BACKGROUND
most organisms exhibit daily rhythms in physiology, metabolism, and behavior that persist in the absence of environmental cues.
in animals, these ~ <dig> hr rhythms are controlled by circadian oscillators that reside in the central nervous system  and/or peripheral tissues.
these oscillators are comprised of interlocked transcriptional feedback loops that regulate rhythmic gene expression within and downstream of the circadian timekeeping mechanism.
in drosophila, the per/tim and clk feedback loops control rhythmic transcription that peaks around dusk and dawn, respectively .
the per/tim feedback loop is initiated during mid-day, when clk/cyc heterodimers bind e-box sequences to activate per and tim transcription  <cit> .
although per and tim mrnas peak around dusk, phosphorylation of per and tim delays their peak accumulation to the late evening and promotes their nuclear localization  <cit> .
after entering the nucleus, per or per-tim heterodimers bind clk to inhibit clk-cyc-dependent transcription  <cit> .
in addition, clockwork orange  is also thought to inhibit per and tim transcription by competing for e-box binding with clk-cyc  <cit> .
per and tim are then degraded after dawn, thus relieving transcriptional inhibition.
clk-cyc initiates the clk feedback loop by binding e-boxes to activate vri transcription  <cit> .
vri accumulates in parallel with vri mrna during early evening and binds to v/p-boxes to repress clk transcription  <cit> .
mutants that disrupt clk-cyc transcriptional activity  exhibit constitutive high levels of clk mrna  <cit> , indicating that clk is activated independent of circadian oscillator function.
since clk-cyc is required to initiate circadian feedback loop function, we hypothesize that the activation of clk and cyc during development determines oscillator cell identity.
locomotor activity rhythms in adults can be synchronized by light-dark cycles in l <dig> larvae, but not in embryos, which indicates that the circadian oscillator is only functional after hatching  <cit> .
circadian oscillator cells are present in lnvs, dn1s and dn2s from l <dig> larval brains based on rhythmic expression of per and tim  <cit> .
since entrainment of oscillators to light is tim dependent, and tim accumulates in concert with per about 6– <dig> h after their respective mrnas , per and tim transcription are expected to be initiated during embryogenesis.
indeed, per mrna is detected in the central nervous system  of embryos  <cit> , which implies that clk and cyc accumulate in presumptive oscillator cells during embryonic development.
to understand oscillator cell development in drosophila, the spatial and temporal expression of clk and per was determined during embryogenesis.
in our previous studies, clk gp <dig> antibody revealed clk expression in circadian oscillator and non-oscillator cell nuclei from adult heads at all times of day  <cit> .
using a newly generated clk antibody we show here that clk is expressed exclusively in circadian oscillator cells, and that detection of clk in non-oscillator cells in a previous study was due to cross-reactivity with dachshund .
during embryonic development per is first expressed in the ventral nerve chord  at es  <dig> and then the brain at es  <dig>  whereas clk is not detected until es  <dig> in brain cells that lack per expression.
these clk-expressing brain cells correspond to lnvs, dn1s and dn2s, and by the end of es  <dig> or early es  <dig> per is detected in lnvs and dn1s but not dn2s.
these results demonstrate that presumptive brain oscillator cells are present before functional oscillators are detected around the transition to larval life, suggest that the delayed appearance of per accumulation in presumptive embryonic dn2s gives rise to the antiphase cycling of per in larval dn2s compared to lnvs and dn1s, and imply that per has a clock-independent function in the vnc and brain in non-oscillator cells of embryos.
RESULTS
clk expression is detected only in oscillator neurons
we previously demonstrated clk immunostaining in all circadian oscillator cells and some non-oscillator cells in adults  <cit> .
one group of non-oscillator cells that showed clk immunostaining was kenyon cells , which are involved in olfactory learning and memory  <cit> .
to characterize clk immunostaining during development, we co-stained l <dig> larval cnss with clk, oscillator cell marker per, and the kc cell marker dac  <cit> .
as expected, we observed clk staining in every per-expressing cell, but also detected clk in every dac-expressing cell .
this surprising correspondence between clk and dac expression in non-oscillator cells suggests a relationship between clk and dac activation: clk and dac are activated by the same activator, clk activates dac, or dac activates clk.
alternately, clk and dac co-immunostaining could also result from cross-reactivity of clk antiserum to dac, though little, if any, sequence identity is evident between these two proteins .
because clk and dac run at a similar apparent molecular weight of ~ <dig> kda on western blots  <cit> , we tested whether our clk antiserum  cross-reacted with in vitro translated dac.
as expected, gp <dig> detected clk on western blots, but this clk antiserum also detected dac .
competition with purified dac protein blocked gp <dig> detection of dac on western blots , confirming dac cross-reactivity.
likewise, incubating l <dig> cnss with purified dac specifically blocks gp <dig> immunostaining in dac expressing non-oscillator cells .
gp <dig> antiserum detects more than the canonical  <dig> to  <dig> lnvs,  <dig> dn2s, and  <dig> dn1s in l <dig> brains blocked with purified dac.
although these additional cells could be due to incomplete blocking by dac, they may also represent dn3s, lnds, and/or l-lnvs, which have been detected previously in l <dig> brains  <cit> .
clk immunostaining in non-oscillator cells is also eliminated in the cns of dac <dig> mutant larvae , which lack dac protein  <cit> .
taken together, these results demonstrate that clk immunoreactivity  in non-oscillator cells is due to cross-reactivity with dac, which implies that clk expression is limited to oscillator cells.
additional clk antisera that we had generated were tested for cross-reactivity to dac on western blots, and clk antiserum gp <dig> showed no cross-reactivity to dac .
based on the lack of clk ir in non-oscillator cells from dac <dig> mutant larvae and l <dig> cnss blocked with dac antigen, we expected gp <dig> to detect clk ir only in oscillator cells.
indeed, clk and per co-immunostained several clusters of cells including slnvs, dn2s and dn1s in l <dig> larvae , but do not detect cells elsewhere in the cns , showing that clk gp <dig> antiserum specifically detects oscillator cells in l <dig> brains.
four additional clusters of brain oscillator neurons are present in adult brains: lnds, l-lnvs, lpns and dn3s  <cit> .
brains from adults collected at zt <dig> were co-immunostained with gp <dig> and per to determine if clk expression is limited to oscillator neurons.
clk ir is also detected exclusively in oscillator cells from adult brains and is reduced or eliminated in brains from clkjrk adults , which express very low levels of truncated clk protein .
these results demonstrate that gp <dig> specifically detects clk, and that clk is expressed exclusively in circadian oscillator cells in wild-type adult brains.
given that clk is detected specifically in brain oscillator neurons in adults and l <dig> larvae, we used clk gp <dig> antiserum to determine when brain oscillator neurons first appear during development.
per is expressed before clk during embryogenesis
to determine whether clk is expressed in presumptive brain oscillator cells, embryos between  <dig> h and  <dig> h old were collected at ct <dig> and co-immunostained with per and clk.
per ir is detected in a segmented pattern along vnc as early as es  <dig> , consistent with previous in situ hybridization results in embryos  <cit> .
per in the vnc encompasses more cells, increases in intensity, and expands into the brain by es  <dig> .
loss of per ir in per <dig> mutant confirms that this ir represents true per expression .
surprisingly, no clk ir is detected in per-expressing cells, indicating that per is not activated by clk-cyc during these early developmental stages.
the clk-cyc independent activation of per is similar to the situation in ovaries, where per expression is not associated with circadian oscillator function  <cit> .
to ensure that clk expression below detectable levels does not activate per at es 12– <dig>  clkjrk embryos were immunostained for per.
these clkjrk embryos show per staining in the brain and vnc identical to that in wild-type embryos at es  <dig> .
clk expression preceeds per expression in presumptive brain oscillator cells during embryogenesis
early in es  <dig>  weak clk immunoreactivity is first detected in brain cells that do not express per .
during mid to late es  <dig>  clk expression becomes stronger and expands to additional cells in the dorsal brain .
in addition, per starts to be detected in clk positive cells in the ventral portion of the brain .
by es  <dig>  clk positive cells form three distinct clusters in each brain hemisphere, with two dorsal cells, four ventral cells, and two cells between and slightly posterior to these dorsal and ventral clusters .
the positions of these clk positive cells are reminiscent of oscillator cells in larvae; a dorsal cluster of dn1s, a ventral cluster of s-lnvs, and a medial cluster of dn2s  <cit> .
although per is expressed in some clk-positive dorsal brain neurons, two cells situated between the most dorsal and ventral clk-expressing brain cells show little or no per expression .
based on their location, these clk positive/per negative cells likely correspond to dn2s.
in larvae, per cycling in dn2s is antiphase compared to lns and dn1s  <cit> , which is consistent with the absence of per expression in the presumptive dn2s of embryos during the late night and early morning.
these results suggest that clk is expressed in lnvs, dn1s, and dn2s starting at es  <dig>  followed by per expression in dn1s and lnvs during late es  <dig> and es  <dig>  the timeline for clk and per expression in embryos is the same whether they are collected at ct <dig>  or ct <dig>  , indicating that clk and per expression are controlled developmentally and are not influenced by the time at which embryos were laid during the circadian cycle.
to confirm that clk is expressed in larval lnvs, dn1s, and dn2s, l <dig> larvae collected at zt <dig> and zt <dig> were immunostained with clk gp <dig> and per antisera.
as expected, clk is expressed in all per-expressing cells at zt <dig>  which includes lnvs and dn1s .
clk is also expressed in the presumed dn2s, which lack per expression at this time.
in contrast, per is co-expressed with clk in dn2s at zt <dig>  but is absent in lnvs and dn1s .
in addition, little or no per ir is detected at either time point in the vnc.
from these experiments, we conclude that clk is constantly expressed only in brain oscillator cells from l <dig> larvae; whereas per is rhythmically expressed only in oscillator cells from l <dig> larval brains with high levels at zt <dig> in lnvs and dn1s and at zt <dig> in dn2s.
discussion and
CONCLUSIONS
clk is expressed exclusively in oscillator cells
clk immunostaining was previously detected in all oscillator cells and many non-oscillator cells from adult brains  <cit> .
clk expression in non-oscillator cells was coincident with that of dac, which is structurally related to the winged helix/forked-head subfamily of helix-turn-helix dna binding proteins  <cit> .
here, we find that clk ir in non-oscillator cells is due to cross-reactivity between clk gp <dig> antiserum and dac .
we also characterized another clk antiserum, gp <dig>  and demonstrated that it does not cross-react with dac on westerns .
immunostaining of adult brains with gp <dig> confirms that clk is expressed only in oscillator neurons .
the oscillator cell-specific expression of clk implies that clk is required for the development and/or function of these cells.
this cell type specificity is consistent with the induction of oscillator cell function, when clk is expressed in ectopic locations  <cit> .
however, clk expression cannot induce ectopic oscillators in any cell type, suggesting that other factors critical for oscillator function are not activated by clk or the clk-dependent developmental programs are incompatible with the development of many cell types.
in the loss-of-function clkjrk mutant, expression of direct clk-cyc target genes, per, tim, vri, and pdp1ε is abolished  <cit> , making it difficult to positively identify oscillator cells.
though peripheral oscillator tissues  apparently develop normally in clkjrk flies, the loss of oscillator neuron markers in clkjrk flies makes it difficult to determine whether these neurons are present.
one exception to this is l-lnvs, which continue to express pdf in clkjrk flies  <cit> .
determining whether clk contributes to oscillator cell development depends on the availability of oscillator cell markers that are expressed independent of clk-cyc or rescue of a loss-of-function clk mutant, upon clk induction in adults.
oscillator cell development
in adults, clk-dependent activation of the feedback regulator per is required for oscillator function  <cit> , thus we expect that clk would be expressed before per during development.
however, per mrna and protein are expressed in the vnc starting at es  <dig> and in the brain at es  <dig> , well before clk is detected in the brain at es  <dig>  this early per expression in the vnc and brain is independent of clk because it persists in the clkjrk mutant and does not overlap with clk later in development.
the role of per in clk negative cells during embryogenesis is unknown, but it is possible that per modulates transcription by targeting other bhlh-pas transcription factors  expressed in these cells  <cit> .
regardless of the role per plays, the lack of obvious developmental defects in per <dig> flies suggests that per is not critical for embryonic development.
in larvae, the intensity of per ir in clk-negative brain cells and the vnc decreases drastically , consistent with previous results  <cit> .
clk can be detected in 2– <dig> cells in each brain hemisphere starting at early to mid es  <dig>  and expands during es  <dig> to approximately eight clk-positive cells in each brain hemisphere .
these cells are spatially segregated into three groups that correspond to larval lnvs, dn2s, and dn1s .
per can be detected in some clk-positive brain cells starting as early as the end of es  <dig> , or about  <dig> h post-fertilization.
during es  <dig>  per ir increases in intensity and encompasses all four lnvs and both dn1s .
the 3– <dig> h delay between clk detection and per detection in embryonic brain cells is similar to the delay between the accumulation of per mrna and protein in adults  <cit> , and suggests that once clk-cyc initiates per transcription in embryos, per accumulation is delayed by the same dbt-dependent per degradation mechanism described in adults.
the initiation of molecular oscillator function at es  <dig> coincides with the existence of light-entrainable oscillators that mediate behavioral rhythms in adults; a  <dig> h light pulse ending  <dig> h before larval hatching didn't synchronize behavioral rhythms of adults, but a  <dig> h light pulse ending at the time of larval hatching did synchronize behavioral rhythms in adults  <cit> .
the initiation of oscillator function by clk in embryos is also consistent with clk's unique ability to initiate oscillator function when expressed in certain ectopic cells  <cit> .
when combined with these previous studies, our results support a model whereby clk expression initiates circadian oscillator function in brain neurons at es  <dig>  and these brain neurons go on to control rhythms in locomotor activity in adults.
clk is expressed in all three groups of oscillator neurons during es  <dig> and es  <dig>  but per is only detected in lnvs and dn1s during this time.
the delayed onset of per accumulation in dn2s is intriguing, considering that the dn2s oscillator is antiphase compared to those in larval lnvs and dn1s  <cit> .
the antiphase cycling of oscillator in dn2s can be brought into phase with oscillators in lnvs and dn1s by expressing cry in dn2s  <cit> , demonstrating that this antiphase cycling is cry-dependent.
whether cry also acts to control antiphase cycling in embryonic dn2s will be investigated.
in any case, our results demonstrate that antiphase cycling of oscillators in dn2s is developmentally regulated and light independent.
the activation and maintenance of clk transcription in developing and adult drosophila is not well understood.
the basic zipper protein pdp1ε is involved in maintaining clk activation in adults  <cit> , but does not appear to be the primary clk activator  <cit> .
one approach to defining clk activators in embryos is to first determine which cells within the drosophila embryonic brain express clk base on co-expression of marker genes  <cit> .
once clk-expressing cells have been identified, transcriptional activators expressed in these cells can be tested singly or in combination for their ability to activate clk.
identifying factors that activate clk in a cell-specific manner will ultimately reveal determinants of oscillator cell fate.

 most organisms exhibit daily rhythms in physiology, metabolism, and behavior that persist in the absence of environmental cues. in animals, these ~ hr rhythms are controlled by circadian oscillators that reside in the central nervous system and/or peripheral tissues. these oscillators are comprised of interlocked transcriptional feedback loops that regulate rhythmic gene expression within and downstream of the circadian timekeeping mechanism. in drosophila, the per/tim and clk feedback loops control rhythmic transcription that peaks around dusk and dawn, respectively. the per/tim feedback loop is initiated during mid-day, when clk/cyc heterodimers bind e-box sequences to activate per and tim transcription. although per and tim mrnas peak around dusk, phosphorylation of per and tim delays their peak accumulation to the late evening and promotes their nuclear localization. after entering the nucleus, per or per-tim heterodimers bind clk to inhibit clk-cyc-dependent transcription. in addition, clockwork orange is also thought to inhibit per and tim transcription by competing for e-box binding with clk-cyc. per and tim are then degraded after dawn, thus relieving transcriptional inhibition. clk-cyc initiates the clk feedback loop by binding e-boxes to activate vri transcription. vri accumulates in parallel with vri mrna during early evening and binds to v/p-boxes to repress clk transcription. mutants that disrupt clk-cyc transcriptional activity exhibit constitutive high levels of clk mrna, indicating that clk is activated independent of circadian oscillator function. since clk-cyc is required to initiate circadian feedback loop function, we hypothesize that the activation of clk and cyc during development determines oscillator cell identity. locomotor activity rhythms in adults can be synchronized by light-dark cycles in l larvae, but not in embryos, which indicates that the circadian oscillator is only functional after hatching. circadian oscillator cells are present in lnvs, dn1s and dn2s from l larval brains based on rhythmic expression of per and tim. since entrainment of oscillators to light is tim dependent, and tim accumulates in concert with per about 6– h after their respective mrnas, per and tim transcription are expected to be initiated during embryogenesis. indeed, per mrna is detected in the central nervous system of embryos, which implies that clk and cyc accumulate in presumptive oscillator cells during embryonic development. to understand oscillator cell development in drosophila, the spatial and temporal expression of clk and per was determined during embryogenesis. in our previous studies, clk gp antibody revealed clk expression in circadian oscillator and non-oscillator cell nuclei from adult heads at all times of day. using a newly generated clk antibody we show here that clk is expressed exclusively in circadian oscillator cells, and that detection of clk in non-oscillator cells in a previous study was due to cross-reactivity with dachshund. during embryonic development per is first expressed in the ventral nerve chord at es and then the brain at es whereas clk is not detected until es in brain cells that lack per expression. these clk-expressing brain cells correspond to lnvs, dn1s and dn2s, and by the end of es or early es per is detected in lnvs and dn1s but not dn2s. these results demonstrate that presumptive brain oscillator cells are present before functional oscillators are detected around the transition to larval life, suggest that the delayed appearance of per accumulation in presumptive embryonic dn2s gives rise to the antiphase cycling of per in larval dn2s compared to lnvs and dn1s, and imply that per has a clock-independent function in the vnc and brain in non-oscillator cells of embryos.  clk expression is detected only in oscillator neurons we previously demonstrated clk immunostaining in all circadian oscillator cells and some non-oscillator cells in adults. one group of non-oscillator cells that showed clk immunostaining was kenyon cells, which are involved in olfactory learning and memory. to characterize clk immunostaining during development, we co-stained l larval cnss with clk, oscillator cell marker per, and the kc cell marker dac. as expected, we observed clk staining in every per-expressing cell, but also detected clk in every dac-expressing cell. this surprising correspondence between clk and dac expression in non-oscillator cells suggests a relationship between clk and dac activation: clk and dac are activated by the same activator, clk activates dac, or dac activates clk. alternately, clk and dac co-immunostaining could also result from cross-reactivity of clk antiserum to dac, though little, if any, sequence identity is evident between these two proteins. because clk and dac run at a similar apparent molecular weight of ~ kda on western blots, we tested whether our clk antiserum cross-reacted with in vitro translated dac. as expected, gp detected clk on western blots, but this clk antiserum also detected dac. competition with purified dac protein blocked gp detection of dac on western blots, confirming dac cross-reactivity. likewise, incubating l cnss with purified dac specifically blocks gp immunostaining in dac expressing non-oscillator cells. gp antiserum detects more than the canonical to lnvs, dn2s, and dn1s in l brains blocked with purified dac. although these additional cells could be due to incomplete blocking by dac, they may also represent dn3s, lnds, and/or l-lnvs, which have been detected previously in l brains. clk immunostaining in non-oscillator cells is also eliminated in the cns of dac mutant larvae, which lack dac protein. taken together, these results demonstrate that clk immunoreactivity in non-oscillator cells is due to cross-reactivity with dac, which implies that clk expression is limited to oscillator cells. additional clk antisera that we had generated were tested for cross-reactivity to dac on western blots, and clk antiserum gp showed no cross-reactivity to dac. based on the lack of clk ir in non-oscillator cells from dac mutant larvae and l cnss blocked with dac antigen, we expected gp to detect clk ir only in oscillator cells. indeed, clk and per co-immunostained several clusters of cells including slnvs, dn2s and dn1s in l larvae, but do not detect cells elsewhere in the cns, showing that clk gp antiserum specifically detects oscillator cells in l brains. four additional clusters of brain oscillator neurons are present in adult brains: lnds, l-lnvs, lpns and dn3s. brains from adults collected at zt were co-immunostained with gp and per to determine if clk expression is limited to oscillator neurons. clk ir is also detected exclusively in oscillator cells from adult brains and is reduced or eliminated in brains from clkjrk adults, which express very low levels of truncated clk protein. these results demonstrate that gp specifically detects clk, and that clk is expressed exclusively in circadian oscillator cells in wild-type adult brains. given that clk is detected specifically in brain oscillator neurons in adults and l larvae, we used clk gp antiserum to determine when brain oscillator neurons first appear during development. per is expressed before clk during embryogenesis to determine whether clk is expressed in presumptive brain oscillator cells, embryos between h and h old were collected at ct and co-immunostained with per and clk. per ir is detected in a segmented pattern along vnc as early as es, consistent with previous in situ hybridization results in embryos. per in the vnc encompasses more cells, increases in intensity, and expands into the brain by es. loss of per ir in per mutant confirms that this ir represents true per expression. surprisingly, no clk ir is detected in per-expressing cells, indicating that per is not activated by clk-cyc during these early developmental stages. the clk-cyc independent activation of per is similar to the situation in ovaries, where per expression is not associated with circadian oscillator function. to ensure that clk expression below detectable levels does not activate per at es 12– clkjrk embryos were immunostained for per. these clkjrk embryos show per staining in the brain and vnc identical to that in wild-type embryos at es. clk expression preceeds per expression in presumptive brain oscillator cells during embryogenesis early in es weak clk immunoreactivity is first detected in brain cells that do not express per. during mid to late es clk expression becomes stronger and expands to additional cells in the dorsal brain. in addition, per starts to be detected in clk positive cells in the ventral portion of the brain. by es clk positive cells form three distinct clusters in each brain hemisphere, with two dorsal cells, four ventral cells, and two cells between and slightly posterior to these dorsal and ventral clusters. the positions of these clk positive cells are reminiscent of oscillator cells in larvae; a dorsal cluster of dn1s, a ventral cluster of s-lnvs, and a medial cluster of dn2s. although per is expressed in some clk-positive dorsal brain neurons, two cells situated between the most dorsal and ventral clk-expressing brain cells show little or no per expression. based on their location, these clk positive/per negative cells likely correspond to dn2s. in larvae, per cycling in dn2s is antiphase compared to lns and dn1s, which is consistent with the absence of per expression in the presumptive dn2s of embryos during the late night and early morning. these results suggest that clk is expressed in lnvs, dn1s, and dn2s starting at es followed by per expression in dn1s and lnvs during late es and es the timeline for clk and per expression in embryos is the same whether they are collected at ct or ct, indicating that clk and per expression are controlled developmentally and are not influenced by the time at which embryos were laid during the circadian cycle. to confirm that clk is expressed in larval lnvs, dn1s, and dn2s, l larvae collected at zt and zt were immunostained with clk gp and per antisera. as expected, clk is expressed in all per-expressing cells at zt which includes lnvs and dn1s. clk is also expressed in the presumed dn2s, which lack per expression at this time. in contrast, per is co-expressed with clk in dn2s at zt but is absent in lnvs and dn1s. in addition, little or no per ir is detected at either time point in the vnc. from these experiments, we conclude that clk is constantly expressed only in brain oscillator cells from l larvae; whereas per is rhythmically expressed only in oscillator cells from l larval brains with high levels at zt in lnvs and dn1s and at zt in dn2s. discussion and  clk is expressed exclusively in oscillator cells clk immunostaining was previously detected in all oscillator cells and many non-oscillator cells from adult brains. clk expression in non-oscillator cells was coincident with that of dac, which is structurally related to the winged helix/forked-head subfamily of helix-turn-helix dna binding proteins. here, we find that clk ir in non-oscillator cells is due to cross-reactivity between clk gp antiserum and dac. we also characterized another clk antiserum, gp and demonstrated that it does not cross-react with dac on westerns. immunostaining of adult brains with gp confirms that clk is expressed only in oscillator neurons. the oscillator cell-specific expression of clk implies that clk is required for the development and/or function of these cells. this cell type specificity is consistent with the induction of oscillator cell function, when clk is expressed in ectopic locations. however, clk expression cannot induce ectopic oscillators in any cell type, suggesting that other factors critical for oscillator function are not activated by clk or the clk-dependent developmental programs are incompatible with the development of many cell types. in the loss-of-function clkjrk mutant, expression of direct clk-cyc target genes, per, tim, vri, and pdp1ε is abolished, making it difficult to positively identify oscillator cells. though peripheral oscillator tissues apparently develop normally in clkjrk flies, the loss of oscillator neuron markers in clkjrk flies makes it difficult to determine whether these neurons are present. one exception to this is l-lnvs, which continue to express pdf in clkjrk flies. determining whether clk contributes to oscillator cell development depends on the availability of oscillator cell markers that are expressed independent of clk-cyc or rescue of a loss-of-function clk mutant, upon clk induction in adults. oscillator cell development in adults, clk-dependent activation of the feedback regulator per is required for oscillator function, thus we expect that clk would be expressed before per during development. however, per mrna and protein are expressed in the vnc starting at es and in the brain at es, well before clk is detected in the brain at es this early per expression in the vnc and brain is independent of clk because it persists in the clkjrk mutant and does not overlap with clk later in development. the role of per in clk negative cells during embryogenesis is unknown, but it is possible that per modulates transcription by targeting other bhlh-pas transcription factors expressed in these cells. regardless of the role per plays, the lack of obvious developmental defects in per flies suggests that per is not critical for embryonic development. in larvae, the intensity of per ir in clk-negative brain cells and the vnc decreases drastically, consistent with previous results. clk can be detected in 2– cells in each brain hemisphere starting at early to mid es and expands during es to approximately eight clk-positive cells in each brain hemisphere. these cells are spatially segregated into three groups that correspond to larval lnvs, dn2s, and dn1s. per can be detected in some clk-positive brain cells starting as early as the end of es, or about h post-fertilization. during es per ir increases in intensity and encompasses all four lnvs and both dn1s. the 3– h delay between clk detection and per detection in embryonic brain cells is similar to the delay between the accumulation of per mrna and protein in adults, and suggests that once clk-cyc initiates per transcription in embryos, per accumulation is delayed by the same dbt-dependent per degradation mechanism described in adults. the initiation of molecular oscillator function at es coincides with the existence of light-entrainable oscillators that mediate behavioral rhythms in adults; a h light pulse ending h before larval hatching didn't synchronize behavioral rhythms of adults, but a h light pulse ending at the time of larval hatching did synchronize behavioral rhythms in adults. the initiation of oscillator function by clk in embryos is also consistent with clk's unique ability to initiate oscillator function when expressed in certain ectopic cells. when combined with these previous studies, our results support a model whereby clk expression initiates circadian oscillator function in brain neurons at es and these brain neurons go on to control rhythms in locomotor activity in adults. clk is expressed in all three groups of oscillator neurons during es and es but per is only detected in lnvs and dn1s during this time. the delayed onset of per accumulation in dn2s is intriguing, considering that the dn2s oscillator is antiphase compared to those in larval lnvs and dn1s. the antiphase cycling of oscillator in dn2s can be brought into phase with oscillators in lnvs and dn1s by expressing cry in dn2s, demonstrating that this antiphase cycling is cry-dependent. whether cry also acts to control antiphase cycling in embryonic dn2s will be investigated. in any case, our results demonstrate that antiphase cycling of oscillators in dn2s is developmentally regulated and light independent. the activation and maintenance of clk transcription in developing and adult drosophila is not well understood. the basic zipper protein pdp1ε is involved in maintaining clk activation in adults, but does not appear to be the primary clk activator. one approach to defining clk activators in embryos is to first determine which cells within the drosophila embryonic brain express clk base on co-expression of marker genes. once clk-expressing cells have been identified, transcriptional activators expressed in these cells can be tested singly or in combination for their ability to activate clk. identifying factors that activate clk in a cell-specific manner will ultimately reveal determinants of oscillator cell fate. 
these feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. these results define the temporal and spatial coordinates of factors that initiate clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that per functions in a different capacity before oscillator cell development is initiated. per is also expressed in non-clk-expressing cells in the embryonic cns starting at es although per expression in clk-negative cells continues in clkjrk embryos, per expression in cells that co-express per and clk is eliminated. the drosophila circadian oscillator is composed of transcriptional feedback loops in which clock-cycle heterodimers activate their feedback regulators period and timeless via e-box mediated transcription. per then accumulates in all clk-expressing cells except presumptive dn2s during late es and es consistent with the delayed accumulation of per in adult oscillator neurons and antiphase cycling of per in larval dn2s. a novel clk antiserum is used to show that clk expression in the larval cns and adult brain is limited to circadian oscillator cells. these data demonstrate that brain oscillator neurons begin development during embryogenesis, that per expression in non-oscillator cells is clk-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. since clk is required to initiate feedback loop function, defining the pattern of clk expression in embryos and larvae will shed light on oscillator neuron development. clk is initially expressed in presumptive small ventral lateral neurons, dorsal neurons s, and dorsal neuron s at embryonic stage and this clk expression pattern persists through larval development.
 the drosophila circadian oscillator is composed of transcriptional feedback loops in which clock-cycle heterodimers activate their feedback regulators period and timeless via e-box mediated transcription. these feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. since clk is required to initiate feedback loop function, defining the pattern of clk expression in embryos and larvae will shed light on oscillator neuron development.  a novel clk antiserum is used to show that clk expression in the larval cns and adult brain is limited to circadian oscillator cells. clk is initially expressed in presumptive small ventral lateral neurons, dorsal neurons s, and dorsal neuron s at embryonic stage and this clk expression pattern persists through larval development. per then accumulates in all clk-expressing cells except presumptive dn2s during late es and es consistent with the delayed accumulation of per in adult oscillator neurons and antiphase cycling of per in larval dn2s. per is also expressed in non-clk-expressing cells in the embryonic cns starting at es although per expression in clk-negative cells continues in clkjrk embryos, per expression in cells that co-express per and clk is eliminated.  these data demonstrate that brain oscillator neurons begin development during embryogenesis, that per expression in non-oscillator cells is clk-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. these results define the temporal and spatial coordinates of factors that initiate clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that per functions in a different capacity before oscillator cell development is initiated. 