engrailed
engrailed is first expressed in early gastrulating embryos, three to four hours after fertilization. The first stripe is just posterior to the deep groove subdividing the head (cephalic furrow). Subsequently, 13 other stripes appear along the longitudinal axis of the embryo. The 14 stripes account for what later will become mandibular, maxillary and labial segments of the head, three thoracic and eight abdominal segments [Images] (Fojse, 1985).
Based on the expression pattern of the segment polarity genes engrailed and wingless during the embryonic development of the larval head, it has been found that the head of Drosophila consists of remnants of seven segments (4 pregnathal and 3 gnathal) all of which contribute cells to neuromeres in the central nervous system. (Schmidt-Ott, 1992).
The proneural genes achaete and scute and the segment polarity genes wingless and engrailed each have limited expression in only a few identifiable and stereotyped clusters of the head. For example, sc appears exclusively in a small part of the protocerebral domain, followed by transient expression in one to two protocerebral neuroblasts. wg is expressed in a total of three patches and engrailed is expressed in domains that are posterior and ventral to the adjacent wg domains. en is expressed in one patch in both the protocerebrum and the deuterocerebrum (Younossi-Hartenstein, 1996).
The domains of engrailed expression in the tail region have been documented. The en patterns in different Bithorax-Complex
Abd-B morphogenetic (m) and regulatory (r) mutants demonstrated that among other activities Abd-B functions to suppress embryonic ventral epidermal structures on the posterior side of A8 to A9. Ventral epidermal structures were not added back into the en pattern in r- or BX-C-
mutants, indicating that although the BX-C functions extend through A10, other non-BX-C genes must be required for development of this segment (Kuhn, 1995).
Engrailed is required for execution of the decision between the glial and neuronal fates in median neuroblasts (MNB) of the ventral nervous system (CNS). Engrailed expression in MNB progeny of the grasshopper is inhibited by injection of antisense oligodeoxynucleotides into the MNB nucleus. This produces a phenotype in which the midline glia do not develop and extra midline neurons are generated. In the absence of en function, midline glial precursors are apparently converted into neuronal precursors (Condron, 1994).
During neurogenesis, the transmembrane protein Patched promotes a wingless-mediated specification of a neuronal precursor cell, NB4-2. Wg, secreted by row 5 cells promotes wingless expression in adjacent row 4 cells; Wg in turn represses gooseberry. Novel interactions of these genes with engrailed and invected
during neurogenesis have been uncovered. While in row 4 cells Ptc represses gsb and wg, in row 5 cells en/inv relieve Ptc repression of gsb by a non-autonomous mechanism that does not involve hedgehog. The non-autonomous mechanism originates in Row 6/7 cells where en/inv engender hedgehog and another unknown secreted signal which acts in turn on adjacent row 5 cells to heighten wingless, and consequently, the expression of gooseberry. This differential regulation of gsb leads to the specification of NB5-3 and NB4-2 identities to two distinct neuroblasts. The row 5, NB5-3, neuroblasts are specified by high levels of gsb, expressed autonomously in row 5. The fate of row 4, NB4.2, requires an absence of gooseberry, assured by Patched repression and Wingless signaling from adjacent row 5 cells. The uncoupling of the ptc-gsb
regulatory circuit by hedgehog and the unknown secreted signal from row 6/7 cells enables gsb to promote Wg expression in row 5 cells. These results suggest that the en/inv->ptc->gsb->wg pathway uncovered here and the hh->wg are distinct pathways that function to maintain the wild-type level of Wg. These results also indicate that Hh is not the only ligand for Ptc and similarly, that Ptc is not the only receptor for Hh (Bhat, 1997).
In many instances, remote signaling involves the transport of secreted molecules. The spread of Wingless within the embryonic epidermis of
Drosophila was examined. Using two assays for Wingless activity (specification of naked cuticle and repression of rhomboid transcription), it was found that Wingless acts at a
different range in the anterior and posterior directions. This asymmetry follows in part from differential distribution of the Wingless protein. Transport or
stability is reduced within engrailed-expressing cells, and farther posteriorward Wingless movement is blocked at the presumptive segment boundary and perhaps
beyond. The role of hedgehog in the formation of this barrier is demonstrated (Sanson, 1999).
It is proposed that asymmetric Wingless distribution ensures the establishment of well-differentiated cell fates on either side of the engrailed domain. Anteriorly, at the wingless source, rhomboid expression is repressed. In contrast, reduced Wingless movement and/or stability within the engrailed domain allows nascent posterior rhomboid expression. Around this time (stage 11), a barrier to Wingless that requires hedgehog signaling forms at the posterior of the engrailed domain and ensures that Wingless does not foray across and repress rhomboid. rhomboid then activates the Egfr pathway within its expression domain and in adjacent cells. It may be that rhomboid itself contributes to barrier formation and thus builds a line of defense against invasion by its repressor. In addition, activation of the EGF pathway by rhomboid would antagonize any Wingless leaking through.
Denticle formation requires transcription of shavenbaby, which is under positive regulation by the Egfr pathway and negative regulation by the wingless pathway. Activated EGFR and the absence of Wingless posterior to the engrailed domain allow shavenbaby expression and hence denticle formation. At the anterior side, converse conditions exist, since Wingless is present at high levels and the Egfr pathway is inactive. Therefore, it is proposed that polarization of Wingless transport by engrailed and hedgehog guarantees the naked fate anterior to the engrailed domain and the denticle fate posteriorly, and thus establishes the anteroposterior polarity of each segment (Sanson, 1999 and references).
In the ventral abdominal region of the Drosophila embryo, wingless is expressed in single cell-wide stripes. To assess the range of Wingless in specifying the naked fate, these stripes were mapped onto the final cuticle pattern. Unexpectedly, the wingless stripes were found to be are eccentric within each expanse of naked cuticle. Naked cuticle is made over a distance of approximately 3-4 cell diameters anterior to the wingless source. In contrast, posterior to it, only the adjoining cells make naked cuticle; these cells are the most anterior of each engrailed stripe. The denticle fate of more posterior engrailed-expressing cells could be explained if they were unable to respond to wingless. Therefore the responsiveness to Wingless of all epidermal cells was assessed. To ask whether Wingless is required beyond one cell diameter posterior to its source, in individual cells the ability to respond to Wingless was removed and the phenotypic consequences was analyzed at single-cell resolution. Armadillo is a downstream effector of Wingless signal transduction and is also a component of Cadherin-based adhesion complexes. Armadillo is titrated out of its signaling pool when Shotgun (DE-Cadherin) is overexpressed, and in the embryonic epidermis, this leads to a phenocopy of a wingless mutation. Overexpressed Shotgun, therefore, blocks the response to Wingless in a cell-autonomous manner. Shotgun was overexpressed in single cells with the "Flp-on Gal4" system. Clones of cells expressing Gal4, which in turn activates expression of Shotgun and GFP, were induced during early stage 11 and scored at the end of embryogenesis. GFP-positive cells within naked regions make ectopic denticles, confirming that wingless signaling is required everywhere naked cuticle is made. All Shotgun-overexpressing cells located within belts make denticle of the type and size expected for their position. These results show that wingless signaling (via Armadillo) is not required in the presumptive denticle belts. Wingless signaling is only required within the naked domain, and this requirement is asymmetric relative to the wingless source (Sanson, 1999).
Two types of mechanisms could account for the asymmetry of wingless action: (1) the wingless protein itself could be unequally distributed in the anterior and posterior directions, or (2) Wingless could be distributed symmetrically, but downstream signaling would be repressed posterior to the source. Immunocytochemistry has revealed an asymmetric distribution of the wingless-containing vesicles within each segment at stage 11. By colabeling with anti-Engrailed, it has been shown that the posterior transition in Wingless distribution occurs at the interface with engrailed-expressing cells. Although Wingless protein can be detected in the wingless domain and anterior to it, only a small number of Wingless-containing vesicles can be seen in the most anterior row of engrailed cells, and none can be seen more posteriorly. Thus, Wingless distribution is asymmetric, and this could explain the asymmetry of wingless action (Sanson, 1999).
It is conceivable that undetectable yet active Wingless is present in cells posterior to engrailed stripes. To demonstrate the absence of active Wingless there, a functional assay was used, based on the finding that wingless signaling represses rhomboid expression. From stage 11 onward, rhomboid is expressed in stripes just posterior to each engrailed domain, and this expression is abolished by continuous and uniform expression of Wingless. Later ectopic expression, induced at late stage 11, inhibits rhomboid transcription only in the midventral region, and if sibling embryos are left to develop, they make ectopic naked cuticle in the same region. Therefore, Wingless can repress rhomboid transcription in the same time window as it specifies naked cuticle. Wingless is not only sufficient for rhomboid repression, it is also necessary since wingless null mutants have an additional rhomboid stripe in each abdominal segment. The position of these extra stripes relative to landmarks in the CNS suggests that they form at the anterior of the domain of extinct engrailed expression, where wingless would normally be expressed. Thus, in the wild type, the presence of Wingless at the anterior of each engrailed stripe maintains the silence of rhomboid expression there. Significantly, rhomboid is expressed posterior to the engrailed domain of wild-type embryos. Therefore, active Wingless is not present in these cells, at least at late stage 11; if it were, rhomboid would not be expressed. These cells are located only two cell diameters posterior to the Wingless source (Sanson, 1999).
The asymmetric distribution of Wingless could be explained by decreased transport/stability either within the engrailed domain or at its posterior edge, where the segment boundary forms. To explore this, wingless was misexpressed directly in the engrailed domain (posterior to endogenous wingless) and it was determined whether the range of Wingless was shifted posteriorly. Wingless was expressed with the engrailed-Gal4 driver in otherwise wild-type embryos. The only effect on the cuticle pattern is the loss of row 1 denticles. Remarkably, no other denticles are lost. In particular, row 2 denticles are present even though they are adjacent to the Wingless-misexpressing cells. Thus wingless expressed at the anterior side of the presumptive segment boundary does not affect the fate of cells on the posterior side. To confirm this finding, rhomboid expression was used as an early molecular marker for the absence of Wingless. In the wild-type larva, rhomboid is expressed in the cells secreting rows 2-4. In en-Gal4/UAS-wg larvae, this expression is unchanged, indicating that the wingless pathway is not operative in the cells immediately posterior to the wingless-misexpressing cells. Thus, it appears that Wingless cannot cross the posterior edge of the engrailed domain. This was verified by looking directly at the distribution of Wingless protein in en-Gal4/UAS-wg embryos. In these embryos, Wingless is present within the domain of wingless misexpression, as expected. However, it is not detectable posterior to the engrailed-expressing cells. It is concluded that a barrier to Wingless protein movement exists at the presumptive segment boundary (Sanson, 1999).
Among various candidate genes, hedgehog was found to be required for the posterior barrier to Wingless. Assaying the range of Wingless in a hedgehog mutant is not straightforward, since wingless expression requires hedgehog signaling. Therefore, wingless expression was maintained artificially in hedgehog null mutants using en-Gal4. Normally the engrailed promoter also turns off in a hedgehog mutant for lack of wingless, but in en-Gal4/UAS-wg; hh- embryos, this is remedied by exogenous Wingless. Thus, a hedgehog-independent positive feedback loop is established between engrailed and wingless, and stripes coexpressing Wingless and Engrailed are obtained. The distribution of the Wingless protein in en-Gal4/UAS-wg; hh- embryos is different from that seen in en-Gal4/UAS-wg control embryos. Wingless spreads posterior to the engrailed domain as if a barrier had been lifted or Wingless movement enhanced. The resulting protein distribution is symmetrical, and this is reflected in the cuticle pattern: in contrast to en-Gal4/UAS-wg embryos, en-Gal4/UAS-wg; hh- embryos lack rows 2-4 and, instead, have an extra expanse of naked cuticle. At the positions where rows 5 and 6 normally form, lies a thin stripe of small denticles. Naked cuticle is specified equally in the anterior and posterior directions, as shown by marking the wingless-expressing cells with GFP. Thus, in the absence of hedgehog, wingless action is symmetric (Sanson, 1999).
Thus, loss of hedgehog signaling increases the range of Wingless. Now it was asked whether the converse is true. To assay the range of Wingless in the presence of excess hedgehog signaling, endogenous wingless must be removed because hedgehog signaling activates wingless expression and this would confuse the assay. Therefore, the en-Gal4/UAS-wg combination was used again, but this time in a wingless mutant background. The sole source of Wingless in these embryos is in the engrailed domain. The wingless mutant phenotype is significantly rescued: the normal alternation of denticle belts and naked cuticle is restored, and many belts are nearly wild type, except for the loss of row 1. In these embryos, the width of the band of naked cuticle is 4-5 cells, and this provides an assay for the anterior range of Wingless. This assay was validated with a version of Wingless expected to act only at short range. If a membrane-tethered form of Wingless is expressed instead of the wild-type protein, an expanse of naked cuticle only 1-2 cells wide is found. This demonstrates that Wingless has to be physically transported from cell to cell to specify a band of naked cuticle of the normal size, and that there is no relay mechanism. Next, the assay was used to find out the effect of increasing hedgehog signaling on the range of Wingless. Increased hedgehog signaling can be achieved either by overexpressing Hedgehog or by removing patched activity. wg- en-Gal4/UAS-wg embryos carrying in addition UAS-hedgehog have significantly narrower naked domains. Likewise, wg- ptc- en-Gal4/UAS-wg embryos have narrow naked bands as well. This suggests that excess hedgehog signaling reduces the range of Wingless, although excess Hedgehog signaling could also induce ectopic rhomboid, which would in turn antagonize Wingless signaling and bring about the loss of naked cuticle (Sanson, 1999).
It is known that Wingless sustains engrailed expression only in adjoining cells, suggesting that Wingless is not readily transported across the engrailed domain. This is supported by the asymmetric distribution of the protein. Immunostaining reveals the presence of Wingless anterior to its source, whereas very little is detected posteriorly; posterior to wingless-expressing cells, in the engrailed domain, some Wingless staining is found but only in the most anterior cells (nearest the Wingless source). Thus, engrailed-expressing cells appear to restrict Wingless movement. Restricted Wingless transport through the engrailed domain could be explained by the downregulation of a specialized transport receptor in the engrailed cells; the existence of such a receptor has been hypothesized. Alternatively, inefficient transport could follow from selective instability of Wingless or its sequestration within the engrailed domain. In wing imaginal discs, the stability and range of Wingless increase in response to overexpression of its receptor Frizzled2. By analogy, and conversely, Wingless might be particularly unstable within engrailed stripes for lack of a receptor there. Alternatively, the surface or extracellular matrix surrounding engrailed cells might trap Wingless and impede its movement. A receptor of the proteoglycan type could possibly mediate this activity. Indeed, in mutants for the gene encoding UDP-glucose dehydrogenase, that lack HSPGs, embryonic engrailed stripes are temporarily widened, implying an increased range of Wingless. Identification of the relevant receptors and their pattern of expression will be required to discriminate between the above alternatives (Sanson, 1999 and references).
Not only is Wingless movement restricted within the engrailed domain, but a barrier seems to exist at its posterior edge. This is especially evident in embryos that ectopically express Wingless in the engrailed domain. In these embryos, Wingless does not specify naked cuticle nor repress rhomboid posteriorly, even in adjacent cells. The lack of response is unlikely to be due to insufficient expression, since en-Gal4 is a robust driver. Also, uniform wingless expression (even at low levels and up to late stage 11) induces uniform naked cuticle (D
The notion that Wingless movement is blocked at the forming segment boundary contrasts with an earlier proposal that Wingless spreads symmetrically. According to this view, posterior to its source, Wingless signaling is antagonized by active EGFR. The Egfr pathway is activated within and near the rhomboid stripe, which lies just posterior to the segment boundary. However, it is proposed that this segmental activation, which requires rhomboid, occurs after formation of the restrictions to Wingless movement. If wingless protein were present in the rhomboid cells at late stage 11, rhomboid expression would not be allowed there since wingless has been shown to repress rhomboid transcription. Subsequent establishment of rhomboid expression would further counteract activation of the Wingless pathway in prospective denticle belts (Sanson, 1999).
It is suggested that two mechanisms restrict posterior Wingless movement. The first restriction occurs within the engrailed domain and is unlikely to be under hedgehog control, since engrailed cells are not thought to respond to Hedgehog. Rather, engrailed could implement this restriction by controlling a gene involved in Wingless transport, sequestration, or stability. By contrast, the barrier at the posterior of the engrailed domain requires hedgehog signaling. Wingless produced ectopically in the engrailed domain of hedgehog mutants is allowed to invade posteriorly located cells and induce naked cuticle there. The finding that the same effects are seen in cubitus interruptus mutants indicates that the hedgehog signaling pathway is involved. The role of the hedgehog pathway is confirmed by "gain-of-function" experiments. Loss of patched results in overactivation of the hedgehog pathway and so does excessive hedgehog expression. Both situations reduce the range of Wingless in the anterior direction as if
the spread of the protein were reduced. It is presumed that, in the wild type, a downstream Hedgehog target is upregulated at the posterior of each
engrailed/hedgehog stripe and this would lead to Wingless destabilization or a block to transport there (Sanson, 1999).
In Drosophila embryos, segment boundaries form at the posterior edge of each stripe of engrailed expression. An HRP-CD2 transgene has been used to follow by transmission electron microscopy the cell shape changes that accompany boundary formation. The first change is a loosening of cell contact at the apical side of cells on either side of the incipient boundary. Then, the engrailed-expressing cells flanking the boundary undergo apical constriction, move inwards and adopt a bottle morphology. Eventually, grooves regress, first on the ventral side, then laterally. Groove formation and regression are contemporaneous with germ band retraction and shortening, respectively, suggesting that these rearrangements could also contribute to groove morphology. The cellular changes accompanying groove formation require that Hedgehog signalling be activated, and, as a result, a target of Ci is expressed at the posterior of each boundary (obvious targets like stripe and rhomboid appear not to be involved). In addition, Engrailed must be expressed at the anterior side of each boundary, even if Hedgehog signalling is artificially maintained. Thus, there are distinct genetic requirements on either side of the boundary. In addition, Wingless signalling at the anterior of the domains of engrailed (and hedgehog) expression represses groove formation and thus ensures that segment boundaries form only at the posterior (Larsen, 2003).
Segmental boundary formation is initiated shortly after germ-band retraction has begun. They are recognizable as periodic indentations in the epidermis that separate cells expressing engrailed at the anterior from those expressing rhomboid at the posterior. To allow identification of cells in electron micrographs, a transgenic membrane marker was devised based on horseradish peroxidase (HRP), which catalyses the production of an electron-dense product from diaminobenzidine (DAB). HRP was fused to the transmembrane protein CD2 so that the marker would outline cells and thus reveal cell shapes. This inert fusion protein was expressed under the control of engrailed-Gal4, so that the membrane of engrailed-expressing cells appears dark under the electron microscope (Larsen, 2003).
Cell shape changes during groove formation were studied in horizontal sections through the ventral aspect of the embryo at the level of parasegment 9 (the boundary between abdominal segments 3 and 4). Groove formation begins shortly after initiation of germ band retraction as a slight splaying between HRP-positive and HRP-negative cells. As this slit matures into the boundary, the cells on either side are referred to as 'groove founder cells'. The groove founder cells further lose contact apically, and a groove forms between them. Subsequently, in any one section, the cell at the anterior of the incipient boundary (the one expressing engrailed) appears to constrict its apical surface. At the same time, it moves toward the interior of the embryo, seemingly pulling neighboring cells along. As boundary formation proceeds, this cell becomes positioned at the bottom of the groove and begins to adopt a bottle shape (apical constriction). The cells neighboring the groove founder cells follow this inward movement, and also display partial apical constriction. The groove continues to deepen, until the bottle cell, which is still HRP positive, ends up three to four cell diameters below the surface of the embryo. This cell remains at the bottom of the groove with its apex constricted until late stage 13, coinciding with the onset of dorsal closure. After this stage, in the ventral region, the groove regresses until stage 15, when it has practically disappeared. At lateral positions, a similar sequence of events is seen, but with two quantitative differences. Lateral grooves dig deeper into the embryo and regress later than ventral ones. In conclusion, groove formation involves specific changes in cell contact between the groove founder cells, apical constriction of the most posterior engrailed-expressing cells, and inward migration of cells surrounding the groove (Larsen, 2003).
The most posterior engrailed-expressing cells display a distinctive behavior during groove formation. So far it has not been possible to track the fate of this cell as the grooves disappear. However, evidence has been obtained that it ceases to express Engrailed around the time when grooves are deepest. Embryos expressing HRP-CD2 under the control of engrailed-Gal4 were stained for HRP and Engrailed protein. As the groove grows deeper, Engrailed and HRP are co-expressed as expected. However, at later stages, Engrailed protein is no longer detectable in the bottle cell, whereas HRP membrane stain remains, presumably because HRP is relatively stable. Thus, during groove formation the most posterior engrailed-expressing cell changes morphology dramatically and, upon completion of this process, stops expressing the Engrailed protein (Larsen, 2003).
Groove formation coincides with germ band retraction as if segments were being compressed, much like an accordion. The first segments to undergo such apparent compression are the most anterior ones and this is where grooves are deepest. Another noteworthy temporal correlation is between the disappearance of grooves and dorsal closure, a process whereby the epidermis spreads dorsally to enclose the whole embryo. Thus, it could be that the need for additional surface area during dorsal closure promotes groove regression. To investigate this further, zipper mutants, which are defective in dorsal closure, albeit with a variable penetrance, were examined. In those zipper mutants that completely fail to undergo dorsal closure, grooves persist longer. For example, ventral grooves can be seen well into stage 15, a stage when the ventral surface of wild-type siblings is relatively smooth. Moreover, at lateral positions, grooves appear to be deeper in zipper mutants (Larsen, 2003).
In conclusion, cells undergo specific morphological changes
at incipient boundaries, especially those cells that line the anterior side of
the boundary (the most posterior engrailed-expressing cells). At the
same time, it may be that global rearrangements within the epithelium also
contribute to groove formation (Larsen, 2003).
Engrailed has both a cell autonomous and
a non-cell autonomous function in the establishment of the compartment
boundary in wing imaginal discs. Although the compartment boundary does not
trace its embryonic origin to segment boundaries, there is
a striking parallel between the two. For segmental grooves
to form, Hedgehog signaling is required in cells at the posterior of the
boundary, even if engrailed expression is artificially maintained at
the anterior side. Conversely, Hedgehog signaling is not sufficient as
exogenous expression of hedgehog in the absence of engrailed
does not lead to groove formation (Larsen, 2003).
It is the cells that line the anterior side of segment
boundaries (the most posterior engrailed-expressing cells) that
undergo the most distinctive behavior during groove formation. This behavior
requires Hedgehog signalling, and yet engrailed-expressing cells are
not responsive to this signal. Therefore, their morphological changes must be
in response to a signal originating from neighboring non-engrailed
expressing cells. This could be achieved through standard paracrine signaling
or by contact-dependent signal mediated by cell surface proteins. Whatever the mechanism, Hedgehog-responsive cells influence the behavior of adjoining
engrailed-expressing cells across the boundary, and crosstalk between
the two cells takes place. This is reminiscent of the situation found during eye morphogenesis; at rhombomere
boundaries cross communication between neighboring rhombomere cells are required for rhombomere formation (Larsen, 2003).
Because boundaries form in the complete absence of Ci
(in ci94), it is concluded that the activator form of Ci is
not required for segment boundary formation. However, no boundary forms in
ciCell mutant embryos, indicating that the presence of
Ci[75] (the repressor) prevents boundary formation. It is suggest therefore that boundary formation requires the expression of a gene (x) that is
repressed by Ci[75] but does not require Ci[155] to be activated. Presumably,
an activator of x is constitutively present but, in the absence of
Hedgehog, it is prevented from activating x expression by Ci[75].
Hedgehog signaling would remove Ci[75] and thus allow activation to occur. Two characterized target genes of Hedgehog (wingless and
rhomboid) follow the same mode of regulation. For example, expression
of wingless in the embryonic epidermis decays in
ciCell but is still present in the complete absence of Ci,
in ci94 embryos (Larsen, 2003).
Although Hedgehog signaling is activated both at the anterior and the
posterior of its source, segment boundaries only form at the posterior. One
reason for this asymmetry is that Wingless signaling represses boundary
formation at the anterior. Indeed, in the absence of Wingless, boundaries are
duplicated, as long as expression of Engrailed and Hedgehog is artificially
maintained. It is concluded that expression of x is repressed by Wingless signalling. Two obvious candidates for x are Rhomboid and Stripe. Genes encoding both these proteins are activated by Hedgehog signaling and repressed by Wingless
signaling and, indeed, both are expressed in cells that line the
segment boundary. To determine if either gene could mediate the role of
Hedgehog in boundary formation, the respective mutants were examined. No effect
on grooves could be seen. It is concluded that neither rhomboid nor stripe is
required for boundary formation although the possibility
that these genes could contribute in a redundant fashion cannot be excluded. Overall the genetic
analysis suggests that additional targets of Hedgehog must be involved in
boundary formation. It will be interesting to find out whether any of these
targets will turn out to be implicated in compartment boundary maintenance as
well (Larsen, 2003).
Although the role of a Hedgehog target gene in boundary
formation has been emphasized, it is clear from this analysis that engrailed also has a
cell-autonomous role. Even though Engrailed
represses ci expression, its role in boundary formation is likely to
involve the transcriptional regulation of another target gene. One possibility is
that Engrailed could be a repressor of x and that boundaries would
form at the interface between x-expressing and non-expressing cells.
However, it is thought that instead, or in addition, Engrailed has a
Hedgehog-independent effect on cell affinity and that this could contribute to
boundary formation. Of note is the observation that
engrailed-expressing cells remain together in small groups even when
boundaries are lost for lack of hedgehog. This suggests that
engrailed-expressing cells have increased affinity for one another.
Thus, Engrailed could specify P specific cell adhesion independently of
Hedgehog. Clearly, future progress will require the identification of
Engrailed target genes that control such preferential affinity and/or
contribute to boundary formation (Larsen, 2003).
Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. This study quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. Mechanical tension was found to be approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632, an inhibitor of Rho-kinase whose main effector is Myosin II. Simulations using a vertex model demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. These results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries (Landsberg, 2009).
A long-standing hypothesis to explain the maintenance of compartment boundaries is based on differential cell adhesion (or cell affinity). Cell adhesion molecules required for the maintenance of compartment boundaries, however, have not been identified. More recently, it has been proposed that actin-myosin-based tension is important for keeping the dorsoventral compartment boundary of the developing Drosophila wing smooth and straight. However, whether a similar mechanism operates at the anteroposterior compartment boundary (A/P boundary) is unclear. Moreover, a physical measurement of differential mechanical tension at compartment boundaries has not been reported. Furthermore, whether and how differential mechanical tension governs cell sorting at compartment boundaries is not well understood (Landsberg, 2009).
To test whether actin-myosin-based tension is increased at the A/P boundary, the levels of Filamentous (F)-actin and nonmuscle Myosin II (Myosin II) were quantified. The A/P boundary in the wing disc epithelium was particularly well defined by the cell bonds located at the level of adherens junctions, indicating that mechanisms maintaining the boundary operate at this cellular level. F-actin and the regulatory light chain of Myosin II (encoded by spaghetti squash, sqh) were increased at these cell bonds along the A/P boundary. Cell bonds displaying elevated levels of Myosin II correlate with decreased levels of Par3 (Bazooka in Drosophila), a protein organizing cortical domains, at the dorsoventral compartment boundary and during germ-band extension in Drosophila embryos. Likewise, Bazooka was decreased at cell bonds along the A/P boundary, indicating a common mechanism of complementary protein distribution of Myosin II and Bazooka. The level of E-cadherin, a component of adherens junctions, was not altered along the A/P boundary (Landsberg, 2009).
To identify signatures of increased tension in the vicinity of the A/P boundary, the morphology of cells were quantitatively analyzed at the level of adherens junctions. Line tension and mechanical properties of cells have been proposed to contribute to cell shape and to influence angles between cell bonds. Line tension associated with adherens junctions, here termed cell bond tension, can be defined as the work, per unit length, performed as a cell bond changes its length. Cell bond tension results from actin-myosin bundles and other structural components at junctional contacts that generate tensile stresses. Wing discs from late-third-instar larvae were stained for E-cadherin and engrailed-lacZ, a marker for the posterior compartment. Cell bonds were identified, and morphological parameters were analyzed. Adjacent anterior and posterior cells (A1 and P1, respectively) displayed a significantly enlarged apical cross-section area compared to cells farther away from the compartment boundary, indicating that apposition of anterior and posterior cells alters specifically the properties of A1 and P1 cells. Angles between adjacent cell bonds along the A/P boundary were larger compared to angles between bonds of the remaining cells and were significantly smaller in mutants for Myosin II heavy chain (encoded by zipper; zip2/zipEbr). Thus, the unique morphology of A1 and P1 cells depends on Myosin II. These data are consistent with an increased Myosin II-based tension of cell bonds located along the A/P boundary (Landsberg, 2009).
Cells on opposite sides of the A/P boundary differ in gene expression. The homeodomain-containing proteins Engrailed and Invected as well as the Hedgehog ligand are only expressed on the posterior side. The Hedgehog signal is transduced exclusively on the anterior side. Hedgehog signal transduction and the presence of Engrailed and Invected are required to maintain this compartment boundary. Whether the altered cell morphology at the A/P boundary could be reproduced by ectopically juxtaposing Hedgehog signaling and non-Hedgehog signaling cells was tested. Clones of cells that expressed Hedgehog from a transgene and that were also mutant for the gene smoothened (encoding an essential transducer of the Hedgehog pathway) were generated. In the P compartment, which is refractory to Hedgehog signal transduction, clones displayed a normal morphology. In the A compartment, a response to Hedgehog that is secreted by the clones is elicited in the surrounding wild-type cells. These clones had a rounder appearance, and at the clone border, but not away from it, apical cross-section area and bond angles were increased. Similarly, juxtaposing cells expressing engrailed and invected with cells that are mutant for these genes resulted in increased apical cross-section area and increased bond angles at the clone border. It is concluded that the morphology that is characteristic of cells at the A/P boundary can be imposed on cells within a compartment by juxtapositioning cells with different activities of Hedgehog signal transduction or Engrailed and Invected (Landsberg, 2009).
Ablating cell bonds generates cell vertex displacements, providing direct evidence for tension on cell bonds. Individual cell bonds were ablated by using a UV laser beam focused in the plane of the adherens junctions. Single-cell bonds were cut, and the displacement of vertices of neighboring cells, visualized by E-cadherin-GFP, was recorded. The P compartment was visualized by expression of GFP-gpi under control of the engrailed gene via the GAL4/UAS system. The increase in distance between the two vertices of the ablated cell bond and the initial velocity of this vertex separation were analyzed. The ratio of initial velocities in response to cell bond ablation is a measure of the tension ratio on these cell bonds. Initial velocity and extent of vertex separation were indistinguishable between anterior (A/A) and posterior (P/P) cell bonds located away from the A/P boundary. This was also the case when specifically cell bonds between the first and second row of anterior cells were ablated. By contrast, ablation of bonds between adjacent anterior and posterior cells (A/P cell bonds) gave rise to a larger vertex separation. This result was not due to the fact that A/P cell bonds have a preferred orientation. Moreover, the initial velocity of ablated A/P bonds was 2.37-fold higher compared to the mean of initial velocities of A/A and P/P bonds. This value provides an estimate of the ratio λ of cell bond tension along the A/P boundary relative to the average tension of cell bonds. In the presence of the Rho-kinase inhibitor Y-27632, the ratio of initial velocity of vertex separation of A/P cell bonds relative to A/A cell bonds was reduced to 1.46. Given that Myosin II is the main effector of Rho-kinase, these results strongly suggest that Myosin II-based tension acting on cell bonds is locally increased along the A/P boundary (Landsberg, 2009).
To quantify λ by an independent method, the displacement field was calculated after laser ablation. Using a vertex model, two populations of adjacent cells were introduced and cell bond ablations were simulated, varying λ between 1 and 4. When λ = 2.5, the vertex displacement, and in particular the anisotropy of displacements, in the simulations closely matched the vertex displacements in the experiment. In the vertex model, λ = 2.5 also resulted in increased bond angles at the interface of the two cell groups, similar to the A/P boundary in the wing disc. Thus, on the basis of two different methods, the data demonstrate that cell bond tension is increased approximately 2.5-fold along the A/P boundary compared to the remaining tissue (Landsberg, 2009).
To test whether a 2.5-fold increase in cell bond tension is sufficient to maintain a compartment boundary, the vertex model was used to simulate the growth of two adjacent cell populations for λ = 1, 2.5, and 4. For λ = 1, the interface between two growing cell populations became increasingly irregular. By contrast, for λ = 2.5 and 4, a well-defined interface was maintained. Moreover, corresponding changes in cell bond tension at borders of simulated clones resulted in the morphology and sorting behavior of cell patches that resembled those of experimental cell clones compromised for Hedgehog signal transduction or Engrailed and Invected activity. The roughness of the interface in the simulations decreased with increasing λ, showing that cell bond tension is sufficient to maintain straight interfaces between growing cell populations. For λ = 2.5, the roughness of the interface was still larger than the roughness of the A/P boundary in wing discs. This suggests that additional mechanisms might contribute to further reduce the roughness of the A/P boundary. Also, because of the uncertainty of the mechanical properties of A1 and P1 cells, which differ from those of the remaining cells, the value of λ, inferred from laser ablation of cell bonds, might be underestimated. Remarkably, the roughness of the A/P boundary could be altered in mutant conditions. In zip2/zipEbr mutant wing discs, the roughness of the compartment boundary was significantly larger than in controls, demonstrating a role for Myosin II in maintaining a sharp and straight A/P boundary (Landsberg, 2009).
In summary, by applying physical approaches and quantitative imaging, this work for the first time demonstrates and quantifies an increase in tension confined to the cell bonds along the A/P boundary. Moreover, simulations show that this increase in tension suffices to maintain a stable interface between two proliferating cell populations. Genetic studies demonstrated that cells of the two compartments differ in their expression profiles and signaling activities. It has therefore been proposed that biophysical properties of cells within the P compartment differ from those within the A compartment, and that such differences could drive cell sorting. When quantifying cell morphology and vertex displacements after laser ablation, no differences were detected in the biophysical properties of cells between the two compartments. However, the two rows of abutting A and P cells show clear differences in biophysical properties from other cells. Most importantly, the cell bond tension along the A/P boundary is increased. Cell divisions in the vicinity of the A/P boundary were randomly oriented in the epithelial plane. Thus, taken together with the simulations, these results suggest a sorting mechanism by which an increased cell bond tension guides the rearrangement of cells after cell division to maintain a straight interface. Increased cell bond tension and the roughness of the A/P boundary depend on Rho kinase activity and Myosin II, indicating a role for actin-myosin-based tension in this process. Because cell bond tension also depends on cell-cell adhesion, differences in the adhesion between A1 and P1 cells as compared to the remaining cells might also contribute to sorting. The heterotypic, but not homotypic, interaction of molecules presented on the surface of A and P cells might trigger the local increase in cell bond tension. Hedgehog signal transduction and the presence of Engrailed and Invected might control the expression of these heterotypically interacting molecules. These data indicate an important role for cell bond tension directing cell sorting during animal development (Landsberg, 2009).
The Drosophila CNS contains a variety of glia, including highly specialized glia that reside at the CNS midline and functionally resemble the midline floor plate glia of the vertebrate spinal cord. Both insect and vertebrate midline glia play important roles in ensheathing axons that cross the midline and secreting signals that control a variety of developmental processes. The Drosophila midline glia consist of two spatially and functionally distinct populations. The anterior midline glia (AMG) are ensheathing glia that migrate, surround and send processes into the axon commissures. By contrast, the posterior midline glia (PMG) are non-ensheathing glia. Together, the Notch and hedgehog signaling pathways generate AMG and PMG from midline neural precursors. Notch signaling is required for midline glial formation and for transcription of a core set of midline glial-expressed genes. The Hedgehog morphogen is secreted from ectodermal cells adjacent to the CNS midline and directs a subset of midline glia to become PMG. Two transcription factor genes, runt and engrailed, play important roles in AMG and PMG development. The runt gene is expressed in AMG, represses engrailed and maintains AMG gene expression. The engrailed gene is expressed in PMG, represses runt and maintains PMG gene expression. In addition, engrailed can direct midline glia to a PMG-like non-ensheathing fate. Thus, two signaling pathways and runt-engrailed mutual repression initiate and maintain two distinct populations of midline glia that differ functionally in gene expression, glial migration, axon ensheathment, process extension and patterns of apoptosis (Watson, 2011).
This paper describes how the Hh morphogen patterns the midline cells to generate two populations of MG with distinct functional properties. The key output of this signaling is the expression of en that imparts PMG cell fate, in part, by repressing runt. In turn, the runt gene maintains AMG fate by repressing en. Thus, morphogenetic signaling and transcriptional regulation lead to AMG and PMG with divergent molecular, morphological and functional differences (Watson, 2011).
At stage 10, the 16 midline cells per segment consist of three equivalence groups of neural precursors, four to six cells each. Notch signaling directs ten of these 16 cells to become MG; the remainder become MPs and the MNB. Thus, Notch represses neuronal development in MG and activates a core set of MG-expressed genes (e.g., wrapper). MG in the anterior of the segment become AMG; those in the posterior of the segment become PMG. Notch signaling by itself is unlikely to influence different MG fates, as expression of activated Suppressor of Hairless in midline cells drives all cells into a MG fate but does not affect their AMG or PMG patterns of gene expression. Thus, additional factors that can direct AMG and PMG cell fates were sought (Watson, 2011).
Previous work demonstrated that hh can pattern midline cells along the A/P axis, and, indeed, this study demonstrates that hh is required for PMG cell fate. The source of Hh is not in the midline, but in the lateral ectoderm in a stripe of cells, collinear with the pair of midline early en+ cells. Hh signals to midline cells posterior to the early en+ cells, inducing en in an additional six to seven cells. These late en+ cells plus the early en+ cells become about four PMG, as well as MP4-6 and the MNB. Misexpression and mutant analyses indicate that hh is required for all PMG gene expression and for repressing AMG expression. hh signaling probably has multiple target genes because hh is required for en and l(1)sc expression, but en does not regulate l(1)sc. Misexpression of hh can activate en expression in anterior MG, and both hh and en misexpression convert these cells functionally into non-ensheathing MG that resemble PMG, results also consistent with observations that ectopic expression of hh and en in midline cells affects AMG differentiation. However, neither hh nor en can activate all PMG gene expression in anterior MG, because neither activates masquerade (mas) expression in anterior MG. The mas gene is expressed transiently at stage 12 in a subset of PMG, suggesting that functionally distinct classes of PMG might exist. Expression of mas might require other signals in addition to hh that are absent in anterior MG (Watson, 2011).
runt is present in AMG and represses en and PMG-specific gene expression. In runt mutant cells that are runt- en+, expression of three genes expressed in only AMG (CG33275, Fhos and nemy) are absent and wrapper is reduced. This could be due to runt repression of en, repression of other genes or activation by runt. In runt mutant cells that are runt- en-, Fhos and nemy are present, wrapper is at high levels, but CG33275 expression is absent. This suggests that runt does not activate expression of Fhos, nemy and wrapper in AMG, but maintains their AMG levels by repressing en. By contrast, runt is required for expression of CG33275, possibly indicating a positive role for runt in AMG differentiation in addition to its repressive role in AMG maintenance. However, CG33275 is most prominently expressed in a subset of AMG closest to the commissures, and this AMG expression could be dependent on additional signals, perhaps from the developing axon commissure. Thus, absence of CG33275 expression in runt mutant embryos could alternatively be due to an effect of runt on developing axons or CNS development (Watson, 2011).
As most AMG gene expression is not dependent on runt, it is proposed that Notch signaling initially induces an AMG pattern of gene expression in all glia and, either simultaneously or soon after, Hh signaling in the posterior of the segment generates PMG. One important downstream target of Notch signaling is likely to be the sim gene, which encodes a bHLH-PAS protein that functions as a DNA-binding heterodimer with the Tango (Tgo) bHLH-PAS protein. During early development, sim is expressed in all midline primordia and is required for midline cell development. However, later in development, sim is restricted to MG and a subset of midline neurons. Genetically, sim expression is absent in embryos mutant for Notch signaling. The sim gene is likely to be an important aspect of MG transcription, because mutation of Sim-Tgo binding sites in the slit and wrapper MG enhancers results in loss of MG expression, and Sim-Tgo binding sites are present in other identified MG enhancers. The Hh morphogen transforms only posterior MG into PMG. It is unknown why hh does not affect anterior MG, but it is likely to be owing to the presence of unknown factors in these cells that inhibit
hh signaling. Since Notch signaling, rather than runt, is primarily required for AMG gene expression, the key role of runt is probably to maintain AMG gene expression by repressing en. Similarly, en functions to maintain PMG gene expression by repressing runt, but also contributes positively to PMG cell fate, as en misexpression confers PMG-like function to AMG (Watson, 2011).
The most striking features of AMG are their ability to migrate around the commissures, ensheath them and extend processes into the axons. The function of PMG is unknown, but they are unable to ensheath the commissures, even though they are in close proximity. One of the major factors influencing AMG-axon interactions is Nrx-IV-Wrapper adhesion. Levels of wrapper expression in AMG are higher than in PMG, and this is likely to be a key determinant of why AMG ensheath commissures, and PMG do not, because loss of wrapper expression results in incomplete migration and ensheathment. Recent work has demonstrated that sim directly regulates wrapper expression, and spitz signaling from axons might also form a positive feedback loop to control wrapper levels and strengthen Nrx-IV-Wrapper interactions. As en genetically reduces wrapper levels in PMG, it will be interesting to determine if this regulation is direct or indirect. Although the control of wrapper levels is likely to be a major factor in AMG-PMG differences and the ability of glia to ensheath axons, other genes whose levels differ between AMG and PMG might also contribute. This illustrates why it will be important to identify target genes and understand better the roles that Notch//Suppressor of Hairless, sim, hh, Ci, en, runt and other MG transcription factors play in regulating MG gene expression and function (Watson, 2011).
The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, a comprehensive analysis was carried out of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, evidence is provided indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deuto-cerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed (Urbach, 2003).
In the trunk neuroectoderm, segment-polarity genes are expressed in
stereotypical segmental stripes, and in NBs that delaminate from these
domains, subdividing each neuromere along the AP axis. In the
pregnathal head region the expression domains of segment polarity genes are less obvious, but analysis of engrailed and wingless expression in the head peripheral ectoderm, and of PNS
mutant phenotypes, support the existence of four pregnathal segments in
Drosophila: the intercalary, antennal, ocular and labral segments (from posterior to anterior). However, the identity and organization of brain structures
deriving from these segments is still obscure. In order to obtain evidence concerning the number and extent of the brain neuromeres, and to map the position of their boundaries, the expression of segment polarity genes, including wingless, hedgehog, gooseberry-distal, engrailed, invected and mirror, was analyzed. The spatiotemporal pattern of their expression was traced in the neuroectoderm and in the NB-layer until stage 11,
when all brain NBs are formed. The data
show that segmental expression is retained for most of the investigated segment polarity genes in both the developing head ectoderm (procephalon) and brain NBs, providing landmarks for the definition of segmental domains within the developing brain NB pattern (Urbach, 2003).
engrailed (en) expression domains in the trunk define the posterior segmental compartments, from which NBs of row 6 and 7 and NB1-2 derive. In the pregnathal head en expression was found as follows: from late stage 8 in the posterior ectoderm of the antennal segment (en antennal stripe; en as) from which four deutocerebral NBs (Dv8, Dd5, Dd9, Dd13) delaminate; from stage 9 in a small ectodermal domain in the posterior part of the ocular segment, the en head spot (en hs), from which two protocerebral NBs (Ppd5, Ppd8) evolve; and from stage 10 in an ectodermal stripe in the posterior intercalary segment (en intercalary stripe; en is), which gives rise to three to four tritocerebral NBs (Tv4, Tv5, Td3, Td5). Furthermore, from stage 11 onwards, En is weakly detected in the anteriormost ectoderm of the procephalon corresponding to the region of the 'anterior dorsal hemispheres' (en dh). About 10 weakly En-positive NBs were identified that delaminate from the en dh. Thus all four pregnathal head segments contribute to the early embryonic brain. The spatial distribution of the En-positive NBs closely corresponds to the en domains of their origin in the ectoderm. This suggests they demarcate the posterior borders of the respective brain neuromeres (Urbach, 2003).
In the trunk, hedgehog (hh) matches en expression. This is also the case for the intercalary segment in the pregnathal head ectoderm. By contrast, the En-positive antennal stripe and head spot are only subfractions of the large hh-lacZ domain, which, between stages 9 and 10, encompasses the antennal segment and the posterior part of the ocular segment. All NBs delaminating from this domain express hh-lacZ. From stage 10 onwards, en expressing NBs maintain a strong hh-lacZ signal, whereas hh-lacZ subsequently diminishes in the neuroectoderm and in NBs between the en antennal stripe and head spot. Additionally, hh-lacZ-expressing NBs positioned dorsally to the en/hh-lacZ-co-expressing Ppd5 and Ppd8 (both NBs demarcating part of the posterior border of the ocular neuromere), appear to prolong the boundary between the deuto- and proto-cerebrum in the dorsal direction (Urbach, 2003).
From late stage 8 onwards, Wingless (Wg) protein is expressed in a neuroectodermal domain spanning a broad area of the ocular and the anterior antennal segment (and in the invaginating foregut). This becomes clearer in En/Wg double labelling at stage 9, revealing that the en hs is localized within this Wg domain. At that stage, Wg is already detectable in about 4-5 protocerebral NBs (Pcd6, Pcd15, Pcd7, Ppd3), derived from the region with strongest Wg expression (which later corresponds to the wg head blob). Furthermore, Wg is faintly expressed in the deutocerebral Dd7 emerging from the antennal part of the Wg domain, which corresponds to the later wg antennal stripe. By stage 10, when the wg head blob is clearly distinguishable from the wg antennal stripe, about 10-12 Wg-positive NBs have emerged from this domain. In addition, a small, spot-like wg domain was found in the intercalary segment from which a single NB (Td4) delaminates. Thus, all three wg domains, the intercalary, antennal and ocular (head blob), contribute to the anlage of the brain. From late stage 9 an additional wg domain is visible in the ectodermal anlage of the clypeolabrum, which is the wg counterpart to the En/Inv-positive region in the 'dorsal hemispheres'. Upon double labelling for either asense or deadpan (both are general markers for neural precursor cells) and wg, in embryos between stage 9 and 11 no NB emerging from the wg labral spot could be detected. By stage 11 the number of wg expressing NBs originating from the ocular head blob has increased to about 16-20, which is more than 25% of the total number of identified protocerebral NBs. Three Wg-positive NBs are identified in the deutocerebrum and one in the tritocerebrum (Urbach, 2003).
A subset of sound-detecting Johnston's Organ neurons (JONs) in Drosophila melanogaster that express the transcription factors Engrailed (En) and Invected (Inv) form mixed electrical and chemical synaptic inputs onto the giant fiber (GF) dendrite. These synaptic connections are detected by trans-synaptic Neurobiotin (NB) transfer and by colocalization of Bruchpilot-short puncta. Misexpressing En postmitotically in a second subset of sound-responsive JONs causes them to form ectopic electrical and chemical synapses with the GF, in turn causing that postsynaptic neuron to redistribute its dendritic branches into the vicinity of these afferents. A simple electrophysiological recording paradigm was introduced for quantifying the presynaptic and postsynaptic electrical activity at this synapse, by measuring the extracellular sound-evoked potentials (SEPs) from the antennal nerve while monitoring the likelihood of the GF firing an action potential in response to simultaneous subthreshold sound and voltage stimuli. Ectopic presynaptic expression of En strengthens the synaptic connection, consistent with there being more synaptic contacts formed. Finally, RNAi-mediated knockdown of En and Inv in postmitotic neurons reduces SEP amplitude but also reduces synaptic strength at the JON-GF synapse. Overall, these results suggest that En and Inv in JONs regulate both neuronal excitability and synaptic connectivity (Pezier, 2014).
See Chris Doe's Hyper-Neuroblast map site for information on the expression of engrailed in specific neuroblasts.
For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.
Continued: Engrailed Developmental Biology part 2/2
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