even-skipped
In the visceral mesoderm, homeotic gene expression depends upon eve function, whereas most other segmentation mutations have only minor effects on position and/or width of the homeotic expression domains in this germ layer. Analysis of pair-rule double mutants indicates that complete loss of homeotic gene activity in the visceral mesoderm, as observed in amorphic eve mutants, correlates with loss of engrailed expression in the epidermis and loss of segmentation. The establishment of parasegment borders, a consequence of eve expression and witnessed by subsequent en expression, is thought to be a necessary precondition for homeotic gene expression in the visceral mesoderm (Tremml, 1989).
The regulation of stem cell division by developmental cues is critical for the assembly and function of
multicellular organisms. Stem cell division in the Drosophila brain is controlled by terribly reduced optic lobes (trol), which is required for activation of proliferation by quiescent neuroblasts at the appropriate stage of larval development. The transcriptional regulator eve is shown to be part of the trol activation pathway by identifying eve
as a dominant enhancer of a weak trol allele, trolb22. Known eve mutations are capable of enhancing the lethality of trolb22 and uncovering a defective neuroblast proliferation phenotype. Additionally,
genetic and molecular analysis reveals that an independent mutation that acts as a dominant enhancer of trol is also an allele of eve. The enhancement of trolb22 lethality can be suppressed by the presence of an eve transgene. Interestingly, extra copies of eve supplied by the eve transgene also enhance trolb22 lethality, suggesting that the level of Eve protein may be critical for neuroblast activation. Finally, activation of neuroblast proliferation is normal in eve4 heterozygotes, suggesting that
the proliferation defect observed in trolb22;eve/+ animals is due to a synergistic interaction (Park, 1998).
Embryos of higher metazoans are divided into repeating
units early in development. In Drosophila, the earliest
segmental units to form are the parasegments.
Parasegments are initially defined by alternating stripes of
expression of the fushi-tarazu and even-skipped genes. How
ftz and eve define the parasegment
boundaries, and how parasegments are lost when ftz
or eve fail to function correctly, has never
been fully or properly explained. It is shown that
parasegment widths are defined early by the relative levels
of ftz and eve at stripe junctions.
Changing these levels results in alternating wide and
narrow parasegments. When shifted by 30% or more, the
enlarged parasegments remain enlarged and the reduced
parasegments are lost. Loss of the reduced parasegments
occurs in three steps: delamination of cells from the
epithelial layer, apoptosis of the delaminated cells and
finally, apoptosis of inappropriate cells remaining at the
surface. The establishment and maintenance of vertebrate
metameres may be governed by similar processes and properties (Hughes, 2001).
Previous studies have shown that ftz and eve are the primary
determinants of parasegmental boundaries and identities (even
versus odd). Until quite recently, it was believed that the two genes
perform these roles relatively late (stages 6-7), and that high
levels and sharp anterior stripe boundaries are crucial. However, when in
the right proportions, the absolute levels of ftz and eve are not
particularly important. ftz and eve first
define the positions of parasegment borders prior to the
completion of cellularization (mid stage 5). At this time, ftz
and eve stripes have a bell-shaped distribution across each
stripe, and the stripes overlap with one another at their edges. It is suggested that
parasegment boundaries occur at the points where stripes
intersect and activity levels are equivalent. If the
activity of one gene is raised while the other remains
unchanged, these positions of equivalency move. The result is an alternating set of narrow and wide parasegments. These shifts become more pronounced with
greater changes in activity or when both genes change in
opposite directions. However, if both gene activities are
increased or decreased at the same time the positions of equivalency do not change, and parasegments remain equal in width (Hughes, 2001).
It is suggested that the transition from overlapping stripe
boundaries to sharp non-overlapping boundaries occurs via a
combination of autoregulatory and mutually antagonistic
functions. For example, if above a certain relative threshold
level, ftz autoregulation dominates over repression by eve,
and ftz expression rises to maximal levels while eve expression
is lost. If below that relative threshold, repression by eve
dominates over ftz autoregulation and ftz expression is lost
while eve rises to maximal levels. The ability of ftz and eve
to autoregulate and to mutually repress one another (directly
or indirectly) has been well documented. Once the
borders of ftz and eve stripes are established, combinatorial
interactions with other segmentation gene products then
determine where downstream targets such as en and wg are
activated or repressed, thereby locking in the positions of the
parasegment boundaries (Hughes, 2001).
ftz and eve pair-rule phenotypes have been described and explained in a number of conflicting ways. The remaining cuticle is
not simply composed of every second parasegment, nor is it
composed of double-width or homeotically re-transformed
segments. A relative decrease in ftz or eve activity causes
a decrease in width of alternate parasegments and a
corresponding expansion of adjacent parasegments. The
smaller parasegments are excised and the enlarged
parasegments retained. Efficient deletion (greater than 90%)
of the reduced parasegments occurs when they are reduced
by 30% or more. Enlarged parasegments are 1.4-1.5 times
wider than normal parasegments. This degree of enlargement
remains the same when levels of ftz or eve are increased
further or when the levels of ftz are reduced to zero (eve
nulls affect all parasegments due to earlier roles). It is suggested
that these maximal widths reflect the edges of stage 5 ftz
and eve stripes, beyond which autoregulation cannot occur. Further expansion of these stripes may be limited by the
actions of other pair-rule or gap gene products. The resulting
larva is composed of half the normal number of segments,
but these are 1.3-1.5 times wider than normal segments,
giving an overall length that is about 65%-75% the length of
a normal larva (Hughes, 2001).
Parasegments are considered to be the first 'compartments' to
form within the embryo. Compartments are fields of cells that originate from a common
group of founder cells and that remain defined in lineage
thereafter. Cells within adjoining compartments do not mix, most
likely due to differential adhesion properties. Compartments are further defined by unique gene
expression patterns (e.g. ftz and eve) that respect their
boundaries. Another property of compartments relevant to this study is
that they are capable of sensing and modulating their size.
Changes in size can be induced by injury, transplantation,
irradiation, or genetic manipulation.
In the case of reductions in size, compensation is most often
in the form of increased cellular proliferation, and when
increased in size, by programmed cell death.
These studies show that parasegments can compensate for
changes in size, but that this ability is relatively limited. Both
reduced and enlarged parasegments showed changes in the
normal numbers of apoptotic events. Dying cells are rarely
seen in the ectoderm of reduced parasegments while higher
than normal numbers are seen in enlarged parasegments. The
numbers of dying cells and the time of onset are proportional
to the degree of parasegment enlargement. These changes,
however, are insufficient to compensate for the changes in
widths induced in this study (Hughes, 2001).
It was also found that changes in mitotic frequency, as an
alternate form of compensation, do not occur. Once
established, the ratio of the number of cells per mutant
parasegment, as compared to wild-type segments, remains
relatively constant until cells in the reduced segments begin to
delaminate. This finding agrees with those obtained previously
by increasing the number of copies of the bicoid gene. Reduced
parasegments in the compacted middle of the embryo fail to
compensate by increasing rates of mitosis. However, these
changes in width were usually subtle enough (<20%) that most
segments were able to recover by reducing their rates of
apoptosis. These results show that once
these changes reach 30% or higher, variations in apoptotic
frequencies can no longer compensate (Hughes, 2001).
One of the most novel and intriguing findings of this study was
the unstable nature of reduced parasegments and the manner
in which they are removed. It was
found that this occurs via a three-step process. First, large
patches of cells move out of the ectodermal layer. Next, they
pinch off from the overlying ectoderm and then programmed
cell death is initiated. Finally, the fused engrailed stripes
remaining at the surface are resolved by late and sporadic
apoptotic events. Although the precise spatial and temporal
details of this process vary between individual embryos and
different mutant backgrounds, the general trends and final
consequences are the same (Hughes, 2001).
The delamination of reduced parasegment cells occurs
primarily during the late stages of germ band retraction. This
coincidence between reduced parasegment delamination and
germ band retraction suggests the possibility that cellular
movement and adhesion may play a prominent role in the
delamination process. During germ band retraction, normal
parasegments are reduced in width by almost half (approx.
11 cells to 7). In reduced parasegments, the corresponding
decrease results in an average width of just 3 cells. This
reduced width means significantly fewer contacts with other
reduced parasegment cells and more contacts with the cells of
neighboring parasegments. This may drive the reduced
parasegment cells to increase homogeneous contacts by
forming spheres, much as observed in imaginal discs when
small clones of anterior compartment identity are formed in the
posterior compartment (Hughes, 2001).
During convergent extension in Drosophila, polarized cell movements cause the germband to narrow along the dorsal-ventral (D-V) axis and more than double in length along the anterior-posterior (A-P) axis. This tissue remodeling requires the correct patterning of gene expression along the A-P axis, perpendicular to the direction of cell movement. A-P patterning information results in the polarized localization of cortical proteins in intercalating cells. In particular, cell fate differences conferred by striped expression of the even-skipped and runt pair-rule genes are both necessary and sufficient to orient planar polarity. This polarity consists of an enrichment of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 protein at the reciprocal D-V cell borders. Moreover, bazooka mutants are defective for germband extension. These results indicate that spatial patterns of gene expression coordinate planar polarity across a multicellular population through the localized distribution of proteins required for cell movement (Zallen, 2004).
Polarized cell movement during convergent extension ultimately derives from the asymmetric localization of proteins that direct cell motility. Interestingly, intercalating cells in the Drosophila germband display a polarized localization of the ectopically expressed Slam protein (Lecuit, 2002). Slam is present in a bipolar distribution that correlates spatially and temporally with intercalary behavior. These observations indicate that Slam can serve as a molecular marker for polarized cell behavior. Pair-rule patterning genes expressed in stripes along the A-P axis are necessary for Slam localization and, conversely, altering the geometry of their expression is sufficient to reorient Slam polarity. An endogenous planar polarity in intercalating cells has been shown to be manifested by the accumulation of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 at D-V cell borders. Moreover, germband extension is defective in bazooka mutant embryos, supporting a model where molecular polarization of the cell surface is a prerequisite for polarized cell movement. Therefore, differences in gene expression along the A-P axis may direct planar polarity in intercalating cells through the creation of molecularly distinct cell-cell interfaces that differ in migratory potential (Zallen, 2004).
Cell movement during germband extension is oriented along the D-V axis, suggesting a mechanism that restricts the productive generation of motility to dorsal and ventral cell surfaces. Molecules that are asymmetrically localized during convergent extension may therefore contribute to the spatial regulation of cell motility. Interestingly, intercalating cells in the Drosophila germband display a polarized localization of the ectopically expressed Slam protein, a novel cytoplasmic factor required for cellularization in the early embryo (Lecuit, 2002). While proteins such as Armadillo/β-catenin are uniformly distributed at the cell surface, ectopic Slam is enriched in borders between neighboring cells along the A-P axis. This polarized Slam population is present in a punctate apical distribution, coincident with the adherens junction component Armadillo/β-catenin. Therefore, intercalating cells have distinct apical junctional domains that differ in their capacity for Slam association (Zallen, 2004).
Interestingly, the polarized distribution of ectopic Slam protein is spatially and temporally correlated with intercalary behavior. Slam polarity is not observed in Stage 6 embryos prior to the onset of intercalation. Slam accumulation at A-P cell borders first appears in late Stage 7, when cells of the germband initiate intercalation, and reaches its full extent during the period of sustained intercalation in Stage 8. In contrast, Slam is uniformly distributed in cells of the head region and the dorsal ectoderm, tissues which do not undergo intercalary movements. These results indicate that the polarized distribution of ectopic Slam protein is specific to intercalating cells and that Slam can therefore serve as a molecular marker for the visualization of polarized cell behavior (Zallen, 2004).
The enrichment of Slam at borders between neighboring cells along the A-P axis is consistent with two modes of localization: Slam could mark one side of each cell in a unipolar distribution, or Slam could localize to both anterior and posterior surfaces in a bipolar pattern. To distinguish between these possibilities, mosaic embryos were generated where Slam-expressing cells were juxtaposed with unlabeled cells, using the Horka mutation to induce sporadic chromosome loss in early embryos. Slam protein accumulates at anterior and posterior boundaries of mosaic clone, indicating that ectopic Slam protein is targeted to both anterior and posterior surfaces of intercalating cells in a symmetric, bipolar distribution. The bipolar localization of ectopic Slam corresponds well with the bidirectionality of cell movement during germband extension, where cells are equally likely to migrate dorsally or ventrally during intercalation. Bipolar motility is also observed during convergent extension in the presumptive Xenopus and Ciona notochords and in Xenopus neural plate cells in the absence of midline structures (Zallen, 2004).
To extend the spatial and temporal correlation between Slam polarity and cell movement, it was asked if this polarized Slam localization is achieved in mutants that are defective for intercalation. Cell intercalation is dependent on the transcriptional cascade that generates cell fates along the A-P axis, in the direction of tissue elongation and perpendicular to the migrations of individual cells. A-P patterning reflects the hierarchical action of maternal, gap, and pair-rule genes. Cell fate differences along the A-P axis are abolished in embryos maternally deficient for the bicoid, nanos, and torso-like genes (referred to as bicoid nanos torso-like mutants), and these mutant embryos do not exhibit intercalary behavior. Ectopic Slam is correctly targeted to the apical cell surface in bicoid nanos torso-like mutants, but fails to adopt a polarized distribution in the plane of the epithelium (Zallen, 2004).
Downstream of the maternal patterning genes, gap genes establish overlapping subdomains along the A-P axis. A quadruple mutant for the gap genes knirps, hunchback, forkhead, and tailless lacks A-P pattern within the germband while retaining terminal structures. This quadruple mutant exhibits severely reduced cell intercalation, and mutant embryos also display a loss of Slam polarity. The absence of planar polarity in A-P patterning mutants correlates with a more hexagonal appearance of germband cells, in contrast to the irregular morphology of wild-type intercalating cells (Zallen, 2004).
In response to maternal and gap genes, pair-rule patterning genes expressed in narrow stripes act in combination to assign each cell a distinct fate along the A-P axis. In particular, the even-skipped (eve) and runt pair-rule genes are essential for germband extension. This strong requirement for eve and runt during germband extension contrasts with the more subtle effects in mutants for other pair-rule genes such as hairy and ftz. Consistent with these defects in intercalation, eve and runt mutants also display aberrant Slam localization. These results establish a correlation between intercalary behavior and the polarized localization of the ectopic Slam marker (Zallen, 2004).
While eve and runt mutants fail to complete germband extension, they extend further than embryos lacking maternal and gap genes, suggesting that some intercalary behavior is retained. Consistent with this possibility, Slam polarity is only partially disrupted in eve and runt mutants. While some cells display an aberrant uniform Slam distribution, in other cells Slam is correctly enriched at A-P cell interfaces. Therefore, the residual intercalation in eve and runt mutants correlates with the establishment of planar polarity in a subset of germband cells (Zallen, 2004).
The disruption of Slam polarity in A-P patterning mutants demonstrates that proper gene expression along the A-P axis is required for planar polarity in intercalating cells. In particular, the Eve and Runt transcription factors are expressed in 7 stripes at the onset of germband extension and 14 stripes as intercalation proceeds. Each cell in the germband is assigned a fate distinct from its anterior and posterior neighbors through the graded and partially overlapping expression of these and other pair-rule genes. Slam preferentially accumulates at contacts between cells with different levels of pair-rule gene activity, suggesting a model where cells concentrate specific proteins at interfaces with neighbors that differ in A-P identity. To directly address this model, mosaic embryos were generated with altered patterns of pair-rule gene expression in order to artificially introduce differences between dorsal and ventral neighbors. It was then asked if Slam protein is aberrantly recruited to these ectopic juxtapositions between different cell types, even at interfaces that are perpendicular to the normal axis of polarity (Zallen, 2004).
The Horka mutation was used to generate embryos that ectopically express Eve or Runt in a mosaic pattern. When these genes are ubiquitously expressed, planar polarity is generally disrupted and Slam displays a more uniform localization. This disruption of Slam polarity correlates with defective germband extension in Eve and Runt overexpressing embryos. The effects of Eve and Runt overexpression are not mimicked by overexpression of other pair-rule proteins such as Paired, Odd-paired, or Sloppy-paired. Moreover, localized sources of Eve or Runt expression direct aberrant patterns of polarity in mosaic embryos. For example, mosaic embryos display circles of Slam polarity that are bordered by ectopic Eve clones. Similarly, Slam polarity in germband cells is diverted from its normal orientation to follow boundaries of Runt misexpression. These results demonstrate that ectopic sites of Eve and Runt expression can reorient Slam polarity at clone boundaries, even when these interfaces are perpendicular to the normal axis of polarity (Zallen, 2004).
In contrast to the reorientation of planar polarity at boundaries of Eve and Runt misexpression, cells distant from the clone often exhibited complex patterns of Slam localization. These patterns may arise from nonautonomous effects of pair-rule gene activity, as well as aberrant cell movements and ectopic folds that form at clone boundaries, suggestive of a disruption in cell adhesion. Therefore mosaic embryos were examined at Stage 7, prior to the onset of cell movement and ectopic fold formation. While early Stage 7 embryos do not normally exhibit Slam polarity, ectopic Eve induces a precocious accumulation of Slam at clone boundaries. In contrast, ectopic Runt only occasionally induces a subtle polarity at Stage 7. The more potent effect of the eve transgene may reflect higher levels of ectopic expression compared to the endogenous eve stripes. These mosaic experiments indicate that differences in gene expression play an instructive role in the generation of planar polarity in intercalating cells. While Eve and Runt are both sufficient for planar polarity, the absence of either gene alone disrupts polarity. However, the defects in eve or runt single mutants may result from a combined disruption of multiple pair-rule genes; loss of eve leads to altered runt expression and vice versa (Zallen, 2004).
Generation of planar polarity by ectopic Eve expression is subject to the same spatial requirements as in wild-type polarity: Eve clones in the head region failed to induce polarity, suggesting that these cells are resistant to Eve-dependent polarization. In contrast, ectopic Runt expression in the head led to a concentration of Slam at clone boundaries, despite the fact that these cells do not normally display Slam polarity or intercalary behavior. These results indicate that in contrast to Eve, Runt can induce planar polarity in head cells, raising the possibility of functional distinctions between Eve and Runt target genes (Zallen, 2004).
The Eve and Runt transcription factors ultimately direct Slam polarity and cell intercalation through the transcriptional regulation of target genes. To identify downstream effectors involved in this process, components of the noncanonical planar cell polarity (PCP) pathway, which is required for convergent extension in vertebrates, were examined. Germband extension occurs normally in the majority of embryos lacking the Frizzled and Frizzled2 receptors. Similarly, germband extension is unaffected in the absence of Dishevelled. Moreover, dishevelled mutants exhibit a normal polarization of the Slam marker. These results demonstrate that molecular and behavioral properties of planar polarity in the Drosophila germband do not require Frizzled or Dishevelled function (Zallen, 2004).
The polarized distribution of ectopic Slam in intercalating cells provides the first clue to a molecular distinction between D-V cell interfaces that generate productive cell motility and A-P interfaces that do not. However, endogenous Slam mRNA and protein are not detected during germband extension, indicating that Slam may not play a functional role in cell intercalation. Slam colocalizes with the Zipper nonmuscle myosin II heavy chain subunit during cellularization and when Slam is ectopically expressed at germband extension (Lecuit, 2002). Therefore, the endogenous distribution of myosin II was examined during germband extension in wild-type embryos. During cell intercalation, myosin II is present in a punctate distribution at the apical cell surface, colocalizing with the adherens junction component Armadillo/β-catenin. In Stage 8 embryos, apical myosin II protein accumulates at interfaces between cells along the A-P axis. Slam can enhance this polarized localization when ectopically expressed (Lecuit, 2002), suggesting that Slam and myosin II may associate with a common localization machinery. Myosin II polarity is not apparent in Stage 6 or early Stage 7 embryos that have not begun intercalation, indicating that the enrichment of myosin II at A-P interfaces is specific to intercalating cells (Zallen, 2004).
The localized distribution of myosin II is not as pronounced as that of ectopic Slam, suggesting that additional asymmetries contribute to the polarization of intercalating cells. To identify such proteins, the localization was examined of components implicated in cell polarity in other cell types. In particular, the PDZ domain protein Bazooka/PAR-3 participates in both apical-basal and planar polarity. Bazooka/PAR-3 also exhibits a polarized distribution in intercalating cells. Bazooka, like myosin II, is present in a punctate apical distribution, coincident with the adherens junction component Armadillo/β-catenin. However, in contrast to the accumulation of myosin II at A-P cell interfaces, Bazooka is enriched in the reciprocal D-V interfaces. Bazooka polarity is specific to intercalating cells, where it first appears at the onset of intercalary movements in late Stage 7. Bazooka polarity is not observed in cells of the head region, which do not undergo intercalation, nor is it observed in germband cells following the completion of germband extension at Stage 9 (Zallen, 2004).
To characterize the relationship between cell shape and the polarized localization of cortical proteins, the orientation of cell borders was measured as an angle relative to the A-P axis (with A-P interfaces closer to 90° and D-V interfaces closer to 0° and 180°). Interfaces from embryos stained for Bazooka and myosin II were ranked according to mean fluorescence intensity as a relative measure of protein distribution. These results illustrate that Bazooka and myosin II are enriched in distinct sets of cell-cell interfaces that adopt largely nonoverlapping orientations relative to the A-P axis. This quantitation confirms the visual impression from confocal images and demonstrates that the molecular composition of a cell surface domain is a reliable predictor of its orientation within the epithelial cell sheet (Zallen, 2004).
The polarized localization of Bazooka is abolished in the absence of A-P patterning information in bicoid nanos torso-like mutant embryos. A similar disruption of myosin II polarity is observed in A-P patterning mutants. The A-P patterning system may therefore mediate cell intercalation through the polarized accumulation of cell surface-associated proteins. Bazooka participates in a conserved protein complex containing the atypical PKC (DaPKC), and DaPKC is also enriched in D-V cell interfaces during germband extension (Zallen, 2004).
To determine whether the polarized Bazooka/PAR-3 protein is functionally required for germband extension, homozygous bazooka (baz) mutant embryos were examined. In zygotic baz mutants, residual Bazooka protein persists from maternal stores and is often, but not always, correctly distributed along the apical-basal and planar axes. Despite this maternal Bazooka contribution, loss of zygotic Bazooka disrupts germband extension. In wild-type embryos, the posterior end of the extended germband is located at 70% egg length from the posterior pole. Of the progeny of bazYD97/+ females and wild-type males, 72% were wild-type-like, 25% were partially defective, and 3% were strongly defective. These results demonstrate that Bazooka is required for normal germband extension (Zallen, 2004).
Bazooka/PAR-3 and the associated DmPAR-6 and DaPKC components also influence epithelial cell polarity along the apical-basal axis. To address the possibility that germband extension defects may occur indirectly as a result of disrupted apical-basal polarity, properties of apical-basal polarity were examined in zygotic baz mutants, where some functions are carried out by maternal gene products. Zygotic baz mutant embryos exhibit several signs of normal apical-basal polarity at gastrulation, including a monolayer epithelial morphology in the germband and the correct distribution of proteins to apical and lateral membrane domains. This is consistent with findings that zygotic baz mutants exhibit proper localization of the Armadillo/β-catenin adherens junction component prior to Stage 10 of embryogenesis. These results demonstrate that properties of apical-basal polarity are established correctly in baz mutant embryos during germband extension, consistent with a direct role for Bazooka in cell movements along the planar axis, independent of its later effects on apical-basal polarity (Zallen, 2004).
The local reorientation of planar polarity in response to Eve and Runt expression argues that planar polarity is generated by cell-cell interactions, rather than a distant polarizing cue. In addition to these local effects of Eve and Runt on planar polarity, Slam polarity frequently adopted a circular pattern in mosaic embryos, even when Eve and Runt were not present along the entire circumference of the circle. This unexpected configuration indicates that polarizing information can propagate from cell to cell downstream of an Eve-dependent signal. A similar relay mechanism is suggested by the swirling patterns of wing hair polarity that persist in Drosophila mutants defective for the PCP signaling pathway. Therefore, mechanisms of cell-cell communication may reinforce local polarizing events in the organization of a two-dimensional cell population (Zallen, 2004).
Planar polarity in Drosophila germband extension is locally established through the concentration of specific proteins at sites of contact between cells with different levels of Eve and Runt expression. Cells can monitor the identity of their neighbors through qualitative or quantitative differences in the activity of cell surface proteins, perhaps through ligand-receptor mediated signaling events or adhesion-based cell sorting. Transcriptional targets of Eve and Runt are therefore likely to include components that mediate intercellular signaling events involved in the transmission of polarizing information during multicellular reorganization (Zallen, 2004).
Axon pathfinding and target choice are governed by cell type-specific responses to external cues. In the
Drosophila embryo, motorneurons with targets in the dorsal muscle field express the homeobox gene even-skipped and
this expression is necessary and sufficient to direct motor axons into the dorsal muscle field. Motorneurons projecting to ventral targets express the LIM homeobox gene islet, which is sufficient to direct axons to the
ventral muscle field (Thor, 1997). Thus, even-skipped complements the function of islet, and together these two genes constitute a
bimodal switch regulating axonal growth and directing motor axons to ventral or dorsal regions of the muscle field (Landgraf, 1999).
After segmentation is complete, Eve expression in the wild-type embryo is very restricted. In the
mesoderm, Eve is expressed in a subset of pericardial cells and muscle DA1. In the CNS, Eve is expressed in approximately 16 cells per
abdominal hemineuromere: medially, the pCC and fpCC interneurons and the aCC and RP2
motorneurons; mediolaterally, four CQ neurons, and laterally, the eight to ten EL interneurons. Of the Eve-expressing
motorneurons, aCC and RP2 innervate dorsal muscles DA1 [muscle number 1] and DA2 [number 2], respectively, and the four CQ neurons are thought to project to other dorsal
muscles (Landgraf, 1999 and references).
The fact that Eve is expressed in several dorsally projecting motorneurons and that aCC and RP2 have
been shown to require Eve function to project to their dorsal targets suggests
that Eve expression might be a common property of motorneurons that project through the ISN into the
dorsal muscle field. To test this idea, the pattern of innervation in late stage 16 embryos was established by retrograde
labeling of motorneurons with DiI and subsequent double labeling with
anti-Eve antibody. Six motorneurons express Eve, and these exclusively innervate dorsal
to dorsolateral muscles. aCC and RP2 innervate muscles DA1 [1] and DA2 [2],
respectively. In addition, muscles DO1-2 [9
and 10], DA3 [3], and LL1 [4] are innervated by the four ventral mediolateral CQ neurons. The CQ neurons correspond to the so-called U neurons by position, morphology, and muscle target. While Eve-expressing motorneurons
exclusively innervate the most dorsal and two dorsolateral targets, there is also an array of four
dorsolateral muscles, DO3-5 [11, 19, and 20] and DT1 [18], that are innervated by
non-Eve-expressing motorneurons (Landgraf, 1999 and references).
To investigate the requirement for Eve for the formation and projection patterns of the Eve-expressing
neurons, a temperature-sensitive eve allele was used to remove Eve protein function at different
times of development. Eve is expressed at 5 hr after egg laying (AEL) in the ganglion mother cells that
give rise to the Eve-positive neurons, of
which aCC and pCC are the first born at 6 hr AEL. At 9.45 to 10 hr AEL, aCC
generates an axon that pioneers the ISN, closely followed by the axons of the U/CQ and RP2 neurons. When Eve function is removed from 5 hr AEL onward, most Eve-expressing neurons form, suggesting that Eve function at this stage is not essential for the generation
of the Eve-positive neurons. However, the dorsal projections of motor axons in these embryos are
always abnormal. The ISN, through which aCC, RP2, and the U/CQ axons project, is arrested
prematurely in the ventral or dorsolateral region of the muscle field, leaving dorsal muscles without
innervation. Removing Eve function at progressively later
times, the occurrence of dorsolateral ISN truncation is increasingly less frequent. By 9-10
hr AEL, that is, as the axons of aCC, RP2, and the U/CQ neurons exit the CNS, removal of Eve function rarely affects formation
of the ISN. It is concluded that Eve function is required in motorneurons (but not their targets) prior to or during the
early phase of ISN formation if their axons are to grow dorsally. By ectopic expression of Eve, Eve has been shown to be sufficient to direct motor axons into the ISN path toward dorsal muscles. Misexpression of Eve in motorneurons, which do
not normally express Eve, is sufficient to alter dramatically their patterns of pathfinding and fasciculation so
as to direct their axons via the ISN to the dorsal muscle field (Landgraf, 1999).
Because ectopic Eve expression causes a dramatic rerouting of those motor axons that would normally
innervate ventral or lateral muscles, the development of motorneuron projections onto ventral and
lateral targets is disrupted. However, there is ample evidence that
in the Drosophila embryo innervation can take place despite misrouting of motor axons, although
often with a delay. While Eve expression dictates the growth properties of motorneurons so that they direct their
axons via the ISN into the dorsal muscle field, the properties that are required for target recognition
appear to be unaffected by ectopic Eve (Landgraf, 1999).
How do transcriptional regulators such as Eve and Islet direct patterns of axonal growth? The
phenotype observed in ectopic Eve embryos (fusion of the main nerve trunks and
failure of secondary nerve branching) is similar to, though more severe than, phenotypes produced in
embryos where general interaxonal adhesion is increased either by overexpression of the homophilic
CAM Fas II or by removal of its antagonist Beaten path (Beat). In such embryos, the two main nerve trunks (SN and ISN)
form, but secondary nerves fail to branch off. A test was carried out to see if ectopic Eve increases interaxonal
adhesion by downregulating the antiadhesive neural CAM antagonist Beat. No
significant changes were seen in the overall pattern or relative levels of BEAT mRNA expression in ectopic Eve embryos. The expression patterns of the
major neural CAMs Fas II, Fas III, and Connectin were examined in ectopic Eve or eve mutant
embryos, but no changes in their expression patterns were detected. To test if Eve might regulate the expression of another (as yet unidentified) neural CAM, it was
reasoned that beat might antagonize interaxonal adhesion mediated by such a CAM, just as beat
antagonizes adhesion mediated by Fas II and Connectin. When Eve and beat are ectopically co-expressed, the ectopic Eve phenotype of
excessive axonal fasciculation is partially rescued.
Thus, Eve directs motor axons to the dorsal region of the muscle field by suppressing expression of
the ventrally directing islet gene and by promoting adhesion to the ISN (Landgraf, 1999).
There is an interesting correlation between the expression of islet homologs in vertebrate and
invertebrate motorneurons. However, while all vertebrate motorneurons express islet-1 and/or islet-2, only a subset of motorneurons express islet in Drosophila
(Thor, 1997). Another subset expresses eve, and there may well be
further subsets expressing other genes that direct axons to different parts of the muscle field. For instance,
the dorsolateral muscles DO3-5 [11, 19, and 20] and DT1 [18] are innervated by at least four
intersegmental motorneurons that express neither eve nor islet. Thus, there may be a third gene that defines the dorsolateral sector of the
muscle field as the target area of these motorneurons. Interestingly though, the axonal
projections of the DO3-5 [11, 19, and 20] and DT1 [18] motorneurons are frequently affected by loss of
Eve function. This suggests that these motorneurons rely on the axons of the Eve-expressing cells for
pathfinding. In addition, motorneurons whose axons project through the SN express neither eve nor
islet, and their growth patterns are likely to be regulated by other genes.
Interestingly, the gene vab-7 in the nematode Caenorhabditis elegans (a homolog of the Drosophila eve gene)
is also expressed in a set of motorneurons that go to dorsal targets, and it is required for their correct
pathfinding (B. Esmaeili and J. Ahringer, personal communication to Landgraf, 1999). Thus, it appears that the function of
eve in directing patterns of motorneuron growth is an ancient one (Landgraf, 1999).
Nervous system-specific eve mutants were created by removing regulatory elements from a 16 kb transgene capable of complete rescue of normal eve function. When transgenes lacking the regulatory element for either RP2+a/pCC, EL or U/CQ neurons were placed in an eve-null background, eve expression was completely eliminated in the corresponding neurons, without affecting other aspects of eve expression. Many of these transgenic flies are able to survive to fertile adulthood. In the RP2+a/pCC mutant flies: (1) both RP2 and aCC show abnormal axonal projection patterns, failing to innervate their normal target muscles; (2) the cell bodies of these neurons are positioned abnormally; and (3) in contrast to the wild type, pCC axons often cross the midline. The Eve HD alone is able to provide a weak, partial rescue of the mutant phenotype, while both the Groucho-dependent and -independent repressor domains contribute equally to full rescue of each aspect of the mutant phenotype. Complete rescue is also obtained with a chimeric protein containing the Eve HD and the Engrailed repressor domain. Consistent with the apparent sufficiency of repressor function, a fusion protein between the Gal4 DNA-binding domain and Eve repressor domains is capable of actively repressing UAS target genes in these neurons. A key target of the repressor function of Eve is Drosophila Hb9 (Extra-extra), the derepression of which correlates with the mutant phenotype in individual eve-mutant neurons. Finally, homologs of Eve from diverse species are able to rescue the eve mutant phenotype, indicating conservation of both targeting and repression functions in the nervous system (Fujioka, 2003).
A regulatory element capable of driving expression in neurons RP2, aCC and pCC has been identified in the eve locus. It was
asked whether this region is necessary for expression in the context of a
transgene capable of complete functional rescue of eve null mutants.
This rescue transgene extends from –6.4 to either +8.6 or +9.2 kb, with either end point providing full function when homozygous, at most chromosomal insertion sites. A transgene construct was used with a deletion in the RP2+a/pCC enhancer region to fully rescue segmentation function, while simultaneously removing it from the RP2, aCC and pCC neurons and their progenitors. In these rescued embryos, eve expression was never observed in RP2 and a/pCC neurons, while
eve expression was normal in EL and U/CQ neurons in all lines.This combination of rescuing transgenes in the
background of a null mutation at the endogenous eve locus is referred to as the RP2 mutant (Fujioka, 2003).
Cell-type-specific U/CQ mutant flies were created analogously, by deleting the U/CQ expression element from the full-length rescue construct and placing the resulting transgene in an eve-null background. EL mutants were similarly made using a deletion of the EL enhancer. These deletions resulted in the loss of detectable expression specifically in either U/CQ or EL neurons, respectively (Fujioka, 2003).
These data indicate that each of the neuronal regulatory elements is not
only sufficient, but is also necessary for eve expression in the
corresponding set of neurons. Somewhat surprisingly, each of these neuronal
specific mutants survived to adulthood, and neither mutant adults nor larvae showed any obvious behavioral abnormalities (Fujioka, 2003).
Although eve expression was eliminated in specific subsets of
neurons in these transgenic lines, marker experiments show the presence of the eve mutant cells, showing that eve function is not required to maintain the activity of the eve neuronal enhancers, and that these neurons still exist without eve function (Fujioka, 2003).
The mutants that were constructed lack all detectable Eve expression either in the combination of RP2 and a/pCC neurons, or in the U/CQ neurons, or in the EL neurons, and the defects in the first of these was analyzed in detail. In this case, despite
significant defects in axonal architecture, individuals were
able to survive to fertile adulthood. In a preliminary analysis, no behavioral abnormalities were detected in either larvae or adult flies. Eve is normally also expressed in the parental GMCs of
RP2, aCC, pCC and the U/CQ neurons, and this expression is also eliminated in these mutants. The removal of Eve from these neuronal lineages does not cause a loss of neurons, showing that eve function in the GMCs is not required for their normal cell division to occur (Fujioka, 2003).
In order to identify the mutant neurons and to analyze the axonal
phenotypes, the eve neuronal elements were used to drive marker gene
expression. Expression in RP2+a/pCC was enhanced using the Gal4-UAS system. In order to be effective, Gal4 activity needed to be increased by
replacing the yeast translational initiation signal in the Gal4 driver
transgene with that from eve, and by multimerizing the enhancer. With these modifications, in combination with a UAS-taulacZ transgene, the axons of RP2, aCC and pCC were clearly marked. Combining the same Gal4 driver with UAS-CD8GFP allowed the marking of axons with membrane-localized GFP. This allowed an examination of how far mutant axons grow towards the muscle field, and visualization of connections to specific target muscles (Fujioka, 2003).
In the absence of eve function, all neuronal enhancer elements
remain active. This suggests that the cells do not completely change their
identities in the absence of Eve, but continue to express the combination of
factors that normally initiate and maintain eve expression, and which
presumably act in concert with Eve to specify the phenotype (Fujioka, 2003).
Using a combination of the constructed cell-specific mutant and marker
transgenes, the morphology of RP2 mutant neurons was analyzed.
The mutant RP2s exhibit a variety of defects in axonal morphology, and in addition they are defective in their ability to migrate to their normal position within the CNS. Although the abnormal
position of mutant RP2s might affect their ability to extend axons normally,
it does not seem to be a primary determinant of whether they extend to the
muscle field, because the position defect is often rescued by the Eve protein without its repressor domains (EveNH, supplied by an additional rescue transgene expressing this protein); and yet, in this case, axons still fail to extend properly. In
addition, there are a few abnormally located RP2s in wild-type embryos, and
they still extend their axons normally. The mutant RP2s seem
to retain some axonal guidance capability, since their axons often recognize
their normal point of exit from the CNS along the ISN, once they 'happen' onto it, although many of these fail to extend further. The RP2 mutant axons
were never observed to extend as far as their normal target muscles, and
indeed, many of them do not exit the CNS. Of those that do exit
the CNS, most appear to be unable to defasciculate from the ISN onto muscle
targets. It is unlikely that the failure of mutant RP2 axons to exit the CNS
by stage 16 is due solely to a delay in either axon outgrowth or recognition
of the nerve roots, because similar percentages of peripherally
projecting RP2 neurons are found in both stage 16 embryos and young first instar larvae (Fujioka, 2003).
The inability of the mutant axons to reach the muscle field is partially
rescued by the Eve HD without its repressor domains. However, in
addition to the HD, the two distinct repressor domains of Eve contribute
strongly to rescue of the mutant phenotype. Interestingly,
complete rescue can also be provided by the Eve HD fused with a heterologous
repressor domain from Engrailed. Therefore, in addition to the functions provided by the DNA-binding HD, a generic repressor function of sufficient strength is required for normal function in these neurons. The fact that the HD alone can provide a detectable degree of rescue, which contrasts with the lack of its ability to rescue segmentation function,
suggests that perhaps competition for binding sites with activators of
downstream target genes plays a more prominent role in Eve function in the
nervous system. Consistent with the requirement for its repressor domains, an Eve-Gal4 fusion protein is able to actively repress a UAS-containing target gene in RP2 neurons (Fujioka, 2003).
The requirement for Eve in axonal guidance is somewhat more stringent in
aCC than in RP2 neurons. Although a significant fraction of mutant RP2s
initially extend axons in the same direction as wild-type RP2s, essentially
none of mutant aCCs do so. In addition, unlike for RP2s, the aCC phenotype is not significantly rescued by either the HD alone or the HD with the N terminus (which provides no detectable repression activity, but might stabilize the protein). In aCC, as in RP2, the phenotype is partially rescued by including either repressor domain, and the Engrailed repressor domain is able to provide full activity. Furthermore, Eve repressor domains are able to actively repress a UAS target gene in aCC neurons.
These data indicate that the primary function of eve in aCC is to
actively repress target genes. The more stringent requirements in aCC versus
RP2 suggest that there may be different target genes in these two motoneurons,
although Drosophila Hb9 is a common target (Fujioka, 2003).
In mutant pCC neurons, in contrast to the wild type, 40% of axons crossed
the ventral midline to the contralateral side. This phenotype is
rescued quite effectively by the HD alone, suggesting that the target gene(s) involved may be passively repressed through a competition for activator binding sites (although more complex possibilities cannot be ruled out). Recent studies have shown that midline crossing is regulated by a complex interplay of responses to attractive and repellent signals secreted by midline cells. One possibility is that the midline crossing phenotype of
mutant pCCs might be caused by derepression of the DCC/Frazzled receptor for
the midline attractant Netrins (Fujioka, 2003).
In addition to the defects in axonal morphology, the cell body position of pCC relative to that of its sibling aCC is apparently randomized in the mutant. It is not known whether this is due to the lack of Eve in aCC, in pCC, or in both. Since other
neurons in the CNS are wild type, it is unlikely to be an effect involving
surrounding neurons. This defect is rescued effectively only when the Eve HD
is accompanied by at least one repressor domain. It is unclear whether the
normally tight control of this characteristic has a role in the subsequent
morphogenesis of the neurons (Fujioka, 2003).
Previous studies using the eve temperature-sensitive allele
eveID19 show that eve is required in
eve-positive motoneurons for proper axonal morphology, including the
ability to reach the dorsal muscle field. The
eveID19 allele contains a point mutation in the HD, and
shows a near-null segmentation phenotype at the restrictive temperature. However, the data indicate that eveID19
is not a true null in the nervous system. This might explain
the differences between the morphological phenotypes of mutant RP2, aCC and
pCC neurons that are observed in the cell-type specific mutant as compared with those seen in in the
eveID19 mutant (Fujioka, 2003).
A significant variation in phenotype with a small change in the level of
function is consistent with an interpretation wherein loss of Eve leads to the absence of a particular subset of neuronal properties. Interestingly, even in the mutant, which has no detectable Eve expression in these lineages, some RP2s as well as some pCCs show several of the characteristics of their wild-type counterparts. It is assumed that the mutant represents the complete null phenotype: this indicates that, to a limited extent, Eve acts in parallel with other factors in the specification of these cell types, rather than being an overall determinant of the cell fate. This notion is also consistent with the fact that the eve regulatory elements, which are specific markers for these cell types, continue to be active in the absence of eve function (Fujioka, 2003).
Drosophila Hb9 and Eve are expressed in a non-overlapping pattern in the wild-type CNS, and ectopic Eve expression represses Hb9, indicating that Hb9 is a target gene of Eve. Hb9 is derepressed in the RP2 mutant in both RP2 and aCC, but not in pCC neurons (the RP2 mutant lacks Eve in all three cell types), showing that there are significant differences in target gene regulation in different neurons, even in those derived from the same GMC (in the case of aCC and pCC) (Fujioka, 2003).
When the Eve HD alone is used to rescue the RP2 mutant, Hb9 is repressed in
many of the RP2 neurons, and this seemingly stochastic repression correlates
with a more normal axonal morphology. However, effective repression,
particularly in aCC, requires active repression domains, with either of the
repressor domains of Eve alone providing partial activity (in the context of
the Eve HD). Although there is a strong correlation in situations of partial
rescue between the axonal phenotypes of individual neurons and derepression of Hb9, this correlation is not 100%. This suggests that there may be other key target genes that mediate Eve neuronal function in addition to Hb9. The level of expression of the antigen (Futsch) of the monoclonal antibody 22C10 is reduced in RP2 and aCC in the absence of Eve. However, the gene encoding this antigen is likely to be an indirect target of Eve, because its expression is activated rather than repressed by Eve (Fujioka, 2003).
Either of the repressor domains of Eve is sufficient to give a similar
degree of partial rescue of each of the phenotypes studied in the
nervous system, including the repression of Hb9, showing that these
repressor domains provide a similar function. In fact, two copies of a
transgene expressing either EveDeltaC or EveDeltaR are able to rescue to a
similar degree as that of one copy of the wild-type transgene. Thus, the recruitment of either of two apparently distinct co-repressors, Groucho or Atrophin, produces the same net result. The two are used in these neurons in an additive fashion to generate the appropriate level of Eve repressor activity, with no apparent target gene specificity (Fujioka, 2003).
A series of inductive signals are necessary to subdivide the
mesoderm in order to allow the formation of the progenitor
cells of the heart. Mesoderm-endogenous transcription
factors, such as those encoded by twist and tinman, seem to
cooperate with these signals to confer correct context and
competence for a cardiac cell fate. Additional factors are
likely to be required for the appropriate specification of
individual cell types within the forming heart. Similar to
tinman, the zinc finger- and homeobox-containing gene
zfh-1 is expressed in the early mesoderm and later in the
forming heart, suggesting a possible role in heart
development. zfh-1 is specifically
required for formation of the even-skipped (eve)-expressing
subset of pericardial cells (EPCs), without affecting the
formation of their siblings, the founders of a dorsal body
wall muscle (DA1). In addition to zfh-1, mesodermal eve
itself appears to be needed for correct EPC differentiation,
possibly as a direct target of zfh-1. Epistasis experiments
show that zfh-1 specifies EPC development independent
of numb, the lineage gene that controls DA1 founder versus
EPC cell fate. The combinatorial control
mechanisms that specify the EPC cell fate in a spatially
precise pattern within the embryo are discussed (Su, 1999).
zfh-1 and the components of the numb pathway are not the only
factors required for specifying EPC or DA1 founder fates (or for
eve expression characteristic of these fates). A transcription
factor encoded by the lethal-of-scute gene is expressed in a
cluster of mesodermal cells out of which the EPC and other
muscle progenitors emerge aided by a laterally inhibitory
mechanism. lethal-of-scute, however, as well as another
transcription factor encoded by the Krüppel gene, which is
expressed in the DA1 (and other muscle) founder cells, are only
weakly required for the corresponding muscles to form. In contrast, the
Drosophila EGF signal transduction pathway plays an essential
role in DA1 specification. For example, in the
absence of the secreted EGF-receptor ligand spitz, the
number of EPCs is normal but nearly all the DA1 muscles fail
to form.
Since DA1 founders and EPCs are likely to derive from
common precursors and the phenotype of spi mutants is the
opposite of zfh-1, it was decided to determine whether or not zfh-1 and spitz function as part of a
common genetic pathway. The phenotype of spitz;zfh-1
double mutants was examined. In these double mutants, neither EPC- nor
DA1-specific eve expression is present, suggesting
that the Egf-r pathway is required for DA1 differentiation
independently of zfh-1. This raises the question of whether or not
Egf-r pathway activation is required for providing the correct
DA1 differentiation context in a way that is reminiscent of zfh-1
function, which provides a context for EPC differentiation. If yes,
it would be expected that spitz, like zfh-1, functions independently
of the numb pathway. Indeed, when numb is mesodermally
overexpressed in spitz mutant embryos, a phenotype similar
to that of spitz;zfh-1 double mutants is observed: neither EPC- and
nor DA1-specific eve expression is observed. Taken together, these results suggest that correct cell
type-specific differentiation depends on both asymmetric
segregation of cell fate determinants during cell division as
well as on the appropriate regional context. In this case, the
context information (zfh-1 or Egf-r activity) does not need to
be originating from a spatially localized source, but may act in
concert with other mesodermal context determinants (e.g.,
tinman) (Su, 1999).
eve is well known for its function in ectodermal
segmentation. eve also
participates in the patterning of the early mesoderm. eve null mutants lack visceral
and cardiac mesoderm altogether. As
described above, zfh-1 is required for the formation and/or
expression of eve in EPCs. Thus, mesodermal eve expression
itself may be needed for correct EPC differentiation. To
address this question, a temperature-sensitive
allele of eve (eveID), which produces a non-functional but
nevertheless antigenic protein at the non-permissive
temperature, was used. When eveID mutant
embryos are shifted to the non-permissive temperature for
2 hours at early stage 11, the number of EPCs (expressing
eve) is drastically reduced: on average only
24% of EPCs are present as compared to wild type. DA1
muscle formation also seems to be affected, but to a lesser
degree. The EPC deficiency is less severe
when the temperature shifts occurs earlier or later in
development. Interestingly, some of the
remaining EPCs in early stage 11 shifted embryos are located
at some distance from the heart tube,
suggesting that eve function during this critical time period
is required for correct differentiation of the EPCs. Thus, in
the absence of eve, the forming EPCs lose their association
with the heart and disappear.
Temperature shifts during the temperature-sensitive period
for EPC formation also affect the overall pericardial cell
population, as seen in zfh-1 mutant embryos, perhaps due in part to the lack of EPCs. In contrast to
the zfh-1 phenotype, however, the number of cardial cells, heart
tube formation and overall body muscle formation are not
significantly affected in early stage 11 shifted eveID embryos, which is not surprising since eve is not expressed in
these tissues during the critical period for EPC formation.
Although eve inactivation at earlier stages also perturbs neural
development (that is, RP2 neurons), in addition to visceral and
somatic muscle formation, stage 11 shifts
show no morphologically detectable CNS defects. It has been concluded that eve, in addition to zfh-1, is required
for the proper differentiation of the EPCs at the time when the
eve progenitors normally appear (Su, 1999).
Since zfh-1 is required for eve expression in the forming EPCs
and eve function is required for EPC differentiation, it was asked if eve function is sufficient to promote EPC
development in the absence of zfh-1. Mesodermal expression
of eve may be autoregulated, as is the case for the eve late stripe
expression element. Thus, eve expression may need to be
activated at least until autoregulation is initiated. The Gal4 system was used to ectopically
express eve in the mesoderm of zfh-1 mutant embryos until
(but not beyond) stage 11. At stage 14/15,
EPC-specific endogenous Eve protein expression was examined. In order to
achieve this, the twist promoter was used to drive Gal4
expression, which in turn drives the
eve cDNA under the control of UAS Gal4-binding sites. This
protocol to overexpress eve in the mesoderm of zfh-1 mutant
embryos partially restores the formation of EPCs.
These results support the hypothesis that eve acts downstream
of zfh-1 and that it is required itself for the proper formation
of EPCs. Since the rescue is partial, it cannot be ruled out that
normally the combination of both zfh-1 and eve functions are
necessary for EPC development. A putative consensus Zfh-1
homeodomain-binding sequence (P3/RCS1) is present within the eve mesodermal enhancer., and the homeodomain of Zfh-1 can bind to the putative
P3/RCSI consensus site in this enhancer. This finding
is consistent with the hypothesis that EPC-specific eve
expression is under the direct control of Zfh-1 (Su, 1999).
A model is provided of the genetic network regulating the specification and
differentiation of the EPC progenitors and their heart and muscle
associated progeny (EPC and DA1). Initially, the spatially coincident
activity of the transcription factor, Tinman, together with the
mesoderm-specific response induced by the patterning signals, Wg
and Dpp, are necessary to specify and position the most dorsal
portion of the mesoderm, which includes the EPC progenitors and
other cardiac precursors. The EPC progenitors then divide and
produce two types of progeny cells under the control of the lineage
gene numb. The daughter cell that inherits Numb protein will
differentiate as the DA1 muscle founder, because the Notch and spdo
encoded functions are inhibited, allowing Egf-r signaling (Spitz) to be
effective (perhaps in conjunction with Eve). In the daughter cell
without Numb, Notch signaling is operational and the transcription
factors Zfh-1 together with and/or mediated by Eve can effectively
contribute the correct differentiation of the EPC fate. Thus, three
levels of information appear to cooperate in the specification of a
particular cell fate: prepatterning or positional information,
asymmetric lineages and tissue context information (Su, 1999).
In Drosophila embryos, founder cells that give rise to cardiac precursors and dorsal somatic muscles derive from dorsally located progenitors. Individual fates of founder cells are thought to be specified by combinatorial code of transcription factors encoded by identity genes. To date, a large number of identity genes have been identified; however, the mechanisms by which these genes contribute to cell fate specification remain largely unknown. Regulatory interactions of ladybird (lb), msh and even skipped (eve), the three identity genes specifying a subset of heart and/or dorsal muscle precursors, have been analyzed. Deregulation of each of them alters the number of cells that express the other two genes, thus changing the ratio between cardiac and muscular cells, and the ratio between different cell subsets within the heart and within the dorsal muscles. Specifically, mutation of the muscle identity gene msh and misexpression of the heart identity gene lb leads to heart hyperplasia with similar cell fate modifications. In msh mutant embryos, the presumptive msh-muscle cells switch on lb or eve expression and are recruited to form supernumerary heart or dorsal muscle cells, thus indicating that msh functions as a repressor of lb and eve. Similarly, overexpression of lb represses endogenous msh and eve activity, hence leading to the respecification of msh and eve positive progenitors, resulting in the overproduction of a subset of heart cells. As deduced from heart and muscle phenotypes of numb mutant embryos, the cell fate modifications induced by gain-of-function of identity genes are not lineage restricted. Consistent with all these observations, it is proposed that the major role of identity genes is to maintain their restricted expression by repressing other identity genes competent to respond positively to extrinsic signals. The cross-repressive interactions of identity genes are likely to ensure their localized expression over time, thus providing an essential element in establishing cell identity (Jagla, 2002).
Ectopically expressed lb has been shown to inhibit eve in the founder cell of the DA1 muscle. This effect may be due to either a specific inhibition of eve by lb or a more general regulatory mechanism of fate specification. Data presented here favour the latter possibility, showing that the gain of lb function affects expression of several identity genes and consequently influences fates of cells in which these genes are expressed. Specifically, embryos that ectopically express lb have an increased number of tin-positive heart cells with a concomitant reduction of dorsal muscles. To demonstrate that the supernumerary cardiac cells result from cell fate switches, rather than from additional proliferation, mshDelta mutants, displaying heart hyperplasia similar to that observed in embryos overexpressing lb, were used. In this particular msh mutant, the presumptive msh-positive muscle cells monitored by lacZ start to express cardiac markers. This suggests that switches from muscular to cardiac fates contribute to heart hyperplasia induced by deregulation of identity genes. Interestingly, the ectopic expression of lb and msh leads to reciprocal phenotypes, and indicates that the identity genes specifically expressed in the heart promote dorsal mesodermal cells to enter the cardiogenic pathway, while the muscle identity genes promote the myogenic pathway. However, more detailed analysis shows that ectopic lb promotes only specific cardiac fates and ectopic msh only specific muscle identities, thus indicating that the identity genes instruct dorsal mesodermal cells to adopt the specific cardiac or muscular fates, rather than make a choice between cardiac and muscular development. This property is particularly well illustrated by the phenotypes generated by the ectopic eve, which is involved in the specification of a subset of heart and dorsal muscle cells and when ectopically expressed promotes specification of supernumerary cells of both types. Moreover, deregulated heart and dorsal muscle identity genes preferentially affect fates of mesodermal cells located in dorsal but not in ventral regions, thus suggesting that the identity gene action is instructive only in a permissive context (Jagla, 2002).
This observation is in complete agreement with the model of competence domain. According to this concept, the high level of Wg and Dpp signals present in the anterodorsal region (under the intersection of Wg and Dpp epidermal domains) provides a major cue that direct mesodermal cells into cardiac or dorsal muscle development. In relation to this model, these data design a new regulatory mechanism that provides a paradigm of how the intrinsic transcription factors and extrinsic signaling molecules converge to specify cell fates (Jagla, 2002).
The findings suggest cross-repressive interactions that occur between transcription factors that specify adjacent and non-overlapping populations of muscle and heart cells. Most likely, in normal development, these interactions have a functional relevance once the progenitor cells segregate, and then continue to play an important role in the next step of cell fate diversification, namely in founder cells. The gain- and loss-of-function experiments presented indicate that the identity genes may function as repressors starting from the progenitor stage onwards. However, the earliest activation of inappropriate identity gene as a result of the loss of function of repressor (in mshDelta embryos) was documented in founder cells (Jagla, 2002).
It is proposed that cross-repressive interactions allow the refinement of the potentially imprecise pattern of identity gene expression induced by the interplay of Wg and Dpp signaling pathways. Wg and Dpp create a permissive context for the development of cardiac and dorsal muscle precursors. In such a context, the transcription factors that specify these two types of cells (e.g. lb, eve and msh) are expected to be activated in all dorsal mesodermal cells. The local restriction of identity gene expression is, however, provided by a combinatorial signaling code mediated by two receptor tyrosine kinases, the Drosophila epidermal growth factor receptor and the Heartless (Htl) fibroblast growth factor receptor. Transient localized activity of these two mesodermal signaling pathways is thought to subdivide the large competence domain into small clusters of equivalent cells from which individual progenitors segregate. Depending on the combination of RTKs activities, the individual identity genes are activated only in a defined equivalence group and in the resulting progenitor. This study defines an additional step to the aforementioned model. It is proposed that the major role of identity genes is to maintain their restricted expression in progenitors and subsequently in founder cells by repressing other identity genes competent to respond positively to Wg and Dpp signals. These cross-repressive interactions are likely to ensure constant localized identity gene expression over time, thus providing a crucial element in establishing cell identity (Jagla, 2002).
In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. To understand how cell fate information residing within founders is translated during differentiation, this study focused on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Muscle protein 20 (Mp20), Paxillin (Pax), and M-spondin (mspo), three genes that regulate actin dynamics and cell adhesion and, as is shown in this study, modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation (Bataillé, 2010).
The myoblast fusion is asymmetric and takes place between founder cells (FCs) and fusion competent myoblasts (FCMs). Previous reports originated the idea that FCMs are not 'naive' myoblasts and contribute to the modulation of fusion process. In contrast, the current results support a view that FCs rather than FCMs carry the instructive information and lead to the conclusion that FCMs do not play an active role in setting the number of fusion events. However, because the spatial distribution of FCMs seems to be nonuniform, it is conceivable that the local distribution of FCMs is coordinated with the requirements of FCs to facilitate fusion process (Bataillé, 2010).
The identity genes lb, slou, and eve are required to specify FCs at the origin of five muscles the DA1, DT1, SBM, VA2, and VT1. This study provides evidence that these identity genes are also required for setting the muscle-specific number of fusions and demonstrates how this identity information is executed. After specification step, FCs fuse, between the embryonic stage 12 and 15, with a determined number of FCMs to generate muscles with a specific number of nuclei. During this time period eve, lb, and slou continue to be expressed in subsets of developing muscles and the data show that they are sufficient to establish the muscle-specific fusion programs in DA1, SBM, and VT1 (11, 7, and 4 nuclei, respectively). Furthermore, slou in combination with other factors contributes to two other programs that end up with seven to eight fusion events in muscles DT1 and VA2. To regulate number of fusion events eve, lb, and slou act by modulating expression of genes involved in dynamics of actin cytoskeleton or cell adhesion. Starting from stage 13, they establish a muscle-specific combinatorial code of expression levels of three targets: Mp20, Pax, and mspo. The combination of expression of the targets leads to the muscle-specific control of the number of fusion events. This notion is supported by the fact that each of identity genes is able to impose at ectopic locations the combinatorial realisator code of Mp20, Pax, and mspo expression, and thus, ectopically execute its fusion program. Given that the code of Mp20, Pax, and mspo is not sufficient to explain fusion programs in all muscles, it is hypothesized that other identity gene targets exist that modulate fusion counting (Bataillé, 2010).
The data support a two-step model of myoblast fusion according to which a muscle precursor is formed between stage 12 and 13 by an initial fusion, and then, between stage 13 and 15, fuses with additional myoblasts until the muscle reaches its final size. The fact that Mp20, Pax, and mspo are expressed from stage 13 suggests that the transition point between the two steps depends not only on the timing of FCM migration but also on the activation of limiting factors such as the identity gene targets which modulate the number of additional fusions. Since no nuclear divisions were observed in FCs or in growing myotubes in any of the genetic contexts analyzed, it can confidently be said that the number of nuclei present in each muscle is determined only by the number of fusion events (Bataillé, 2010).
Specification of FCs requires combinatorial code of activities of identity genes. This study shows that the same identity genes play instructive roles in subsequent muscle-type-specific differentiation process. Importantly, the data enlighten the fact that the identity genes are not equivalent and have distinct, context-dependent mode of action. eve, lb, and slou are sufficient to set the fusion programs in DA1, SBM, and VT1 muscles; however, in VA2 and DT1 slou functions in a different way and seems not to have a decisive role in this process. Because the specification of the VA2 and DT1 FCs also involves functions of Poxm, Kr, and ap, it is hypothesized that they act together with slou in setting fusion programs of VA2 and DT1. This raises an important question about hierarchy of identity genes during execution of muscle identity programs and their roles in acquisition of specific properties of muscles such as number of nuclei, attachment points, and innervation (Bataillé, 2010).
The data presented in this study demonstrate that the number of fusion events in developing muscles is regulated by a muscle-specific combinatorial realisator code of identity gene targets. In contrast to the previously identified fusion genes acting in all muscles, the identified identity targets, Mp20, Pax, and mspo, display muscle-type specific expression and modulate fusion in a muscle-type-specific manner proportionally to the level of their expression. The loss and gain of function of each of them lead to subtle fusion phenotypes indicating that the range of fusion events controlled by these three candidates is limited. Indeed, the loss of function of Mp20 results in loss of two nuclei in a subset of muscles, whereas its overexpression induces the recruitment of maximum two FCMs. A similar range of defects in number of fusion events is observed in Pax and mspo mutant embryos indicating that they influence fusion process at the same level (Bataillé, 2010).
Mp20 encodes a cytoskeletal protein displaying restricted expression in adult muscles and sharing sequence homology with the lineage-restricted mouse proteins SM22alpha, SM22beta, and NP25. These proteins contain calponin-like repeats, and, in mammals, interact with F-actin and participate in the organization of the actin cytoskeleton. In Drosophila S2R cells, the RNAi knockdown of Mp20 induces a phenotype of round and nonadherent cells supporting its role in regulation of fusion process (Bataillé, 2010 and references therein).
The second candidate, Pax (DPxn37), is a scaffold protein that recruits structural and signaling molecules to the sites of focal adhesion. Pax has been shown to be involved in the actin cytoskeleton organization, cell adhesion, cell migration, and cell survival. In the developing Drosophila muscles, Pax protein localizes at muscle-tendon junctions suggesting that it may play a role in muscle attachment. The current analyses of Pax mutant embryos do not reveal muscle-tendon adhesion defects but show discrete myoblast fusion phenotypes, which correlate with differential muscle-specific expression of Pax. The role of Pax in modulating fusion is consistent with previously described implications of Pax interacting proteins, including ARF6 in myoblast fusion in both Drosophila and vertebrates, and FAK in vertebrates (Bataillé, 2010 and references therein).
Finally, mspo belongs to the F-Spondins, a conserved family of ECM proteins, which maintain cell-matrix adhesion in multiple tissues. In vertebrates, F-Spondins have context-dependent effects on axon outgrowth and cell migration. As Mp20, Pax, and Mspo are expressed in FC cells and growing myotubes, one possibility is that they modify the spreading and/or motility of FC protrusions required to attract FCMs. Alternatively, by modulating actin cytoskeleton, Mp20, Pax, and Mspo may also influence the stability of adhesion between the growing muscle and the FCM creating permissive conditions or blocking the progression of fusion process (Bataillé, 2010).
The muscle-type-specific regulation of fusion programs by the identity genes and their targets raises an intriguing question of how this regulation is executed from the mechanistic point of view. Because different levels of expression of Mp20, Pax, and mspo correlate with different fusion programs in both wild-type and genetically manipulated embryos, it was thought that by following kinetics of fusion in small and big muscles insights would be gained into how the fusion programs are modulated. It turns out that the rate of fusion is proportional to the size of muscle, meaning the number of fusion events, thus revealing that the identity genes acting via their targets set up the frequency of fusion events. Accordingly, loss and gain of function of identity genes and their targets identified here results in modulations of fusion programs by accelerating or slowing down the fusion rate. This finding provides insights into mechanistic understanding of muscle-type-specific regulation of fusion process and raises an important question about whether this mechanism is broadly conserved (Bataillé, 2010).
Development of a multicellular organism requires precise
coordination of cell division and cell type determination.
The selector homeoprotein Even skipped (Eve) plays a very
specific role in determining cell identity in the Drosophila
embryo, both during segmentation and in neuronal
development. However, studies of gene expression in eve
mutant embryos suggest that eve regulates the embryonic
expression of the vast majority of genes. Genetic interaction and phenotypic analysis is presented showing that
eve functions in the trol pathway to regulate the onset of
neuroblast division in the larval CNS. Surprisingly, Eve is
not detected in the regulated neuroblasts, and culture
experiments reveal that Eve is required in the body, not the
CNS. Furthermore, the effect of an eve mutation can be
rescued both in vivo and in culture by the hormone
ecdysone. These results suggest that eve is required to
produce a trans-acting factor that stimulates cell division
in the larval brain (Park, 2001).
Several genes have been identified that affect neuroblast
proliferation, including anachronism (ana), terribly reduced optic lobes (trol) and eve. trol was originally identified in
a genetic screen for abnormal larval brain morphology that
was due to defective patterns of neuroblast proliferation in
the larval brain. Mutations in trol
cause a dramatic decrease in the reactivation of proliferation
from mitotic quiescence. Recent studies
suggest that trol may regulate this reactivation of neuroblast
proliferation by stimulating the G1/S transition through
upregulation of Cyclin E (CycE) expression. Several studies on trol and ana have led to the hypothesis that trol is required to overcome the repression of neuroblast cell division imposed by ana. eve was identified in a screen for enhancers of a hypomorphic allele trol. Mutations in eve enhanced both the trol proliferation phenotype and the associated lethality, indicating that eve may regulate transcription of cell cycle genes in the trol pathway (Park, 2001).
Analysis of explants has shown that ecdysone
enables activation of neuroblast division and can
substitute for larval extract. Furthermore,
addition of ecdysone does not rescue the proliferation
phenotype of cultured trol mutant brains, implying that
ecdysone acts upstream of trol. Thus, ecdysone can overcome the lack of eve-induced
activity in extracts of mutant flies. Interestingly, almost
complete rescue is obtained when animals are fed
ecdysone from 16-20 hours posthatching, indicating that
the time between ecdysone action and S phase entry is at most
four hours (Park, 2001).
The genetic interaction between eve and trol has all the
characteristics expected for two components of a common
pathway: (1) the eve;trol interaction is not allele specific and
the known functional domains of Eve are implicated in the
interaction; (2) the strength of the interaction mirrors the
strength of the eve allele in segmentation; (3) eve mutants
themselves have the predicted proliferation phenotype; and (4)
neuroblasts arrested in trol;eve double heterozygotes can be rescued by
expression of CycE, as can the neuroblasts arrested in a strong
trol mutant. The latter is especially revealing, as induction of
CycE expression in trol mutants results in the activation of cell
division only in the number of neuroblasts appropriate to
the developmental stage of the induction. That is, not all mitotically quiescent neuroblasts are
arrested at the same cell cycle phase, and the extent to which
CycE is a limiting factor is developmentally controlled.
Therefore, as in embryonic segmentation and determination of
neuronal identity, eve appears to function in a specific genetic
pathway to affect the behavior of specific cells at specific
times (Park, 2001).
However, Eve is not detectable in regulated neuroblasts at
any time during first instar. Furthermore, eve function is not
required within the larval CNS, but is required within the larval
body from which extracts are prepared. Moreover, low levels
(10%-20%) of extract made from eve plus animals will not
support activation of neuroblast division while higher
concentrations will. This
concentration dependence indicates that eve does not inhibit
production of a trans-acting proliferation repressor that is
produced at higher levels in a eve mutant, since dilution of such
a repressor would allow neuroblast division at lower rather than
higher extract concentrations. These results strongly suggest
that eve function is required for the production of a trans-acting
factor that stimulates neuroblast division (Park, 2001).
Is ecdysone the trans-acting factor produced in response to
eve? Ecdysone can rescue eve-dependent proliferation defects
both in vivo and in vitro, but not the
proliferation defect of trol mutants in vitro.
This suggests that ecdysone acts upstream of trol, as would
be expected if it is the eve-dependent trans-acting signal, and
trol acts within the receiving cells. However, while the
ecdysone receptor has been detected in a few neurosecretory
cells of the first instar CNS, it has not been detected in
neuroblasts. This may indicate that only
a few high-affinity receptors are required to transduce the
ecdysone signal, or that ecdysone acts indirectly through the
products of the neurosecretory cells. However, as Eve is not
detectable in the neurosecretory cells in wild-type brain lobes, it is unlikely that the added ecdysone rescues mutant animals by compensating for a loss of Eve activity in those
cells. In each of these cases, eve could be acting through
ecdysone production. Alternatively, ecdysone may act
through a parallel pathway to that stimulated by an
(unknown) eve-dependent signal. While the relationship
between eve and ecdysone is not yet clear, it seems likely that
eve is required for the production of an organismal-level
trans-acting signal that is specifically required to stimulate
larval neuroblast proliferation (Park, 2001).
Recently, double-stranded RNA (dsRNA) has been found to be a potent and specific inhibitor of gene activity in the nematode Caenorhabditis elegans (Fire, 1998). The potential of dsRNA to interfere with the function of genes in Drosophila, termed RNA inhibition or RNAi) has been investigated. Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested. dsRNA corresponding to four genes with previously defined functions was introduced. dsRNA is shown to potently and specifically inhibits the activities of wg, fushi tarazu (ftz), even-skipped (eve), and tramtrack (ttk). The reception mechanism of the morphogen Wingless was determined using dsRNA. Interference of the frizzled and Drosophila frizzled 2 genes together produces defects in embryonic patterning that mimic the loss of wingless function. Interference with the function of either gene alone has no effect on patterning. Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. These results demonstrate that dsRNA interference can be used to analyze many aspects of gene function (Kennerdell, 1998).
To determine whether
dsRNA-mediated interference can occur in Drosophila, RNA was synthesized in vitro, allowed to anneal,
and then injected into syncytial blastoderm embryos. The ftz and eve genes were chosen for initial characterization of this method based on several criteria. Both genes are required for embryonic segmentation. Transcription of ftz and eve begins
approximately 90 to 120 min after egg laying, which corresponds to a time 10 to 60 min after dsRNA injection. Although both genes function in the first few
hours of embryogenesis, null mutant animals survive to the end of embryogenesis and exhibit segmentation
defects in their cuticle. Finally, mutants with reduced activity of either ftz or eve produce increasingly severe phenotypes, such that a semiquantitative relationship exists between genotype and phenotype. Antisense and sense RNAs for each gene were synthesized and annealed. Injection of either ftz- or eve-annealed RNA into wild-type embryos effectively interfers with gene activity as demonstrated by cuticle phenotypes characteristic of ftz or eve mutants. In contrast, antisense or sense RNAs injected separately have an order-of-magnitude weaker interference activity than annealed RNA. When a decreasing amount of ftz-annealed RNA is injected, interference activity declines also, though interference was still detectable at
the lowest dose. The abundance of each RNA strand at this dose was calculated to be about 2
million molecules per injected embryo. Assuming uniform distribution of RNA, the original injected material
is diluted to about 30 molecules per cell. Thus, dsRNA is a robust inhibitor of gene activity in
Drosophila, comparable in its potency to that observed in C. elegans (Kennerdell, 1998).
The phenotypes produced by ds-ftz and ds-eve RNAs are highly specific. Injected animals exhibit
cuticle defects indistinguishable from ftz and eve loss-of-function mutants. The phenotypes
vary significantly among individuals, possibly due to variability in the injected dose. At high doses of
ds-ftz RNA, the majority of animals exhibit the null mutant phenotype. At lower doses of ds-ftz RNA, the majority of animals exhibit localized or patchy
interference. This localized phenotype is consistent with loss of ftz activity. Even within a
group of animals given the same dose, variation in phenotype is apparent. Some ds-eve RNA-treated
animals exhibit a lawn of denticles characteristic of the known null mutant, while
the remaining animals exhibit a complete pair-rule phenotype or localized pair-rule phenotype characteristic of partial loss of eve function. Since both ftz and eve are expressed in
cells spanning 60% the embryo's length, the complete phenotypes observed indicate that interference can occur
in cells throughout the embryo. The observed interference is at the level of gene expression. Little or no endogenous Ftz protein is observed
in embryos injected with ds-ftz RNA. In contrast, embryos injected with buffer exhibit a
normal pattern of Ftz protein expression (Kennerdell, 1998).
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