of Torso target genes, tll and hkb. Furthermore, consistent with this model is the observation that Krz and MEK apparently compete for ERK when all three proteins are co-expressed in S2 cells (Tipping, 2010).

Interaction assays using mutated forms of Krz and ERK indicate that the conformations of both proteins have an effect on their binding affinity. On binding to an activated GPCR, the arrestin molecule undergoes a dramatic conformational change that can be mimicked by specific mutations (Gurevich, 2004). In immunoprecipitation experiments it was observed that such 'pre-activated' form of Krz (R209E) has a much greater affinity for ERK, compared with the wild-type Krz protein, and that this higher affinity is also observed for the equivalent mutant of human β-arrestin2. This suggests that the ERK-binding ability of β-arrestin may be affected by its conformation, but it is unknown at present whether any upstream signals convert Krz into an activated form in the embryo. Overexpression of Krz-R209E using the da-GAL4 driver did not result in any observable phenotype and could rescue zygotic loss of krz, suggesting that it retains most of the functions of wild-type Krz (data not shown) (Tipping, 2010).

It was observed that the conformation of ERK itself has a large effect on its interactions with Krz. In the binding experiments, activated forms of ERK bind Krz (and human β-arrestin2) with lower affinity, compared with wild-type inactive ERK. Moreover, mutations in the TEY motif, which render ERK constitutively inactive, also lower its affinity for Krz, which is at a first glance a surprising result. However, previous studies have shown that both types of mutations in the TEY motif, which is a part of the activation loop, increase disorder in the lip region and cause a conformational change in the ERK molecule that makes it different from the basal state. It is therefore speculated that the activation loop may be involved in mediating an interaction of ERK with β-arrestin. Consistent with the current results, deviation of ERK structure from the basal state would decrease its association with β-arrestin (Tipping, 2010).

Other studies have reported formation of protein complexes containing β-arrestins and an activated form of ERK. It is possible that in those experimental conditions other binding partners, such as Raf or the activated receptor, assist in stabilizing the complex of MAP kinases with β-arrestin. This study has shown that although Krz can bind to the Drosophila homologues of both MEK and Raf, overexpression of Krz does not increase production of dpERK by the MAPK cascade downstream of activated RTKs, but instead appreciably inhibits it in the absence of overexpressed Raf. The data do not rule out a possibility that Krz may still promote ERK activation in other biological contexts, particularly downstream of activated GPCRs, but this question awaits further investigation (Tipping, 2010).

Interestingly, the sequestration mechanism of ERK inhibition described in this study is different from the effects of Krz on Notch. Previous studies have shown that Krz inhibits Notch activity by forming a ternary complex with Deltex and the Notch receptor. Formation of this complex increases Notch turnover and thereby downregulates Notch signalling (Mukherjee, 2005). No change was observed in ERK turnover in the presence of wild-type overexpressed Krz, suggesting that Krz is unlikely to be involved in the regulation of ERK stability. However, given the versatility of molecular functions displayed by β-arrestins, it is possible that there are other, as yet uncharacterized mechanisms by which Krz controls signalling downstream of RTKs (Tipping, 2010).

The inhibitory effects of Krz on ERK activity are not limited to the Torso pathway and early embryogenesis, but are also observed in other tissues and at later developmental stages. Thus, broadening of the dpERK patterns activated by EGFR and Btl was observed in krz maternal mutant embryos. An increase in the overall levels of dpERK during mid-to-late embryogenesis was also detected on western blots. Later in development, ERK is activated by EGFR in the wing and both EGFR and Sevenless in the eye. Genetic data suggest that Krz also inhibits ERK activity in these tissues during larval development. A broad involvement of Krz in inhibiting ERK activity suggests that Krz has a general inhibitory role to limit the activity of different RTKs in Drosophila development (Tipping, 2010).

In addition to its effects on RTK signalling, it was observed that Krz has an important role in limiting the activity of the Toll receptor, which specifies the development of the ventral structures. Other studies have reported that mammalian β-arrestins can downregulate NF-κB signalling by binding and stabilizing the NF-κB inhibitor IκBα. The inhibitory effects of Krz on Dorsal may involve a similar mechanism. It was observed that Krz can directly bind to the Drosophila orthologue of IκBα, Cactus, suggesting that the mechanism of NF-κB inhibition by β-arrestins at the level of IκBα may be conserved. Consistent with this finding, a decrease was detected in the level of the Cactus protein in krz maternal mutants at 0-4 h of development, which may explain the observed expansion of the nuclear gradient of Dorsal in these mutants. It is still unclear why expansion of Dorsal nuclear localization is more pronounced in the posterior half of the embryo (Tipping, 2010).

In the developing embryo, the Torso and Toll pathways do not work in isolation, but are involved in cross-regulatory interactions on certain common targets, such as zen. zen is repressed by nuclear Dorsal in the ventral part of the embryo, and relieved of this repression (de-repressed) by the signalling activity of Torso emanating from the embryo poles. The molecular mechanism of this de-repression is still unknown. It was observed that loss of krz shifts the balance of the effects of Torso on Toll, which results in an inappropriate expansion of zen expression at the embryo poles. It is speculated that Krz helps Torso to achieve a precise level of de-repression of zen by limiting the activity of ERK. Krz is thus able to control the separate activities of the Torso and Toll pathways (reflected in its effects on tll, hkb, twi, and rho), as well as regulate common Torso and Toll targets such as zen. For such pathways that are engaged in cross-regulatory interactions, Krz ensures that a proper level of signalling activity from one pathway reaches the other. This function adds an important new mechanism to understanding of the ways in which signalling pathways are coordinately regulated during development (Tipping, 2010).

A ubiquitous distribution of Krz in the embryo agrees with the dysregulation of multiple pathways observed in krz mutant animals. As overexpression of Krz does not cause any obvious defects, the level of Krz itself is not limiting for the regulation of signalling. Instead, Krz apparently makes other signalling co-factors limiting for their respective pathways, essentially working as a molecular 'sponge' to prevent pathway hyperactivity. Specificity of Krz function is likely to be determined by its selective interactions with specific pathway co-factors. Maternal loss of krz function thus affects multiple developmental signalling pathways, resulting in an accumulation of defects that ultimately lead to severe morphological abnormalities such as a disruption of gastrulation movements. By analysing the effects of loss of krz on individual pathways in vivo, this study has been able to show its role in the regulation of RTK and Toll signalling. Future studies will likely reveal other pathways and levels of regulation that are under the control of the Drosophila β-arrestin Krz (Tipping, 2010).

Serpin facilitates tumor-suppressive cell competition by blocking Toll-mediated Yki activation in Drosophila

Normal epithelial tissue exerts an intrinsic tumor-suppressive effect against oncogenically transformed cells. In Drosophila imaginal epithelium, clones of oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg) are eliminated by cell competition when surrounded by wild-type cells. In this study, a genetic screen in Drosophila identified Serpin5 (Spn5), a secreted negative regulator of Toll signaling, as a crucial factor for epithelial cells to eliminate scrib mutant clones from epithelium. Downregulation of Spn5 in wild-type cells leads to elevation of Toll signaling in neighboring scrib cells. Strikingly, forced activation of Toll signaling or Toll-related receptor (TRR) signaling in scrib clones transforms scrib cells from losers to supercompetitors, resulting in tumorous overgrowth of mutant clones. Mechanistically, Toll activation in scrib clones leads to c-Jun N-terminal kinase (JNK) activation and F-actin accumulation, which cause strong activation of the Hippo pathway effector Yorkie that blocks cell death and promotes cell proliferation. These data suggest that Spn5 secreted from normal epithelial cells acts as a component of the extracellular surveillance system that facilitates elimination of pre-malignant cells from epithelium (Katsukawa, 2018).

Clones of oncogenic polarity-deficient cells are actively eliminated from Drosophila imaginal epithelium when surrounded by normal tissue, indicating the existence of intrinsic tumor-suppression mechanism by cell competition. The present study shows that normal epithelial cells secrete Spn5 to facilitate the tumor-suppressive cell competition by antagonizing Toll signaling activation in polarity-deficient cells. Elevation of Toll signaling in polarity-deficient cells transforms them from losers to supercompetitors, which leads to tumorous overgrowth of mutant tissue. Thus, Spn5 acts as a component of the extracellular surveillance system that eliminates oncogenic cells by cell competition. It is not known at this stage why scrib cells are more sensitive to loss of spn5 to upregulate Toll signaling compared to surrounding wild-type cells. One possible mechanism that drives Toll activation in scrib cells would be JNK activation, which was shown to be sufficient to activate Toll signaling (Katsukawa, 2018).

Interestingly, it has been shown that activation of TRR signaling in losers of Myc- or Minute-induced cell competition causes losers' death through nuclear factor κB (NF-κB)-mediated induction of cell death gene hid or rpr, respectively. Consistent with this report, it has been shown in Drosophila larval fat bodies that activation of Toll signaling leads to inactivation of Yki, which may cause hid- or rpr-mediated cell death because one of the important Yki targets is a caspase inhibitor diap1. These observations intriguingly indicate that Toll signaling has opposite roles in different types of cell competition; while Toll activation promotes elimination of losers in Myc- or Minute-induced cell competition, it suppresses elimination of polarity-deficient losers in tumor-suppressive cell competition. Importantly, however, in both cases, Toll or TRR signaling seems to act as an oncogenic signaling that promotes expansion of pre-malignant winner clones within the tissue. Consistent with these findings in Drosophila, it has been reported that upregulation of Toll-like receptors (TLRs) is associated with tumor growth and progression in some human cancers. In addition, one of the human orthologs of Drosophila Spn5, SpnA5, has been shown to inhibit breast cancer growth and metastasis, and its expression level is decreased in renal cell carcinoma and sarcoma. These observations, together with the data from Drosophila genetics, suggest that Toll signaling drives tumorigenesis by promoting supercompetition of oncogenic cell clones (Katsukawa, 2018).

The mechanism by which Toll activation in polarity-deficient cells leads to Yki activation is an important open question for future studies. One possible mechanism is co-activation of JNK and Ras signaling in Toll-activated scrib cells, as these two pathways have shown to cooperate to induce Yki activation through F-actin accumulation and Wts inactivation. Interestingly, it has been shown in mammalian systems that the TLR signaling activates JNK signaling and that several TLRs activate EGFR-Ras signaling upon immune response. Given that signaling molecules identified in Drosophila are all conserved, similar Toll-mediated regulation of tumorigenesis could be involved in human cancer (Katsukawa, 2018).

The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner

The Toll pathway regulates the cellular response to infection via the transcriptional upregulation of antimicrobial peptides. In Drosophila, apart from its role in innate immunity, this pathway has also been reported to be important for the elimination of loser cells in a process referred to as cell competition, which can be locally triggered by secreted factors released from winner cells. This work provides evidence that the inhibition of Toll signaling not only increases the fitness of loser cells, but also bestows a clonal growth advantage on wild-type cells. This growth advantage depends on basal infection levels since it is no longer present under axenic conditions but exacerbated upon intense pathogen exposure. Thus, the Toll pathway functions as a fine-tuned pro-apoptotic and anti-proliferative regulator, underlining the existence of a trade-off between innate immunity and growth during development (Germani, 2018).

These findings reveal that in a non-sterile environment cells deficient for the Toll-mediated immune response grow better than immunocompetent wild-type cells. This growth difference depends on the level of infection: it is null in axenic conditions and enhanced by addition of pathogens. Most likely, therefore, it is driven by different levels of Toll pathway activity. Importantly though, these different levels must occur in cell populations that cohabitate in the same tissue, as no growth effects were detected in organs entirely programmed to exhibit elevated or reduced Toll signaling. These findings can therefore be explained by a model in which cells with lower Toll pathway activity profit from, and grow faster than, nearby cells with higher activity. Conversely, cell clones with higher Toll signaling levels (e.g., by overexpressing Toll receptors) are eliminated from the tissue via apoptosis and delamination when surrounded by cells with lower levels (Germani, 2018).

These experiments indirectly suggest that the local effects on clonal growth depend on a systemic response to infection. Likely therefore, the active form of Spätzle is produced in distant organs and reaches the wing disc through the haemolymph. However, it has recently been proposed that wing discs locally produce Spätzle and the serine proteases responsible for its activation. Via an increased secretion of these factors, Myc OE cells may be able to induce Toll-dependent apoptosis in neighboring loser cells. It is therefore possible that local and systemic sources of Spätzle co-exist. Since infection is the initial trigger for the aforementioned effects in cell competition and clonal growth, it will be interesting to investigate whether the local production of Spätzle and serine proteases depends on a systemic response to infection (Germani, 2018).

The findings also underline the important awareness that this study is carried out in non-anthropic environments. Experimental results can dramatically differ because of complex and often unexpected consequences of immune responses. This aspect has been emphasized with experiments conducted in mice in recent years. This study shows that analogous issues can affect Drosophila research (Germani, 2018).

Finally, the results also reveal a phenotypic connection between the two fundamental processes of innate immunity and cell growth at the cellular level. The previous findings that Dorsal induces the transcription of the pro-apoptotic gene rpr and that the Drosophila Toll pathway cross-talks with the growth controlling Hippo signaling pathway also suggest a potential mechanistic connection for the observations. The evolutionary implications can be viewed in the light of the life history theory, which seeks to explain natural selection on the basic assumption that environmental resources are limited and organisms establish trade-offs between processes such as reproduction, growth and immunity. Allocating resources into a costly trait like immunity occurs at the expense of other important processes, such as organismal growth. In agreement with this theory, this study has experimentally shown that a cellular trade-off exists between innate immunity and clonal growth during development (Germani, 2018).

Characterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor

The Drosophila Toll-1 receptor is involved in embryonic development, innate immunity, and tissue homeostasis. Currently, as a ligand for the Toll-1 receptor, only Spatzle (Spz) has been identified and characterized. It has been previously reported that Drosophila larva-derived tissue extract contains ligand activity for the Toll-1 receptor, which differs from Spz based on the observation that larval extract prepared from spz mutants possessed full ligand activity. This study demonstrates that Spz5, a member of the Spz family of proteins, functions as a ligand for the Toll-1 receptor. Processing of Spz5 by Furin protease, which is known to be important for ligand activity of Spz5 to Toll-6, is not required for its function to the Toll-1 receptor. By generating a spz5 null mutant, it was further shown that the Toll-1 ligand activity of larva-derived extract is mainly derived from Spz5. Finally, a genetic interaction was found between spz and spz5 in terms of developmental processes. This study identified a novel ligand for the Drosophila Toll-1 receptor, providing evidence that Toll-1 is a multi-ligand receptor, similar to the mammalian Toll-like receptor (Nonaka, 2018).

This study has demonstrated that Spz5 is most likely the protein in charge of the Toll pathway-stimulating activity in larva-derived tissue extract, which was reported in a previous paper (Kanoh, 2015). Since it was not possible to examine a direct effect of Spz5, i.e., spz5-expressing cell extracts in the Drs reporter assay with DL1 cells because of the technical difficulty in purifying Spz5, it can be at most concluded that Spz5 was required for novel Toll-1 ligand activity in the larval extract based on the mutant experiment (Nonaka, 2018).

The Spz family of proteins is known to become active via protease cleavage. Spz is cleaved by Easter or SPE, Spz3 by Easter, and Spz5 by Furin proteases. This study, however, showed that Spz5 activity in terms of the Toll-1 ligand does not require processing by Furin, and Spz5 is likely to function in a full-length form, which was suggested by the result that Furin cleavage site-lacking Spz5 (Spz5R284G) retained the Toll-1 ligand activity. This implied that Spz5 might work as a DAMP that is released by cell damage or necrotic death, because proteolytic processing is required for secretion of Spz5 from cells. Because this study demonstrated that Spz5 is not involved in pinching-induced activation of innate immunity in larvae, Spz5 might function in a different physiological or pathological context, which should be pursued in future studies (Nonaka, 2018).

It is widely appreciated that homozygous mutant flies from loss-of-function Toll-1 alleles are scarce but those from Spz can be substantially obtained. Because spzΔ8 must be a null allele, it would be possible that the Toll-1 receptor might have other ligands during developmental processes (though this could be simply accounted for by the amount of their maternal mRNAs). In the current study, it was observed that spz5 single mutant flies were homozygous viable, but the double mutants for spz and spz5 produced fewer homozygous progenies. This implied that Spz5 might have a role in development with Spz. Elucidating when and how Spz5 functions during development will be of interest (Nonaka, 2018).

Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction

Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In Drosophila melanogaster, PGCs from embryos maternally compromised for germ cell-less (gcl) misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. This study demonstrates that the somatic determinant of female fate, Sex-lethal (Sxl), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso (torso(Deg)) can activate Sxl transcription in PGCs, whereas simultaneous loss of torso-like (tsl) reinstates the quiescent status of gcl PGCs. Intriguingly, like gcl mutants, embryos derived from mothers expressing torso(Deg) in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction (Colonnetta, 2021).

Following fertilization, a Drosophila embryo undergoes 14 consecutive nuclear divisions to give rise to the cellular blastoderm. While the initial nuclear divisions take place in the center of the embryo, the nuclei begin to migrate toward the periphery around nuclear cycle (NC) 4-6 and reach the cortex at NC9/10. Even before bulk nuclear migration commences, a few nuclei move toward the posterior of the embryo, enter a specialized, maternally derived cytoplasm known as the pole plasm, and induce the formation of pole buds (PBs). The centrosomes associated with these nuclei trigger the release of pole plasm constituents from the posterior cortex and orchestrate precocious cellularization to form the primordial germ cells (PGCs), the progenitors of the germline stem cells in adult gonads. Unlike pole cell nuclei, somatic nuclei continue synchronous divisions after they reach the surface of the embryo until NC 14 when they cellularize (Colonnetta, 2021).

The timing of cellularization is not the only difference between the soma and PGCs. Although newly formed PGCs divide after they are formed, they undergo only one or two asynchronous divisions before exiting the cell cycle. Another key difference is in transcriptional activity. Transcription commences in the embryo during NC 6-7 when a select number of genes are active. Transcription is more globally upregulated when the nuclei reach the surface, and by the end of NC 14, zygotic genome activation (ZGA) is complete. This transition is marked by high levels of phosphorylation of residues Serine 5 (Ser5) and Serine 2 (Ser2) in the C-terminal domain (CTD) of RNA polymerase II. By contrast, in newly formed PGCs, transcription is switched off, and PGC nuclei have only residual amounts of Ser5 and Ser2 CTD phosphorylation. Moreover, and consistent with their transcriptionally quiescent status, other changes in chromatin architecture that accompany ZGA are also blocked in PGCs (Colonnetta, 2021).

Three different genes, nanos (nos), polar granule component (pgc), and germ cell-less (gcl), are known to be required for establishing transcriptional quiescence in newly formed PGCs. The PGCs in embryos derived from mothers carrying mutations in these genes fail to inhibit transcription, and this compromises germ cell specification and disrupts germ cell migration. (As these are maternal effect genes, embryos derived from nos/pgc/gcl mothers display the resulting mutant phenotypes and will be referred to as nos/pgc/gcl here onwards.) Interestingly, these three genes share only a few targets, suggesting overlapping yet distinct mechanisms of action. Nos is a translation factor and thus must block transcription indirectly. Together with the RNA-binding protein Pumilio (Pum), Nos interacts with recognition sequences in the 3'-untranslated regions (3'UTRs) of mRNAs and inhibits their translation. Currently, the key mRNA target(s) that Nos-Pum repress to block transcription is unknown; however, in nos and pum mutants, PGC nuclei display high levels of Ser5 and Ser2 CTD phosphorylation and activate transcription of gap and pair-rule patterning genes and the sex determination gene Sex-lethal (Sxl). pgc encodes a nuclear protein that binds to the transcriptional elongation kinase p-TEFb, blocking Ser5 CTD phosphorylation. In pgc mutant pole cells, Ser5 phosphorylation is enhanced, as is transcription of several somatic genes, including genes involved in terminal patterning (Colonnetta, 2021).

While the primary function of nos and pgc appears to be blocking ZGA in PGCs, gcl has an earlier function, which is to turn off transcription of genes activated in somatic nuclei prior to nuclear migration. Targets of gcl include two X-chromosome counting elements (XCEs), scute (sc/sis-b) and sisterless-a (sis-a), that function to turn on the sex determination gene, Sxl, in female soma. gcl embryos not only fail to shut off sis-a and sis-b transcription in PBs, but also show disrupted PGC formation. In some gcl embryos, PGC formation fails completely, while in other embryos only a few PGCs are formed. In this respect, gcl differs from nos and pgc, which have no effect on the process of PGC formation, but instead interfere with the specification of PGC identity (Colonnetta, 2021).

Studies by Leatherman (2002) suggested that the defects in PGC formation in gcl mutant embryos are linked to failing to inhibit somatic transcription. That study found that when PBs first form during NC 9 in wild-type (WT) embryos, levels of CTD phosphorylation PB are only marginally less than in nuclei elsewhere in the embryo. However, by NC 10, there was a dramatic reduction in CTD phosphorylation even before PBs cellularize. By contrast, in gcl mutant embryos, about 90% of the NC 10 PB nuclei had CTD phosphorylation levels approaching that of somatic nuclei. Moreover, this number showed an inverse correlation with the number of PGCs in blastoderm stage gcl embryos. Whereas WT blastoderm embryos have >20 PGCs per embryo, gcl embryos had on average just three PGCs under their culturing conditions. Interestingly, expression of the mouse homologue of Gcl protein, mGcl-1, can rescue the gcl phenotype in Drosophila (Leatherman, 2000). Supporting the conserved nature of the involvement of Gcl during transcriptional suppression, a protein complex between mGcl-1 and the inner nuclear membrane protein LAP2β is thought to sequester E2F:D1 to reduce transcriptional activity of E2F:D1 (Colonnetta, 2021).

The connection Leatherman postulated between failing to turn off ongoing transcription and defects in PGC formation in gcl mutants is controversial and unresolved. This model predicts that a non-specific inhibition of polymerase II should be sufficient to rescue PGC formation in gcl embryos. However, the PGC formation defects seen in gcl embryos are not rescued after injection of the RNA polymerase inhibitor, α-amanitin. Since α-amanitin treatment disrupted somatic cellularization without impacting PGC formation in WT embryos, it was concluded that it effectively blocked polymerase transcription. On the other hand, subsequent experiments by Pae (2017) raised the possibility that inhibiting transcription in pole cell nuclei is a critical step in PGC formation. The Pae paper showed that Gcl is a substrate-specific adaptor for a Cullin3-RING ubiquitin ligase that targets the terminal pathway receptor tyrosine kinase, Torso, for degradation. The degradation of Torso would be expected to prevent activation of the terminal signaling cascade in PGCs. In the soma, Torso-dependent signaling activates the transcription of several patterning genes, including tailless, that are important for forming terminal structures at the anterior and posterior of the embryo. Thus, by targeting Torso for degradation, Gcl would prevent the transcriptional activation of terminal pathway genes by the MAPK/ERK kinase cascade in PGCs. Consistent with this possibility, simultaneous removal of gcl and either the Torso ligand modifier, torso-like (tsl) or torso resulted in rescue of germ cell loss induced by gcl. Surprisingly, however, Pae (2017) was unable to observe a similar rescue of gcl phenotype when they used RNAi knockdown to compromise components of the MAP kinase cascade known to act downstream of the Torso receptor. Based on these findings, they proposed that activated Torso must inhibit PGC formation via a distinct non-canonical mechanism that is both independent of the standard signal transduction pathway and does not involve transcriptional activation (Colonnetta, 2021).

The current study has revisited these conflicting claims by examining the role of Gcl in establishment/maintenance of transcriptional quiescence. The studies of Leatherman (2002) indicated that two of the key X chromosomal counting elements, sis-a and sis-b, were inappropriately expressed in gcl PBs and PGCs. Since transcription factors encoded by these two genes function to activate the Sxl establishment promoter, Sxl-Pe, in somatic nuclei of female embryos, their findings raised the possibility that Sxl might be ectopically expressed in PBs/PGCs of gcl embryos. This study shows that in gcl embryos, Sxl transcription is indeed inappropriately activated in PBs and newly formed PGCs. Moreover, ectopic expression of Sxl in early embryos disrupts PGC formation similar to gcl. Supporting the conclusion that Sxl is a biologically relevant transcriptional target of Gcl, PGC formation defects in gcl embryos can be suppressed either by knocking down Sxl expression using RNAi or by loss-of-function mutations. As reported by Pae (2017), this study found that loss of torso-like (tsl) in gcl embryos suppresses PGC formation defects. However, consistent with a mechanism that is tied to transcriptional misregulation, rescue is accompanied by the reestablishment of transcriptional silencing in gcl PGCs. Lending further credence to the idea that transcription misregulation plays an important role in disrupting PGC development in gcl embryos, this study found that expression of a mutant form of Torso that is resistant to Gcl-dependent degradation (hereafter referred to as torsoDeg: Pae, 2017) ectopically activates transcription of two Gcl targets, sis-b and Sxl, in PB and PGC nuclei. In addition, stabilization of Torso in early PGCs also mimics another gcl phenotype, the failure to properly sequester key PGC determinants in PBs and newly formed PGCs (Colonnetta, 2021).

gcl differs from other known maternally deposited germline determinants in that it is required for the formation of PBs and PGCs. gcl PGCs exhibit a variety of defects during the earliest steps in PGC development. Unlike WT, gcl PGCs fail to properly establish transcriptional quiescence. While other genes like nos and pgc are required to keep transcription shut down in PGCs, their functions only come into play after PGC cellularization. By contrast, gcl acts at an earlier stage beginning shortly after nuclei first migrate into the posterior pole plasm and initiate PB formation. In gcl PBs, ongoing transcription of genes that are active beginning around nuclear cycle 5-6 is not properly turned off. This is not the only defect in germline formation and specification. As in WT, the incoming nuclei (and the centrosomes associated with the nuclei) trigger the release of the pole plasm from the posterior cortex. However, instead of sequestering the germline determinants in PBs so that they are incorporated into PGCs during cellularization, the determinants disperse into the soma where they become associated with the cytoplasmic territories of nearby somatic nuclei. There are also defects in bud formation and cellularization. Like the release and sequestration of germline determinants, these defects have been linked to the actin cytoskeleton and centrosomes (Colonnetta, 2021).

Two models have been proposed to account for the PGC defects in gcl mutants. In the first, Leatherman (2002) attributed the disruptions in PGC development to a failure to turn off ongoing transcription. The second argues that the role of gcl in imposing transcriptional quiescence is irrelevant. Instead, the defects are proposed to arise from a failure to degrade the Torso receptor. In the absence of Gcl-dependent proteolysis, high local concentrations of the Tsl ligand modifier at the posterior pole would activate the Torso receptor. According to this model, the ligand-receptor interaction would then trigger a novel, transcription-independent signal transduction pathway in PBs and PGCs that disrupts their development. These conflicting models raise several questions. Does gcl actually have a role in establishing transcriptional quiescence in PBs and PGCs? If so, is this activity relevant for PB and PGC formation? Is the stabilization of Torso in gcl mutants responsible for the failure to shut down transcription in PBs and PGCs? If not, does gcl target a novel, transcription-independent but Torso-dependent signaling pathway? Is the stabilization of Torso responsible for some of the other phenotypes that are observed in gcl mutants? These studies have addressed these outstanding questions, leading to a resolved model of Gcl activity and function (Colonnetta, 2021).

Shutting off transcription is, in fact, a critical function of Gcl protein. As previously documented by Leatherman, this study found that several of the key X-linked transcriptional activators of Sxl-Pe are not repressed in newly formed PBs and early PGC nuclei, and Sxl-Pe transcription is inappropriately activated in the presumptive germline. Previous studies found that ectopic expression of Sxl in nos mutants disrupts PGC specification. In this case, the specification defects in nos embryos can be partially rescued by eliminating Sxl activity. The same is true for gcl mutants: elimination or reduction in Sxl function ameliorates the gcl defects in PGC formation/specification. Conversely ectopic expression of Sxl early in embryogenesis mimics the effects of gcl loss on PGC formation. Importantly, the role of Gcl in inhibiting Sxl-Pe transcription is not dependent upon other constituents of the pole plasm. When Gcl is ectopically expressed at the anterior of the embryo, it can repress Sxl. This observation is consistent with the effects of ectopic Gcl on the transcription of other genes reported by Leatherman et al., 2002. Since the rescue of gcl by eliminating the Sxl gene or reducing its activity is not complete, one would expect that there must be other important gcl targets. These targets could correspond to one or more of the other genes that are misexpressed in gcl PB/PGCs. Consistent with this possibility, transcriptional silencing in gcl PBs/PGCs is reestablished when terminal signaling is disrupted by mutations in the tsl gene. On the other hand, it is possible that excessive activity of the terminal signaling pathway also adversely impacts some non-transcriptional targets that are important for PB/PGC formation and that transcriptional silencing in only part of the story (see below) (Colonnetta, 2021).

Pae (2017) showed that mutations in the Gcl interaction domain of Torso (torsoDeg) stabilize the receptor and disrupt PGC formation. Consistent with the notion that Torso receptor is the primary, if not the only, direct target of gcl, they found that mutations in the Torso ligand modifier, tsl, or RNAi knockdown of torso rescued the PGC formation defects in gcl embryos. As would be predicted from those and the current findings, ectopic expression of the TorsoDeg protein induces the inappropriate transcription of sis-b and Sxl-Pe in PBs and newly formed PGCs. Thus, the failure to shut down ongoing transcription in gcl PBs and PGCs must be due (at least in part) to the persistence of the Torso receptor in the absence of Gcl-mediated degradation. Corroborating this idea, the ectopic activation of transcription in gcl PGCs is no longer observed when the terminal signaling pathway is disrupted by the removal of tsl. Taken together, these data strongly suggest that the establishment/maintenance of transcriptional silencing in PBs is a critical function of Gcl (Colonnetta, 2021).

Since RNAi knockdowns of terminal pathway kinases downstream of torso did not rescue gcl mutants, Pae (2017) postulated that the Tsl-Torso receptor interaction triggered a novel, non-canonical signal transduction pathway that disrupted PGC development. If that suggestion is correct, then the activation of sis-b and Sxl-Pe in PBs/PGCs in gcl and torsoDeg embryos would be mediated by this novel terminal signaling pathway. The results of the current study are ambiguous. Consistent with the suggestion of Pae, 2017, GOF mutations in MEK, a downstream kinase in the Torso signaling pathway, did not activate Sxl-Pe transcription in pole cells. However, an important caveat is that the GOF activity of MEK variants that was tested is likely not equivalent to the activity from the normal Torso-dependent signaling cascade. As the pole plasm contains at least two other factors that help impose transcriptional quiescence, the two GOF MEK mutants that were tested may simply not be sufficient to overcome their repressive functions. Two observations are consistent with this possibility. First, like torsoDeg, this study found that MEK^E203K induces Sxl-Pe expression in male somatic nuclei. The same is true for a viable GOF mutation in Torso: it can induce ectopic activation of Sxl-Pe in male somatic nuclei, but is unable to activate Sxl-Pe in PGCs. Second, a key terminal pathway transcription target tailless is not expressed in gcl mutant PBs/PGSs even though the terminal pathway should be fully active. This is also true for embryos expressing torsoDeg and the two GOF MEK proteins. For these reasons, it cannot be unambiguously determined if it is the canonical terminal signaling pathway or another, noncanonical signaling pathway downstream of Torso that is responsible for the expression of sis-b, Sxl-Pe, and other genes in gcl mutant PB/PGCs (Colonnetta, 2021).

There are also reasons to think that the canonical Torso signal transduction cascade must be inhibited for proper PGC formation. One of the more striking phenotypes in gcl mutants is the dispersal of key germline mRNA and protein determinants into the surrounding soma. A similar disruption in the sequestration of pole plasm components is observed not only in torsoDeg embryos but also in MEK^E203K and MEK^F53S embryos. Thus, this gcl phenotype would appear to arise from the deployment of the canonical Torso receptor signal transduction cascade, at least up to the MEK kinase. However, this result does not exclude the possibility that the Tsl->Torso->ERK pathway has other non-transcriptional targets that, like Sxl-Pe expression, can also interfere with PB/PGC formation. If this was the case, it could potentially explain why global transcriptional inhibition failed to rescue the PGC defects in gcl embryos. In this respect, a potential-if not likely-target is the microtubule cytoskeleton. In previous studies, it was found that the PB and PGC formation defects as well as the failure to properly sequester critical germline determinants in gcl arise from abnormalities in microtubule/centrosome organization. Preliminary imaging experiments indicate that centrosome distribution of torsoDeg PBs is also abnormal, suggesting that inappropriate activation of the terminal signaling pathway perturbs the organization or functioning of the microtubule cytoskeleton and/or centrosomes. Such a mechanism would also be consistent with the dispersal of germline mRNA and protein determinants in torsoDeg and GOF MEK embryos. While further experiments will be required to demonstrate microtubule and centrosomal aberrations in torsoDeg and GOF MEK embryos, a role for a receptor-dependent MEK/ERK signaling cascade in promoting centrosome accumulation of γ-tubulin and microtubule nucleation has been documented in mammalian tissue culture cells. It is thus conceivable that MEK/ERK signaling has a similar role in Drosophila PB nuclei and PGCs. It will be important to determine if Torso-dependent activation of MEK/ERK can perturb the behavior or organization of centrosomes and/or microtubules in early embryos, and, if so, whether the influence can alter the pole plasm RNA anchoring and/or transmission. Taken together, the current data reveal a mutual antagonism between the determinants that specify germline versus somatic identity. Future studies will focus on how and when during early embryogenesis such feedback mechanisms are activated and calibrated to establish and/or maintain germline/soma distinction (Colonnetta, 2021).

Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning

The relationship between chromatin organization and gene regulation remains unclear. While disruption of chromatin domains and domain boundaries can lead to misexpression of developmental genes, acute depletion of regulators of genome organization has a relatively small effect on gene expression. It is therefore uncertain whether gene expression and chromatin state drive chromatin organization or whether changes in chromatin organization facilitate cell-type-specific activation of gene expression. In this study, using the dorsoventral patterning of the Drosophila melanogaster embryo as a model system, evidence is provided for the independence of chromatin organization and dorsoventral gene expression. Tissue-specific enhancers are defined and linked to expression patterns using single-cell RNA-seq. Surprisingly, despite tissue-specific chromatin states and gene expression, chromatin organization is largely maintained across tissues. The results indicate that tissue-specific chromatin conformation is not necessary for tissue-specific gene expression but rather acts as a scaffold facilitating gene expression when enhancers become active (Ing-Simmons, 2021).

Previous studies produced conflicting results regarding the relationship between gene expression, chromatin state and 3D chromatin organization. This study set out to understand this relationship in the context of embryonic development in Drosophila. Using the well-studied dorsoventral patterning system, it was shown that, despite significant differences in chromatin state and gene expression between tissues along the dorsoventral axis of the embryo, chromatin conformation is largely maintained across tissues. This suggests that cell-type-specific gene regulation does not require cell-type-specific chromatin organization in this context. Nevertheless, developmentally regulated genes and enhancers are organized into chromatin domains. It is suggested that this organization plays a permissive role to facilitate the precise regulation of developmental genes (Ing-Simmons, 2021).

Use was made of maternal effect mutations in the Toll signaling pathway, which lead to embryos that lack the usual patterning of the dorsoventral axis and have long been used as a system to study the specification of mesoderm (Toll^10B), neuroectoderm (Toll^rm9/rm10) and dorsal ectoderm (gd⁷) cell fates as well as the regulation of tissue-specific gene expression. However, these embryos are still under the influence of anterior-posterior patterning signals and do not show completely uniform cell identities. This study sought to investigate heterogeneity of cell identity at the single-cell level by using single-cell gene expression profiling. This revealed that certain cell types are indeed maintained in all three Toll pathway mutants, including pole cells and other terminal region cell identities, hemocytes and trachea precursor cells. However, heterogeneity of gene expression is reduced in the mutants, as shown by the loss of cells assigned to mesoderm clusters in gd⁷ and Toll^10B embryos and the depletion of ectoderm subsets in each of the mutants. These datasets showcase the advantages of measuring cellular heterogeneity at the single-cell level and provide a useful resource for further characterization of these embryos and investigation of the regulation of dorsoventral patterning (Ing-Simmons, 2021).

Although the gd⁷, Toll^10B and Toll^10B embryos still have heterogeneous gene expression profiles, nevertheless, there are clear differences in chromatin state and overall gene expression between these embryos. This study expanded on previous studies by identifying putative enhancers specific to neuroectoderm in addition to dorsal ectoderm and mesoderm. This allowed the identification of tissue-specific putative enhancer-gene pairs, which correspond well with known dorsoventral patterning enhancers and genes that are differentially expressed (DE across the dorsoventral axis. These regulatory elements and their target genes are located inside chromatin domains, distinct from the enrichment of housekeeping genes at domain boundaries. This is in line with previous results that suggest that 3D chromatin domains act as regulatory domains (Ing-Simmons, 2021).

This domain organization is maintained across tissues, even in cases in which there are significant changes in the local chromatin state and gene expression. This is consistent with earlier results from Hi-C experiments carried out in anterior and posterior embryo halves, which also showed no differences, and with previous studies in Drosophila cell lines and other systems, which suggested that domains are widely conserved across different tissues and even different species. To explain this maintenance of organization across cell lines, it was proposed that active chromatin, especially at broadly expressed genes, is responsible for partitioning the genome into domains. It has been proposed that compartmentalization of active and inactive chromatin, at the level of individual genes, underlies the formation of insulated chromatin domains. This model predicts that, when a developmentally regulated gene is active, its domain would merge with or have increased interactions with neighboring domains containing active genes, such as broadly expressed housekeeping genes. The results do not support this model, as this study found no evidence that differences in domain structure are driven by changes in chromatin state or by active expression of developmentally regulated genes. By contrast, this supports the idea that, similar to mammalian domain architecture, additional factors, such as insulator proteins, modulate domain organization in Drosophila. Therefore, based on current data, it is not believed that active transcription is the key determinant of 3D chromatin organization in this system (Ing-Simmons, 2021).

While overall and locus-specific chromatin organization are maintained across tissues, Hi-C and Micro-C analyses identify a small number of examples of regions that do have changes in organization. However, at these loci, there is no clear relationship between changes in organization and changes in chromatin state or expression, and the vast majority of developmentally regulated loci in this system do not have changes. It will be important for future studies to further investigate these loci to understand what drives these rare changes (Ing-Simmons, 2021).

This study also investigated chromatin organization at the level of enhancer-promoter interactions. Previous studies produced conflicting results about whether these interactions are correlated with tissue-specific activation of gene expression. No evidence was found for widespread enrichment of interactions between enhancers and their target promoters, including in tissues where they are active. This is in contrast with previous studies using 3C approaches that have found evidence of enriched enhancer-promoter interactions, which may precede or correlate with transcriptional activation. Notably, Ghavi-Helm (2019) found that a subset of Drosophila long-range enhancer-promoter pairs do form stable interactions that are enriched above local background19. While these loops are visible in the dataset presented in this study, the results suggest that such loops are not likely to be the primary mechanism of promoter regulation during Drosophila development, perhaps because most enhancers are close to their target promoters. Many stable loops in the Drosophila genome are instead associated with polycomb-mediated repression (Ing-Simmons, 2021).

Hi-C provides information about the average conformation across a population of hundreds of thousands of nuclei, which contain dynamic ensembles of different 3D conformations. While the scRNA-seq results indicate that the mutant embryos contain a range of different cell types, it is believed that the results indicate that the 3D chromatin structures in these cell types are drawn from the same population of possible conformations. This is supported by results from a recent study analyzing the structure of the Doc and sna loci in Drosophila embryos using Hi-M, a high-resolution single-cell imaging approach. Strikingly, this orthogonal technique also reveals chromatin organization that is consistent across different tissues in the embryo, despite differential expression of these genes. Imaging-based approaches directly measure spatial proximity between genomic loci, whereas Hi-C and Micro-C rely on cross-linking to detect chromatin interactions. Therefore, care must be taken when comparing these approaches. Nevertheless, both approaches indicate that genome organization is maintained across different tissues in this system (Ing-Simmons, 2021).

The results are consistent with several recent studies in mammals as well as in Drosophila, which provide evidence that stable enhancer-promoter contacts are not always required for gene activation. This is in line with models in which transient or indirect contacts with a regulatory element are sufficient to activate transcription, such as through the formation of nuclear microenvironments or phase-separated condensates (Ing-Simmons, 2021).

Together, the results indicate that differential chromatin organization is not a necessary feature of cell-type-specific gene expression. It is proposed that chromatin organization into domains instead provides a scaffold or framework for the regulation of developmental genes during and after the activation of zygotic gene expression. This may help render developmental enhancers 'poised' for timely regulation of target genes upon receipt of appropriate cellular signals. Other mechanisms of priming have been described, including paused polymerase (Pol) II at promoters and pioneer factors bound to poised enhancers. Feedback effects, such as downstream modification of chromatin state and additional mechanisms, including looping between polycomb-bound elements and segregation of active and inactive chromatin, may then act as layers on top of the initially established domain structure (Ing-Simmons, 2021).

Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila

Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. This study shows that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, this study defines a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP (Shields, 2022).

Since the discovery of the original Toll gene in Drosophila (Toll-1 hereafter), a large number of Toll-related genes have been identified in both insects and mammals. The Drosophila genome encodes a total of 9 Toll-related genes, including Toll-1, while mammalian genomes encode between 10 and 13 Toll-like receptors (TLRs). TLRs are single-pass transmembrane proteins that upon ligand stimulation usually trigger a conserved intracellular signaling pathway, culminating in the activation of NF-κB transcription factors. Initially identified as an essential gene for dorsoventral patterning in the early Drosophila embryo, Toll-1 was later also found to be a critical component for innate immunity. In this function, Toll-1 signaling via the NF-κB transcription factors Dorsal and Dorsal-related immunity factor (Dif) induces the expression of anti-microbial peptides (AMPs) that mediate innate immunity. A role in innate immunity has also been demonstrated for all mammalian TLRs. Likewise, Drosophila Toll-8 (aka Tollo) regulates immunity in the trachea. Toll-7 may regulate anti-viral responses, albeit through an NF-κB-independent mechanism. However, for the remaining Toll-related proteins in Drosophila, a function in innate immunity has not been clearly demonstrated (Shields, 2022).

Of particular interest is Toll-9 in Drosophila because it behaves genetically most similar to Toll-1, and its intracellular TIR domain is most closely related to those of the mammalian TLRs. Overexpression of Toll-9 results in the production of AMPs, which led to the proposal that Toll-9 might be involved in innate immunity. However, a loss-of-function analysis with a defined Toll-9 null allele did not confirm this prediction. Nevertheless, although Drosophila Toll-9 does not appear to be directly involved in regulating expression of AMPs, it has been implicated together with a few other Toll-related proteins in cell competition, an organismal surveillance program that monitors cellular fitness and eliminates cells of reduced fitness (losers). Depending on the type of cell competition, Toll-9 participates in the expression of the pro-apoptotic genes reaper and hid in loser cells, triggering their elimination . Therefore, it has been proposed that the function of Toll signaling for elimination of bacterial pathogens by AMPs and for elimination of unfit cells by pro-apoptotic genes bears a conceptual resemblance between innate immunity and cell competition (Shields, 2022).

Apoptosis is an evolutionarily well-conserved process of cellular suicide mediated by a highly specialized class of Cys-proteases, termed caspases. In Drosophila, the pro-apoptotic genes reaper and hid promote the activation of the initiator caspase Dronc (caspase-9 homolog in Drosophila). Dronc activates the effector caspases DrICE and Dcp-1 (caspase-3 and caspase-7 homologs), which trigger the death of the cell. However, caspases not only induce apoptosis but can also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), during which caspases in apoptotic cells promote the proliferation of surviving cells independently of their role in apoptosis. Early work has shown that AiP requires the initiator caspase Dronc (Shields, 2022).

To reveal the mechanism of AiP, this study expressedthe pro-apoptotic gene hid and the apoptosis inhibitor p35 simultaneously using the ey-Gal4 driver (referred to as ey > hid,p35) which drives expression in the larval eye disc anterior to the morphogenetic furrow (MF) . p35 encodes a specific inhibitor of the effector caspases DrICE and Dcp-1 but does not block the activity of Dronc. In ey > hid,p35-expressing discs, the apoptotic pathway is activated by hid expression but blocked downstream because of DrICE and Dcp-1 inhibition by P35, rendering cells in an 'undead' condition. Although apoptosis is inhibited in undead tissue, AiP still occurs because of non-apoptotic signaling by the initiator caspase Dronc, triggering hyperplasia of the anterior portion of the larval eye imaginal discs at the expense of the posterior eye field, which is reduced in size. Combined, these effects result in overgrowth of the adult head capsule, but a reduction or even absence of the eyes in the adult animal (Shields, 2022).

Using the undead model, this study showed that AiP is mediated by extracellular reactive oxygen species (ROS) generated by the NADPH oxidase Duox. ROS trigger the recruitment of hemocytes, Drosophila immune cells most similar to mammalian macrophages, to undead imaginal discs. Hemocytes in turn release signals that stimulate JNK activity in undead cells, which then promotes AiP. In addition, JNK can also induce the expression of hid, thus setting up an amplification loop in undead cells which continuously signals for AiP (Shields, 2022).

Given that undead cells are abnormal cells with potentially altered cellular fitness and that signaling by Toll-related proteins surveilles cellular fitness, this study examined if signaling by Toll-9 has an important function for undead AiP. This study shows that Toll-9 is strongly up-regulated in undead cells and is necessary for the overgrowth of undead tissue. Overexpression of Toll-9 with p35 induces all hallmarks of undead AiP signaling, including Duox-dependent ROS generation, hemocyte recruitment and JNK signaling. Mechanistically, genetic evidence is provided for a heterologous interaction between Toll-9 and Toll-1, which engages the canonical Toll-1 signaling pathway to promote nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces the expression of reaper and hid. This activity contributes to the establishment of the feedback amplification loop that signals continuously for AiP. In conclusion, although Toll-9 does not appear to have an important function in innate immunity, it appears to be involved in the expression of pro-apoptotic genes such as reaper and hid in stress situations such as cell competition and undead AiP (Shields, 2022).

Toll-9 is the most closely related Drosophila TLR compared with mammalian TLRs, but a biological function of Toll-9 has not been clearly defined. All mammalian TLRs are involved in innate immunity; therefore, the close homology has led to the prediction that Drosophila Toll-9 also participates in innate immunity. However, in Toll-9 mutants, the basal as well as bacterially induced AMP production is not affected, leading to the conclusion that Toll-9 is not involved in innate immunity (Narbonne-Reveau, 2011). Nevertheless, instead of eliminating foreign pathogens, previous work has shown that Toll-9 together with several other Toll-related receptors participates in elimination of unfit cells during cell competition. This is achieved through the expression of the pro-apoptotic gene rpr. This study demonstrates that Toll-9 has a similar rpr- and hid-inducing function during AiP, thereby adding to the database of Toll-9 function (Shields, 2022).

To identify the mechanism by which Toll-9 participates in AiP, advantage was taken of the observation that misexpression of Toll-9 is sufficient to induce overgrowth of ey > p35 animals. There are multiple aspects of this phenotype that are worth being discussed. First, key for many of the observations presented in this paper is the presence of P35, a very efficient inhibitor of the effector caspases DrICE and Dcp-1. In the absence of P35, overexpression of Toll-9 does not induce any obvious phenotypes in eye discs or adult heads. The exact reason for this P35 dependence is currently unknown, but it has also been observed upon misexpression of other genes involved in AiP, such as Myo1D, Toll-1, and SpzAct. The only known function of P35 is to inhibit DrICE and Dcp-1. Therefore, one possible explanation for the P35 dependence is that these caspases cleave and inactivate an as yet unidentified component of the AiP network, possibly to block inappropriate AiP under normal conditions. In the presence of P35, the AiP-blocking activity of DrICE is inhibited and with the addition of an AiP-inducing stimulus such as misexpression of Toll-9, AiP is engaged and can trigger tissue overgrowth. Second, the data show that ectopic p35,Toll-9 co-expression triggers overgrowth through a similar mechanism as hid,p35 co-expression. This includes Dronc activation, Duox-generated ROS, hemocyte recruitment, and JNK activation. These similarities allow placing the function of Toll-9 into the AiP network (Shields, 2022).

Third, mis-expressed Toll-9 can genetically interact with Toll-1. This interaction results in nuclear translocation of Dorsal and is dependent on Myd88, Tube, and Pelle, all canonical components of the intracellular Toll-1 signaling pathway. Importantly, the nuclear translocation of Dorsal and the p35,Toll-9-induced overgrowth is also dependent on Toll-1, suggesting that the activation of the Myd88/Tube/Pelle pathway is directly triggered by Toll-1 and not by Toll-9. Mechanistically, Toll-9 may activate Toll-1 either directly through hetero-dimerization or mediated by additional factors. Future work will be necessary to identify the molecular mechanism of the Toll-9/Toll-1 interaction (Shields, 2022).

Fourth, the outcome of the Toll-9/Toll-1 interaction is the expression of the pro-apoptotic genes reaper and hid. Because Toll-9 expression is strongly up-regulated in undead cells, these data suggest that Toll-9-induced expression of reaper and hid in undead cells is setting up an amplification loop. The cause of the strong transcriptional upregulation of Toll-9 in undead cells is unknown, but it requires JNK activity. The Toll-9 amplification loop contributes to the strength of undead signaling during AiP and propels the overgrowth of the tissue (Shields, 2022).

Fifth, another important question is how Toll signaling becomes activated during AiP. Toll-1 is activated by the ligand Spatzle during embryogenesis and the immune response. Spz requires proteolytic processing for activation, which during the immune response is mediated by the Ser-protease Spatzle-processing enzyme (SPE). Consistently, SPE RNAi can suppress both the ey > p35,Toll-9INTRA and ey > hid,p35-induced overgrowth phenotypes. SPE RNAi suppressed ey > p35,Toll-9INTRA, which lacks the extracellular domain and should be insensitive to a ligand. Thus, the suppression of ey > p35,Toll-9INTRA suggests that SPE does not act through Toll-9 but instead on Toll-1. This result is consistent with a recent finding that Toll-9 RNAi cannot suppress apoptosis induced by a dominant active SPE (SPEAct) transgene. Although that there is an unknown Toll-9 ligand cannot be ruled out, Toll-9 may not need to be activated by a ligand. Toll-9 naturally carries an amino acid substitution in the cysteine-rich extracellular domain similar to the gain-of-function Toll-11 mutant. Indeed, Toll-9 behaves as a constitutively active receptor in cell culture assays. As TLRs can form homo- and heterodimers, it is possible that the constitutive activity of Toll-9 and the strong transcriptional upregulation of Toll-9 together with ligand stimulation of Toll-1 by Spz is sufficient for the activation of the Toll-1/Toll-9 complex. However, it remains unknown how SPE becomes activated during AiP (Shields, 2022).

With these considerations in mind, the following model for Toll-9 function during undead AiP emerges. The initial stimulus for AiP is the Gal4-induced expression of hid and p35, which leads to the activation of Dronc. Because of P35, Dronc cannot induce apoptosis but instead activates Duox for generation of ROS. ROS attract hemocytes which release signals for JNK activation in undead cells. JNK signaling directly or indirectly induces Toll-9 transcription. Up-regulated Toll-9 interacts with Toll-1, and after Spz ligation the Myd88/Tube/Pelle pathway triggers the nuclear accumulation of Dorsal and potentially Dif. These factors transcriptionally induce reaper and hid expression setting up the feedback amplification loop, which maintains and propels AiP and overgrowth (Shields, 2022).

One other interesting question to examine in the future will be how the intracellular pathway of Toll-1 signaling including Dorsal and Dif can induce different target genes under different conditions. For dorsoventral patterning of the Drosophila embryo, Dorsal induces the expression of twist and snail). During immune responses in the fat body, it promotes the expression of AMP genes as well as Kennedy pathway genes for the synthesis of phospholipids, while during cell competition and AiP which occur in larval imaginal discs, pro-apoptotic genes hid and rpr are induced. One potential answer to this question is that the specificity of Toll-1 signaling may be modified by the interaction with Toll-9. Although this interaction occurs at the plasma membrane, it also might influence the activity in the nucleus. It will also be interesting to examine if Toll-1 can interact with some or all of the other Toll-related receptors in Drosophila and how this interaction might influence the specificity of the transcriptional outcome. Although Toll-9 in Drosophila does not appear to be required for innate immunity, on the basis of its non-essential function to induce AMP production during bacterial infection (Narbonne-Reveau, 2011), the currenrt work and work by others (Meyer, 2014) reveals that Toll-9 may have a function during stress responses which involves expression of pro-apoptotic genes such as rpr and hid. That was demonstrated previously for cell competition and now for undead AiP, indicating potential similarities between cell competition and undead AiP. At first, such similarities appear to be at odds with the common dogmas that proliferating winner cells trigger apoptosis of loser cells, while during AiP, apoptotic cells induce proliferation of surviving cells. However, it has also been reported that loser cells have a much more active role during cell competition and can promote the winner status of cells with increased fitness. Therefore, there appear to be significant similarities between cell competition and undead AiP. The common denominator for both systems is the expression of pro-apoptotic genes. These responses have different outcomes in both systems. During cell competition, this response involves the death of the loser cells. During undead AiP, it sets up the amplification loop known to operate in undead cells, which propels hyperplasia and tissue overgrowth (Shields, 2022).

This work was performed largely under undead conditions (i.e., in the presence of the effector caspase inhibitor p35, which is not an endogenous gene in Drosophila). In reality, however, in the absence of p35, effector caspases are also activated in apoptotic cells, which will eventually lead to the death of the cell. Therefore, the question arises as to how apoptosis and AiP are linked to allow compensatory proliferation under normal conditions. Recently, evidence has been presented that certain apoptotic cells (dying enterocytes in the adult intestine) can adopt a transiently undead-like state that enables them to signal for AiP before they are dying and removed. The transiently undead-like state is achieved by transient localization of Dronc to the plasma membrane, which might serve as a non-apoptotic compartment of the cell. In that way, apoptotic cells, before they die, can trigger AiP in a p35-independent manner. Therefore, in future research, it will be important to examine if the Toll-9/Toll-1 interaction sets up a similar amplification loop in transiently undead enterocytes (Shields, 2022).

Earlier Summaries

Toll is a transmembrane receptor and a member of the 12 gene dorsal group responsible for dorsoventral polarity in the fly. The ligand for Toll is Spätzle, and immediate targets include Pelle, Tube, Dorsal and Cactus. These proteins are held in a state of readiness while in the unfertilized egg. They are primed to carry out the transition from egg to zygote after fertilization occurs. Spätzle is available only in the ventral portion of the egg in the extracellular perivitelline space; when subjected to proteolysis, Spätzle becomes an active ligand for Toll.

Toll signals are first picked up by Tube, a protein in contact with the cell membrane. The signal is transduced to Pelle and subsequently to Cactus, which until its destruction holds Dorsal in the cytoplasm. With Cactus's bond destroyed, Dorsal enters the nucleus where it can serve as activator and repressor of genes involved in dorso-ventral polarity.

Toll is required zygotically in the development of a number of tissues, but Spätzle has not been documented as the ligand in these circumstances. Toll's mammalian homolog is Interleukin-1 (IL-1) receptor, involved in the activation of the immune response. Similarly, the Toll-Cactus-Dorsal system in the fly also activates an immune response.

The extracellular region of the Toll protein does not bear any similarity to the extracellular ligand-binding portion of the IL-1 receptor. Instead, it carries leucine rich repeats (LRR) that are found in molecules as diverse as proteoglycans, adhesion molecules, enzymes, tyrosine kinase receptors and G-protein coupled receptors. LRRs in the fly are found in Toll, Chaoptin and Connectin adhesion molecules and the Slit secreted protein. All these have roles in cell differentiation, morphogenetic events and the migration of cells and axons (Ollendorff, 1994).

The localized activation of Toll was first suggested based on the results of injection of wild-type cytoplasm into mutant flies. Wherever the Toll rescuing activity occurs defines the region from which ventral structures arise. Rescuing activity is not localized ventrally, but distributed uniformly in the wild-type embryo. This implies that the Toll gene product is normally present throughout the embryo but its activity is somehow restricted to ventral regions (Anderson, 1985 and Hashimoto, 1991). It is now understood that the ventral stimulation of Toll is caused by Spätzle, activated only on the ventral side of the egg (Morisato, 1994 and Roth, 1994).

Toll is dynamically expressed later in development by the embryonic musculature. Growth cones of RP3 and other motoneurons normally grow past muscle cells expressing Toll on their surface and innervate more distal muscle cells (muscles 6 and 7), which have down-regulated their Toll expression. The RP3 growth cone likely responds positively to Fasciclin III, an Ig-like cell adhesion molecule expressed on the target muscle cells, but still manages to avoid targeting errors in embryos lacking Fas III. Toll protein preferentially accumulates at muscle-muscle contact sites or "clefts," (the apposition between muscle cells). Later, the Toll-positive ventral muscle cells gradually lose Toll. Late-arriving growth cones innervate the clefts just as Toll expression becomes undetectable (Rose, 1997).

Reciprocal genetic manipulations of Toll proteins can produce reciprocal RP3 phenotypes. In Toll null mutants, the RP3 growth cone sometimes innervates the wrong muscle cells, including those that are normally Toll-positive. In contrast, heterochronic misexpression of Toll in the musculature leads to the same growth cone reaching its correct target region but delaying synaptic initiation. It is proposed that Toll acts locally to inhibit synaptogenesis of specific motoneuron growth cones and that both temporal and spatial control of Toll expression is crucial for its role in development (Rose, 1997).

The LRR (leucine-rich repeats) motif is shared by a number of other Drosophila cell surface molecules: Connectin, Chaoptin, 18-wheeler, Kekkon and Toll-like, as well as mammalian neural receptors, such as NLRR-1 and GARP. Structural analyses indicate that the LRR motifs could mediate protein-lipid as well as homophilic and heterophilic protein-protein interactions. The structurally related Connectin protein, when ectopically expressed in some of the ventral muscle cells, can function as a repulsive signal to motoneuron growth cone. All this evidence suggests a pivotal role for LRR proteins in axon guidance (Rose, 1997 and references).

GENE STRUCTURE

Bases in 5' UTR - 574

Bases in 3' UTR - 1257

PROTEIN STRUCTURE

Amino Acids - 1097

Structural Domains

A 5.3 kb poly(A)+ ovarian transcript of Toll was purified by hybrid selection with cloned DNA. The sequence of cDNAs suggests that the Toll protein is an integral membrane protein with a cytoplasmic domain and a large extracytoplasmic domain. The putative extracytoplasmic domain contains two blocks (for a total of 15 repeats) of a 24 amino acid, leucine-rich sequence found in both human and yeast membrane proteins. The transmembrane domain is between residues 804 and 828. There are 17 potential glycosylation sites and 17 cysteine residues in 3 clusters (Hashimoto, 1988).

The Toll protein has sequences held in common with the human membrane receptor platelet glycoprotein 1b (Gp1b). These sequences in Toll form disulphide linked extracellular domains that are important for the binding of ligands in the perivitelline space of the embryo. Expression of Toll protein induced in a non-adhesive cell line promotes cellular adhesion, a property held in common with the related Drosophila glycoprotein Chaoptin. Toll protein in such aggregates accumulates at sites of cell-cell interaction, a characteristic displayed by other cellular adhesion molecules (Keith, 1990).

Unusual properties are found for a synthetic LRR peptide derived from the sequence of the Drosophila membrane receptor Toll. In neutral solution the peptide forms a gel revealed by electron microscopy to consist of extended filaments approximately 8 nm in thickness. As the gel forms, the circular dichroism spectrum of the peptide solution changes from one characteristic of random coil to one associated with beta-sheet structures. Molecular modelling suggests that the peptide forms an amphipathic structure with a predominantly apolar and charged surface. Based on these results, models for the gross structure of the peptides filaments and a possible molecular mechanism for cellular adhesion are proposed. The finding that Toll-LRR forms intramolecular ß-sheet structures supports the view that LRRs can participate in protein-protein interactions and homotypic cellular adhesion. It could be that LRRs expressed on the cell surface are initially of disordered structure and that interactions with similarly disordered LRRs on an adjacent cell causes the formation of an extended and stable intermolecular ß structure. Such a mechansim could provide a molecular basis for cellular adhesion mediated by LRRs (Gay, 1991).

date revised: 2 February 2023  

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