The first PTD transcripts can be detected during cellular blastoderm stage in two broad stripes in the lateral neurogenic region. As gastrulation [Image] proceeds, P1 transcription is progessively restricted to a row of ectodermal cells, at first three cells wide, then one cell wide. These cells belong to the tracheal anlagen. Additional P1 transcripts are detected in the head region.

From stage 12 through 14, pointed P1 expression is seen in longitudinal glia cells (Klambt, 1993). P2 transcripts are present in the precellular stage at the anterior tip of the embryo, but later, P2 transcripts are located mostly in the mesoderm. At stage 12, P2-driven pointed expression is shut off in the mesoderm and turned on at the midline of the CNS, corresponding to midline glia (Klambt, 1993).

Ectopically expressed pointed P1 forces additional CNS cells to enter the glial-differentiated pathway. Additional glial-like cells are often flanked by cells ectopically expressing neuronal antigen, that is, glial cells are able to induce neuronal antigens in neighboring cells (Klaes, 1994).

The transcription factors encoding genes tailless (tll), atonal (ato), sine oculis (so), eyeless (ey) and eyes absent (eya), and Efgr signaling play a role in establishing the Drosophila embryonic visual system. The embryonic visual system consists of the optic lobes primordium, which, during later larval life, develops into the prominent optic lobe neuropiles, and the larval photoreceptor (Bolwig's organ). Both structures derive from a neurectodermal placode in the embryonic head. Expression of tll is normally confined to the optic lobe primordium, whereas ato appears in a subset of Bolwig’s organ cells that are called Bolwig’s organ founders. Phenotypic analysis of tll loss- and gain-of-function mutant embryos using specific markers for Bolwig’s organ and the optic lobe, reveals that tll functions to drive cells to an optic lobe fate, as opposed to a Bolwig’s organ fate. Similar experiments indicate that ato has the opposite effect, namely driving cells to a Bolwig’s organ fate. Since tll and ato do not regulate one another, a model is proposed wherein tll expression restricts the ability of cells to respond to signaling arising from ato-expressing Bolwig’s organ pioneers. The data further suggest that the Bolwig’s organ founder cells produce Spitz (the Drosophila TGFalpha homolog) signal, which is passed to the neighboring secondary Bolwig’s organ cells where it activates the Epidermal growth factor receptor signaling cascade and maintains the fate of these secondary cells. The regulators of tll expression in the embryonic visual system remain elusive, no evidence for regulation by the 'early eye genes' so, eya and ey, or by Egfr signaling is found (Daniel, 1999).

Epidermal growth factor receptor is activated in midline regions of the head neurectoderm, in particular in the anlage of the visual system. Moreover, increased and/or ectopic activation of Egfr results in a 'cyclops' phenotype very similar to what has been described for ectopic tll expression. Egfr signaling has been shown to be required in both chordotonal organs and compound eye for the inductive signaling triggered by ato expression. Two questions raised by these observations have been investigated: (1) is Egfr signaling required for tll expression in the optic lobe and (2) is Egfr signaling involved in the recruitment of the secondary (non-atonal-expressing) Bolwig’s organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for the presence of Egfr-relevant mRNAs or proteins: Rhomboid mRNA, which would be expected to be present only in the signaling cells, and phosphorylated MAPK, Pointed and Argos mRNAs, which would be expected to be expressed in all cells receiving an Egfr-mediated signal. In stage 12 embryos, rho is expressed only in the small group of Bolwig’s organ founder cells (the same cells expressing ato). In contrast, activated (phosphorylated) MAPK is present in a larger cluster of cells including the entire Bolwig’s organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both known to be switched on in cells receiving the Spi signal, are expressed at the same stage throughout the entire Bolwig’s organ primordium. These gene expression and MAPK activation patterns are consistent with the idea that the Spi signal is activated by rho in the Bolwig’s organ founders and passed to the neighboring secondary Bolwig’s organ cells where it activates the Egfr signaling cascade. Supporting this view, only 3-4 photoreceptor neurons are found in the Bolwig’s organ of embryos lacking rho or spi; furthermore, the size of the posterior lip of the optic lobe is reduced in such embryos. The fact that absence of secondary Bolwig’s organ cells in rho or spi mutant embryos can be rescued by blocking cell death in the background of a deficiency that takes out the reaper complex of genes indicates that the Spi signal is not necessary for the specification (recruitment) of secondary Bolwig’s organ cells, but rather, for their maintenance (Daniel, 1999).

Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct tracheal migration

Branching morphogenesis is a widespread mechanism used to increase the surface area of epithelial organs. Many signaling systems steer development of branched organs, but it is still unclear which cellular processes are regulated by the different pathways. The development of the air sacs of the dorsal thorax of Drosophila was used to study cellular events and their regulation via cell-cell signaling. Two receptor tyrosine kinases play important but distinct roles in air sac outgrowth. Fgf signaling directs cell migration at the tip of the structure, while Egf signaling is instrumental for cell division and cell survival in the growing epithelial structure. Interestingly, Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct migration, suggesting that both cytoskeletal and nuclear events are downstream of receptor activation. Ras and the Mapk pathway are also needed for Egf-regulated cell division/survival, but Pointed is dispensable (Cabernard, 2005).

The air sac of the dorsal thorax grows from a bud that arises during the third larval instar from a wing disc-associated tracheal branch. To illustrate the development of the air sac, a GFP trap line was used that rather ubiquitously expressed membrane bound GFP; tracheal cells were counterstained with an mRFP1-moesin construct under the direct control of the trachea-specific breathless (btl) enhancer. From the early to late third instar stage, a bud-like structure grows out of the transverse connective and spreads on the wing imaginal disc epithelium; this outgrowth corresponds to the primordium of the air sac of the dorsal thorax (Cabernard, 2005).

It has been proposed that the air sac of the dorsal thorax forms de novo from a small group of wing imaginal disc cells, and that the resulting sac subsequently generates a tracheal lumen by an unknown process (Sato, 2002). Since, in the early Drosophila embryo, the lumen arises from an epithelial invagination via cell migration, it was asked whether the cells in the growing air sac are epithelial in nature with a clear apical/basal polarity. For this purpose, a Dα-Catenin-GFP (Dα-Cat-GFP) fusion construct was expressed in the developing air sac and the distribution of GFP from early to late third instar larvae was analyzed. Dα-Cat-GFP labels the adherens junctions (AJs) of epithelial cells. Clearly, the growth of the air sac was accompanied from the early stages onward by an out-bulging of an AJ network, suggesting that most or all of the cells in the growing bud were epithelial in nature, and that a luminal space was generated at the apical side of the epithelial tracheal cells during outgrowth. To confirm this interpretation, use was made of the recent identification of a protein, Piopio (Pio), which is apically secreted into the tracheal lumen in the embryo (Jazwinska, 2003). Indeed, the prospective luminal space in the outgrowing air sac is filled with Pio protein, demonstrating that the air sacs consist of a sac-like epithelial sheet, generating a luminal space as they grow (Cabernard, 2005).

To test whether all cells maintained an apical-basal polarity during air sac budding, single tracheal cells were labelled by using a recently developed assay system that allows for the visualization (and manipulation) of individual tracheal cells in vivo (Ribeiro, 2004). When this scenario was used in the presence of a UAS-Dα-Catenin-GFP chromosome, it was found that, in virtually all cases, such individually labeled air sac cells contacted the lumen and formed AJs with neighboring cells, even when these cells were located at the tip of the outgrowing air sac. The same conclusion was reached when the expression of GFP-moesin was analyzed in single air sac cells; cells at the tip made clear contact with the lumen. Therefore, it is concluded that the air sac is sculpted from an epithelial cell layer, which expands and at the same time generates an apical luminal space filled with secreted proteins (Cabernard, 2005).

It was of interest to better understand how Ras can be used in the same tissue at the same time for different cellular processes. egfr mutant cells can contribute to the tip of the growing air sac, although the clones are relatively small. In the stalk of the air sac, cells lacking Egfr often appeared fragmented, a sign of cell death. Indeed, when egfr mutant cells were sustained with anti-Drice, a marker for apoptotic cells, a strong accumulation of this protein was found. When p35, a viral antiapoptotic protein, was expressed in egfr mutant cell clones, these clones grew to larger sizes and were able to populate the air sac tip at a significantly higher frequency than in the absence of p35. These experiments establish that Egfr is dispensable for migration, and that migration is exclusively triggered by one of the two RTKs, Btl/Fgfr. The experiments also demonstrate that, during the growth phase, Btl/Fgfr signaling is dispensable for cell division; clones can grow to large sizes, although they fail to populate the tip. This same result was obtained with two other components, which are exclusively used by the Fgfr signaling pathway in the air sac (and not the Egfr pathway), namely, Dof and Pointed. Thus, migration and cell division are controlled by two different RTKs, but both RTKs signal via the activation of Ras and the Map kinase pathway to regulate these different cellular outcomes (Cabernard, 2005).

How does Ras control cell migration in the tip and cell division in the remaining air sac? To start to address these questions, whether high levels of constitutive active Ras were compatible with directional cell migration was tested and RasV12 was expressed in wild-type tissue in small cell clones. Interestingly, such clones expanded considerably and grew to large sizes in the center of the air sac or in the stalk, resulting in bulgy outgrowths; however, clones expressing RasV12 never contributed to the tip of the air sac. This finding suggests that unrestricted levels of Ras in a cell perturb its capacity to read out the migratory cues (presumably the Bnl/Fgf ligand); wild-type cells were apparently much better in taking up the leading position. In line with this interpretation, expression of an activated version of Btl (Torso-Btl/Fgfr) also resulted in bulky outgrowths. In addition, cells expressing the chimeric Btl receptor never populated the tip. Quite in contrast, activated Egfr (Egfr fused to a lambda dimerization site) was not able to perturb air sac guidance, but it also triggered higher division rates in clones, generating bulgy outgrowths (Cabernard, 2005).

To test whether single cells expressing activated receptor constructs changed their behavior with regard to cytoskeletal dynamics, the expression of either the activated version of Fgfr or Egfr was induced in early third instar stages and the behavior of such cells was analyzed with live imaging of cultured discs. Cells in the stalk of the air sac expressing activated Fgfr showed extremely dynamic cytoskeletal activity and formed large lamelipodia extending away from the air sac, similar to cells at the tip. Quite in contrast, cells expressing activated Egfr did not show increased lamelipodia formation, and their basal side remained relatively inactive (Cabernard, 2005).

Since the expression of constitutive active versions of the two different RTKs during air sac growth had different effects, whether the endogenous receptors activated the Ras/Mapk pathway to different levels in wild-type air sacs was investigated. In order to monitor the strength of Mapk signaling, an antibody recognizing the double-phosphorylated form of Erk, dpErk were used. Indeed, high levels of dpErk was detected in the nucleus of tip cells; lower dpErk levels were found in the cells in the center of the air sac, and dpErk was mostly cytoplasmic (Cabernard, 2005).

From all of the above-mentioned data, it is concluded that air sac development makes use of two distinct RTKs to control directed organ extension via cell migration (Fgfr) and organ growth via cell division (Egfr). This study carefully analyzed air sac outgrowth from early to late stages, by using a number of different markers labeling either membranes or AJs of individual air sac cells, or the apical luminal compartment. It was found that the thoracic air sac is modeled out of the existing tracheal epithelium, and that a luminal space is generated by the migration of a few cells away from the cuticle of the existing tracheal branch; the luminal space is then expanded by increasing the cell number in the sac-like epithelial structure via cell division. During this process, all cells remain within the epithelium and only round up when they divide. Even those cells that send out filopodia and lamelipodia and migrate in the direction of Bnl/Fgf remain embedded within the epithelium, contact the lumen, and form AJs with their neighbors. Thus, the directed outgrowth of the thoracic air sac during larval development is very similar to the budding of tracheal branches in the early embryo, in that epithelial cells form extensions from the basal side, ultimately resulting in cell movement toward the Fgf ligand. In contrast, during tubule formation of MDCK cells in culture, cells initially depolarize and migrate to form chain-like structures before they repolarize and form the luminal cavity; tubulogenesis is thus accompanied with partial epithelial-to-mesenchymal as well as mesenchymal-to-epithelial transitions. The tube-forming process has been subdivided into different stages such as cyst, extension, chain, cord, and tubule. In the case of the MDCK model system, growth factors have been proposed to trigger branching by inducing a dedifferentiation that allows the monolayer to be remodeled via cell extension and chain formation. Similar to the MDCK system, it was found in Drosophila that growth factor signaling induces the formation of cellular extensions, the first sign of outgrowth. Also, in both systems, cell division is an integral part of the process, but it occurs randomly throughout the structure and not locally at the point of outgrowth. However, two different RTKs are used in the air sac to control extension (migration) and cell division, and chain and chord stages are not observed. It thus appears that both similarities and differences exist between these different cellular systems (Cabernard, 2005).

It has already been reported that cells divide during air sac formation. The cell division rates have been semiquantified and it was found that the elongating structure does not grow preferentially at the tip. The genetic analysis demonstrates that the Egfr is essential for cells to divide and survive efficiently in the air sac. Egfr signals via Ras and the Mapk pathway, but it does not require the Pnt transcription factor to regulate cell division. It is not yet known which ligand activates Egfr, and whether expression of this ligand is induced at early stages of development by Fgf signaling. As shown before (Sato, 2002), the complete lack of Fgfr signaling results in the absence of air sacs; Fgf signaling might thus be used at the onset of the budding process to initiate or trigger cell division, but it is clearly dispensable in later stages. Since cells in the tracheal branch, which gives rise to the air sac primordium, also divide in the absence of Fgf signaling, it is possible that the role of Fgf signaling consists in generating an outgrowth via directed cell movement, triggering cell division indirectly (Cabernard, 2005).

Interestingly, a recent study addressing the role of GDNF/Ret signaling in kidney branching morphogenesis in vivo has shown that ret mutant cells (which are unable to respond to GDNF) can contribute to the primary outgrowth of the ureteric bud, but are excluded from the ampulla that forms at its tip. Apparently, a Ret-dependent proliferation of tip cells under the influence of GDNF controls branch outgrowth. This study found that in Drosophila, in the developing air sac, cells lacking Fgfr are also excluded from the tip. However, evidence is provided that Fgf signaling is translated into directed migration in the leading structure and not into a local increase in cell proliferation. The isolation and cultivation of wing imaginal discs allows for using 4D imaging to document cell behavior during air sac growth. It was found that numerous tip cells extend long filopodia and lamelipodia, similar to the findings reported earlier (Sato, 2002). Tip cells not only produce extensions, but they indeed change their respective position with time, and move forward over the substrate in the direction of the filopodia/lamelipodia. Thus, tip cells are clearly motile and migrate in the direction of Bnl/Fgf. Cell clones incapable of responding to different families of ligands were produced and marked and were examined with regard to their capacity to populate the air sac tip. Among the receptors analyzed, only Btl/Fgfr was strictly required for cells to populate the leading tip of the air sac. Considering the observation that cells in the tip actively migrate, that Btl/Fgfr signaling is required for tracheal cell migration in the embryo, that tracheal cells migrate to ectopic sources of Bnl/Fgf in the embryo and the larva (Sato, 2002), and that cells form numerous filopodia and lamelipodia upon constitutive activation of the Fgf signaling pathway, it is concluded that Fgf steers cell migration in the tip of the air sac and leads to its directional outgrowth on the surface of the wing imaginal disc. The demonstration that the MARCM system can be used to analyze gene function with regard to cell migration in the developing air sac prompted an investigation of the role of Ras and the Mapk pathway in Fgf-directed cell movement (Cabernard, 2005).

Using the MARCM system, it was found that Ras activation is essential for cells to migrate at the tip of the air sac. The requirement for Cnk and Ksr strongly suggests that one important branch downstream of Ras in the control of cell migration is the Mapk pathway. This interpretation is supported by the somewhat surprising finding that the transcription factor Pnt is also strictly required for cell migration. In the Drosophila embryo, genes regulated by Fgf signaling at the transcriptional level and essential for migration have not been identified so far; although both pnt itself and blistered/DSrf are targets of Fgf signaling with important functions in tracheal morphogenesis, they are not required for migration. One possible target of Fgf signaling in the dorsal air sac cells might be the btl/fgfr gene itself. Attempts were made to rescue the pointed defects in air sac development by supplementing a btl transgene under the control of UAS sequences. It was found that even when Btl/Fgfr is provided by the transgenes, pnt mutant clones do not reach the tip. A second gene that might have been a transcriptional target of Pointed is dof; however, it was found that Dof protein is still present in pnt mutant clones (Cabernard, 2005).

The results demonstrate that the outgrowth of the dorsal air sac along the underlying wing imaginal disc is controlled by Btl/Fgfr and Egfr. Fgf signaling is required for directional outgrowth via cell migration, and Egf signaling is required for organ size increase sustaining cell division/cell survival. Both signals use the Ras/Mapk pathway to elicit their cellular responses. To what extent these two pathways regulate different downstream targets is not known at present. However, this study shows that Pointed is only required downstream of Fgf signaling in the control of directed cell migration, and not downstream of Egf signaling in the control of cell division/survival. Since the activation of the Map kinase pathway is much stronger in the cells at the tip as compared to the cells in the central portion or in the stalk of the air sac (according to the levels of dpErk), it is thought that the local availability of the Bnl/Fgf ligand results in a local signaling peak. Egf signaling in more central and proximal cells does not result in a strong activation of the Map kinase pathway, yet this activation is apparently sufficient to control cell division and survival. The independent regulation of cell migration and cell division by two different RTKs might be even more important in later stages of dorsal air sac development, when the growing tip is yet farther away from the main body of the air sac. It will be interesting to find whether other growing branched tissues use similar mechanisms to uncouple directional expansion and size increase (Cabernard, 2005).

A comparative study of Pointed and Yan expression reveals new complexity to the transcriptional networks downstream of receptor tyrosine kinase signaling

The biochemical regulatory network downstream of receptor tyrosine kinase (RTK) signaling is controlled by two opposing ETS family members: the transcriptional activator Pointed (Pnt) and the transcriptional repressor Yan. A bistable switch model has been invoked to explain how pathway activation can drive differentiation by shifting the system from a high-Yan/low-Pnt activity state to a low-Yan/high-Pnt activity state. Although the model explains yan and pnt loss-of-function phenotypes in several different cell types, how Yan and Pointed protein expression dynamics contribute to these and other developmental transitions remains poorly understood. Toward this goal this study used a functional GFP-tagged Pnt transgene (Pnt-GFP) to perform a comparative study of Yan and Pnt protein expression throughout Drosophila development. Consistent with the prevailing model of the Pnt-Yan network, numerous instances were found where Pnt-GFP and Yan adopt a mutually exclusive pattern of expression. However, many examples were also found of co-expression. While some co-expression occurred in cells where RTK signaling is presumed low, other co-expression occurred in cells with high RTK signaling. The instances of co-expressed Yan and Pnt-GFP in tissues with high RTK signaling cannot be explained by the current model, and thus they provide important contexts for future investigation of how context-specific differences in RTK signaling, network topology, or responsiveness to other signaling inputs, affect the transcriptional response (Boisclair Lachance, 2014).


In the differentiation of photoreceptors in the eye imaginal disc, pointed is activated by MAP Kinase through the sevenless pathway. The signal for the Sevenless receptor is Bride of sevenless.

Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function

Maintenance of hematopoietic progenitors ensures a continuous supply of blood cells during the lifespan of an organism. Thus, understanding the molecular basis for progenitor maintenance is a continued focus of investigation. A large pool of undifferentiated blood progenitors are maintained in the Drosophila hematopoietic organ, the larval lymph gland, by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. This study examined the function of the Drosophila fibroblast growth factor receptor (FGFR), Heartless, a critical regulator of early lymph gland progenitor specification in the late embryo, during larval lymph gland hematopoiesis. Activation of Heartless signaling in hemocyte progenitors by its two ligands, Pyramus and Thisbe, is both required and sufficient to induce progenitor differentiation and formation of the plasmatocyte-rich lymph gland cortical zone. Two transcriptional regulators were identified that function downstream of Heartless signaling in lymph gland progenitors, the ETS protein, Pointed, and the Friend-of-GATA (FOG) protein, U-shaped, which are required for this Heartless-induced differentiation response. Furthermore, cross-talk of Heartless and target of rapamycin signaling in hemocyte progenitors is required for lamellocyte differentiation downstream of Thisbe-mediated Heartless activation. Finally, the Drosophila heparan sulfate proteoglycan, Trol, was identified as a critical negative regulator of Heartless ligand signaling in the lymph gland, demonstrating that sequestration of differentiation signals by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance that is of potential relevance to many other stem cell niches (Dragojlovic-Munther, 2013).

bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression
Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. This study demonstrates that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). Evidence is provided that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. The results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins (Li, 2017).

This study demonstrates that similar to the canonical Notch signaling, Dpn maintains the identity and self-renewal of type II NBs at least in part by inhibiting Erm expression. Loss of Dpn leads to the ectopic activation of erm in type II NBs and that removing Erm not only prevents the transformation of dpn mutant or Dpn knockdown type II NBs into type I-like NBs but also largely inhibits their premature termination of self-renewal. The results from gel-shift assays and reporter assays provide evidence to support that Dpn and E(spl) proteins suppress Erm expression by directly binding to at least two of the three putative bHLH-O binding sites in the erm enhancer (Li, 2017).

Although Dpn and canonical Notch signaling could function through a similar mechanism, these factors do not appear to be completely functionally redundant as previously suggested. First, during early 1st instar larval stages when type II NBs are still quiescent, the maintenance of type II NBs may mainly rely on Dpn in that Notch is not activated in quiescent type II NBs, as evidenced through the findings showing that the loss of Dpn at early 1st instar larval stages leads to ectopic Erm-mediated transformation and the premature loss of type II NBs. Second, after reactivation of type II NBs, both Dpn and Notch signaling are required to suppress the ectopic Erm expression in type II NBs because both the loss of Dpn and the components of the canonical Notch signaling pathways alone lead to ectopic Erm expression in type II NBs. However, the Notch signaling likely plays a dominant role in suppressing ectopic Erm expression and maintaining type II NBs. It has been previously shown that the loss of components of the canonical Notch pathway, including E(spl) proteins, leads to ectopic Erm expression and the transformation and premature loss of type II NBs, despite the presence of Dpn in the NBs, whereas the knockdown of Dpn after the reactivation of NBs only results in the weak ectopic activation of erm but not transformation or premature loss of type II NBs. Therefore, Dpn and Notch signaling may not be completely functionally redundant in suppressing the ectopic Erm expression or maintaining type II NBs, and their functions might be dependent on developmental stages. Furthermore, a recent study reported that Klu could also bind to the R9D11 enhancer to repress the expression of Erm. Thus, type II NBs likely utilize multiple mechanisms to ensure that erm will not be prematurely activated (Li, 2017).

Previous studies suggested that all E(spl) proteins share similar DNA sequences. However, results from the present study suggest that this similarity may not always be the case. Gel-shift assays show that only members of the E(spl) family, including E(spl)mγ, mβ, mδ, m3, and m7, can bind to the bHLH-O binding sites in the erm regulatory region, whereas the other two, E(spl)m5 and m8, cannot. The difference in their DNA binding specificity is consistent with differences in the amino acid sequences of their bHLH domains and their overexpression phenotypes in type II NB lineages. Therefore, although multiple E(spl) proteins have been shown to be expressed in larval NBs and at least two of them, E(spl)mγ and m8, are activated by Notch, these E(spl) proteins may bind to different DNA sequences and regulate the expression of different target genes, which may in turn determine their functional specificity (Li, 2017).

In contrast to the maintenance of type II NBs, the maturation of imINPs requires the activation of erm by PntP1 and shutdown of Dpn expression and Notch signaling. It has previously been shown that the loss of Erm or aberrant activation of dpn or Notch signaling in imINPs both lead to the dedifferentiation of imINPs and overproliferation of type II NBs. However, the functional relationship between the activation of erm and the absence of Dpn or Notch signaling in imINPs has never been established. This study demonstrates that the absence of Dpn and Notch signaling is essential for the activation of erm and subsequent Erm-mediated maturation of INPs. First, the results show that aberrant activation of dpn or Notch signaling inhibits the activation of erm in imINPs. Second, maintaining Erm expression in imINPs largely blocks the overproliferation of type II NBs resulting from the misexpression of E(spl) or Dpn proteins, suggesting that one main reason for the dedifferentiation of imINPs caused by Dpn or E(spl) overexpression is the suppression of Erm. However, the overproliferation of type II NBs resulting from the overexpression of Nintra or Numb knockdown can only be partially suppressed by concomitant Erm expression. Therefore, in addition to functioning through the canonical pathway to activate E(spl) expression, Notch may also act through noncanonical pathways, such as the mTORC2/Akt pathway, to regulate type II NB proliferation (Li, 2017).

How does Erm promotes INP maturation and prevents the dedifferentiation of imINP? It has previously been suggested that Erm prevents the dedifferentiation of INPs by activating pros expression and attenuating the response of INPs to self-renewing factors such as Dpn and E(spl) proteins. However, two pieces of evidence argue against this notion. First, the loss of Pros only induces the overproliferation of INPs but not the dedifferentiation of imINPs into type II NBs . Second, Erm is only expressed in imINPs, which do not express Dpn or E(spl) proteins. In the present study, evidence is provided demonstrating that Erm likely promotes INP maturation in part by inhibiting the expression and/or function of PntP1. These results show that the overproliferation of type II NBs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins, which leads to suppression of Erm expression, could be significantly inhibited by knocking down PntP1. These data strongly argue that the dedifferentiation of imINPs and generation of extra type II NBs resulting from the loss of Erm is in part due to de-repression of PntP1 expression and/or function in imINPs, which is consistent with the PntP1 function in specifying type II NBs and suppressing the activation of ase. Similar to other Ets family proteins that are commonly involved in tumorigenesis, PntP1 may also activate the expression of cell cycle regulators that promote nonproliferative imINPs to enter the cell cycle and initiate unrestricted tumorigenic overproliferation. However, PntP1 may not be the only target of Erm in imINPs. As shown in a recent study, in addition to PntP1, Erm also directly inhibits the expression of Grh-O in imINPs (Janssens, 2017). Therefore, Erm likely promotes the maturation of INPs by regulating the expression/function of multiple target genes (Li, 2017).

In conclusion, this study demonstrates here that similar to Notch signaling, Dpn maintains the identity and self-renewal of type II NBs in part by inhibiting Erm expression. Whereas in imINPs, the absence of Dpn and E(spl) proteins allows PntP1-mediated activation of erm, which in turn promotes INP maturation by inhibiting the expression and/or function of PntP1 and Grh-O in imINPs. Thus, the present study elucidates the mechanistic details of the maintenance of type II NBs and maturation of INPs (Li, 2017).

pointed: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Effects of Mutation | References

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