Klar α, β and γ are expressed throughout Drosophila development
Previous Western and immunolocalization studies have detected Klar expression in specific embryonic, larval, and adult tissues 
, but no comprehensive developmental time course has yet been reported. In addition, detection of the protein by immunostaining makes it challenging to identify which isoform is expressed, in part because available antibodies each recognize two of the isoforms 
(see also ).
We therefore designed primer sets to distinguish the mRNAs for the three isoforms by RT-PCR (purple in ). We used primer pairs in exons 14 and 17 to detect Klar α (predicted product: 550 bp), in exons 14 and 15ext to detect Klar β (predicted product: 450 bp), and in exons G and 17 to detect Klar γ (predicted product 450 bp).
For all three probes, we detected amplification products of the correct size across many developmental stages (). For adults, we detected robust signal in both males and females as well as in fractions enriched for bodies, heads, and legs. Signal was also detected throughout development, from embryos through larvae and pupae, though the signal among the three probes was divergent.
During embryonic development, the Klar isoforms displayed a dynamic expression pattern (). We collected embryos for 2 hours and aged the collection for various times. Most of the RNAs present in 0–2 hours embryos are generated during oogenesis, and previous in-situ
and Western analysis had indeed revealed that Klar β message and protein are maternally provided 
. Changes in older embryos would reflect degradation of such maternal pools and/or new transcription. While the Klar β probe yielded similar signal at all three time points, signal for Klar α and especially Klar γ increased dramatically over time, implying new transcription for these isoforms during the first 6 hours of embryogenesis (). Published microarray studies with probes not designed to distinguish isoforms confirm that Klar expression is very dynamic during development, especially during pupal stages 
. This pattern implies that Klar is required for specific, developmentally controlled processes, and thus likely plays roles beyond its characterized functions.
Zygotic expression of Klar is in part due to a previously uncharacterized transcription start site
The changes in Klar α and Klar γ signal () suggest that Klar transcription is highly regulated during early embryogenesis. A careful analysis of the temporal and spatial pattern of this expression might reveal new processes in which Klar plays a role. However, teasing out the contribution of new mRNA and protein expression in early embryos is challenging because there is substantial maternal deposit of Klar message and protein. This maternal pool might mask the contribution of new expression.
By analyzing females lacking Klar expression, it is possible to remove this maternal pool. Zygotes derived from such females and wild-type males should still be able to express Klar zygotically, and thus they should allow pinpointing the spatial and temporal contribution of zygotically generated Klar. In particular, we employed females homozygous for the klarYG3
allele, a deletion of the Pα/ β
promoter (). It indeed abolishes Klar α and β protein expression in ovaries; Klar γ expression remains intact 
. Early embryos derived from klarYG3
mothers and fathers also lack detectable Klar α or β messages (as detected by in situ hybridization 
and by RT-PCR (, hr sample)) or Klar protein 
Identification of the Pδ/ε promoter.
Unexpectedly, when we examined later stages of the same genotype, we detected RT-PCR signal with both our Klar α and β probes (). This result suggests that at least some of the zygotic expression of Klar detected in the wild type is due to an as-yet uncharacterized klar promoter. It appears to drive expression of messages with two different 3′ ends.
To determine if this expression leads to the production of Klar proteins, we examined klarYG3
embryos by immunostaining with Klar-M, a Klar-specific antibody that recognizes an epitope in exon 9 
(see also ). In early embryos until cellularization, we detected no signal above background (), as previously described 
. However, during gastrulation and germ-band extension, the signal increased dramatically.
If this antibody reactivity indeed represents a novel form of Klar not dependent on the Pα/β
promoter responsible for α/β expression, then it is presumably driven by a more proximal promoter, located somewhere between exon 0 (deleted in klarYG3
) and exon 9 (the epitope recognized by antibody Klar-M). We therefore examined a distinct klar
) allele that is due to a translocation in which the chromosome is broken between exons 4 and 5 () 
. In embryos of this genotype, we detected no signal above background even during germ-band extension (). This result demonstrates that the signal observed in klarYG3
is indeed Klar specific, and suggests that expression is due to a promoter that is upstream of the klarmBP
chromosomal break point.
The klarmBP allele allowed us to perform a variant of the experiment initially imagined since this allele on its own abolishes both maternal and zygotic Klar expression. We therefore crossed klarmBP mothers to klarYG3 fathers and examined their embryos. We observed similar Klar expression pattern as when both mothers and fathers were homozygous for klarYG3, demonstrating that this allele zygotically expressed a form of Klar ().
Zygotic klar expression results in new Klar protein isoform(s)
The translation start for Klar α and β is located in exon 2, more than 30 kb downstream of the transcription start site for the α and β messages and the region deleted in klarYG3
(). It is therefore conceivable that a promoter upstream of exon 2 drives the Klar expression we observed in klarYG3
, resulting in production of the same protein isoforms previously described. Alternatively, a more proximal promoter and the use of different translation starts would result in Klar proteins lacking N-terminal regions present in Klar α and β.
By Western analysis of early wild-type embryos, Klar is detected as a series of proteins of varying molecular weight, with the major band of apparent molecular weight >250 kDa representing Klar β 
. The identity of the minor bands is unknown, but they all originate from transcription starting at the canonical α/β promoter, since they are absent in klarYG3
embryos; likely, they are derived from full-length Klar β by degradation or proteolytic processing.
To address the identity of the zygotically expressed Klar, we first compared protein samples from wild-type embryos of various ages by Western analysis, using Klar-M (). The 0–6 hr sample is predominated by the major Klar β band, as seen before 
. However, in samples of older embryos multiple additional bands of various sizes were detected, including a prominent band of ~215 kDa in 6–12 hr embryos (). This band was absent in klarmBP
embryos but present in klarYG3
embryos, and thus represents a form of Klar not expressed from the canonical α/β promoter (). Its apparent molecular weight is significantly shorter than that of the Klar β present in early embryos. This band also does not represent Klar α (whose molecular weight is even larger than that of Klar β) or Klar γ (~75 kDa, and is not detected by Klar-M antibody). Thus, it apparently represents a novel Klar isoform distinct from α, β and γ. The Klar-C antibody also recognizes a band of similar size in 6–12 hr embryos, a band absent in klarmBP
(data not shown); thus, this novel isoform (or at least one such isoform) apparently encompasses exon 9 (Klar-M epitope) and exon 18 (Klar-C epitope) ().
Mapping a promoter for the new Klar isoforms
Attempts to isolate cDNAs for the novel Klar isoform proved unsuccessful; however, that is not surprising given that we also were not successful at amplifying early exons of the Klar β message even in the wild type. Presumably, the low expression level and great length of these klar messages make recovery of 5′ exons particularly difficult.
However, our analysis suggested an alternative strategy for identifying the transcription start site of this unknown isoform: First, since the novel isoform shares exons 9 and 18 with Klar α, but has a much shorter apparent molecular weight by Western analysis, it presumably lacks N-terminal sequences relative to Klar α, and thus its translation likely starts downstream of exon 2. Second, this isoform is absent in klarmBP animals, and thus transcription for it likely starts upstream of the breakpoint of klarmBP ().
We therefore examined the region between exon 2 and exon 5 for potential promoters (), using the McPromoter prediction program 
. At the highest sensitivity setting, only one potential transcription start site, located ~15 kb downstream of exon 4, was predicted above threshold (marked as promoter Pδ/ε
in ). Two independent P-element insertions (EY13781
) have been reported within 1 kb of this potential transcription start site, consistent with the fact that P elements tend to insert into promoter regions 
. The genomic region downstream of the predicted promoter (called exon D in the following) is indeed transcribed: in various RNA seq datasets, this region shows robust signal above background 
. In addition, the exon D-exon 5 junction has been found by RNA-seq analysis (FlyBase ID FBsf0000106532), and we recovered a piece of cDNA containing exon D, exon 5, and exon 6 sequences from an embryonic cDNA library (data not shown).
Conceptual translation of exon D revealed numerous stop codons in all three reading frames, suggesting that this exon is part of the 5′ UTR. In addition, neither exons 5 nor 6 had start codons in the reading frame corresponding to Klar α and β. This analysis predicts that protein isoforms expressed from the hypothetical Pδ/ε promoter start with amino acid 630 (in exon 7) of the canonical Klar α sequence and, compared to Klar α and β, lack N-terminal sequences of ~ 68 kDa, consistent with the difference in apparently mobility we detect on Westerns (). We designate this promoter Pδ/ε because it can apparently drive expression of two different messages, provisionally called δ and ε, with alternative 3′ ends ().
To abolish expression from the Pδ/ε promoter, we employed imprecise-excision of the EY13781 P-element and recovered allele klarSC2 that removes 2040 bp of genomic sequence, including the predicted promoter. By Western analysis, both klarSC2 and the original P insertion (in the following referred to as klarSC1) greatly reduce – and possibly entirely abrogate – expression of the ~215 kDa Klar form in embryos in germ-band extension (), while leaving expression of Klar β intact ().
Klar expression from the Pδ/ε promoter is widespread during Drosophila development
In the wild type, Klar protein expression (as detected by Klar-M or Klar-C staining) displays complicated temporal and spatial patterns throughout development 
. In stage 5 embryos, Klar signal accumulates around the central yolk, while during germ-band extension, Klar is widely distributed, and is particularly strong in a repeating pattern suggestive of the developing nervous system (). In third instar larvae, Klar is detected in the brain (enriched in brain lobes, ) and in eye imaginal discs (enriched in regions posterior to the morphogenetic furrow, ). This signal is Klar-specific, as it is absent in klarmBP
animals. In ovaries, Klar is abundant in follicle cells, nurse cells, and oocytes 
. For example, in early egg chambers (), Klar-C signal is present strongly around the nuclei of follicle cells (white arrowheads) and nurse cells (yellow arrowheads). In this case, klarmBX13
serves as negative control, as klarmBP
animals retain expression of the γ isoform 
In all these tissues, Klar-M and Klar-C show partially overlapping and partially distinct patterns (compare, for example, , top and bottom panels), reflecting the expression of different mixes of isoforms. For example, in stage 5 embryos, Klar-M signal is strong, but Klar-C signal is close to background, since here Klar expression is dominated by Klar β 
. And in the nurse cells of early egg chambers, Klar-M signal (not shown) is much less distinct than Klar-C signal 
(see also ); this is in part because of the expression of Klar γ, an isoform recognized by Klar-C, but not Klar-M (). This contribution is, for example, apparent from the comparison of klarmBP
animals (): the former retain expression of the γ isoform, the latter do not.
In klarYG3 animals, the wild-type Klar expression pattern is partially abolished. In stage 5 embryos, Klar staining is almost completely abolished, while in stage 10/11 embryos it looks grossly normal (). In third instar larvae, Klar-M signal is detected throughout eye discs, with less obvious posterior enrichment than in the wild type (), and prominently in the brain (). And in early egg chambers, Klar-C still detected prominent signal in follicle cells ().
Animals homozygous for either klarSC1 or klarSC2 displayed a distinct loss of Klar expression. In embryos during stage 10/11, overall Klar signal was much reduced, (). In larval eye discs, Klar-M staining was strong anteriorly, but reduced in the rest of the disc (), and in the larval brain, large rings suggestive of perinuclear signal were absent (). And in early egg chambers, perinuclear signal in follicle cells was reduced, while perinuclear signal in nurse cells was robust (). A comparison to the klarYG3 pattern suggests that Klar signal in the wild type is a combination of expression from the Pα/β and the Pδ/ε promoters.
RT-PCR analysis on adult samples supports this conclusion (). In the wild type, we detected robust signal with all of our three probes (exon 14/17, exon 14/15ext, exon G/17). Klar γ expression (as detected by the G/17 probe) is apparently unaffected in klarYG3 and klarSC2 animals, as expected, since the lesions in these alleles are far from Pγ. 14/17 signal was reduced in both klarYG3 and klarSC2 animals, suggesting that in the wild type it detects a mixture of Klar α and Klar δ. 14/15ext signal was unaltered in klarSC2 and abolished in klarYG3 animals; apparently, Klar ε makes a negligible contribution to overall Klar levels in adults.
Phenotypes associated with disruption of the new Klar isoforms
Animals homozygous for the klarSC2
allele are viable and fertile, and we have not noticed any obvious developmental defects. Because Klar α is crucial for nuclear migration in larval photoreceptors 
and the new Klar isoforms are co-expressed with Klar α in eye discs (), we examined the positioning of photoreceptor nuclei in klarSC2
eye discs (). Eye discs were fixed, stained for Elav to reveal photoreceptor nuclei, and apical and basal sections of the tissues were imaged. In the wild type, all nuclei are apical, arranged in a regular pattern 
. Disruption of Klar α with the klarYG3
allele results in a disrupted apical pattern, and accumulation of nuclei in basal sections, indicating disrupted nuclear migration (). Similar disruption is seen with other klar
a premature stop codon in exon 8 (klarmBX14
) (). In contrast, klarSC2
eye discs were indistinguishable from the wild type, displaying a regular apical pattern and absence of nuclei in basal sections (). Thus, the new isoforms expressed from the Pδ/ε
promoter are apparently not essential for proper nuclear positioning in eye discs.
Ectopic expression of Klar δ/ε, but not their absence, causes nuclear mispositioning in eye imaginal discs and oocytes.
These novel Klar isoforms may act in a redundant pathway, or their lack may have subtle effects not detectable as gross deficiencies. In such cases, it is often informative to ectopically express the molecule of unknown function to uncover potential biological activities. We took advantage of the fact that the transposable element insertion of klarSC1 carries a UAS element (a binding site for the transcription factor Gal4) as well as a basal promoter and thus should allow expression of neighboring sequences in a Gal4-dependent manner. Indeed, when combining klarSC1 with tissue-specific Gal4 drivers, we observed increased Klar-M and Klar-C signal by immunostaining in the relevant tissues (, and data not shown).
In particular, we employed the Gal4 drivers elav-Gal4 and GMR-Gal4 to force ectopic expression in developing photoreceptors. Resulting adults had rough eyes (data not shown), indicating problems with eye development. Staining for photoreceptor nuclei in eye discs or co-expression of a nuclear targeted GFP revealed severe disruption of nuclear positioning: many photoreceptor nuclei were found in basal sections, and distribution of nuclei in apical sections was irregular (). Nuclear mislocalization was highly penetrant when using the GMR-Gal4 driver (42 out of 42 eye discs examined had disruptions similar to ). These defects are reminiscent of Klar α loss-of-function mutations. No nuclear mispositioning was observed in animals homozygous for klarSC1 or animals carrying only the Gal4 drivers (data not shown).
Dramatic alteration of nuclear positioning was also observed when we drove ectopic expression in the female germ line, using the matα4-Gal4-VP16 driver (). In the wild type, the oocyte nucleus is located posteriorly in stage 6 egg chambers and relocates to the dorsal anterior corner in stages 7 and 8 
. Proper nuclear positioning depends on the microtubule motors kinesin-1 and cytoplasmic dynein 
, but is not altered in any of the klar
mutants examined to date 
, including klarSC2
(data not shown). Ectopic expression from the EY13781
element resulted in drastically increased Klar levels, as judged by Klar-C immunostaining () and frequent displacement of the oocyte nuclei away from the anterior-dorsal corner (in about half of the stage 10 egg chambers: 12 out of 24 oocytes examined). Thus, ectopic expression of Klar isoforms from the Pδ/ε
promoter can drastically affect nuclear position in multiple tissues.