The Interactive Fly
Genes involved in tissue and organ development
Circadian rhythms are entrained by light to follow the daily solar cycle. Drosophila uses at least three light input pathways for this entrainment: (1) cryptochrome, acting in the pacemaker cells themselves, (2) the compound eyes, and (3) extraocular photoreception, possibly involving an internal structure known as the Hofbauer-Buchner eyelet, which is located underneath the compound eye and projects to the pacemaker center in the brain. Although influencing the circadian system in different ways, each input pathway appears capable of entraining circadian rhythms at the molecular and behavioral level. This entrainment is completely abolished in glass60j;cryb double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome (Helfrich-Forster, 2001).
Extraocular photoreception is involved in the circadian systems of many organisms, but in most of them the structures and molecules involved are unknown or barely characterized. The present study demonstrates that nonretinal photoreceptors are involved in entrainment of Drosophila's circadian clock: fruit flies utilize at least a tripartite light-input pathway -- one pathway makes use of cryptochrome; a second acts through norpA-dependent photoreceptors in the compound eyes (perhaps also the ocelli), and a third pathway, which acts independently of norpA and cry gene functions, seems likely to involve the extraretinal Hofbauer-Buchner eyelets or other extraocular photoreceptors located in the brain (Helfrich-Forster, 2001).
These separate clock-input pathways influence the circadian system in different ways. Light input through CRY mainly entrains the evening peak of behavioral activity. Retinal and extraretinal eye structures predominantly synchronize the morning peak of activity. In spite of their different effects on behavioral rhythmicity, each light-input pathway alone seems capable of entraining the locomotor rhythm in a rather normal manner when the other one is impaired. Only when all three input routes -- those subserved by CRY, the compound eyes/ ocelli, and extraretinal eye structures are absent is the circadian system of the fly unable to respond to light. This is so at the cellular and at the behavioral level, thus revealing that fruit flies entrainment to multiple light-input pathways for adapting their circadian clock to the cyclic environmental LD changes. Interestingly, similar findings have recently been described for mice, for which depletion of retinal photoreceptors results in almost complete circadian blindness (Helfrich-Forster, 2001).
How Drosophila's multiple photoreceptors might interact remains a mystery but is likely related to the task, faced by many organisms, of extracting time-of-day information from dawn and dusk. During natural twilight, the quality of light changes in three important respects: the amount of light, its spectral composition, and the direction of incoming light (i.e., the position of the sun). These photic parameters all change in a systematic way at twilight times ['Heavenly shades of night are falling, its twilight time' (The Platters, 1958)]; all could be used by the circadian system throughout times of changing photic conditions at dawn and dusk, thus forming a versatile input system that subserves daily adjustments of the rhythms pace. Using different rhodopsins in addition to CRY permits the fly systems to scan all the way from the UV into the red (Helfrich-Forster, 2001).
The observation that the different pathways have at least some overlapping cellular targets provides a first hint about how the fly's different photoreceptors communicate: PER cycling in the s-LNv brain cells can be at the very least a tripartite light-input pathway -- one of them makes use of cryptochrome; a second pathway acts through internal eye structures (in the glass mutant). Similarly, in the absence of CRY and functional external eyes (in the norpAP41;cryb double mutant), the same neurons are acts independently of norpA and cry gene functions, synchronized by extraretinal photoreceptors. Such multiple input aimed at a single cell type would allow the organism to integrate the incoming light (Helfrich-Forster, 2001).
Among the multiple photoreceptors that could contribute to circadian photoreception in Drosophila, the contributions of the external eyes and of CRY function have been demonstrated with the help of the norpAP41;cryb doubly mutant flies that show entrainment defects, which are much more severe than those exhibited by either mutant alone. Nevertheless, this double-mutant type is not completely blind in the circadian sense, compared with the effects of the gl60j;cryb combination revealed in this study. Most norpAP41;cryb individuals are still able to entrain to LD cycles (Helfrich-Forster, 2001).
There are at least two possible explanations for these findings: cryb is not a loss-of-function mutation (indeed, this is a missense mutant), or other norpA-independent photoreceptors feed into the clock. The fact that flies could be generated that are totally circadian blind favors the second hypothesis (Helfrich-Forster, 2001).
The H-B eyelets send their projections directly to the accessory medulla and are therefore anatomically well suited to transmit light signals to the LNv pacemaker cells. The fact that, in the absence of CRY, PER cycling in the s-LN cells is nicely entrained also favors this hypothesis. The H-B eyelets express the photopigment Rhodopsin 6 and thus seem to have photoreceptive properties. The input pathway via Rhodopsin 6 might utilize the flys norpA-independent phospholipase C (which is expressed in many neurons in its phototransduction cascade (Helfrich-Forster, 2001).
Further candidates for circadian photoreceptors (revealed in this study) are the clock-gene-expressing dorsal neurons called DN1 cells. Like the photoreceptor cells of the compound eyes, the ocelli and the H-B eyelets, the DN1s appear to be eliminated by the gl60j mutation. Similar to the H-B eyelets, the DN1s send axonal projections toward the LNvs and could entrain the latter through this anatomical pathway. Furthermore, disconnected mutant flies, which largely lack the LNv but not the DN1 cells, are able to entrain to LD cycles. However, it is not known whether the DN1s express a photopigment or have photoreceptive properties like those exhibited by the H-B eyelets (Helfrich-Forster, 2001).
In spite of the inability of the gl60j;cryb double mutant to entrain to LD cycles, the behavior of such flies was still modified by the altered environmental conditions. This modulation of the activity level is interpreted as direct effects of light/radiant energy on locomotion that bypass the circadian system. Light-related energy often exerts such direct (or masking) effects on physiological parameters, including behavior. There are possibilities to distinguish real entrainment from masking: (1) after a phase shift of the LD cycle, the circadian rhythm often takes several transient cycles to reentrain to the new LD regime, whereas masking follows the new light schedule immediately (in Drosophila, transients can be observed at very low light intensities or in photoreceptor mutants like cryb; (2) after transfer into constant darkness, masking disappears immediately whereas an entrained rhythm starts to free run from the phase it had established in LD, and (3) masking is independent of a functional circadian clock -- for example, it occurs in animals deprived of their clock, such as squirrel monkeys suffering from lesions of their suprachiasmatic nucleus arrhythmic per0 mutants of Drosophila (Helfrich-Forster, 2001).
In gl60j;cryb, prominent masking is observed at the highest light intensities employed (1000 lux). The sudden increase of the activity level after lights off and phase delay of the LD cycle did not show any transients after the 8 hr phase shift. Furthermore, this apparently forced activity disappears immediately after transfer into DD and independent of a functional clock, owing to its presence in these doubly mutant flies, which exhibit apparent rhythmicity in constant darkness. Moreover, no entrained cycling of clock protein levels was observed in these gl60j;cryb flies, demonstrating that the forced behavior is neither the consequence of molecular clock gene cyclings nor a nonphotic Zeitgeber that could influence the circadian clock (Helfrich-Forster, 2001).
In summary, the circadian blindness of flies expressing both glass and cryptochrome mutations is due to elimination of all photoreceptor cells that participate in entraining the circadian system. Similar complex light entrainment pathways may also exist in vertebrates. Interestingly, cryptochromes, certain opsins located in the retina, and standard photoreceptor cells
are candidates for participating in the circadian photoreception of mammals. Thus, rather than having an exclusive photopigment for entrainment of circadian rhythms, the situation in mammals could be similar to that in Drosophila: multiple photoreceptors share the workload involved in transmitting the principle environmental Zeitgeber to the circadian clock (Helfrich-Forster, 2001).
Many invertebrates have supplementary extraocular photoreceptors that often are implicated in circadian rhythms. An extraretinal group of candidate photoreceptors in the fruit fly, Drosophila melanogaster, has been revealed previously at the posterior margin of the compound eye by using a photoreceptor-specific monoclonal antibody, but it never has been characterized. This study reports the fine structure of this cell cluster reported by Hofbauer and Buchner, which is called "eyelet," as well as the further candidacy of their visual pigment and neurotransmitter. Eyelet forms a specialized, pigmented organ with cells that have numerous microvilli arranged into coherent rhabdomeres. The presence of rhabdomeric microvilli is a defining feature of a photoreceptor, reported here for the first time in eyelet. The rhabdomeres exhibit Rh6 opsin-like immunoreactivity, which provides evidence that the photoreceptors are functional: they fail to immunostain with antibodies against NINAE (Rh1), Rh4, or Rh5. The photoreceptors have been shown previously to exhibit histamine-like immunoreactivity, but they also stain with a monoclonal antiserum raised against Drosophila choline acetyltransferase (ChAT), suggesting that the photoreceptors not only may contain histamine but also can synthesize acetylcholine. A ChAT-immunoreactive axon bundle originating from eyelet terminates in the cortex of the anterior medulla. This bundle also is seen with reduced silver stains. Electron microscopic examination revealed four axon profiles of similar size in this bundle, indicating that eyelet contains at least four photoreceptors. The pathway of eyelet's axon bundle coincides with the precocious pathway of Bolwig's nerve that arises from the larval organ of sight. The origin and possible function of eyelet are discussed (Yasuyama, 1999).
Circadian rhythms can be entrained by light to follow the daily solar cycle. In
adult flies a pair of extraretinal eyelets expressing
immunoreactivity to Rhodopsin 6 each contains four photoreceptors located
beneath the posterior margin of the compound eye. Their axons project to the
region of the pacemaker center in the brain with a trajectory resembling that of
Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Kruppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. It is thus demonstrated that the adult eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LNv), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues to be expressed in the adult eyelet. Action spectra of entrainment were compared in different fly strains: in flies lacking compound eyes but retaining the adult eyelet (so1), lacking both compound eyes and the adult eyelet (so1;gl60j), and retaining the adult eyelet but lacking compound eyes as well as Cryptochrome (so1;cryb). Responses to phase shifts suggest that, in the absence of compound eyes, the eyelet together with Cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in the eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring (Helfrich-Forster, 2002).
The fly possesses five photoreceptors and/or photopigments that appear to be involved in light reception and synchronization of the circadian clock: (1) the compound eyes, (2) the ocelli, (3) the Hofbauer-Buchner eyelets, (4) the blue-light photopigment cryptochrome, and (5) unknown photopigments in the clock-gene-expressing dorsal neurons. To understand the contributions of these photoreceptors and photopigments to synchronization, this study monitored the flies' activity rhythms under artificial long and short days. It was found that all the different photoreceptors and photopigments contribute significantly to entrainment under each photoperiod, but the compound eyes are especially important for entrainment to extreme photoperiods. The compound eyes are, furthermore, necessary for adjusting the phase of the activity rhythm, for distinguishing long days from constant light, and for the normal masking effects of light--namely, promotion of activity by lights-on and inhibition of activity by darkness. Cryptochrome is important for period lengthening under long days, although it is more important for entrainment to short days than to long days and is, furthermore, important for aftereffects of the photoperiod on the internal clock. The specific roles of the remaining photoreceptors are more difficult to assess (Rieger, 2003).
Extraretinal photoreception is a common input route for light resetting signals into the circadian clock of animals. In Drosophila melanogaster, substantial circadian light inputs are mediated via the blue light photoreceptor Cryptochrome (Cry) expressed in clock neurons within the brain. The current model predicts that, upon light activation, Cry interacts with the clock proteins Timeless (Tim) and Period (Per), thereby inducing their degradation, which in turn leads to a resetting of the molecular oscillations within the circadian clock. This study investigated the function of another putative extraretinal circadian photoreceptor, the Hofbauer-Buchner eyelet (H-B eyelet), located between the retina and the medulla in the fly optic lobes. Blocking synaptic transmission between the H-B eyelet and its potential target cells, the ventral circadian pacemaker neurons, impaired the flies' ability to resynchronize their behavior under jet-lag conditions in the context of nonfunctional retinal photoreception and a mutation in the Cry-encoding gene. The same manipulation also affected synchronized expression of the clock proteins Tim and Per in different subsets of the clock neurons. This shows that synaptic communication between the H-B eyelet and clock neurons contributes to synchronization of molecular and behavioral rhythms and confirms that the H-B eyelet functions as a circadian photoreceptor. Blockage of synaptic transmission from the H-B eyelet in the presence of functional compound eyes and the absence of Cry also results in increased numbers of flies that are unable to synchronize to extreme photoperiods, supplying independent proof for the role of the H-B eyelet as a circadian photoreceptor (Veleri, 2007).
Circadian rhythms are orchestrated by a master clock that emerges from a network of circadian pacemaker neurons. The master clock is synchronized to external light/dark cycles through photoentrainment, but the circuit mechanisms underlying visual photoentrainment remain largely unknown. This study reports that Drosophila has eye-mediated photoentrainment via a parallel pacemaker neuron organization. Patch-clamp recordings of central circadian pacemaker neurons reveal that light excites most of them independently of one another. Light-responding pacemaker neurons were shown to send their dendrites to a neuropil called accessary medulla (aMe), where they make monosynaptic connections with Hofbauer-Buchner eyelet photoreceptors and interneurons that transmit compound-eye signals. Laser ablation of aMe and eye removal both abolish light responses of circadian pacemaker neurons, revealing aMe as a hub to channel eye inputs to central circadian clock. Taken together, this study demonstrates that the central clock receives eye inputs via hub-organized parallel circuits in Drosophila (Li, 2018).
Light is one of the most important factors regulating rhythmical behavior of Drosophila melanogaster. It is received by different photoreceptors and entrains the circadian clock, which controls sleep. The retina is known to be essential for light perception, as it is composed of specialized light-sensitive cells which transmit signal to deeper parts of the brain. This study examined the role of specific photoreceptor types and peripheral oscillators located in these cells in the regulation of sleep pattern. Sleep was shown to be controlled by the visual system in a very complex way. Photoreceptors expressing Rh1, Rh3 are involved in night-time sleep regulation, while cells expressing Rh5 and Rh6 affect sleep both during the day and night. Moreover, Hofbauer-Buchner (HB) eyelets which can directly contact with s-LN (v) s and l-LN (v) s play a wake-promoting function during the day. In addition, this study showed that L2 interneurons, which receive signal from R1-6, form direct synaptic contacts with l-LN (v) s, which provides new light input to the clock network (Damulewicz, 2020).
The transcription factors encoding genes tailless (tll), atonal (ato), sine oculis (so),
eyeless (ey) and eyes absent (eya), and Efgr signaling play a role in
establishing the Drosophila embryonic visual system. The
embryonic visual system consists of the optic lobe
primordium, which, during later larval life, develops into
the prominent optic lobe neuropiles, and the larval
photoreceptor (Bolwig's organ). Both structures derive
from a neurectodermal placode in the embryonic head.
Expression of tll is normally confined to the optic lobe
primordium, whereas ato appears in a subset of Bolwig's
organ cells that are called Bolwig's organ founders.
Phenotypic analysis of tll loss- and gain-of-function
mutant embryos using specific markers for Bolwig's
organ and the optic lobe, reveals that tll functions to drive cells to an
optic lobe fate, as opposed to a Bolwig's organ fate. Similar
experiments indicate that ato has the opposite effect,
namely driving cells to a Bolwig's organ fate. Since tll and ato do not regulate one another, a model is proposed wherein tll expression restricts the ability of cells
to respond to signaling arising from ato-expressing
Bolwig's organ pioneers. The data further suggest that the
Bolwig's organ founder cells produce Spitz (the Drosophila
TGFalpha homolog) signal, which is passed to the neighboring
secondary Bolwig's organ cells where it activates the Epidermal growth factor receptor
signaling cascade and maintains the fate of these secondary
cells. The regulators of tll expression in the embryonic
visual system remain elusive: no
evidence for regulation by the 'early eye genes' so, eya and
ey, or by Egfr signaling is found (Daniel, 1999).
The Drosophila visual system comprises the adult compound eye,
the larval eye (Bolwig's organ) and the optic lobe (a part of the brain). All of these
components are recognizable as separate primordia during late
stages of embryonic development. These components originate from a small,
contiguous region in the dorsal head ectoderm. During the
extended germband stage, the individual components of the
visual system can be distinguished morphologically as well as by
spatially localized expression of the homeobox gene so and the adhesion molecule Fas II. Initially centered as an unpaired, oval domain
straddling the dorsal midline, the anlage of the visual
system subsequently elongates in the transverse axis and narrows
in the anteroposterior axis. By late gastrulation (stage 8), the
anlage occupies two bilaterally symmetric stripes that are anterior and
adjacent to the cephalic furrow. The domain of so
expression at this stage contains two regions with a high
expression level [olex (the external fold of the optic lobe) and olin]. Only these two regions
will ultimately give rise to the optic lobe and Bolwig's organ; the
so-positive cells dorsal and posterior to these domains will either form
part of the dorsal posterior head epidermis (dph) or undergo apoptotic cell death.
During the extended germband stage, the
anlage of the visual system expands further
ventrally until, around stage 10, it reaches the
equator (50% in the dorsoventral axis) of the
embryo. Shortly thereafter, olin, the
portion of the anlage that will give rise to most of
the optic lobe and Bolwig's organ, reorganizes
into a placode of high cylindrical epithelial cells
that differ in shape from the surrounding more
squamous cells of the head ectoderm.
During stage 12, this placode starts to invaginate,
forming a V-shaped structure with an anterior lip
(olal) and a posterior lip (olpl). Bolwig's organ,
which consists of a small cluster of sensory
neurons, derives from the basal part of the
posterior lip and can be recognized during stage
12 as a distinct, dome-shaped protrusion. During stage 13,
invagination of the optic lobe separates it from the
head ectoderm; only the cells of Bolwig's organ
remain in the ectoderm. The ectodermal
region olex is also internalized and forms
an external 'cover' of the optic lobe; many cells
of this population undergo apoptosis (Daniel, 1999).
The tll gene is expressed in a dynamic
pattern in the protocerebral neurectoderm. In the posterior,
this region overlaps part of the anlage of the
visual system, in particular that part that will give rise
to the anterior lip of the optic lobe. The
anterior lip of the optic lobe upregulates
expression of tll during stage 12. In
addition, the posterior lip of the optic lobe,
does not expressed tll at an earlier stage,
now switches on this gene.
Expression of tll in the posterior lip is patchy,
with some cells expressing the gene at a higher
level than others. The Bolwig's organ
primordium does not express tll. During later
embryonic stages and during larval
development, tll expression remains
strong in the optic lobe, but is never detected
in the Bolwig's organ. Also the primordium of
the eye disc, which expresses tll during larval
stages, is devoid of this expression
during embryonic development (Daniel, 1999).
tll controls a switch between optic lobe
and Bolwig's organ cell fate.
Loss of zygotic tll activity results in an absence
of most of the protocerebrum of the brain
(Younossi-Hartenstein, 1997). In addition, the visual
system of the late tll embryo shows a dramatic phenotype,
namely the transformation of optic lobe into Bolwig's organ. In wild-type embryos, the neuronal marker 22C10 (see Futsch)
labels 12 neurons and their axons that project towards the optic
lobe. In tll mutant embryos, the number of cells in the Bolwig's
organ is dramatically increased (by a factor of 2-3),
while the optic lobe, marked by anti-Crumbs or a
PlacZ insertion in so, is absent.
Use of antibodies to FasII and Crb, which label the apical
surface of the optic lobe, and not that of the Bolwig's organ,
allowed an analysis of how the phenotype unfolds in the tll
mutant embryo. Abnormalities first become apparent during
stage 11, when FasII expression increases strongly in the domain
of the anterior lip of the optic lobe placode, a region that
normally ceases to express FasII. In spite of this
abnormal expression, the optic lobe placode appears to
invaginate normally. As a results, in stage 13 tll mutant embryos,
a Crb-positive vesicle can be seen subjacent to the head
epidermis. During
stage 14, all cells of this aberrant vesicle activate expression of
the neuronal marker 22C10 and lose Crb expression, revealing that these cells are Bolwig's organ cells.
Overexpression of tll under the control of a heat-shock
promoter has an effect opposite that seen in the absence of
tll activity. An
additional consequence of tll overexpression is that the optic
lobes become located more dorsally and fused in the dorsal
midline. This 'cyclops' phenotype most likely arises
because the dorsomedial cells, which normally
die or form part of the head epidermis, now express optic lobe
markers and become an integral part of the optic lobe.
The
results of both loss and gain of tll expression are consistent
with the interpretation that tll is required to drive cells of the
anlage, which would otherwise become photoreceptor neurons
of Bolwig's organ, to develop as optic lobe cells (Daniel, 1999).
atonal is expressed in and required for the development of Bolwig's organ. ato is expressed in the head in
several small cell clusters, one of which is a group of three to
four cells that is part of the Bolwig's organ primordium.
Expression of ato in this domain begins during stage 11 and continues until stage 12. Initially, a group of
6-8 cells faintly expresses ato. By stage 12, their number has
decreased to 3 cells. During this period, ato-expressing cells
can be seen as a small group of cells within the dome-shaped
Bolwig's organ primordium. Loss of ato function results in the
absence of Bolwig's organ. Thus, similar to what has
been demonstrated for the compound eye, even though only a
small subset of photoreceptors actually expresses ato, lack of
ato function results in absence of all photoreceptors.
Since Bolwig's organ is enlarged in a tll mutant background,
it was asked whether tll inhibits ato expression. The number and pattern of ato-positive cells in tll mutants is found to be
normal. These results suggest that tll functions in
parallel with, or downstream of ato in the development of the
Bolwig's organ/optic lobe primordium (Daniel, 1999).
Epidermal growth factor receptor is activated in midline
regions of the head neurectoderm, in particular in the anlage
of the visual system. Moreover,
increased and/or ectopic activation of Egfr results in a
'cyclops' phenotype very similar to what has been described for
ectopic tll expression. Egfr signaling has been shown to be
required in both chordotonal organs and compound eye
for the inductive signaling triggered by ato expression. Two questions raised by these observations have been investigated:
(1) is Egfr signaling required for tll expression in the optic
lobe and
(2) is Egfr signaling involved in the recruitment
of the secondary (non-atonal-expressing) Bolwig's
organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for
the presence of Egfr-relevant mRNAs or proteins:
Rhomboid mRNA, which would be expected to be present
only in the signaling cells, and phosphorylated
MAPK, Pointed and Argos mRNAs, which would be
expected to be expressed in all cells receiving an
Egfr-mediated signal. In stage 12 embryos, rho is
expressed only in the small group of Bolwig's organ
founder cells (the same cells expressing ato).
In contrast, activated (phosphorylated) MAPK is
present in a larger cluster of cells including the entire
Bolwig's organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both
known to be switched on in cells receiving the Spi
signal, are expressed at the same stage throughout the
entire Bolwig's organ primordium.
These gene expression and MAPK activation
patterns are consistent with the idea that the Spi signal
is activated by rho in the Bolwig's organ founders and
passed to the neighboring secondary Bolwig's organ
cells where it activates the Egfr signaling cascade.
Supporting this view, only 3-4 photoreceptor neurons
are found in the Bolwig's organ of embryos lacking
rho or spi; furthermore, the size of the
posterior lip of the optic lobe is reduced in such
embryos. The fact that absence of
secondary Bolwig's organ cells in rho or spi mutant
embryos can be rescued by blocking cell death in the
background of a deficiency that takes out the reaper
complex of genes indicates that the Spi signal is not necessary
for the specification (recruitment) of secondary Bolwig's organ
cells, but rather, for their maintenance (Daniel, 1999).
While the maternal patterning systems that regulate
tll during its blastoderm expression have been
determined, the genes required
to turn on tll at a later stage in the visual system are
not known. Candidates are the 'early eye genes', so,
eya and ey, which encode transcription factors
expressed in the embryonic visual system and in the
larval eye disc in front of the morphogenetic furrow. The expression of these genes was analyzed in the visual system
anlage, and tll expression was examined in embryos mutant
for each of these genes. tll expression
in the optic lobe does not depend on any of these three genes.
It was also concluded that ey and so, which have been shown
to interact with each other during eye disc determination, must act independently in embryonic
visual system development, since they are expressed in those
primordia in non-overlapping patterns (Daniel, 1999).
Although so is expressed initially in the entire visual system
anlage, during later stages its expression
becomes increasingly restricted to subsets of visual system
progenitors. Thus, during stage 11, when a morphologically
distinct optic lobe placode first becomes visible, the domain of
so expression retreats to the posterior lip of this placode; slightly
later its expression is limited to only the Bolwig's organ, where
it is maintained until stage 13. eya expression is
initiated during the late blastoderm stage in a trapezoidal field
in the dorsomedial head region that includes the visual system
anlage, as well as progenitors of the medial brain. Beginning during gastrulation (stage 6/7), the eya
domain becomes divided into an anterior stripe and a narrow
posterior stripe immediately anterior to the cephalic furrow that
widens laterally; this posterior domain will become part of the
posterior lip of the optic lobe, including Bolwig's organ. eya expression continues in the optic lobe until stage 12 and
in Bolwig's organ until stage 13.
Embryos that lack either so or eya exhibit defects in the
portions of the visual system where these genes are expressed.
In both mutants, development proceeds normally until stage
11, when the posterior lip of the optic lobe (olpl) would
normally start to invaginate. In eya and so embryos,
invagination of the optic lobe placode does not take place and
differentiation markers characteristic of the lobe are not expressed.
In conclusion, so and eya, although
expressed coincidental with tll, are not required for its
activation. ey plays no role in the embryonic visual system (Daniel, 1999).
Since tll is a nuclear receptor transcription factor, it must
function to block the effect of signaling from the founder cells
(which is mediated at least in part by Egfr)
at the transcriptional level. In the posterior of the blastoderm
stage embryo, tll has been shown to function directly as both
a repressor (of Kruppel, knirps and Ubx) and as an activator
(of hunchback). Additional activator effects
of tll (not yet demonstrated to be direct) have been shown for
brachyenteron at the posterior of the blastoderm embryo, and
for the proneural gene lethal of scute in the brain. Thus, in the optic
lobe, tll could repress genes that would in its absence be
activated by Egfr signaling, and/or activate genes that would
block receipt, or execution, of the signal (Daniel, 1999 and references).
How to build and maintain a reliable yet flexible circuit is a fundamental question in neurobiology. The nervous system has the capacity for undergoing modifications to adapt to the changing environment while maintaining its stability through compensatory mechanisms, such as synaptic homeostasis. This study describes findings in the Drosophila larval visual system, where the variation of sensory inputs induces substantial structural plasticity in dendritic arbors of the postsynaptic neuron and concomitant changes to its physiological output. Furthermore, a genetic analysis has identified the cyclic adenosine monophosphate (cAMP) pathway and a previously uncharacterized cell surface molecule as critical components in regulating experience-dependent modification of the postsynaptic dendrite morphology in Drosophila (Yuan, 2011).
Proper functions of neuronal circuits rely on their fidelity, as well as plasticity, in responding to experience or changing environment, including the Hebbian form of plasticity, such as long-term potentiation, and the homeostatic plasticity important for stabilizing the circuit. Activity-dependent modification of neuronal circuits helps to establish and refine the nervous system and provides the cellular correlate for cognitive functions, such as learning and memory. Multiple studies have examined synaptic strength regulation by neuronal activity, whereas to what extent and how the dendritic morphology may be modified by neuronal activity remain open questions (Yuan, 2011).
The model organism Drosophila melanogaster has facilitated genetic studies of nervous system development and remodeling. Notwithstanding the relatively stereotyped circuitry, flies exhibit experience-induced alterations in neuronal structures and behaviors such as learning and memory). In a study of experience-dependent modifications of the Drosophila larval CNS, it has been found that different light exposures dramatically altered dendritic arbors of ventral lateral neurons [LN(v)s], which are postsynaptic to the photoreceptors. Unlike the visual activity-induced dendrite growth in Xenopus optic tectum, extending the light exposure of Drosophila larvae reduced the LN(v)s' dendrite length and functional output, a homeostatic plasticity for compensatory adaptation to alterations in sensory inputs. It was further shown that the cyclic adenosine monophosphate (cAMP) pathway and an immunoglobulin domain-containing cell surface protein, CG3624, are critical for this sensory experience-induced structural plasticity in Drosophila CNS (Yuan, 2011).
In Drosophila larvae, Bolwig's organ (BO) senses light, and its likely postsynaptic targets are LN(v)s. As the major circadian pacemaker, LN(v)s are important for the entrainment to environmental light-dark cycles and larval light avoidance behavior. In the larval brain, Bolwig's nerve (BN), the axonal projection from BO, terminates in an area overlapping the dendritic field of LN(v)s. Using the FRT-FLP system [in which DNA sequences flanked by flippase recognition targets (FRT) are snipped out by flippase (FLP)] along with three-dimensional (3D) tracing, the dendritic arbor of individual LN(v) neurons were labeled and analyzed. Then potential synaptic connections were demonstrated between BN and LN(v)s using the GRASP [green fluorescent protein (GFP) reconstitution across synaptic partners] technique to drive expression of one-half of the split GFP in the BN by means of Gal4/UAS and expression of the other half of the split GFP in LN(v)s via LexA/LexAop. The proximity of putative synaptic connections between BN and LN(v)s' dendrites reconstituted GFP fluorescence for photoreceptors expressing either rhodopsin 5 (Rh5) or rhodopsin 6 (Rh6) in BO, which suggested that both groups of photoreceptors may have synaptic connections with LN(v)s (Yuan, 2011).
To test whether LN(v)s can be activated by BN inputs through light stimulation, calcium imaging was performed using GCaMP3 transgenic flies with the larval brain-eye preparation, which included BO, BN, developing eye disks, the larval brain, and ventral nerve cord. Because BO senses blue and green light, the confocal laser at 488 nm (blue) and 543 nm (green) were used to stimulate these larval photoreceptors. LN(v)s' axon terminals displayed a relatively stable baseline of GCaMP3 fluorescence and, upon light stimulation, yielded large calcium responses, which increased with the greater intensity and longer duration of the light pulses (Yuan, 2011).
Recent studies suggest that Cryptochrome (CRY) in adult large LN(v)s senses light and elicits neuronal firing. In larvae, however, severing BN abolished light-induced calcium responses in LN(v)s. The loss-of-function mutation of NorpA (no-receptor-potential A), encoding a phospholipase C crucial for phototransduction, also eliminated these calcium responses, which indicated that light-elicited responses in LN(v)s are generated via phototransduction in larval photoreceptors rather than as a direct response to light by LN(v)s (Yuan, 2011).
In animals with BO genetically ablated, the dendritic field of LN(v) is absent. To test whether BO is required for LN(v)s' dendrite maintenance, the expression of cell death genes rpr and hid was induced in BO after synapse formation, and the LN(v) dendrite length was also found to be greatly reduced. Whereas physical contacts with BN or growth-promoting factors released from presynaptic axons could be important for LN(v)s' dendrite maintenance, it is also possible that synaptic activity from BN promotes LN(v) dendrite growth, as suggested by previous studies. To explore the latter scenario, newly hatched larvae were provided with different visual experiences through various light regimes—including the standard 12 hours of light and 12 hours of dark daily cycle (LD); constant darkness (DD) for sensory deprivation; constant light (LL) for enhanced light input; 16-hour light and 8-hour dark cycle, mimicking a long day; and 8-hour light and 16-hour dark cycle, mimicking a short day. The dendrite morphology of LN(v)s of late third instar larvae was examined. Whereas different light exposure had no detectable effects on larval developmental timing, increasing light exposure reduced the total dendrite length of individual LN(v) neurons, with the longest dendrite in constant darkness and the shortest dendrite length in constant light condition. Thus, not only is the LN(v) dendrite dependent on the presence of presynaptic nerve fibers, its length is modified by the sensory experience in a compensatory fashion, whereby an increase in sensory inputs causes a reduction in the dendrite length and vice versa (Yuan, 2011).
Whereas adult LN(v)s alter their axon terminal structures in a circadian cycle-controlled fashion, no difference was found in dendrite morphology of LN(v)s from larvae collected at four different time points around the clock, which indicated that circadian regulation is not involved in the light-induced modification of LN(v) dendrites. Under regular light-dark conditions, LN(v) dendrites expanded as the larval brain size increased from the second to the third instar stage. However, the dendrite length of the LL group increased at a significantly slower rate than the DD group. It thus appears that light exposure retards the growth of LN(v) dendrites throughout the larval development (Yuan, 2011).
To test the contribution of different light-sensing pathways, loss-of-function mutations of Cry (cry01) or NorpA (norpA36) and of double mutants lacking both Rh5 and Rh6 (rh52;rh61) were examined. Although wild-type and cry01 larvae displayed differences in their dendrite length when exposed to constant darkness versus constant light, such light-induced changes were absent in the rh52;rh61 double mutant and the norpA36 mutant. Thus, similar to the calcium response to light, light-induced modification of LN(v) dendritic structure requires visual transduction mediated by rhodopsin and NorpA in BO but not Cry function in LN(v)s (Yuan, 2011).
To manipulate the level of synaptic activity, the BO excitability was weither increased by expressing the heat-activated Drosophila transient-receptor-potential A1 (dTrpA) channel, or transmitter release from BN was reduced through a temperature-sensitive form of the dominant-negative dynamin, Shibirets (Shits). These manipulations eliminated light-induced modification of LN(v) dendrites at 29°C. Reducing BO activity by means of Shits caused dendrite expansion, as if the animal detected no light, whereas increasing BO activity by means of the dTrpA channel resulted in reduction of LN(v) dendrites, a process reminiscent of constant light exposure (Yuan, 2011).
Whether intrinsic LN(v) neuronal activity drives modification of its dendrite morphology was further tested by expression of either the sodium channel NaChBac to increase excitability or the potassium channel Kir2.1 to reduce excitability. LN(v)s expressing Kir2.1 showed reduced or no calcium responses upon light stimulation. In contrast, LN(v)s expressing NaChBac displayed numerous peaks in GCaMP3 signals in the presence or absence of light stimulation, indicative of elevated spontaneous activities. Upon examining LN(v) dendrites, it was found that neuronal excitability of the LN(v) was inversely proportional to its dendrite length (Yuan, 2011).
These results obtained using genetic approaches agreed with findings in experiments with different environmental light conditions. They suggested that LN(v)'s dendritic structures are modified according to its neuronal activity, which varies with light-induced synaptic inputs (Yuan, 2011).
To test whether synaptic contacts of BN on LN(v)s are modified by light, synapses formed by BN with EGFP (enhanced green fluorescent protein)-tagged Cacophony (Cac-EGFP) were marked, because Cacophony is a calcium channel localized at presynaptic terminals and its distribution correlates with the number of synapses. Close association was found of Cac-EGFP-expressing structures with LN(v)s' dendritic arbors. Compared with regular light-dark conditions, constant darkness increased, whereas constant light reduced, the total intensity of Cac-EGFP, which suggested that light modified not only dendritic arbors of LN(v)s but also the number of synaptic contacts impinging on LN(v) dendrites (Yuan, 2011).
Next, using calcium imaging, whether there are light-induced functional modifications of LN(v)s was examined. Increased light exposure caused LN(v)s to be less responsive. Conversely, sensory deprivation in constant darkness increased LN(v)s' sensitivity to light. Thus, in contrast to stable synaptic responses observed in synaptic homeostasis, light-induced responses of central neurons postsynaptic to photoreceptors in the Drosophila larval visual circuit have a dynamic range, modifiable by sensory experiences and positively correlated to the dendrite length (Yuan, 2011).
In dunce1, a loss-of-function mutant of the fly homolog of 3'5'-cyclic nucleotide phosphodiesterase, the LN(v)s' dendrite length was comparable among LD, LL, and DD groups. Reducing dunce gene expression specifically in LN(v)s through RNA interference (dncIR) resulted in a similar indifference of LN(v)s' dendrite size to the light exposure, which implicated a cell-autonomous action of dunce in LN(v) neurons (Yuan, 2011).
To explore the possibility that the elevated cAMP level caused by the dunce mutation interfered with dendrite plasticity, tests were performed for the involvement of downstream components of the cAMP pathway, including the catalytic subunit of protein kinase A (PKAmc), which up-regulates cAMP signaling, and a dominant-negative form of the cAMP response element-binding protein (CREBdn), which inhibits cAMP-induced transcription activation. Expression of either transgene specifically in LN(v)s obliterated their ability to adjust dendrite length under different light-dark conditions. Calcium imaging further revealed that the expression of PKAmc or CREBdn eliminated changes of LN(v)s' light responses produced by different light-dark conditions. Thus, the cAMP pathway regulates both structural and functional plasticity of LN(v)s (Yuan, 2011).
The screen for mutants with defective LN(v) dendritic plasticity also identified babos-1, a mutant with a P-element insertion near the transcriptional start site of CG3624, a previously uncharacterized immunoglobulin domain-containing cell surface protein. The LN(v) dendrite length of babos-1 mutant larvae was comparable to controls in LD and LL but has no compensatory increase in DD. Similar phenotypes were found in larvae expressing an RNAi transgene targeting CG3624 in LN(v)s. Moreover, flies carrying a hypomorphic allele of CG3624, CG3624[KG05061], also showed defective light-induced dendritic plasticity, which was fully rescued by expressing the UAS-CG3624 transgene specifically in LN(v)s. Thus, the function of this immunoglobulin domain-containing protein in LN(v)s is important for the dendrite expansion in constant darkness (Yuan, 2011).
Bioinformatic analyses suggest that CG3624 is a cell surface protein containing an N-terminal signal peptide, extracellular immunoglobulin domains followed by a transmembrane helix, and a short C-terminal cytoplasmic tail. CG3624 is widely expressed in the nervous system throughout development. Its specific requirement for the adjustment of LN(v)s' dendrite length in constant darkness suggests that elevation or reduction of sensory inputs likely invokes separate mechanisms for compensatory modifications of central neuronal dendrites (Yuan, 2011).
A functioning nervous system must have the capacity for adaptive modifications while maintaining circuit stability. This study of the Drosophila larval visual circuit reveals large-scale, bidirectional structural adaptations in dendritic arbors invoked by different sensory exposure. Whereas the circuit remains functional with modified outputs, this type of homeostatic compensation may modify larval light sensitivity according to its exposure during development and could facilitate adaption of fly larvae toward altered light conditions, such as seasonal changes. The observations also suggest shared molecular machinery between homeostasis and the Hebbian plasticity with respect to the cAMP pathway and indicate the feasibility of genetic studies of experience-dependent neuronal plasticity in Drosophila (Yuan, 2011).
Animal development is coupled with innate behaviors that maximize chances of survival. This study shows that the prothoracicotropic hormone (PTTH), a neuropeptide that controls the developmental transition from juvenile stage to sexual maturation, also regulates light avoidance in Drosophila melanogaster larvae. PTTH, through its receptor Torso, acts on two light sensors (the Bolwig's organ and the peripheral class IV dendritic arborization neurons) to regulate light avoidance. PTTH was found to concomitantly promote steroidogenesis and light avoidance at the end of larval stage, driving animals toward a darker environment to initiate the immobile maturation phase. Thus, PTTH controls the decisions of when and where animals undergo metamorphosis, optimizing conditions for adult development (Yamanaka, 2013)
Animal development is associated with multiple primitive, innate behaviors, allowing inexperienced juveniles to choose an environment that maximizes their survival fitness before the transition to adulthood. In insects, this transition is timed by a peak of ecdysone production induced by the prothoracicotropic hormone (PTTH). In the larval brain of Drosophila, PTTH is produced by two pairs of neurosecretory cells projecting their axons onto the prothoracic gland (PG), where ecdysone
is produced. Transition to adulthood is associated with drastic changes in larval behavior: Feeding larvae remain buried in the food,
whereas wandering larvae (at the end of larval development) crawl out and find a spot where they immobilize and pupariate. Mechanisms allowing proper coordination of these behavioral changes with the developmental program remain elusive (Yamanaka, 2013)
Two pairs of neurons in the central brain were recently reported to control larval light avoidance. Using specific antibodies to PTTH, this study established that these neurons labeled by the NP0394-Gal4 and NP0423-Gal4 lines correspond to the PTTH-expressing neurons. Moreover, silencing the ptth gene by using NP0423-Gal4 or a ubiquitous driver (tub-Gal4) impaired light avoidance, indicating that PTTH itself controls this behavior. PTTH activates Torso, a receptor tyrosine kinase whose
knockdown in the PG prevents ecdysone production and induces a developmental delay. In contrast, knocking down torso in the PG did not cause any change in light avoidance, indicating that the role of PTTH in ecdysteroidogenesis is functionally distinct from its role in light avoidance behavior (Yamanaka, 2013)
Because in Drosophila the PTTH-producing neurons only innervate the PG, it was reasoned that PTTH is secreted into the hemolymph and reaches the cells or organs involved in light avoidance.
Consistent with this, inactivation of PTTH-expressing neurons affects light avoidance with 8 to 10 hours delay, arguing against PTTH neurons projecting directly on their target cells to control light avoidance. PTTH peptide is present in the PTTH-expressing neurons throughout larval development and shows a marked increase before wandering, correlating with the rapid increase of ecdysteroidogenesis at this stage. Using an enzyme-linked immunosorbent assay (ELISA), it was found that PTTH is readily detected in the hemolymph with a fluctuation pattern similar to that of its accumulation in the PTTH-expressing neurons. Furthermore, hemolymph PTTH levels were significantly decreased upon RNA interference (RNAi)-mediated knockdown of ptth in the PTTH-expressing neurons, suggesting that in addition to the paracrine control of ecdysteroidogenesis in the PG, PTTH also carries endocrine function (Yamanaka, 2013)
Pan-neuronal knockdown of torso (elav>torso-RNAiGD) recapitulates the loss of light avoidance observed upon torso ubiquitous knockdown (tub>torso-RNAiGD), suggesting that PTTH acts on neuronal cells to control light avoidance. The potential role of torso was specifically tested
in two neuronal populations previously identified as light sensors in Drosophila larvae: (1) the Bolwig's organ (BO) and (2) the class IV dendritic arborization (da) neurons tiling the larval body wall. An enhancer trap analysis of torso, as well as in situ hybridization using a torso antisense probe, confirmed torso expression in class IV da neurons. In parallel, torso transcripts were detected by means of quantitative reverse transcription polymerase chain reaction in larval anterior tips containing the BO, and their levels were efficiently knocked down by using the BO-specific drivers Kr5.1-Gal4 and Rh5-Gal4, demonstrating torso expression in the BO. The knockdown of torso in the BO (Kr5.1>torso-RNAiGD and GMR>torso RNAiGD) or in the class IV da neurons (ppk>torso-RNAiGD) abolished larval light avoidance (motoneurons serve as a negative control: OK6>torso-RNAiGD). Knocking down torso in both neuronal populations (ppk>, GMR>torso-RNAiGD) mimicked the effect observed with the BO driver or class IV da neuron driver alone. A similar loss of light avoidance was observed when these neurons were separately inactivated by expressing the hyperpolarizing channel Kir2.1 (GMR>Kir2.1 and ppk>Kir2.1), suggesting that both of these light sensors are necessary for light avoidance behavior. Down-regulation of PTTH/Torso signaling did not lead to any neuronal morphology or locomotion defect, further indicating its direct effect on light sensing. The knockdown of torso in class IV da neurons or in the BO had no effect on the pupariation timing. Taken together, these results indicate that PTTH/Torso signaling is required for light avoidance behavior in two distinct populations of light-sensing neurons and that this function is separate from its role in controlling developmental progression (Yamanaka, 2013)
Drosophila light-sensing cells use photosensitive opsins that upon exposure to light, activate transient receptor potential (TRP) cation
channels, thus depolarizing the membrane and triggering neural activation. Although the BO and class IV da neurons use different photosensitive molecules and TRP channels, one can assume that PTTH/Torso signaling regulates the phototransduction pathway through a similar mechanism in both types of neurons. Immunohistochemical detection of Rh5, the opsin involved in light avoidance behavior in the BO, showed no difference in protein level in torso mutant background. PTTH/Torso signaling knockdown did not change the expression level of Gr28b, a gustatory receptor family gene that plays an opsin-like role in class IV da neurons. These results strongly suggest that PTTH affects signaling components downstream of the photoreceptors (Yamanaka, 2013)
The neural activity of the light sensors was investigated using the calcium indicator GCaMP3 for live calcium imaging. torso mutant class IV da neurons showed a 25% reduction of their response to light as compared with that of control. This was accompanied by a loss of light avoidance, indicating that such partial reduction of the GCaMP3 signal corresponds to a reduction of neural activity strong enough to exert a behavioral effect. Indeed, blocking the firing of class IV da neurons by using TrpA1-RNAi caused a similar 25% reduction of the GCaMP3 signal and behavioral effect. This suggests that in da neurons, PTTH/Torso signaling exerts its action upstream of TrpA1 channel activation. Accordingly, a strong genetic interaction was observed between torso and TrpA1 mutants for light preference. A genetic interaction between torso and Rh5 mutants was also detected, further supporting that PTTH/Torso signaling affects a step in phototransduction between the photoreceptor molecule and the TRP channel. Collectively, these data are consistent with the notion that PTTH/Torso signaling acts to facilitate TRP activation downstream of photoreceptor-dependent light sensing (Yamanaka, 2013)
A previous study suggested that larval photophobic behavior diminishes at the end of larval development, perhaps facilitating larval food exit and entry into the wandering phase. The present finding and the increase of PTTH at the beginning of the wandering stage appear to contradict such a hypothesis. Indeed, a sustained larval light avoidance mediated by PTTH was detected that persisted through the wandering stage. These results imply that wandering behavior is triggered by a signal distinct from light preference. Consistent with this notion, the timing of wandering initiation in ppk>torso-RNAiGD or Kr5.1>torso-RNAiGD larvae was found comparable with that of control animals, despite the fact that these animals are not photophobic (Yamanaka, 2013)
As found in other insects, wandering is either directly or indirectly triggered by PTTH- induced ecdysone production. Therefore, concomitant PTTH-mediated photophobicity could ensure that wandering larvae maintain a dark preference for pupariation site, providing better protection from predators and dehydration during the immobile pupal stage. To test this hypothesis, a light/dark preference assay was developed for pupariation. When exposed to a light/dark choice, larvae indeed showed a strong preference to pupariate in the dark. This behavior was abolished either by inactivating PTTH-expressing neurons (ptth>Kir2.1), by silencing ptth in the PTTH-expressing neurons (NP0423>ptth-RNAi, dicer2), or by introducing a torso mutant background (torso[e00150]/[1]). Dark site preference for pupariation was observed in Drosophila populations collected in the wild, confirming that this innate behavior was selected in a natural environment (Yamanaka, 2013)
This work illustrates the use of a single biochemical messenger, PTTH, for the concomitant control of two major functions during larval development. PTTH establishes a neuroendocrine link between distinct neuronal components previously shown to be involved in light avoidance. In contrast to previous interpretations but consistent with another study, this study showed that wandering is independent of light preference and that PTTH maintains a strong light avoidance response through to the time of pupariation. High levels of circulating PTTH during the wandering stage could reinforce the robustness of light avoidance, which might otherwise be compromised by active roaming. This eventually promotes larvae to pupariate in the dark, a trait potentially beneficial for ecological selection. PTTH is thus at the core of a neuroendocrine network, promoting developmental progression and appropriate innate behavioral decisions to optimize fitness and survival (Yamanaka, 2013)
The avoidance of light by fly larvae is a classic paradigm for sensorimotor behavior. This study used behavioral assays and video microscopy to quantify the sensorimotor structure of phototaxis using the Drosophila larva. Larval locomotion is composed of sequences of runs (periods of forward movement) that are interrupted by abrupt turns, during which the larva pauses and sweeps its head back and forth, probing local light information to determine the direction of the successive run. All phototactic responses are mediated by the same set of sensorimotor transformations that require temporal processing of sensory inputs. Through functional imaging and genetic inactivation of specific neurons downstream of the sensory periphery, this study has begun to map these sensorimotor circuits into the larval central brain. It was found that specific sensorimotor pathways that govern distinct light-evoked responses begin to segregate at the first relay after the photosensory neurons (Kane, 2013).
The sensorimotor structure of larval phototaxis is given in the following model.
Larvae modulate their frequency of turning based on
temporal changes in light intensity. In continuously varying
gradients, this modulation results in longer runs in
favorable directions. Larvae also bias their turn size,
executing larger heading changes following runs toward nonpreferred
directions and smaller heading changes following runs
in the preferred direction. Larvae use temporal headsweeps
as probes to explore local luminosity gradients and to
identify the preferred direction for successive runs. The initial
head-sweep direction is unbiased, but larvae are more likely to
accept head-sweeps toward the preferred direction (Kane, 2013).
These strategies all rely on decoding temporal variations in the
amount of light incident on the Bolwig's organ (BO). The BO and the surrounding
cephalopharyngeal sclerites transduce directional light information
to temporal variations of light intensity. On directional
lightscapes, larval head-sweeps change the
amount of light incident on the BO, whereas the apparent intensity
remains constant during straight, forward runs; hence,
turn magnitude and direction are biased, but turn frequency is not (Kane, 2013).
The larva contains two photosensory structures, the BO and the class IV md neurons. The BO
is the main photosensory structure, contributing to phototaxis
across the full range of ecologically relevant light intensities, and is specifically required for direction-based
phototaxis and dark-induced pausing. Multidendritic neurons
contribute to light-evoked turning at very high intensities. Photosensory information
obtained from the BO is translated into behavior more
rapidly than photosensory information obtained from md neurons. Transitions in behavioral state driven by either photosensory
structure are based on temporal comparisons, but the
location of the BO in the 'pigment cup' formed by the cephalopharyngeal
sclerites uniquely allows decoding of incident light
direction. Taken together, the results establish the sensorimotor
structure of larva phototaxis. Moreover, the results appear to
settle a controversy in Drosophila larval phototaxis,
explaining the apparent tropism in direction-based phototaxis as
a joint product of the anatomy of the light-sensing organ and
temporal comparisons used during intensity-based phototaxis.
For BO-mediated visual responses, this study has been able able to map
the relative contributions of the lateral neurons (LNs) and the fifth LN to the
photosensory response. These neurons represent the first relay in
the BO-mediated visual response. Although no evidence was found
that the BO-mediated visual response involves spatial
comparisons between the activities of the two neurons, the first
relay maintains the laterality of BO output, i.e., the neurons in
the first relay on the left side of the brain receive input only from
the left BO, and the neurons in the first relay on the right side
receive input only from the right BO. If signal-averaging or direct
comparisons between the outputs of the BO occur, they occur in
deeper layers (Kane, 2013).
Interestingly, the roles of neurons in the first relay begin to
segregate for the distinct types of photosensory response. It was
found that the fifth LN is essential for dark-induced pausing. The LNs were found to be essential for phototaxis away from directional
illumination, with a partial contribution of the fifth LN.
The LNs partly contribute to light-induced increases
in turning rate. These results, suggesting that sensorimotor pathways
for different components of the overall photosensory response
diverge at the first relay, are a first step toward mapping
the circuit-level basis of phototaxis in the Drosophila larva (Kane, 2013).
In addition to the compound eyes, most insects possess a set of three dorsal ocelli that develop at the vertices of a triangular cuticle patch, forming the ocellar complex. The wingless and hedgehog signaling pathways, together with the transcription factor encoded by orthodenticle, are known to play major roles in the specification and patterning of the ocellar complex. Specifically, hedgehog is responsible for the choice between ocellus and cuticle fates within the ocellar complex primordium. However, the interaction between signals and transcription factors known to date do not fully explain how this choice is controlled. This study shows that this binary choice depends on dynamic changes in the domains of hedgehog signaling. In this dynamics, the restricted expression of engrailed, a hedgehog-signaling target, is key because it defines a domain within the complex where hh transcription is maintained while the pathway activity is blocked. The Drosophila Six3, Optix, is expressed in and required for the development of the anterior ocellus specifically. Optix would not act as an ocellar selector, but rather as a patterning gene, limiting the en expression domain. These results indicate that, despite their genetic and structural similarity, anterior and posterior ocelli are under different genetic control (Dominguez-Cejudo, 2015).
The dorsal adult head of Drosophila derives from the dorsal-anterior region of the eye-antennal imaginal disc. In addition, this disc gives rise to the remaining head capsule, the eyes, the antennae and the maxillary palps. The dorsal head is patterned by the dynamic interplay between orthodenticle [otd; also known as ocelliless (oc)], which encodes an Otx family transcription factor, and the wingless (wg; the fly Wnt1 homolog) and hedgehog (hh) signaling pathways . The result of this patterning is the allocation of the dorsal head, which lies in between the eyes, into three territories (from lateral to medial): orbital cuticle, frons and ocellar complex (OCx). The OCx comprises three small and structurally simple eyes termed the ocelli that are located at the vertices of a triangular patch of cuticle, the so-called interocellar cuticle, which also harbors a set of stereotypical bristles. Ocelli are widespread in insects, where they play a number of roles, including flight stabilization and as movement detectors triggering the escape response (Dominguez-Cejudo, 2015).
Work in past years has aimed at defining the functional relationships between wg, hh and otd during the process of dorsal head patterning in the disc. This work has expanded understanding of the general mechanisms by which the conserved Wnt and Hh signaling pathways interact and of the development and evolution of the eyes and dorsal head of arthropods, and has helped in establishing parallels between head patterning across phyla. For instance, members of both the Wnt and Otx gene families are involved in anterior head/neural tube patterning in both invertebrates and vertebrates (Dominguez-Cejudo, 2015).
The development of the OCx is a typical example of regional specification, in which the OCx progenitor field is further subdivided to give rise to the three ocelli and the interocellar cuticle. One of the earliest steps during the development of the Drosophila head is the initiation of otd expression by wg. In late embryos, all cells of the eye-antennal disc primordium, which can be marked by the expression of eyeless (ey), express Otd. During larval development Otd expression progressively disappears from the disc, and is only maintained in its dorsal anterior region, where wg is expressed. Otd in turn is required to activate hh transcription. This results, in the early third instar (L3) disc, in the coexpression of wg, hh and otd in the prospective OCx region. However, wg transcription is next repressed in the prospective OCx to allow the development of the ocelli and the interocellar cuticle and bristles; otherwise, these structures fail to develop and are replaced by frons, a more lateral type of cuticle (Dominguez-Cejudo, 2015).
During mid and late L3, the OCx region becomes further subdivided into three domains: the central domain transcribes hh and becomes the interocellar cuticle (IOC) region, while two adjacent domains express eyes absent (eya) and sine oculis (so), which encodes a Six1/2 transcription factor, and will become the anterior (a) and posterior (p) ocelli (OC) (Blanco, 2010; Brockmann, 2011). The mechanism by which this aOC-IOC-pOC pattern is controlled by hh has recently been investigated (Aguilar-Hidalgo, 2013) and relies on the differential activation by the Hh signaling pathway of two Hh target genes: eya and the homeobox transcription factor engrailed (en). Hh first activates eya throughout the OCx region; then, en is turned on in a more restricted domain, which results in the attenuation of the Hh signaling pathway and the concomitant loss of eya from these cells. Thus, the central region, expressing en and devoid of eya, becomes the IOC, whereas the remaining flanking eya-expressing domains become the retina-producing OC (Dominguez-Cejudo, 2015).
However, a central question that remains to be answered for a comprehensive understanding of ocellar specification is how the changes in hh signaling domains are regulated during development, as this signaling morphogen plays a major role in controlling the specification and patterning of the OCx structures. This study has followed the regulatory steps that lead from the onset of hh expression to the establishment of its final expression domain, and defines how these steps are interconnected in a gene regulatory network. Two transcriptional repressors, encoded by en and the Drosophila Six3/6 gene Optix, are key players in this network (Dominguez-Cejudo, 2015).
The relative simplicity of the OCx makes it an ideal system with which to study in detail the mechanisms involved in the specification and patterning of a visual structure. Previous work had described the functional relationships between wg, hh and otd during the specification of the dorsal head, the region where the OCx forms. The outcome of these interactions, a wg-cleared OCx region, allows the subsequent specification of the ocellar structures, namely the three ocelli (an eya/so-dependent structure) and the intervening interocellar cuticle (an en-dependent structure), by the Hh signaling pathway. However, it is not clear how this aOC-IOC-pOC pattern is generated. More specifically, since this pattern depends on hh, the question is to understand how hh controls alternative fate decisions in this region. This work has shown that the hh signaling domain changes during this process and that this change is essential for proper ocellar development. This dynamics depends on the establishment of a feedback loop with the hh signaling pathway target en, which, in turn, is restricted in its expression domain by the action of the Drosophila Six3/6 homolog Optix (Dominguez-Cejudo, 2015).
During the first half of L3 two Hh-related events occur. First, wg transcription clears from the prospective ocellar region. This is mediated by high Otd levels, which are achieved through the activation of an otd autoregulatory enhancer [oc7 (Blanco, 2009)] by Hh signaling. Second, and parallel to the wg clearing, low levels of eya expression are induced throughout the whole OCx (Aguilar-Hidalgo, 2013). During the second half of L3, though, the initially uniform expression domain of hh fades away to become restricted to its central region, associated with the activation of en. This central domain becomes the non-retinal interocellar cuticle (IOC), where en represses the transduction of the hh signal. This change in hh expression pattern, which defines the aOC-IOC-pOC domain organization in the disc and the structure of the adult OCx, occurs through transcriptional changes. In particular, the maintenance of hh in the central domain depends on en. Therefore, after en expression is turned on by hh signaling (Aguilar-Hidalgo, 2013), en feeds back positively on hh transcription to maintain high hh expression levels. In the prospective IOC, the expression of en represses hh signaling transduction and the initial expression of eya is lost. In the adjacent regions, though, eya expression is maintained at high levels through an autoregulatory loop that involves so (Brockmann, 2011). In addition, a potential non-autonomous contribution of Hh, produced at the IOC region, cannot be excluded. The en-to-hh maintenance function that is described in this study in the OCx resembles the well-established role of en as a hh transcriptional activator in other contexts, such as the embryo segmental stripes and the posterior compartment of the wing disc, and could constitute a regulatory module that is deployed in several developmental contexts, such as the developing head (Dominguez-Cejudo, 2015).
It was further demonstrated that the peripheral reduction of the hh domain is due to transcriptional regulation rather than cellular rearrangements. This raises the question of how the reduction of hh transcription outside the IOC region occurs. The most likely possibility is that a hh activator is lost as the development of the OCx region progresses through L3. It is noted that hyperactivation of the wg canonical pathway in the OCx region results in an expansion of the hh-Z domain. If wg were required to activate hh, the indirect negative-feedback loop that results in the wg clearing from the OCx region would also result in the loss of its activating action on hh and the loss of hh transcription. This would be prevented only in places where en was expressed. This hypothesis (wg being required for hh expression in the OCx) is supported by the fact that, in the embryonic head, wg activates hh expression. Nevertheless, it seems contradictory to previous reports in which, using a temperature-sensitive wg allelic combination, the reduction of wg signaling activity resulted in an enlargement of the OC and the IOC. However, this result could be reconciled with wg acting as a hh activator. Early during L3, wg would activate hh transcription while simultaneously preventing the expression of en and eya, two targets of hh (Blanco, 2009). Later in L3, high levels of Otd, produced after the activation of the oc7 enhancer, result in the transcriptional repression of wg in the prospective OCx region. This repressive step leaves the wg expression domain restricted to more lateral regions, where the frons and the orbital cuticle will be specified. Removing wg function during this late period, would allow the activation of Hh targets eya and en in a broader domain due to the non-autonomous action of secreted Hh. Since en maintains hh transcription at high levels, late removal of wg should result in an enlarged hh expression domain and an increase in the overall size of the OCx, as observed. It is also important to mention that previous work has shown that the Iroquois Complex (Iro-C) genes araucan and caupolican participate in the restriction of the OCx to the medial region (Yorimitsu, 2011; Dominguez-Cejudo, 2015).
Key to the establishment of OCx patterning is where en becomes expressed. In contrast to hh transcription, which changes over time, that of en is stable once initiated in the prospective IOC. Optix is expressed in a dorsal anterior strip in the eye disc, contained within the otd domain, that abuts posteriorly the en domain. The results suggest that Optix is partially responsible for setting up this anterior border of en. Since en, by acting as a Hh pathway repressor, prevents eya transcription, such an expansion is the most likely cause of the effects on the aOC. The definition of a clear-cut Optix/en border might be further refined by mutually repressive interactions, as indicated by two results: overexpressing Optix in the IOC results in the downregulation of en and, reciprocally, the overexpression of en in the prospective aOC represses Optix. Therefore, as en is initially activated by hh, the anterior limit of the en domain may result from the integration of activator and repressor inputs provided by hh signaling and Optix, respectively, a limit that might be further refined by reciprocal repression of Optix by en. It is hypothesized that a similar mechanism sets the posterior border of the en expression domain to allow for an en-free, hh-receiving domain that becomes specified as the pOC. Potential candidates for the posterior anti-repressor are the Sp genes buttonhead (btd) and Sp1, and hunchback (hb). These genes are expressed in the preoptic region of the embryonic head of all arthropods. However, neither Sp1 nor btd is expressed in the prospective OCx region of the eye disc. hb expression was checked using an anti-Hb antibody and no expression was found in L3 eye-antennal discs. Therefore, the nature of the posterior regulator(s) involved remains to be determined (Dominguez-Cejudo, 2015).
The results indicate that, despite their structural similarity and shared requirement of otd, hh signaling and eya activation, the patterning of the aOC and pOC are under different genetic control. This might be expected, as only the anterior ocellar patches fuse during metamorphosis to form the adult aOC. In fact, evidence for this difference in genetic control had previously been obtained in population selection experiments in Drosophila suboscura. In that study, the authors used an ocelliless (oc) mutant population that showed loss of OCx structures, including the aOC and pOC, with variable penetrance. Through breeding, they were able to establish independent sublines in which the aOC, but not the pOC (or vice versa), were preferentially lost, even when these flies were still carrying the otd mutation. In light of these results, it was proposed that, on top of a common precursor for OC and bristle (i.e. cuticle) determined by otd, an additional 'system' would control the amount of ocellar or cuticle precursors, and this system would differ along the anterior-posterior axis. In this context, Optix would not instruct an ocellar fate but rather control the amount of anterior ocellus precursor cells within the OCx primordium, thus acting as a pre-patterning gene (Dominguez-Cejudo, 2015).
The expression pattern and function of six3/Optix have been studied in Drosophila embryos. In both insects, six3/Optix expression is restricted to the head region, and includes the clypeolabrum and maxillary segment in Drosophila and the labral and middle head regions in Tribolium. Accordingly, six3/Optix mutant Drosophila larvae show reduced or absent labral-derived head skeletal elements, such as the labral organ and the maxillary segment-derived mouth hooks in Drosophila and loss of the labrum and anterior vertex bristle in Tribolium. This study has shown that in Drosophila the expression domain of Optix in eye discs, which runs along the anteriormost medial disc region, maps to the anterior medial dorsal head, where it is required. In addition, OptixNP2631>OptixRNAi adults show defects in the clypeal skeleton, recapitulating the defects seen in larvae. Therefore, all these results point to six3/Optix as a medial head-patterning gene also during eye-antennal disc development. However, it was not been able to detect Optix expression in the embryonic primordium of the eye-antennal disc, marked with the disc primordium marker ey-Z and using either an anti-Optix antiserum or the OptixNP2631-GAL4 line. The expression of all available regulatory constructs associated with the Optix locus generated by the Janelia Project was studied. Neither of the lines expressed in the eye disc showed overlapping expression with the embryonic eye primordium. Therefore, and provided that a low number of Optix-expressing cells in the eye-antennal disc primordium was not missed, the expression of Optix in the head primordium is likely to be initiated during larval life (Dominguez-Cejudo, 2015).
The mechanisms that initiate Optix expression in the most anterior region of the dorsal head need to be investigated further. The results also raise the question of whether en, otd and hh might be similarly engaged in adult head patterning in other insects (Dominguez-Cejudo, 2015).
Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. This paper analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3mum diameter) alternating with thin segments (>0.1mum). Loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs (Hartenstein, 2019).
ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either light increments or decrements. Despite lacking the anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in the larval visual system, focus was placed on investigating the physiological properties and behavioral relevance of larval visual interneurons. The ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes a cholinergic interneuron (cha-lOLP) and hyperpolarizes a glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Galphao signaling produces the sign-inversion required for OFF detection in glu-lOLP, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, these studies identify the molecular and circuit mechanisms underlying ON vs. OFF discrimination in the Drosophila larval visual system (Qin, 2019).
ON and OFF selectivity, the differential neuronal responses elicited by signal increments or decrements, is an essential component of visual computation and a fundamental property of visual systems across species. Extensive studies of adult Drosophila optic ganglia and vertebrate retinae suggest that the construction principles of ON and OFF selective pathways are shared among visual systems, albeit with circuit-specific implementations. Anatomically, dedicated neuronal pathways for ON vs. OFF responses are key features in visual circuit construction. Specific synaptic contacts are precisely built and maintained in laminar and columnar structures during development to ensure proper segregation of signals for parallel processing. Molecularly, light stimuli elicit opposite responses in ON and OFF pathways through signaling events mediated by differentially expressed neurotransmitter receptors in target neurons postsynaptic to the photoreceptor cells (PRs). This has been clearly demonstrated in the mammalian retina, where light-induced changes in glutamatergic transmission activate ON-bipolar cells via metabotropic glutamate receptor 6 (mGluR6) signaling and inhibit OFF-bipolar cells through the actions of ionotropic AMPA or kainate receptors. In the adult Drosophila visual system, functional imaging indicates that ON vs. OFF selectivity emerges from visual interneurons in the medulla. However, despite recent efforts in transcriptome profiling and genetic analyses, the molecular machinery mediating signal transformation within the ON and OFF pathways has not yet been clearly identified (Qin, 2019).
Unlike the ~6000 PRs in the adult visual system, larval Drosophila eyes consist of only 12 PRs on each side. Larval PRs make synaptic connections with a pair of visual local interneurons (VLNs) and approximately ten visual projection neurons (VPNs) in the larval optic neuropil (LON). VPNs relay signals to higher brain regions that process multiple sensory modalities. Despite this simple anatomy, larvae rely on vision for negative phototaxis, social clustering, and form associative memories based on visual cues. How the larval visual circuit effectively processes information and supports visually guided behaviors is not understood (Qin, 2019).
Recent connectome studies mapped synaptic interactions within the LON in the first instar larval brain, revealing two separate visual pathways using either blue-tuned Rhodopsin 5 (Rh5-PRs) or green-tuned Rhodopsin 6 (Rh6-PRs). Rh5-PRs project to the proximal layer of the LON (LONp) and form direct synaptic connections with all VPNs, whereas Rh6-PRs project to the distal layer of the LON (LONd) and predominantly target one cholinergic (cha-lOLP) and one glutamatergic (glu-lOLP) local interneurons. The two PR pathways then converge at the level of VPNs (Qin, 2019).
These connectome studies also revealed potential functions for cha- and glu-lOLP. The pair of lOLPs, together with one of the VPNs, the pOLP, are the earliest differentiated neurons in the larval optic lobe and are thus collectively known as optic lobe pioneer neurons (OLPs). Besides relaying visual information from Rh6-PRs to downstream VPNs, the lOLPs also form synaptic connections with each other and receive neuromodulatory inputs from serotonergic and octopaminergic neurons, suggesting that they may act as ON and OFF detectors. This proposal is further supported by recent studies on the role of the Rh6-PR/lOLP pathway in larval movement detection and social clustering behaviors. However, it remains unclear how the lOLPs support differential coding for ON and OFF signals without anatomical separation at either the input or output level (Qin, 2019).
This study investigated the lOLPs' physiological properties and determined the molecular machinery underlying their information processing abilities. Functional imaging studies revealed differential physiological responses towards light increments and decrements in cha-lOLP and glu-lOLP, indicating their functions in detecting ON and OFF signals. Furthermore, it was found that light-induced inhibition on glu-lOLP is mediated by mAchR-B/Gαo signaling, which generates the sign inversion required for the OFF response and encodes temporal information between the cholinergic and glutamatergic transmissions received by downstream VPNs. Lastly, genetic manipulations of glu-lOLP strongly modified the physiological responses of VPNs and eliminated dark-induced pausing behaviors. Together, these studies identify specific cellular and molecular pathways that mediate OFF detection in Drosophila larvae, reveal functional interactions among key components of the larval visual system, and establish a circuit mechanism for ON vs. OFF discrimination in this simple circuit (Qin, 2019).
The Drosophila larval visual circuit, with its small number of components and complete wiring diagram, provides a powerful model to study how specific synaptic interactions support visual computation. Built on knowledge obtained from connectome and behavioral analyses, the current physiological and genetic studies revealed unique computational strategies utilized by this simple circuit for processing complex outputs. Specifically, the results indicate that ON vs. OFF discrimination emerges at the level of the lOLPs, a pair of second-order visual interneurons. In addition, the essential role is demonstrated of glu-lOLP, a single glutamatergic interneuron, in meditating OFF detection at both the cellular and behavior levels and identify mAchR-B/Gαo signaling as the molecular machinery regulating its physiological properties (Qin, 2019).
Functional imaging studies using genetically encoded calcium and voltage indicators provide valuable information regarding the physiological properties of synaptic interactions among larval visual interneurons and projection neurons. However, optical recording approaches have certain technical limitations, including the kinetics and sensitivities of the voltage and calcium sensors, as well as the imaging and visual stimulation protocols. In addition, although glu-lOLP displays a biphasic response towards the light stimulation, calcium reductions and increases for only the initial set of physiological characterizations were quantified. Compared to the delayed calcium rise, the light-induced calcium reductions have low amplitudes and high variabilities, possibly due to the half-wave rectification of the intracellular calcium previously described in adult visual interneurons. For the genetic experiments, focus was placed on evaluating the activation of glu-lOLP, which is reflected by the increase of intracellular calcium signals that lead to neurotransmitter release (Qin, 2019).
To process light and dark information in parallel, both mammalian and adult fly visual systems utilize anatomical segregation to reinforce split ON and OFF pathways. In the larval visual circuit, however, almost all VPNs receive direct inputs from both cha-lOLP and glu-lOLP as well as the Rh5-PRs. Therefore, the response signs of the VPNs cannot be predicted by their anatomical connectivity to ON and OFF detectors. Based on the cumulative evidence obtained through genetic, anatomical, and physiological studies, it is proposed that temporal control of inhibition potentially contributes to ON vs. OFF discrimination in larvae. While cha-lOLP displays clear ON selectivity, the OFF selectivity in glu-lOLP is strengthened by the extended suppression of its light response by mAchR-B/Gαo signaling. This temporal control may also produce a window of heightened responsiveness in cha-lOLP and ON-VPNs towards light signals, similar to the case in mammalian sensory systems where the temporal delay of input-evoked inhibition relative to excitation sharpens the tuning to preferred stimuli. Together, the temporal separation between cholinergic and glutamatergic transmission could reinforce the functional segregation in the VPNs and lead to distinct transmissions of ON and OFF signals. Although further functional validations are needed, temporal control of inhibition provides an elegant solution that may be of general use in similar contexts where parallel processing is achieved without anatomically split pathways (Qin, 2019).
The connectome study identified ten larval VPNs which receive both direct and filtered inputs from two types of PRs and transmit visual information to higher brain regions, including four LNvs (PDF-LaNs), five LaN, nc-LaN1, and two pVL09, VPLN, and pOLP17. Based on these studies on LNvs and pOLP, it is expected the functional diversity in VPNs generated by differential expression of neurotransmitter receptors or molecules involved in electric coupling will be observed. Besides basic ON vs. OFF discrimination, VPNs are also involved in a variety of visually guided behaviors. The temporal regulation of their glutamatergic and cholinergic inputs as well as the local computation within the LON are among potential cellular mechanisms that increase the VPNs' capability to process complex visual information. Further physiological and molecular studies of the VPNs and behavioral experiments targeting specific visual tasks are needed to elucidate their specific functions (Qin, 2019).
Besides the similarities observed between larval lOLPs and the visual interneurons in the adult fly visual ganglia, an analogy can be drawn between lOLPs and interneurons in mammalian retinae based on their roles in visual processing. Cha-lOLP and glu-lOLP carry sign-conserving or sign-inverting functions and activate ON- or OFF-VPNs, respectively, performing similar functions as bipolar cells in mammalian retinae. At the same time, lOLPs also provide inhibitory inputs to either ON- or OFF-VPNs and thus exhibit the characteristics of inhibitory amacrine cells. The dual role of lOLPs is the key feature of larval ON and OFF selectivity, which likely evolved to fulfill the need for parallel processing using limited cellular resources (Qin, 2019).
Lastly, these studies reveal signaling pathways shared between mammalian retinae and the larval visual circuit. Although the two systems are constructed using different neurochemicals, Gαo signaling is responsible for producing sign inversion in both glu-lOLP and the ON-bipolar cell. In mGluR6-expressing ON-bipolar cells, light increments trigger Gαo deactivation, the opening of TrpM1 channels, and depolarization. In larval glu-lOLP, how light induces voltage and calcium responses via mAchR-B signaling has yet to be determined. Gαo is known to have functional interactions with a diverse group of signaling molecules including potassium and calcium channels that could directly link the light-elicited physiological changes in glu-lOLP. Genetic and physiological studies in the larval visual circuit will facilitate the discovery of these target molecules and contribute to the mechanistic understanding of visual computation (Qin, 2019).
A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. This study shows that the Hofbauer-Buchner eyelets, located between the retina and the medulla in the fly optic lobes, differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, it was shown that the large LNvs interact with subsets of "evening cells" to adjust the timing of the evening peak of activity in a day length-dependent manner. This work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms (Schlichting, 2016).
Circadian clocks create an endogenous sense of time that is used to produce daily rhythms in physiology and behavior. A defining characteristic of a circadian clock is a modest deviation of its endogenous period from the 24.0 h period of daily environmental change. For example, the average human clock has an endogenous period of 24 h and 11 min. Thus, to maintain a consistent phase relationship with the environment, the human clock must be sped up by 11 min every day. A sensitivity of the circadian clock to environmental time cues (zeitgebers) ensures that circadian clocks are adjusted daily to match the period of environmental change. This process, called entrainment, is fundamental to the proper daily timing of behavior and physiology. For most organisms, daily light/dark (LD) cycles are the most salient zeitgeber (Schlichting, 2016).
Although most tissues express molecular circadian clocks in animals, the clock is required in small islands of neural tissue for the presence of sleep/activity rhythms and many other daily rhythms in physiology. Within these islands, a circadian clock neuron network (CCNN) functions as the master circadian clock. Subsets of neurons within the CCNN receive resetting signals from photoreceptors, and physiological connections between these neurons and their clock neuron targets ensure light entrainment of the CCNN as a whole (Schlichting, 2016).
In both mammals and insects, the CCNN receives light input from multiple photoreceptor types. In Drosophila, the CCNN is entrained by photoreceptors in the compound eye, the ocelli, the Hofbauer-Buchner (HB) eyelets, and by subsets of clock neurons that express the blue light photoreceptor Cryptochrome (CRY). Understanding how multiple light input pathways modulate the CCNN to ensure entrainment to the environmental LD cycle is critical for understanding of the circadian system and its dysfunction when exposed to the unnatural light regimens accompanying much of modern life (Schlichting, 2016).
This study investigates the physiological basis and circadian role of a long-suspected circadian light input pathway in Drosophila: the HB eyelets. These simple accessory eyes contain four photoreceptors located at the posterior edges of the compound eyes and project directly to the accessory medullae (AMe), neuropils that support circadian timekeeping in insects. In Drosophila, the AMe contain projections from ventral lateral neurons (LNvs), important components of the CCNN that express the neuropeptide pigment dispersing factor (PDF), an output required for robust circadian rhythms in locomotor activity. The axon terminals of the HB eyelets terminate near PDF-positive LNv projections and analysis of visual system and cry mutants reveals a role for the HB eyelet in the entrainment of locomotor rhythms to LD cycles, but how the eyelets influence the CCNN to support light entrainment is not well understood (Schlichting, 2016).
This study presents evidence that this circadian light input pathway excites the small LNvs (s-LNvs) and acts to phase-dependently advance free-running rhythms in sleep/activity while inhibiting the large LNvs (l-LNvs). This work reveals that input from external photoreceptors differentially affects specific centers within the fly CCNN. Furthermore, it was shown that, under long summer-like days, the l-LNvs act to modulate subsets of so-called evening cells to delay the onset of evening activity. These results reveal a neural network underlying the photoperiodic adjustment of sleep and activity (Schlichting, 2016).
The experiments described in this study lead to two unexpected findings regarding the network properties of circadian entrainment in Drosophila. First, the l-LNvs govern the phase of evening peak of activity through PdfR-dependent effects on evening cells that bypass the s-LNvs. Although previous work has implicated the l-LNvs in the control of evening peak phase, the current results are the first to provide evidence that there is a direct connection between the l-LNvs and evening cells within the AMe and that this connection mediates the photoperiodic adjustment of sleep and activity in the fly. Second, the HB eyelets light input pathways, long implicated in circadian entrainment, have opposing effects on the l-LNvs and s-LNvs, inhibiting the former and exciting the latter. These results reveal not only a differential effect of a light input pathway on specific nodes of the CCNN but also establish that light from extraretinal photoreceptors can have synergistic or antagonistic effects on CRY- and compound eye-mediated light responses, depending on the clock neuron target in question (Schlichting, 2016).
Both the l-LNvs and s-LNvs express the blue light circadian photoreceptor CRY, the expression of which renders neurons directly excitable by light entering the brain through the cuticle. How such CRY-mediated light input interacts with input from external photoreceptors is not well understood, although it is known that each system alone is sufficient for the entrainment of locomotor rhythms. Genetic evidence suggests that the HB eyelets have relatively weak effects on circadian entrainment: flies with functional eyelets that lack compound eyes, ocelli, and CRY entrain relatively poorly to LD cycles relative to flies with functional eyes or CRY. The small phase responses of locomotor rhythms to HB eyelet excitation further supports a relatively weak effect of the eyelet on free-running locomotor rhythms (Schlichting, 2016).
The LNvs are critical nodes in the CCNN and are closely associated with input pathways linking the central brain to external photoreceptors. Work on the LNvs has provided evidence for a division of labor among the l-LNvs and s-LNvs: the l-LNvs are wake-promoting neurons that acutely govern arousal and sleep independently of the s-LNvs, whereas the s-LNvs act as key coordinators of the CCNN to support robust circadian timekeeping. Anatomical and genetic evidence has long supported the notion that the dorsal projections of the s-LNvs represent the key connection between the LNvs and the remaining components of the CCNN. However, a smaller body of work has suggested that the l-LNvs also contribute to the entrainment of sleep/activity rhythms under LD cycles. The Pdf knockdown and PdfR rescue experiments under long day conditions indicate that, as the day grows longer, the l-LNvs play a greater role in the timing of the evening peak. Moreover, the effects of PDF released from the l-LNvs are mediated not by the PDF receptive s-LNvs but rather by the fifth s-LNvs and a subset of the LNds, the NPF and ITP coexpressing LNds in particular (with some influence of the other PDF-receptor positive LNds). These same neurons were recently identified as evening cells that are physiologically responsive to PDF but relatively weakly coupled to LNv clocks under conditions of constant darkness. The results suggest that the l-LNvs differentially modulate the NPF/ITP-positive evening oscillators as a function of day length, producing stronger PDF-dependent delays under long day conditions through increased release of PDF from the l-LNvs, thereby delaying the evening activity peak. Thus, the l-LNvs mediate their effects on the evening peak of activity through their action on the NPF/ITP-positive subset of evening oscillators. The proposed PDF release from the l-LNvs under long days requires their activation via CRY and/or the compound eyes via ACh release from lamina L2 interneurons. It is hypothesized that the inhibitory influence of the HB eyelets ceases under long days allowing the compound eyes and CRY to maximally excite the l-LNvs. Indeed, previous work has established that the compound eyes are especially important for adapting fly evening activity to long days. Furthermore, several studies have suggested that the compound eyes signal to the l-LNvs leading to enhanced PDF release and a slowing-down of the evening oscillators. A recent paper measuring Ca2+ rhythms in the different clock neurons in vivo supports this view (Liang, 2016): Ca2+ rhythms in the l-LNvs peak in the middle of the day, unlike the s-LNvs, which display Ca2+ peaks in the late night/early morning. It is suggested that this phasing is produced by the inhibition of l-LNvs by the eyelets in the morning, followed by the excitation of the l-LNvs by the compound eyes and CRY. Interestingly, the only other clock neuron classes to display Ca2+ increases during the day are the LNds and fifth-sLNv, which phase lag the l-LNvs by ~2.5 h and display peak Ca2+ levels in the late afternoon, a time that coincides with the evening peak of activity (Liang, 2016). It is proposed that the relative coordination of Ca2+ rhythms between the l-LNvs and the LNds/fifth-sLNv is produced by the connection this study has identified between these neurons and the action of the eyelet and visual system on the l-LNvs (Schlichting, 2016).
Recent work has revealed that evening activity is promoted directly by the evening oscillator neurons and that the mid-day siesta is produced by the daily inhibition of evening oscillators by a group of dorsal clock neurons (Guo, 2016). It is proposed that the connections described in this study govern the timing of the evening peak of activity through the PDF-dependent modulation of the molecular clocks within the evening oscillator neurons, although PDF modulation likely results in the excitation of target neurons, which would promote evening activity. The results reveal new and unexpected network properties underlying the entrainment of the circadian clock neuron network to LD cycles. Excitatory effects of light on the LNvs are differentially modulated by the HB eyelets via cholinergic excitation of the s-LNvs and histaminergic inhibition of the l-LNvs. The work further reveals PDF-dependent modulatory connections in the AMe between the l-LNvs and the s-LNvs and, most surprisingly, between the l-LNvs and a small subset of evening oscillators. This work indicates that the latter connection is critical for the adjustment of evening activity phase during long, summer-like days. This network model of entrainment reveals not only how CRY and external photoreceptors interact within specific nodes of the CCNN, but also how photoreception is likely to drive changes in CCNN output in the face of changing day length (Schlichting, 2016).
In order to provide organisms a fitness advantage, circadian clocks have to react appropriately to changes in their environment. High light intensities (HI) play an essential role in the adaptation to hot summer days, which especially endanger insects of desiccation or prey visibility. This study shows that solely increasing light intensity leads to an increased midday siesta in Drosophila behavior. Interestingly, this change is independent of the fly's circadian photoreceptor cryptochrome (CRY), and solely caused by a small visual organ, the Hofbauer-Buchner (HB) eyelets. Using receptor knockdowns, immunostaining, as well as recently developed calcium tools, the eyelets were shown to activate key core clock neurons, namely the s-LNvs, at HI. This activation delays the decrease of PER in the middle of the day and propagates to downstream target clock neurons that prolong the siesta. Together a new pathway is shown for integrating light intensity information into the clock network, suggesting new network properties and surprising parallels between Drosophila and the mammalian system (Schlichting, 2019).
The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, these findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye (Zhou, 2016).
Visual perception of the environment is mediated by specialized photoreceptor (PR) neurons of the eye. Each PR expresses photosensitive opsins, which are activated by a particular wavelength of light. In most insects, the visual system comprises a pair of compound eyes that are mainly associated with motion, color or polarized light detection, and a triplet of ocelli that are thought to be critical during flight to detect horizon and movements. It is widely believed that the evolutionary diversification of compound eye and ocelli in insects occurred from an ancestral visual organ around 500 million years ago. Concurrently, opsin genes were also duplicated to provide distinct spectral sensitivities to different PRs of compound eye and ocelli. In the fruit fly Drosophila melanogaster, Rhodopsin1 (Rh1) and Rh2 are closely related opsins that originated from the duplication of a single ancestral gene. However, in the visual organs, Rh2 is uniquely expressed in ocelli whereas Rh1 is uniquely expressed in outer PRs of the compound eye. It is currently unknown how this differential expression of Rh1 and Rh2 in the two visual organs is controlled to provide unique spectral sensitivities to ocelli and compound eyes. This study shows that Homothorax (Hth) is expressed in ocelli and confers proper rhodopsin expression. Hth was shown to control a binary Rhodopsin switch in ocelli to promote Rh2 expression and repress Rh1 expression. Genetic and molecular analysis of rh1 and rh2 supports that Hth acts through their promoters to regulate Rhodopsin expression in the ocelli. Finally, this study also showed that when ectopically expressed in the retina, hth is sufficient to induce Rh2 expression only at the outer PRs in a cell autonomous manner. It is therefore proposed that the diversification of rhodpsins in the ocelli and retinal outer PRs occurred by duplication of an ancestral gene, which is under the control of Homothorax (Mishra, 2021).
Aguilar-Hidalgo, D., Dominguez-Cejudo, M. A., Amore, G., Brockmann, A., Lemos, M. C., Cordoba, A. and Casares, F. (2013). A Hh-driven gene network controls specification, pattern and size of the Drosophila simple eyes. Development 140: 82-92. PubMed ID: 23154412
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Blanco, J., Pauli, T., Seimiya, M., Udolph, G. and Gehring, W. J. (2010). Genetic interactions of eyes absent, twin of eyeless and orthodenticle regulate sine oculis expression during ocellar development in Drosophila. Dev Biol 344: 1088-1099. PubMed ID: 20580700
Brockmann, A., Dominguez-Cejudo, M. A., Amore, G. and Casares, F. (2011). Regulation of ocellar specification and size by twin of eyeless and homothorax. Dev Dyn 240: 75-85. PubMed ID: 21104743
Damulewicz, M., Ispizua, J. I., Ceriani, M. F. and Pyza, E. M. (2020). Communication Among Photoreceptors and the Central Clock Affects Sleep Profile. Front Physiol 11: 993. PubMed ID: 32848895
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Guo, F., Yu, J., Jung, H. J., Abruzzi, K. C., Luo, W., Griffith, L. C. and Rosbash, M. (2016). Circadian neuron feedback controls the Drosophila sleep--activity profile. Nature 536: 292-297. PubMed ID: 27479324
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Li, M. T., Cao, L. H., Xiao, N., Tang, M., Deng, B., Yang, T., Yoshii, T. and Luo, D. G. (2018). Hub-organized parallel circuits of central circadian pacemaker neurons for visual photoentrainment in Drosophila. Nat Commun 9(1): 4247. PubMed ID: 30315165
Liang, X., Holy, T. E. and Taghert, P. H. (2016). Synchronous Drosophila circadian pacemakers display nonsynchronous Ca(2)(+) rhythms in vivo. Science 351: 976-981. PubMed ID: 26917772
Mishra, A. K., Fritsch, C., Voutev, R., Mann, R. S. and Sprecher, S. G. (2021). Homothorax controls a binary Rhodopsin switch in Drosophila ocelli. PLoS Genet 17(7): e1009460. PubMed ID: 34314427
Qin, B., Humberg, T. H., Kim, A., Kim, H. S., Short, J., Diao, F., White, B. H., Sprecher, S. G. and Yuan, Q. (2019). Muscarinic acetylcholine receptor signaling generates OFF selectivity in a simple visual circuit. Nat Commun 10(1): 4093. PubMed ID: 31501438
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Schlichting, M., Menegazzi, P., Lelito, K. R., Yao, Z., Buhl, E., Dalla Benetta, E., Bahle, A., Denike, J., Hodge, J. J., Helfrich-Forster, C. and Shafer, O. T. (2016). A neural network underlying circadian entrainment and photoperiodic adjustment of sleep and activity in Drosophila. J Neurosci 36: 9084-9096. PubMed ID: 27581451
Schlichting, M., Menegazzi, P., Rosbash, M. and Helfrich-Forster, C. (2019). A distinct visual pathway mediates high light intensity adaptation of the circadian clock in Drosophila. J Neurosci. PubMed ID: 30606757
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Yorimitsu, T., Kiritooshi, N. and Nakagoshi, H. (2011). Defective proventriculus specifies the ocellar region in the Drosophila head. Dev Biol 356: 598-607. PubMed ID: 21722630
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Zhou, Q., DeSantis, D. F., Friedrich, M. and Pignoni, F. (2016). Shared and distinct mechanisms of atonal regulation in Drosophila ocelli and compound eyes. Dev Biol 418: 10-16. PubMed ID: 27565023
Genes involved in organ development
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