BIOLOGICAL OVERVIEW

Mothers against dpp was discovered (Raftery, 1995) in a search for elements of the decapentaplegic signaling pathway in Drosophila (for review, see Raftery, 1999). When given the problem of identifying elements in a biochemical pathway, a geneticist will usually take a genetic approach, one that has worked in previous attempts in other systems. The tactic is to disclose enhancers which exacerbate known mutations. An enhancer is a mutation in the sought-for gene that engenders a more severe phenotype than that caused by the mutation of a single, already characterized gene. Thus a search was carried out for mutations that produce a more severe phenotype (an "enhancement") when combined with known dpp mutations. Mad mutations can be placed in an allelic series based on the relative severity of the maternal effect enhancement of weak dpp alleles, thus explaining the name Mothers against dpp.

Two types of experiments were carried out. The first was designed to reveal mutations in genes expressed in zygotes that exacerbate the phenotype of embryos that have a limiting amount of DPP. Mutations that act as enhancers of such DPP limited embryos cause embryonic lethality. The possibility that some of the gene products involved in DPP signaling might be supplied during oogenesis necessitated a second experiment looking for failure to recover progeny (that is, embryonic lethals) from mothers exhibiting a limiting amount of DPP. The two experiments yielded several kinds of enhancers: 1) new dpp alleles, which along with an already limited quantity of DPP cause embryonic lethality; 2) mutations in tolloid, a gene whose product is involved in DPP processing; 3) mutations in screw whose protein product is a ubiquitously expressed member of the TGF-beta family required for specification of dorsal cell fates in the Drosophila embryo; 4) mutations in Media, another gene involved in DPP signaling, and 5) mutations in Mad. Mutations in Mad, when interacting with limiting DPP levels, produce a defective amnioserosa (see also [Image]), the extra-embryonic membrane comprising the dorsal-most cells in early embryos (Raftery, 1995).

Mad mutations are also enhancers of mutant dpp appendage phenotypes. Thus Mad mutants produce a further reduction in wing blade size, a slight reduction in the eye, and loss of tarsal claws. These Mad mutant phenotypes exhibit a close correspondence to dpp mutant phenotypes. Homozygous Mad mutant larvae also show midgut defects and a greatly reduced gastric caecae. DPP signaling from visceral mesoderm to midgut endoderm is required for proper extension of the gastric caecae in parasegment 4 and for the induction of the homeotic gene labial in the adjacent endoderm of parasegment 7. Homozygous Mad mutant embryos lack labial expression and have defects in midgut constriction engendered by labial expression. Other imaginal disc derived structural defects are evident in homozygous Mad mutants, including heldout wings, split notum, loss of distal leg segments, duplications of the third antennal segment and defects in female genitalia (Sekelsky, 1995).

To date there is no indication that the Drosophila MAD protein is nuclear: antibody staining experiments indicate a cytoplasmic localization. Neverless there is clear indication that a human MAD homolog enters the nucleus upon BMP2 signaling (Hoodless, 1996).

A simple experiment was carried out to see if MAD acts upstream or downstream of DPP. DPP was ubiquitously expressed in Mad mutants. If Mad acts upstream of DPP, then ubiquitous expression of DPP should result in labial induction in the midgut endoderm independently of Mad. In fact labial does not get induced in Mad mutants even when dpp is expressed ubiquitously, indicating that MAD acts downstream of dpp. When MAD is artifically expressed in mesoderm, it fails to rescue labial induction in embryos otherwise deficient in MAD, but artificial expression of MAD in endoderm does rescue labial induction (Newfeld, 1996). Thus MAD appears to be a component in DPP signaling acting downstream of dpp in cells that are the recipients of DPP signaling.

Daughters against dpp (Dad), whose transcription is induced by Dpp shares, weak homology with Drosophila Mad, a protein required for transduction of Dpp signals. Dad is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule, and in fact ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad. In contrast to Mad or the activated Dpp receptor, whose overexpression hyperactivates the Dpp signaling pathway, overexpression of Dad blocks Dpp activity. Dpp target gene optomotor blind is absent in Dad-overexpressing cells. Expression of Dad together with either Mad or the activated receptor rescues phenotypic defects induced by either protein alone. Dad can also antagonize the activity of a vertebrate homolog of Dpp, bone morphogenetic protein, as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. It is concluded that the pattern-organizing mechanism governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Dad. This feedback loop appears to be conserved in vertebrate development (Tsuneizumi, 1997).

Drosophila Medea encodes a homolog of Smad4. Smad4 is relatively divergent from other vertebrate Smads and does not appear to be regulated by signal-dependent phosphorylation. However, overexpression of vertebrate Smad4 stimulates TGF-beta and activin responses. Smad4 associates with Smad1 (the mammalian homolog of Mad) in response to BMP2/4 or with Smad2 in response to TGF-beta, and dominant negative Smad4 blocks both BMP and activin responses. These observations have generated a model in which Smad4 is essential for signal transduction by all TGF-beta family members through its interaction with phosphorylated receptor-regulated Smads. Medea functions downstream of Dpp; complete removal of the Medea gene product causes the same embryonic phenotype as dpp null mutations. Mad undergoes signal-dependent translocation to the nucleus in the absence of Medea; in contrast, Medea is localized in the cytoplasm and requires Mad in order to accumulate in the nucleus. Specific mutations identified in strong alleles of Medea disrupt either Medea interaction with Mad or nuclear translocation of the Mad/Medea complex. Thus, interaction with Mad and nuclear import are critical for Medea function. However, unlike Mad, Medea is not required for expression of all Dpp-dependent genes and in its absence intracellular Dpp signaling rapidly attenuates with distance from the Dpp source. It is propose that the presence of Medea in heteromeric nuclear complexes with Mad modifies or enhances Dpp signaling (Wisotzkey, 1998).

Collaboration between Smads and a Hox protein in target gene repression

Hox proteins control the differentiation of serially iterated structures in arthropods and chordates by differentially regulating many target genes. It is yet unclear to what extent Hox target gene selection is dependent upon other regulatory factors and how these interactions might affect target gene activation or repression. Two Smad proteins, effectors of the Drosophila Dpp/TGF-ß pathway, that are genetically required for the activation of the spalt (sal) gene in the wing, collaborate with the Hox protein Ultrabithorax (Ubx) to directly repress sal in the haltere. The repression of sal is integrated by a cis-regulatory element (CRE) through a remarkably conserved set of Smad binding sites flanked by Ubx binding sites. If the Ubx binding sites are relocated at a distance from the Smad binding sites, the proteins no longer collaborate to repress gene expression. These results support an emerging view of Hox proteins acting in collaboration with a much more diverse set of transcription factors than has generally been appreciated (Walsh, 2007).

The activation of sal in the wing and its repression in the haltere are regulated by a 1.1 kb CRE, sal1.1 (Galant, 2002). Previous studies have shown that sal1.1 is directly repressed by Ubx in the haltere (Galant, 2002). In order to test whether Mad/Med binds to and directly represses the activity of the sal1.1 CRE in the haltere, candidate Mad/Med binding sites were sought in the sal1.1 CRE. One candidate Mad/Med binding site, M1 (5'-AGACGGGCAC-3'), was identified that lies between Ubx binding sites 5 and 6 in sal1.1, using binding site prediction and electrophoretic mobility shift assays (EMSAs). The sequence of M1 deviates somewhat from published Mad/Med silencer consensus binding sites (5'-AGAC-5 bp-GNCGYC-3') (Gao, 2005; Pyrowolakis, 2004), and Mad and Med bound with >10-fold and >25-fold lower affinities, respectively, to the M1 site than to the bam (Gao, 2005) and brk (Pyrowolakis, 2004) silencer elements (Walsh, 2007).

In order to test whether Mad/Med bound specifically to the M1 site, a series of point mutations were introduced within the M1 site, and their effect on protein binding was examined in vitro. Of four point mutations to the M1 site, the single mutation at position 808 reduced the binding of a Med fusion protein (GST-MedMH1) to M1 as compared with the wild-type sequence. The remaining three point mutations did not affect the affinity of GST-MedMH1 for the probe. These results suggest that Med might contact the sequence 5'-AGAC-3' in sal1.1. By contrast, the four individual point mutations each decreased, but did not abolish, binding of a Mad fusion protein (GST-MadN) in vitro, with the point mutation at bp 814 having the strongest effect. The weaker effect of the individual point mutations in M1 on Mad binding affinity in vitro is likely to be due to the affinity of MadN for both 5'-AGAC-3' Smad sites and GC-rich sequence. Combining these four mutations (sal798-824 kM1) had the greatest effect on GST-MadN binding to the probe. This analysis of individual point mutations indicates a putative orientation for a Mad/Med compound-binding site in the sal1.1 CRE (Walsh, 2007).

Most importantly, in transgenic flies, each point mutation of M1 introduced into an otherwise wild-type sal1.1 reporter construct caused derepression of the reporter gene lacZ in the haltere imaginal disc. The strength of derepression correlates with the decreased affinity of Mad for its binding site with the pm814 mutation, the strongest point mutation in vitro, showing the strongest level of derepression in vivo. Full derepression was observed when all four point mutations were combined into a sal1.1 reporter construct. No effect of mutations in M1 were observed on sal1.1-driven reporter gene expression in the wing as compared with the wild-type sal1.1 element or with endogenous sal expression, indicating that this site is not required for gene activation in the wing or haltere disc. Together, the biochemical, reporter gene and genetic evidence indicate that Mad/Med/Shn are directly required for sal repression in the haltere imaginal disc (Walsh, 2007).

This study demonstrates that Mad/Med and Ubx bind to adjacent sites in the sal1.1 CRE and that each protein is required for the direct repression of sal expression in the haltere. Furthermore, the sequence and spacing of Ubx and Smad binding sites are highly conserved and their proximity is required for target gene repression in the haltere. Because no evidence was found that these proteins interact directly, it is suggested this is an example of 'collaboration' or target gene co-regulation without direct cooperative interaction. These results have general implications for understanding how Hox proteins regulate diverse sets of target genes in animal development (Walsh, 2007).

The direct role for Smads in the repression of sal in the haltere is surprising in the light of previous genetic and molecular studies that had indicated that the Dpp pathway and Mad/Med were involved in sal activation in the wing. No direct evidence was found that this is the case and the fact that sal is activated in Mad and Med clones in the haltere indicates that sal is activated independently of Mad/Med in the flight appendages. The requirement for Mad/Med/Shn in shaping the pattern of sal expression in the wing appears to be indirect -- the protein complex represses the expression of brk, a repressor of sal, in cells in the central region of the developing wing and thereby permits sal expression (Walsh, 2007).

The Mad-Med-Shn complex is also active within cells in the central region of the haltere as a consequence of Dpp signaling. However, whereas sal is expressed and the sal1.1CRE is active in the wing, sal and the sal1.1 CRE are repressed in the haltere. These observations raise the question of how the Mad-Med-Shn complex selectively represses sal in the haltere but not in the wing disc? The results suggest that there are two key determinants in the selective repression of sal in the haltere. The first is collaboration with Ubx, which is expressed in the haltere and not in the wing disc. The second key determinant might be the affinity of Mad/Med binding to the sal CRE (Walsh, 2007).

The different responses of the brk and sal genes to Mad/Med/Shn suggests how the different affinities of proteins for binding sites might determine how available transcriptional regulatory inputs are integrated by CREs. Mad/Med binding to the brk CRE is of high affinity (Pyrowolakis, 2004) and apparently sufficient to impart repression, whereas that to the sal CRE is of much lower affinity and insufficient to impart repression in the wing. In the haltere, although Mad-Med-Shn or Ubx binding are alone insufficient, they act together either via simultaneous or sequential occupancy of their binding sites to repress sal (Walsh, 2007).

The requirement for two or more regulators to act together to control gene expression, i.e. combinatorial regulation, is fundamental to the generation of the great diversity of gene expression patterns by a finite set of transcription factors. Several previous studies have revealed the dual requirement for Hox and Smad functions for the activation of a target gene. Studies have suggested a general combinatorial mechanism for gene activation in which apparently separate transcriptional inputs act synergistically in gene activation and, in at least one case, the Hox response element and Dpp response element are separable. In this study, however, a requirement was observed for strict evolutionary conservation of the close topology of Hox and Smad binding sites in the sal CRE. It is suggested that collaboration is a distinct mode of combinatorial regulation in which two or more regulatory proteins must bind to nearby sites, but not necessarily to each other (Walsh, 2007).

The integration of Hox and Smad inputs could work through a number of possible mechanisms in the absence of direct physical interaction. One appealing possibility that might explain the requirement for the close proximity of binding sites is that Ubx and Mad-Med-Shn might interact with, and could therefore cooperatively recruit, the same co-repressor(s) for the repression of sal. Alternatively, if Mad-Med-Shn and Ubx bind sequentially to sal1.1, they might recruit different co-repressors and thereby orchestrate the assembly of a co-repressor complex. A third possibility is that because the Ubx and Mad/Med sites are embedded within a larger block of conserved regulatory DNA sequence in the sal1.1 CRE, the binding of other interacting transcription factors might also be involved in the repression of sal by Ubx and Mad-Med-Shn (Walsh, 2007).

These and recent results raise the question of whether collaboration is a general feature of target gene selection by Hox proteins. It is suggested that collaboration might be a widespread requirement for Hox function in vivo. This proposal is prompted by three observations: (1) Hox proteins alone have low DNA-binding specificity; (2) some, and perhaps all, Hox proteins might act as both repressors and activators; (3) Hox proteins regulate a great diversity of target genes that are also regulated by other transcription factors. In order to be such versatile regulators, it would be too great a constraint to require that Hox proteins always interact cooperatively with the diverse repertoire of transcription factors with which they act. Indeed, it may be argued that too much weight has been ascribed to the cooperative binding of Hox proteins and co-factors to DNA (Walsh, 2007).

Previously, much attention has focused on Exd and Hth, which interact with Hox proteins and bind cooperatively to DNA, thereby increasing Hox DNA-binding selectivity. However, it was only recently shown that the binding of these complexes alone was not sufficient to regulate target gene expression. Rather, Hox-Exd-Hth collaborate with and require the segmentation proteins Slp and En to repress the target gene Dll. This study has shown that the Exd- and Hth-independent target gene repression of sal requires collaboration between Ubx and Mad-Med-Shn. Although still a tiny sample of target genes, cases of transcription factors of various structural types acting as collaborators with Hox proteins are now available. The picture of Hox proteins relying on dedicated interacting co-factors such as Exd and Hth is expanding to a larger pool of collaborating transcription factors that modulate target gene selection (Walsh, 2007).

Indeed, collaboration might be the key to another unresolved mystery of the Hox proteins - the regulation of Hox protein activity. Some Hox proteins appear to act in both gene activation and repression; this is certainly the case for Ubx. This versatility would appear to be crucial to their role as sculptors of major features of body patterns, but how does the same transcription factor act positively in some contexts but negatively in others? There is evidence to suggest that the identity of the collaborating proteins and/or CRE sequences determines the 'sign' of Hox action (Walsh, 2007).

For instance, there is no evidence that the mere binding of Hox-Exd-Hth to a site determines the sign of Hox activity. These co-factors are involved in both Hox target gene activation (e.g., dpp in the midgut) and target repression (e.g.,Dll in the embryonic abdomen). But, in the latter case, En and Slp, two proteins that each harbor motifs for interaction with the co-repressor Groucho, are required collaborators for Dll repression. The roles of En and Slp in this instance might not be so much a matter of facilitating Hox target selection, but rather in regulating the sign of the output of the collaboration (Walsh, 2007).

Similar to the Hox proteins, the Smads can either activate or repress target genes. Furthermore, it has been demonstrated that the topology of Smad binding sites on DNA appears to be critical for determining whether a target gene is activated or repressed. In Drosophila, the topology of Mad and Med binding sites is critical for the recruitment of the co-repressor Shn. The recruitment of Shn was shown here to be necessary for sal repression. These two examples suggest that the positive or negative regulatory activity of a Hox protein depends on the context of surrounding binding sites and how they influence the activity of collaborating factors (Walsh, 2007).

The dependence of Hox proteins upon co-factors and collaborators indicates that, at the molecular level, Hox proteins are not 'master' regulatory proteins that dictate how target genes behave. Rather, they exert their great influence by virtue of their simple binding specificity, broad domains of expression and versatile, collaborative properties (Walsh, 2007).

Nemo kinase interacts with Mad to coordinate synaptic growth at the Drosophila neuromuscular junction

Bone morphogenic protein (BMP) signaling is essential for the coordinated assembly of the synapse, but little is known about how BMP signaling is modulated in neurons. This study shows that the Nemo (Nmo) kinase modulates BMP signaling in motor neurons. nmo mutants show synaptic structural defects at the Drosophila melanogaster larval neuromuscular junction, and providing Nmo in motor neurons rescues these defects. Nmo and the BMP transcription factor Mad can be coimmunoprecipitated, and a genetic interaction was found between nmo and Mad mutants. Moreover, this study demonstrated that Nmo is required for normal distribution and accumulation of phosphorylated Mad in motor neurons. Finally, the results indicate that Nmo phosphorylation of Mad at its N terminus, distinct from the BMP phosphorylation site, is required for normal function of Mad. Based on these findings, a model is proposed in which phosphorylation of Mad by Nmo ensures normal accumulation and distribution of Mad and thereby fine tunes BMP signaling in motor neurons (Merino, 2009).

These findings point to a model in which Nmo phosphorylation of Mad promotes its accumulation in the nuclei of motor neurons and thereby ensures effective BMP signaling at the NMJ. nmo mutant larvae show a significant aberration in the accumulation and/or distribution of p-Mad in motor neurons, with elevated levels of p-Mad at the NMJ and decreased levels of p-Mad in the nuclei of motor neurons. In addition, when Mad is mutated at its phosphorylation site for Nmo (MadS25A), it shows an expression pattern that qualitatively resembles that of p-Mad in nmo mutants, with more accumulation at the NMJ and less accumulation in the nucleus compared with wild-type Mad. Consistent with the importance of this phosphorylation, MadS25A fails to rescue synaptic structural defects in Mad mutants effectively. These observations suggest that phosphorylation of Mad by Nmo most likely modulates Mad's function by regulating its distribution and accumulation in motor neurons. Based on these findings, it is tempting to conclude that the reduction in the number of NMJ synaptic boutons in nmo mutants is, to a large extent, caused by the failure of p-Mad to signal to the nucleus effectively. In support of this, a strong trans-heterozygous interaction was observed between nmo and Mad; synaptic defects in nmo mutants can be partially rescued by overexpression of a constitutively active form of Tkv (Tkv-act). Although these findings provide strong support for this model, it cannot be ruled that Nmo is involved in other processes that contribute to the growth of synaptic boutons at the NMJ (Merino, 2009).

In contrast to its critical role in the regulation of synaptic structure, Nmo does not play an important role in the regulation of synaptic function; in the absence of nmo, quantal content remains at normal levels. Consistently, it was found that the MadS25A transgene is capable of rescuing the severe electrophysiological defects of Mad mutants as efficiently as a wild-type Mad transgene. Previously, it has been suggested that structural growth and the homeostasis of neurotransmitter release at the NMJ have different requirements for BMP signaling. Similarly, the current findings highlight the differential requirements for the regulation of synaptic structure and synaptic strength via BMP signaling. Interestingly, although overexpression of Nmo in motor neurons does not influence synaptic structural growth, it does cause a significant reduction in neurotransmitter release. This observation is consistent with those made by Zeng (2007), showing an antagonistic effect of Nmo gain-of-function on Mad in the wing imaginal discs. Nevertheless, although this observation shows a potential for Nmo to act as a negative regulator of Mad, the findings argue against a significant negative regulatory role for Nmo in motor neurons under normal physiological conditions (Merino, 2009).

It is proposed that Nmo exerts its action primarily by modulating Mad's retrograde movement from the NMJ to the nucleus. Nmo has been implicated in the regulation of Mad nuclear export in heterologous cells (Zeng, 2007); however, the current study found no evidence for changes in Mad nuclear export as a consequence of loss of nmo. In contrast, Nmo was found to be required for accumulation of p-Mad in the nuclei of motor neurons. In the absence of nmo, p-Mad levels increase at the NMJ and decrease in the nuclei of motor neurons, suggesting that Nmo is required for normal translocation/trafficking of Mad from the NMJ to the nucleus (Merino, 2009).

Finally, consistent with previous findings (Zeng, 2007), overexpression of Nmo can reduce the proportion of Mad concentration in nuclei versus that in cell bodies of motor neurons. Based on the phenotypic consequences of nmo loss- and gain-of-function, it appears that normal growth of synaptic structures at the NMJ depends on continuous and efficient BMP signaling from the NMJ to the nuclei of motor neurons and is less sensitive to the residence time of Mad in the nucleus. However, it appears that regulation of neurotransmitter release is more sensitive to the residence time of Mad in the nucleus and less dependent on the continuous retrograde signaling from the NMJ. These findings highlight the importance of Nmo phosphorylation of Mad at serine 25 in this process; however, a comprehensive understanding of the regulation of Mad trafficking and movement dynamics in different cellular compartments will require future studies (Merino, 2009).

Finally, an intriguing possibility would be the involvement of the Wg pathway in the regulation of Mad dynamics through Nmo. Nmo has been implicated in the Wg pathway during wing development and has been shown to be a transcriptional target of Wg. As Wg has been shown to participate in the regulation of synaptic growth at the NMJ, it would be tempting to envisage a role for Wg in the regulation of Nmo transcription in motor neurons and thus a link between the Wg and BMP pathways in the regulation of synaptic growth and function at the NMJ (Merino, 2009).

Subtle changes in motif positioning cause tissue-specific effects on robustness of an enhancer's activity

Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, the ability to relate information on motif occupancy to function from sequence alone is limited. This study engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, it was possible to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood (Erceg, 2014).

While quantifying the activity of a simple 'two-TF motif' CRM (pMad-Tin), the results show that enhancer activity can exhibit very different sensitivity to motif organization in one tissue compared to another. Several mechanisms could account for this interesting effect, including different concentrations of the TF (i.e. pMad or Tin) in the different tissues, the availability of tissue-specific co-factors, or tissue-specific priming of the enhancer, which may increase the ease by which the enhancer is activated (Erceg, 2014).

An elegant dissection of the endogenous sparkling enhancer has demonstrated that completely rearranging the relative order and spacing of TF binding sites could switch its cell type-specific activity from cone cells to photoreceptors in the eye. In comparison, the changes in motif organisation introduced in the current study were much more subtle such that the relative order of motifs was completely preserved. Yet only changing the spacing or orientation of motifs altered the robustness of enhancer activity in a tissue-specific manner. This result indicates that small insertions or deletions in CRMs, that do not affect the TF motifs themselves, could still have significant effects on gene expression in one tissue while having no effect in another. A study examining the activity of neuroectoderm enhancers between Drosophila species supports this model, where reduced spacing between Dorsal and Twist sites results in broader neuroectodermal stripes of CRM activity, while increased motif spacing resulted in progressively narrower stripes. Studies of both endogenous enhancers and the synthetic CRMs described in this study provide compelling evidence that the exact positioning of motifs within CRMs is crucial for the robustness of their activity in one tissue, while it may be largely dispensable in another. Different cell types can therefore interpret the same motif content of a given enhancer in different manners (Erceg, 2014).

The Drosophila heart is composed of two cell types, cardioblasts and pericardial cells, each of which requires the integration of many regulatory proteins for proper specification and diversification. A characterized pericardial enhancer, eve MHE, for example, contains pMad and Tin binding sites in addition to sites for dTCF, Twi, Ets proteins, and Zfh1. Given this complexity, it was surprising that a simple element built from pMad and Tin sites alone was sufficient to drive expression in the heart, albeit at a later developmental stage. This analyses indicate that this activity is due to cooperativity binding between Tin and pMad, facilitated by a very specific motif arrangement. Using crystal structure data from close homologues of pMad, the two TFs interaction on DNA were modelled, using a similar range of motif spacing. This 3D structural model indicates that it is possible for the DNA binding domains of these two proteins to both bind to DNA at a 2 bp spacing and to physically interact at a 2 bp and 4 bp spacing, but not at 6 bp spacing. Although done by homologue mapping, this structural data is consistent with the functional analyses of CRM activity, and further supports direct DNA binding cooperativity between these two TFs (Erceg, 2014).

It is interesting to note, that although pMad and Tin sites are sufficient to drive expression in the heart from stage 13 to 14 (when placed in a limited motif arrangement), nature appears to use other enhancer configurations to regulate this critical function. There are two important aspects to this finding. First, heart activity arising from CRMs containing pMad and Tin sites alone is not robust. The enhancers are on 'the edge' of activation, where subtle changes in motif positioning or enhancer location switch activity between embryos and within embryos. Second, endogenous enhancers that are bound only by pMad and Tin - with no known input from other factors - direct expression in the dorsal mesoderm and not in the heart, at stage 10. In the synthetic situation, pMad and Tin sites also drive robust expression in the dorsal mesoderm, in addition to variable weak expression in the heart. Therefore, although pMad and Tin sites alone are sufficient to drive heart activity in limited motif contexts, this mechanism is most likely not robust enough to be generally used to drive heart expression in vivo. This is consistent with recent studies showing that heart enhancer activity is elicited by the collective action of many TFs, which can occupy enhancers with considerable flexibility in terms of their motif content and configuration. The pMad-Tin synthetic elements uncovered a very simple, although not very robust, alternative mechanism to regulate heart activity, and represent a nice example of how combinatorial regulation can lead to emergent expression profiles more than the simple sum of its parts (Erceg, 2014).

The expression of key developmental genes is generally buffered against variation in genetic backgrounds and environmental conditions. This may occur at many levels including RNA polymerase II pausing and the presence of partially redundant enhancers. However, robust expression may also be buffered by the motif content within an enhancer to ensure a stable regulatory function. CRMs, for example, often include additional binding sites to those that are minimal and necessary. In the context of the pMad-Tin synthetic CRMs, the motif organization can also act to ensure robust activity. The results demonstrate that even in situations where the composition of motifs and their relative arrangement are maintained, subtle changes in the spacing between the motifs could have dramatic effects on enhancer output. Interestingly, this effect seems to be very tissue-specific, with some tissues maintaining robust activity whilst others lost all enhancer activity (Erceg, 2014).

Taken together, the data presented in this study demonstrate that subtle alterations in motif organization can affect the ability of different tissues to 'read' an enhancer, which in turn may allow each tissue to fine-tune enhancer activity based on fluctuations in its molecular components (Erceg, 2014).

The Mediator CDK8-Cyclin C complex modulates Dpp signaling in Drosophila by stimulating Mad-dependent transcription

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. To identify upstream regulators or downstream effectors of CDK8, a dominant modifier genetic screen was performed in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. 26 genomic loci were identified whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led to identification of genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFβ, or Transforming Growth Factor-β) signaling pathway. It was observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFβ signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, this analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila (Li, 2020).

To study the function and regulation of CDK8 in vivo, a genetic system was developed that yields robust readouts for the CDK8-specific activities in developing Drosophila wings. These genetic tools provide a unique opportunity to perform a dominant modifier genetic screen, allowing identification multiple components of the Dpp/TGFβ signaling pathway that can genetically interact with the CDK8-CycC complex in vivo. Subsequent genetic and cellular analyses reveal that CDK8, CycC, and six additional subunits of the Mediator complex, as well as CDK9 and Yki are required for the Mad-dependent transcription in the wing discs. In addition, CDK8 can directly interact with the linker region of Mad. These results have extended the previous biochemical and molecular analyses on how different kinases and transcription cofactors modulate the Mad/Smad-activated gene expression in the nucleus. Further mapping of specific genes uncovered by other deficiency lines may also open up the new directions to advance understanding of the conserved function and regulation of CDK8 during development (Li, 2020).

The Mediator complex functions as a molecular bridge between gene-specific transcription factors and the RNA Pol II general transcription apparatus, and diverse transactivators have been shown to interact directly with distinct Mediator subunits. However, it is unclear whether all Mediator subunits are required by different transactivators to regulate gene expression, or whether Mediator complexes composed of fewer and different combinations of Mediator subunits exist in differentiated tissues or developmental stages. Gene-specific combinations of the Mediator subunits may be required in different transcription processes, as not all Mediator subunits are simultaneously required for all transactivation process. For instance, ELK1 target gene transcription requires Med23, but lacking Med23 does not functionally affect some other ETS transcription factors, such as Ets1 and Ets2 . Similarly, Med15 is required for the expression of Dpp target genes, but does not appear to affect the expression of EGFR (epidermal growth factor receptor) and Wg targets in Drosophila (Li, 2020).

It has been previously reported that the Med15 subunit is required for the Smad2/3-Smad4 dependent transcription, as its removal from the Mediator complex abolishes the expression of Smad-target genes and disrupts Smad2/3-regulated dorsal-ventral axis formation in Xenopus embryos. Further biochemical analyses showed that increased Med15 enhances, while its depletion decreases, the transcription of Smad2/3 target genes, and that the Med15 subunit can directly bind to the MH2 domain of Smad2 or Smad3. In Drosophila, loss or reduction of Med15 reduced the expression of Dpp targets, resulting in smaller wings and disrupted vein patterning (mainly L2). It was also observed that depletion of Med15 or CDK8 reduces the expression of a Mad-target gene. These observations support the idea that CDK8 and Med15 play a conserved and positive role in regulating Mad/Smad-activated gene expression (Li, 2020).

Aside from Med15 and CDK8, it remains unclear whether other Mediator subunits are also involved in Mad/Smad-dependent transcription. This study identified six additional Mediator subunits that are required for the Mad-dependent transcription, including CycC, Med12, Med13, Med23, Med24, and Med31. Interestingly, aside from Med23 and Med24 being specific to metazoans, counterparts of the other six subunits are not essential for cell viability in the budding yeast. The similar effects of the four CKM (CDK8 kinase module) subunits on Mad-activity suggest that they may function together to stimulate Mad-dependent transcription. It is noted that depletion of seven Mediator subunits, Med7, Med8, Med14, Med16, Med17, Med21, and Med22, severely disrupts the morphology of the wing discs, making it difficult to assay their effects on the transcriptional activity of Mad in vivo. Consistently, all corresponding subunits, except Med16, are critical for cell viability in the budding yeast. In contrast, reducing expression of the 15 remaining subunits of the Drosophila Mediator complex did not significantly alter the expression of a Mad-dependent reporter. Med1 and Med25 are loosely associated to the small Mediator complex in human cell lines. A caveat for these negative results is that depleting these subunits using the existing RNAi lines may not be sufficient to affect sal-lacZ expression, even though the majority of these transgenic RNAi lines can generate severe phenotypes in the eye, wing, or both. Further analyses are necessary to validate these negative data in the future. Taken together, the results indicate that not all Mediator subunits are required for the expression of the Mad-target genes that were tested in the developing wing discs (Li, 2020).

Interestingly, Yki/YAP, which can function as a transcriptional co-factor for Mad/Smad, was also reported to associate with several subunits of the Mediator complex to drive transcription. Specifically, Med12, Med14, Med23, and Med24 were identified from a YAP IP-mass spectrometry sample in HuCCT1 cells. Med23 was also reported to regulate Yki-dependent transcription of Diap1 in wing discs. In the current study, Yki, Med12, Med23, and Med24 were also required for Mad-dependent transcription of sal-lacZ. Although the exact molecular mechanisms of how Yki interacts with certain Mediator subunits remain unclear, it is plausible that Yki may further strengthen the binding between Mad and Med15 through interactions with other subunits such as Med12, Med23, and Med24 (Li, 2020).

Based on biochemical analyses of the Smad1 phosphomutants and cell biological analyses using cultured human epidermal keratinocytes (HaCaT cells), several kinases including CDK8, CDK9, and ERK2 were shown to phosphorylate serine residues (Ser, or S) within the linker region of pSmad1 at S186, S195, S206, and S214, or the equivalent sites in pSmad2/3/5. These modifications were proposed to regulate positively Smad1-dependent transcriptional activity. Of these sites, S206 and S214 are both conserved from Drosophila to humans. In addition, studies using Xenopus embryos and cultured L cells suggest that MAPKs may phosphorylate the linker region of Smad1 (including S214) and lead to its degradation. Nevertheless, analyses with Drosophila embryos and wing discs indicate that S212 (equivalent to human pSmad1 S214) is phosphorylated by CDK8, while S204 (unique in Drosophila) and S208 (equivalent to human pSmad1 S210) are phosphorylated by Sgg/GSK3. These studies suggest the following model in explaining how Smads activate the expression of their target genes and how this process is turned off: after Smads are phosphorylated at their C-termini and translocated into the nucleus, CDK8 and CDK9 (potentially also MAPKs) act as the priming kinases to further phosphorylate pSmads in the linker region at S206 and S214. This may facilitate the interaction between pSmads and transcriptional cofactors such as YAP, stimulating the expression of Smads target genes. Overexpression of Yki in Drosophila wing disc increases the expression of the vgQE-lacZ reporter, which validates the role of Yki/YAP in activating Mad/Smad1-dependent gene expression in vivo. Subsequently, pSmads are further phosphorylated by GSK3 within the linker region at T202 and S210, which may facilitate Smad1/5 binding to E3 ubiquitin ligases such as Smurf1 and Nedd4L, causing the degradation of Smads through the ubiquitin-proteasome pathway (Li, 2020).

Although this model is still rather speculative, it serves as a conceptual framework to explain how transactivation of Smads is coupled to its degradation, similar to other transcriptional activators. It is challenging to determine whether these kinases act redundantly or sequentially for different phosphorylation sites, the exact orders of these phosphorylation events, as well as their biological consequences in vivo. Moreover, it remains unexplored whether these regulatory mechanisms are conserved during evolution. The importance of these issues is highlighted by the critical role of TGFβ signaling in regulating the normal development of metazoans and the dysregulation of this pathway in a variety of human diseases such as cancers (Li, 2020).

The precise spatiotemporal activation of the Dpp signaling pathway in the wings discs is critical for proper formation of the stereotypical vein patterns in Drosophila. This model system provides an ideal opportunity to dissect the dynamic regulation of the Mad-activated gene expression in the nucleus. Indeed, depleting CDK8 in wing discs reduces expression of the Mad-dependent sal-lacZ reporter, suggesting that CDK8 positively regulates Mad-dependent transcription. This is consistent with the effects of CDK8 on Smad1/5-dependent transcription in mammals. Depleting CDK8 does not affect the phosphorylation of Mad at its C-terminus as revealed by pMad immunostaining, nor does it affect the physical interaction between CDK8 and the linker region of Mad, supporting the idea that CDK8 may only affect subsequent phosphorylation of Mad, presumably within the linker region (Li, 2020).

Besides CDK8-CycC, depleting CDK9-CycT also decreases the expression of the sal-lacZ reporter, supporting the notion that CDK8-CycC and CDK9-CycT may play non-redundant roles in further phosphorylating pMad in the nucleus. However, no effects of depletion of CDK7 or MAPKs on sal-lacZ expression were observed, suggesting that their role in regulating the transcriptional activity of Smads may not be conserved in Drosophila. Alternatively, the two MAPK/ERK homologs, Rolled and ERK2, may act redundantly in regulating Mad-dependent transcription. Lastly, depleting Sgg/GSK3 in the dorsal compartment of the wing disc increases the size of this compartment, yet the expression level of the sal-lacZ reporter is similar to the ventral compartment. These observations are consistent with previous reports that phosphorylations of Mad/Smad in the linker regions by CDK8-CycC and Sgg/GSK3 regulate the level and range of Mad-dependent gene expression (Li, 2020).

Together with the previous reports, the data support that CDK8-CycC and CDK9-CycT may phosphorylate pMad at the linker region, which may facilitate the binding between Yki and Mad. It is speculated that this interaction may synergize the recruitment of the Mediator complex, presumably at least through the interaction between its Med15 subunit and the MH2 domain of Mad (see Model of Mad/Smad-dependent transcription activation through the CKM and the Mediator complex.). Alternatively, Yki may also facilitate the recruitment of the whole Mediator complex through its interactions with Med12, Med23, and Med24. The synergistic interactions among Mad, Yki, the Mediator complex, and RNA Pol II may be required for the optimal transcriptional activation of the Mad-target genes (Li, 2020).

One of the challenges is to illustrate the dynamic interactions between these factors and diverse protein complexes that couple the transactivation effects of Mad/Smads on gene transcription with their subsequent degradation at the molecular level. Smad3 phosphorylation strongly correlates with Med15 levels in breast and lung cancer tissues; together, they potentiate metastasis of breast cancer cells. Thus, it will be important to test whether additional Mediator subunits that were identified in Drosophila play similar roles in mammalian cells. It will also be interesting to determine whether a partial Mediator complex, composed of a subset of the Mediator subunits, exists and regulates Mad/Smad-dependent gene expression. Furthermore, detailed biochemical analyses may yield mechanistic insights into how CDK8 and Med15 act in concert in stimulating the Mad/Smad-dependent gene expression (Li, 2020).

Wing pouch-specific alteration of CDK8 activity results in two major phenotypes: disrupted vein patterns and altered size of wing blades. While the effects on wing size and cell numbers can be explained by the role of CDK8 in regulating cell proliferation through E2F1, the effects of CDK8 on vein patterning are more complex. The stereotypical wing vein patterns in adult flies are gradually defined by elaborated spatiotemporal interplays among different signaling pathways, including Dpp, EGFR, Hedgehog (Hh), Notch (N), and Wingless (Wg), in the developing wing discs. During the larval and pupal stages, these signaling pathways and their downstream transcriptional targets coordinately control the cell proliferation and differentiation of cell in different parts of the wing disc to form individual veins (Li, 2020).

It is noteworthy that varying CDK8 activities has different effects on different veins: gain of CDK8 causes the loss of the L3 and L4 veins, but the vein patterns of L2 and L5 appear thicker and more diffusive; while the ectopic veins caused by reduction of CDK8 are mainly intertwined with the L2 and L5 veins. This analyses on the genetic interactions between CDK8 and the components of the Dpp signaling pathway led to the discovery of the role of the Mediator complex in Mad-stimulated transcription of sal. However, there is a gap in understanding ohow reduced expression of sal in wing discs is linked to the vein defects in adult wings. It is known that salm and salr (spalt-related), two members of the spalt gene family that encode zinc-finger transcriptional repressors, function downstream of the Dpp signaling pathway during development of the central part of the wing. Depletion of either salm or salr alone resulted in ectopic vein formation around L2 in adult wings, yet depletion or loss of both salm or salr caused loss of vein phenotype. In addition, elimination of L2 in ventral-anterior and ectopic L5 in dorsal-posterior were observed in salm/salr clones at different region of the wing. These observations suggest that the dosage of salm and salr in wing discs does not have a linear relationship with the wing vein patterning at the adult stage (Li, 2020).

Interestingly, it is known that the CKM complex regulates the transcriptional activities of the key transcription factors of these pathways, including N-ICD downstream of N signaling and Mad/Smad proteins. In addition, Med12 (Kohtalo, or Kto in Drosophila) and Med13 (Skuld, or Skd in Drosophila) subunits of the CKM interact with Pangolin (the lymphoid-enhancing factor (LEF)/T cell factor (TCF) homolog in Drosophila), the key transcription factor downstream of Wg signaling, through the transcriptional cofactors such as Pygopus, Legless, and Armadillo. In mammalian cells, Med12 is also known to regulate the activities of Gli proteins, the key transcription factors downstream of Hh signaling. Furthermore, the Mediator subunit Med23 interacts with ETS (E-twenty six transcription factor) proteins, a family of key transcription factors downstream of the EGFR signaling pathway. However, whether CDK8-CycC also regulates TCF-, ETS- or Gli-dependent transcription is still not understood. Nevertheless, these studies in other biological contexts suggest that the effects of CDK8 on wing vein patterning are not likely solely through the Dpp signaling pathway. Therefore, it is speculated that the potential interactions between CDK8 and the aforementioned signaling pathways may contribute to these differential effects on distinct veins. Further analyses of these cross-talks, as well as further mapping of other Df lines that modify the CDK8-specific vein phenotypes, may yield the insights into the molecular and dynamic mechanisms underlying these vein phenotypes (Li, 2020).

To understand how dysregulated CDK8-CycC contributes to a variety of human cancers, it is essential to elucidate the function and regulation of CDK8 in vivo. Given that the CDK8-CycC pair and other subunits of the Mediator complex are conserved in almost all eukaryotes, Drosophila serves as an ideal model system to identify both the upstream regulators and the downstream effectors of CDK8 activity in vivo. The dominant modifier genetic screen is based on the wing vein phenotypes caused by specific alteration of CDK8 activity in the developing wing disc, which serves as a unique in vivo readout for the CDK8-specific activities in metazoans. This screen led to the identification of 26 genomic regions that include loci whose haplo-insufficiency could consistently modify CDK8-CycC depletion or CDK8-overexpression phenotypes. Identification of Dad and genes encoding additional components of the Dpp signaling pathway provides a proof of principle for this approach. Since each of the chromosomal deficiencies uncovers multiple genes, further mapping of the relevant genome regions is expected to identify the specific genetic loci encoding factors that may function either upstream or downstream of CDK8 in vivo. It is hoped that further analyses of the underlying molecular mechanisms in both Drosophila and mammalian systems will advance understanding of how dysregulation of CDK8 contributes to human diseases, thereby aiding the development of therapeutic approaches (Li, 2020).

Mad dephosphorylation at the nuclear pore is essential for asymmetric stem cell division

Stem cells divide asymmetrically to generate a stem cell and a differentiating daughter cell. Yet, it remains poorly understood how a stem cell and a differentiating daughter cell can receive distinct levels of niche signal and thus acquire different cell fates (self-renewal versus differentiation), despite being adjacent to each other and thus seemingly exposed to similar levels of niche signaling. In the Drosophila ovary, germline stem cells (GSCs) are maintained by short range bone morphogenetic protein (BMP) signaling; the BMP ligands activate a receptor that phosphorylates the downstream molecule mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) accumulates in the GSC nucleus and activates the stem cell transcription program. This study demonstrates that pMad is highly concentrated in the nucleus of the GSC, while it quickly decreases in the nucleus of the differentiating daughter cell, the precystoblast (preCB), before the completion of cytokinesis. A known Mad phosphatase, Dullard (Dd), is required for the asymmetric partitioning of pMad. Mathematical modeling recapitulates the high sensitivity of the ratio of pMad levels to the Mad phosphatase activity and explains how the asymmetry arises in a shared cytoplasm. Together, these studies reveal a mechanism for breaking the symmetry of daughter cells during asymmetric stem cell division (Sardi, 2021).

The Drosophila female germline stem cell (GSC) is an excellent model to study niche-stem cell interaction because of its well-defined anatomy and abundant cellular markers. At the tip of each ovariole, two to three GSCs adhere to a cluster of niche cells, known as cap cells (CCs). A bone morphogenetic protein (BMP) ligand, Decapentaplegic (Dpp), is secreted by CCs and is an essential factor for GSC maintenance. Dpp binds to the serine-threonine kinase receptor Thickveins (Tkv) expressed on GSCs. Activated Tkv then phosphorylates Mothers against decapentaplegic (Mad) at its two C-terminal phosphorylation sites. Phosphorylated Mad (pMad) forms a heterodimer with Medea (Med), and the complex enters the nucleus and directly binds to the promoter of the differentiation factor bag of marbles (bam) to down-regulate its transcription. The down-regulation of bam is essential for GSC self-renewal (Sardi, 2021).

It has been hypothesized that the niche signaling rapidly decreases in one of the GSC daughters, precystoblast (preCB), as it is displaced away from the CC niche. Multiple studies have defined mechanisms for fine-tuning Dpp signal strength and range. However, it is still unclear how Mad, the immediate downstream molecule of Tkv, is initially regulated during GSC division (Sardi, 2021).

This study demonstrates that pMad rapidly reaches different levels in the dividing GSCs after mitosis but before the completion of cytokinesis. Its level in the nuclei of future GSCs remains high, while its level in the nuclei of preCBs decreases. Upon activation of the niche signal receptor Tkv kinase, its substrate, Mad, is phosphorylated near the plasma membrane and then travels throughout the cytoplasm and enters the nucleus. After mitosis, GSC takes several hours to complete cytokinesis, and the abscission occurs during DNA synthesis (S) phase. Since preCB shares its cytoplasm with GSC during this time, pMad can travel freely between the cytoplasm of two daughters. How can their nuclei have different levels of pMad (Sardi, 2021)?

This study shows that the previously identified Mad phosphatase Dullard (Dd) plays an essential role in the formation of the sharply different pMad levels in GSC and preCB. It was demonstrated that Dd interacts with Mad at the nuclear pores, where it may directly or indirectly dephosphorylate pMad. Dd itself does not exhibit asymmetric localization in GSC and preCB, but, as mathematical modeling indicates, the unbiased dephosphorylation by evenly distributed Dd combined with the phosphorylation of Mad biased toward the niche (due to local activation of Tkv on the niche side) is sufficient to explain the observed pMad asymmetry (Sardi, 2021).

In summary, these results provide a mechanism by which a self-renewal program is confined to the stem cells during asymmetric division (Sardi, 2021).

During asymmetric stem cell division, two daughter cells acquire different cell fates. The Drosophila ovarian niche differentially activates an GSC and a preCB because of the distinct position of these cells, the former adjacent to the niche and the latter displaced from the niche. What is the initial cause of the difference? While many factors have been identified that can amplify and/or ensure the already existent niche signal difference, it has been unknown how Mad, the immediate downstream molecule of the niche ligand, is initially regulated during GSC division (Sardi, 2021).

Mad is phosphorylated by active receptors at the side of GSC near the niche and then travels throughout the cytoplasm before entering the nucleus. The GSC-preCB pairs continue to share the cytoplasm for at least several hours after mitosis. Consistent with previous studies, this study observed that Mad diffuses throughout the cytoplasm of the GSC and preCB. This study found, however, that during this phase, the pMad levels in the nuclei of the GSC and preCB are already asymmetric (G1/S pMad asymmetry). It was determined that local activation of the kinase at the niche-GSC contact site is not sufficient for G1/S pMad asymmetry. Moreover, neither increasing the activity of kinase nor compromising the rate of Mad degradation affected G1/S pMad asymmetry. It was discovered that the Mad phosphatase, Dd, which dephosphorylates Mad at the nuclear pores in both GSC and preCB cells, is an essential factor in the formation of early asymmetric partitioning of pMad (Sardi, 2021).

To gain insight into the mechanisms responsible for the observed G1/S asymmetry, a mathematical model was formulated that included all major factors governing the spatiotemporal dynamics of pMad, and was constrained by the experimental data obtained in this study. The model suggests that a combination of the asymmetrically localized activated kinases, which reside at the site of contact between the GSC plasma membrane and the niche, together with sufficiently active phosphatase that is distributed symmetrically between nuclear envelopes of the GSC and preCB, is sufficient to explain the experimentally observed asymmetry of pMad levels in the GSC-preCB pairs (Sardi, 2021).

Analysis of the model revealed that for pMad asymmetry to occur, it is necessary that the positive and negative regulators of pMad (the kinases and phosphatases) be separated in space, as this brings about spatial gradients of pMad. However, this condition is not sufficient, as the gradients could be shallow. The modeling showed, furthermore, that the steepness of pMad gradients is exclusively determined by the interplay of two factors: pMad dephosphorylation that steepens the gradients and pMad diffusion in the cytoplasm that levels them out. Active regulation of the pMad diffusivity, which is determined by protein size and effective viscosity of the cytoplasm, is unlikely. This makes the phosphatase activity an essential factor determining pMad asymmetry. To a lesser degree, the asymmetry also depends on the permeability of nuclear pores to pMad export from the nucleus, since the dephosphorylation of pMad occurs inside the nucleus (Sardi, 2021).

In summary, this study identifies and explains a mechanism by which a stem cell can rapidly set up an initial asymmetry with respect to an extrinsic signal thus providing a conceptual framework for understanding the dynamics of niche-stem cell signaling (Sardi, 2021).

RNA-binding FMRP and Staufen sequentially regulate the coracle scaffold to control synaptic glutamate receptor and bouton development

Both mRNA-binding Fragile X Mental Retardation Protein (FMRP) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. This study tested whether these RNA-binding proteins (RBPs) act jointly in a common mechanism. Both dfmr1 and staufen mutants, and trans-heterozygous double mutants, were shown to display increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNAi, a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, this study showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and this study confirmed that Staufen binds to coracle mRNA. FMRP and Staufen were shown to act sequentially to co-regulate postsynaptic Coracle expression, and show Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, this study found dfmr1, staufen and coracle mutants elevate neurotransmission strength. FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development (Song, 2022).

This study reveals the mechanism of the established FMRP negative regulation of postsynaptic GluRIIA receptors and presynaptic bouton formation in the Drosophila FXS disease model. Specifically, the mRNA-binding FMRP-positive translational regulator binds to staufen mRNA as predicted, within the postsynaptic cell. Consequently, both dfmr1 and staufen mutants share the elevated GluRIIA level and bouton number phenotypes based on a common postsynaptic pathway function, and genetically interact as trans-heterozygotes to reproduce these phenotypes. Staufen acts as a dsRBP to bind coracle mRNA as predicted; both dfmr1 and staufen mutants exhibit elevated postsynaptic Coracle levels, and genetically interact as trans-heterozygotes to reproduce this phenotype. Coracle acts as a GluRIIA-binding anchoring scaffold within the postsynaptic domain to regulate local receptor accumulation (Chen, 2005). Consequently, dfmr1, staufen and coracle mutants all increase NMJ synaptic functional differentiation to elevate neurotransmission strength. Finally, the elevated postsynaptic GluRIIA levels mediate retrograde BMP receptor trans-synaptic signaling that induces pMad to drive new presynaptic bouton development. dfmr1, staufen and coracle mutants all exhibit elevated presynaptic pMad levels, thereby linking the postsynaptic GluRIIA accumulation and presynaptic supernumerary bouton formation defects shared by all of these mutants (Song, 2022).

The staufen mutant increased synaptic Coracle levels, GluRIIA levels and bouton number are all internally consistent. In a previous study, opposite phenotypes were measured in staufenHL/Df(2R)Pcl7B, which reduces another 14 genes in heterozygous deficiency, including loci involved in neuronal development (e.g. grh, nopo). Importantly, this study similarly found reduced synaptic protein levels and bouton number in staufenHL/Df(2R)Pcl7B, suggesting that heterozygosity of one or more of the neighboring genes impairs synaptic development. However, this study showed that a staufen RNAi that reduces transcript levels by ~90% replicates the staufen mutant NMJ phenotypes of increased GluRIIA levels and synaptic bouton numbers. This was also replicated with a second, independent staufen RNAi line. Moreover, this study showed that the effect is entirely restricted to postsynaptic muscle RNAi, with no effect from presynaptic neuron RNAi, consistent with restricted postsynaptic Staufen function. In addition, postsynaptic staufen rescue of the staufen mutant restored normal synaptic bouton formation, with OE reducing GluRIIA levels in staufen mutants and rescuing GluRIIA levels in dfmr1 mutants. Both staufen mutants and postsynaptic staufen RNAi also share the arrested supernumerary satellite bouton development characterizing dfmr1 null mutants. These many independent lines of evidence confirm the results, and are consistent with the known parallel FMRP role in restricting GluRIIA levels and synaptic bouton formation (Song, 2022).

To regulate Staufen, FMRP binds staufen mRNA and protects targeted staufen transcripts from degradation. FMRP contains at least three distinct RNA-binding domains (RBDs), and Staufen has five RBDs. Staufen reportedly binds a specific RNA hairpin structure formed by long 3' UTRs, but RIP shows that Staufen also binds mRNAs that are not predicted to generate this secondary structure. Although the decreased staufen mRNA levels in both dfmr1 mutants and muscle-targeted dfmr1 RNAi are predicted to be due to the lack of FMRP binding, it is also possible that other unregulated interactors cause the downregulated staufen mRNA expression (Shah et al., 2020). Localized labeling with an anti-Staufen antibody has been reported in the postsynaptic NMJ, which can be confirmed, but it was not possible to reduce labeling in staufen hypomorphic mutants. Therefore Staufen labeling was not shown in the current study. Moreover, western blots have been reported with the same anti-Staufen antibody; however, attempts were unsuccessful. Therefore qPCR was used to measure staufen mRNA levels. Staufen binds to coracle mRNA, but does so in a non-selective manner. This result is consistent with Staufen acting as a very broad spectrum dsRBP, and suggests that Staufen likely acts with a translational regulator partner to generate specificity. FMRP is very well established to partner with other RBPs to mediate the translational regulation of its target transcripts (Song, 2022). ------

The postsynaptic Coracle scaffold acts in a GluRIIA local anchoring mechanism, presumably to link the receptors to the underlying actin cytoskeleton (Chen, 2005). The jointly elevated Coracle and GluRIIA levels in both dfmr1 and staufen mutants are consistent with this scaffold function. Because the dfmr1/+; staufen/+ trans-heterozygotes share this correlated Coracle and GluRIIA upregulation in the postsynaptic domain, a single common signaling pathway is indicated. Coracle also restricts terminal branching development in peripheral sensory neurons. Both coracle mutants and sensory neuron-targeted coracle RNAi also display increased dendritic branch and termini numbers. These phenotypes are similar to the expanded NMJ terminals and increased synaptic bouton development reported in this study. Importantly, both coracle loss of function (mutants and muscle-targeted RNAi) and gain of function (muscle-targeted OE) increase postsynaptic GluRIIA levels and generate supernumerary boutons. Likewise, the knockdown and OE of many other similar scaffolds are known to cause phenocopying defects. Some examples include the muscle chaperone UNC-45, the tight junction scaffold zonula occludens-1 (ZO-1) and synaptic UNC-13. Indeed, both coracle loss and OE similarly cause increased dendritic crossing in Drosophila sensory neurons, similar to the phenocopy of developmental defects reported in this study. Combining the roles of postsynaptic FMRP-Staufen-Coracle in GluRIIA clustering, it was reasoned that this pathway must be a regulatory determinant of synaptic functional development (Song, 2022).

Removing FMRP, Staufen and Coracle strongly enhances functional synaptic differentiation and NMJ neurotransmission strength. This is consistent with expectations from the postsynaptic GluRIIA accumulation in all of these mutants. Elevated GluRIIA levels are well known to be associated with increased evoked functional responses and prolonged channel open times. A GluRIIA pore sequence (MQQ) critically required for the Drosophila channel Ca2+ permeability is conserved in mammalian receptors. This selectivity allows Ca2+-dependent participation in spontaneous (mEJC) and evoked (EJC) neurotransmission. Although enhanced evoked EJC amplitudes are typically accompanied by mEJC alterations, this study found that mEJC amplitude and frequency are unchanged in both the staufen and coracle mutants, and show only minimal changes in the dfmr1 mutants. Classically, both evoked and spontaneous neurotransmission were thought to be mediated by the same vesicles; however, more recent evidence has indicated that spontaneous and evoked neurotransmission have distinct machinery and vesicle pools. Postsynaptic receptors can be segregated into different compartments that are activated by either spontaneous or evoked release. This work supports this growing body of evidence for differential regulation. Importantly, GluRIIA has unique functions, modulating both presynaptic glutamate release and presynaptic bouton development (Song, 2022).

The dfmr1, staufen and coracle mutants all showed upregulated presynaptic pMad correlated with postsynaptic activated GluRIIA accumulation. GluRIIA activation triggers presynaptic pMad signaling via BMP receptors surrounding active zones, which, in turn, stabilizes GluRIIA receptors in the postsynaptic domains. This trans-synaptic signaling mechanism induces new presynaptic bouton development. The targeted postsynaptic RNAi for all three genes confirms this intercellular link. Synaptic BMP signaling involves both the type I serine/threonine kinase receptors and the type II receptor Wit. Although BMP ligand Glass bottom boat (Gbb) signaling via Wit presynaptic receptors is well established at the NMJ to modulate synaptogenesis, the mechanism of presynaptic bouton formation induced by activated GluRIIA signaling does not involve canonical BMP signaling via Gbb. In the dfmr1 mutants, it is suggested that postsynaptic GluRIIA accumulation induces presynaptic bouton development via non-canonical GluRIIA-Wit trans-synaptic retrograde signaling. Similarly, the muscle postsynaptic glypican Dally-like protein (Dlp) negatively regulates NMJ synaptic development by inhibiting this same non-canonical BMP pathway through decreased activated GluRIIA expression. Postsynaptic GluRIIA clustering can thus trigger presynaptic bouton formation, although supernumerary boutons do not always induce reciprocal GluRIIA changes. It is concluded that an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulates intertwined structural and functional glutamatergic synapse development (Song, 2022).

GENE STRUCTURE

Bases in 5' UTR -346

Bases in 3' UTR - 937

PROTEIN STRUCTURE

Amino Acids - 455

Structural Domains

MAD and its homolog in vertebrates (Smad1) are essential for signaling in DPP and BMP-2/4 pathways and can elicit biological responses characteristic of BMP-2/4 (Newfeld, 1996). SMAD proteins share a high degree of homology in their amino-terminal MH1 (Mad homology) and carboxy-terminal MH2 domains. The MH2 domain is considered the effector domain, whose activity is opposed by its physical interaction with the MH1 domain. SMAD4 is a central signaling molecule of several TGFbeta-related pathways. In contrast to the pathway-restricted SMADs, SMAD4 rapidly associates with both SMAD1 in response to BMPR-1 signaling and SMAD2 in response to TbetaR-I and ActR-IB signaling. Unlike SMAD4, SMAD1 and SMAD2 contain consensus phosphorylation sites for receptor type I Ser/Thr kinases within their MH2 domains. The model emerging from recent biochemical and crystallographic studies implies that phosphorylation of the receptor-regulated SMADs relieves them of the MH1 inhibitory effect, allowing their interaction with SMAD4 and subsequent translocation to the nucleus (Sirard, 1998 and references).

Signal transduction specificity in the transforming growth factor-beta (TGF-beta) system is determined by ligand activation of a receptor complex, which then recruits and phosphorylates a subset of SMAD proteins, including Smads 1 and 2. In vertebrates, Smad1, and presumably its close homologs Smad5 and Smad8, are phosphorylated by BMP receptors and mediate BMP responses. Smad2 and its close homolog Smad3 are phosphorylated by TGF-beta receptors and mediate TGF-beta and activin responses. In Drosophila, Mad (a close homolog of Smad1) mediates the effects of the BMP-like factor, Dpp. After phosphorylation by receptors, Smads 1 and 2 associate with Smad4 and move into the nucleus where they regulate transcription. A discrete surface structure has been identified in Smads 1 and 2 that mediates and specifies their receptor interactions. This structure is the L3 loop, a 17 amino acid region that protrudes from the core of the conserved SMAD C-terminal domain. The L3 loop sequence is invariant among TGF-beta and bone morphogenetic protein (BMP)-activated SMADS, but differs at two positions between these two groups. Swapping these two amino acids in Smads 1 and 2 induces a gain or loss, respectively, in their ability to associate with the TGF-beta receptor complex and causes a switch in the phosphorylation of Smads 1 and 2 by the BMP and TGF-beta receptors, respectively. A full switch in phosphorylation and activation of Smads 1 and 2 is obtained by swapping these two amino acids while, in addition, swapping four amino acids near the C-terminal receptor phosphorylation sites. These studies identify the L3 loop as a determinant of specific SMAD-receptor interactions, and indicate that the L3 loop, together with the C-terminal tail, specifies SMAD activation (Lo, 1998).

date revised: 2 January 2023

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