Krüppel like factors (KLFs) are conserved transcription factors that have been implicated in many developmental processes including differentiation, organ patterning, or regulation of stem cell pluripotency. This study reports the generation and analysis of loss-of-function mutants of Drosophila Klf6/7, the luna gene. luna mutants are associated with very early embryonic defects prior to cellularization at the syncytial stage and cause DNA separation defects during the rapid mitotic cycles resulting in un-coupled DNA and centrosome cycles. These defects manifest themselves, both in animals that are maternally homozygous and heterozygous mutant. Surprisingly, luna is only required during the syncytial stages and not later in development, suggesting that the DNA segregation defect is linked to centrosomes, since centrosomes are dispensable for later cell divisions (Weber, 2014).

Krüppel like factors are highly conserved throughout the animal kingdom and have been implicated in many developmental processes such as differentiation, organ patterning, regulation of pluripotency, and human diseases. They encode Zinc finger containing transcription factors, which bind DNA and regulate various cellular processes as transcriptional activators or repressors. In evolutionary tree analyses KLF6 clusters with KLF7 and luna is a close homolog in Drosophila and Daphnia (Weber, 2014).

KLF6 is known as a ubiquitously expressed activator associated with proliferation, apoptosis, the hematopoietic system, and various cancers in vertebrates (McConnell, 2010). KLF7, also known as ubiquitously expressed Krüppel like factor (UKLF), is known to regulate sensory neuron development (Laub, 2005) and is involved in fat metabolism (McConnell, 2010). The mouse and zebrafish animal model systems established a KLF6 function in a developing organism (Matsumoto, 2006: Zhao, 2010). In the mouse, knockout of Klf6 causes developmental arrest due to failure of erythropoiesis and angiogenesis, and Klf6-/- embryonic stem (ES) cells show proliferation defects (Matsumoto, 2006). In zebrafish, morpholino based knockdown revealed that Klf6/copeb is essential for the proliferation of endoderm derived tissues (Zhao, 2010). KLF7 knockout mice die shortly after birth due to neuronal defects (Weber, 2014).

In Drosophila, early development is characterized by 14 synchronous nuclear divisions in a syncytium, the fertilized egg. The first 9 divisions take place before the onset of zygotic transcription at which point the nuclei migrate to the periphery of the embryo. These processes are solely driven by maternal contribution. De Graeve (2003) has shown that RNA interference for luna aborted development in 50% of the animals prior to gastrulation with large vacuoles forming in the egg yolk and hence coined the gene name luna. This approach also affected later developmental stages in Drosophila as did over expression of luna. However these experiments did not address or reveal for which cellular processes and during which time of development luna function was essential (Weber, 2014)

This study reports the generation of loss-of-function mutants in the Drosophila luna gene and shows that independent alleles and RNA interference cause the same phenotypic effect. Phenotypic analyses reveal that luna function is solely required at early developmental stages during the syncytial divisions, prior to cellularization, and is maternally contributed. Most prominently, luna mutants cause DNA separation defects during the early nuclear divisions, while centrosomes proceed their cycling. Hence, it is concluded that luna is required for the synchronization of nuclear DNA and centrosome cycles (Weber, 2014).

luna mutants die at early embryonic stages likely due to nuclear division defects due to non-segregation of DNA at the syncytial stage, prior to cellularization and before the start of gastrulation. The Drosophila phenotype is reminiscent of the mouse Klf6^-/- defects, where under-proliferation of hematopoietic cells in the yolk sac is the cause of early lethality (Matsumoto, 2006). Also, D'Astolfo (2008) shows that KLF6 is a positive regulator of cell cycle progression, and an anti-apoptotic factor, by silencing of KLF6 via siRNA in several cultured cell lines. These data are consistent with the Drosophila loss-of-function effects in early embryos, where arrested division cycles were observed. Moreover, Racca (2011) describes human KLF6 localization in the syncytium of the human trophoblast in cell culture, another example where KLF6 is active in a syncytial tissue, like in the early Drosophila embryo (Weber, 2014).

luna expression has been shown to be maternally loaded into the egg. Since all early regulators of these division cycles stem from maternal contribution, the timing of the luna requirement and phenotype is consistent with that. This study shows that luna function is required for the synchronization of DNA and centrosome replication, starting in the embryonic syncytium. The most frequent phenotypic defects range from non segregation of DNA, thick DNA connections between 2 prophase-like structures, which are associated each with two centrosomes that are already primed to initiate the next cycle of nuclear division; to asynchronous division cycle figures, and, more rarely, trespassing mitotic spindles and nuclear fall out. In particular, the fused DNA structures associated with 4 centrosomes seem to indicate that DNA replication or segregation is failing, while the centrosomes are ready for the next division cycle in luna mutant embryos. These defects were readily visible in mutant embryos from heterozygous mothers, in homozygous mutant eggs, generated via germline clones in the ovary, and in 20%-30% of maternal RNAi treated embryos (Weber, 2014). As luna mutant alleles manifest themselves as 'early zygotic lethal like', tissue patches mutant for luna were generated in developing eyes and wings. Surprisingly, such mutant clones did not display any defects in cell cycle rate or junctional/adhesion property, as assayed by the respective markers (phosphorylated histone H3 and DE-cadherin staining). Moreover, mutant adult eye clones did not reveal any phenotypic defects in cell morphogenesis, cell fate, or cellular patterning. These data indicate that luna function is dispensable or redundant for later zygotic development (Weber, 2014)

Since KLF6 is thought to be a tumor suppressor, several established cancerogenic and tumorigenic fly models were used to test if luna was able to ameliorate or modulate these effects. Eyeful, an eye over-growth scenario forming ectopic eye tissue in any location of the fly, and Notch signaling induced overproliferation of pluripotent eye tissue were not modifiable by luna (Weber, 2014).

Expression levels were checked in over-expression experiments in developing eye tissue by Western analysis. An antibody against human KLF6 recognized both over-expressed human KLF6 and Drosophila Luna protein, in addition to endogenous Luna on blots. In developing Drosophila tissue however, endogenous Luna protein was not detectable by these means, it could not be confirmed by Western analysis that Luna levels were reduced in embryos from luna heterozygous mothers. Generally, the expression levels of the KLF's need to be tightly regulated, as over-expression of both Luna and hKlf6 interferes with normal development. This is not unexpected, as KLFs are known transcriptional activators (or repressors) and increasing their levels is likely to interfere with various downstream transcriptional programs and targets. According to Western blot analysis, Drosophila Luna over-expression was several times the endogenous level. Similarly, misexpression/over-expression of other Klf family members in the fly eye, e.g. the founding member of this transcription factor family Krüppel itself, causes rough eyes and mis-specified photoreceptor cells (Weber, 2014).

How do the luna loss-of-function defects relate to the phenotypes of other mutants/genes acting at that stage? The heterozygous maternal effect observed in luna was also reported for the epigenetic regulators of the Polycomb group (PcG) of genes, e.g., polyhomeotic (ph), Additional sex combs (Asc), Posterior sex combs (Psc) and Polycomb (Pc) itself and polo, scant double mutants with both sets of genes also affecting the early syncytial division cycles. Whereas embryos from heterozygous mothers of polo, scant double mutants cause a wider array of phenotypes, mutations in the Polycomb group genes look identical to luna loss of function. Therefore, whether PcG heterozygous embryos displayed changes in Luna protein levels was tested, but no such changes were detectable (Weber, 2014).

Several other genes show similar phenotypes, including mutations in Non-muscle Myosin/spaghetti squash and xpd, but these show full maternal requirement for the early nuclear division cycles. However, the uncoupling of DNA replication and centrosome duplication as observed in luna has been described for microcephalin (MCPH1), except that in MCPH1 mutants centrosomes were also observed to detach leading to monopolar, multipolar or acentrosomal spindles, an effect not seen in luna mutants. Taken together, luna might affect DNA status, which then leads to the secondary effect of uncoupled centrosome cycles. If luna were to affect the centrosomal structure alone, the DNA segregation defects should not be seen, as such phenotypes are not reported for genes essential for integral centrosome function such as centrosomin. Nevertheless, the fact that luna is only required during the syncytial stages and not later in development indicates that the DNA segregation defect is linked to the centrosomes, since centrosomes are dispensable for later cell divisions (Weber, 2014).

Both phenotypes, DNA segregation defects and asynchronous divisions, occur most frequently in luna. It is speculated that the formation of DNA bridges is the primary defect and asynchronous divisions arise from unresolved and therefore delayed divisions. Similarly, nuclear fall out, a response to improperly segregated DNA could be a secondary effect (Weber, 2014).

luna mutations do not fully present themselves as dominant female sterile, as stocks can be propagated over a balancer and have 37%-50% lethal offspring, with 8%-20% lethal at preblastoderm stages. Poly comb group mutations in Pc, Psc and ph manifest a similar effect (Weber, 2014).

Many experimental approaches to better understand luna function are precluded because of the syncytial 'zygotic lethality behaviour' of luna loss-of-function mutants, likely a compound effect of maternal and zygotic requirements. Further studies at the syncytial blastoderm stage of embryogenesis should be possible via the recently published technique of live imaging of in vitro explantsand these could provide insight on the precise connection between luna loss-of-function and the early processes of syncytial nuclear divisions (Weber, 2014).

The Kruppel-like transcription factors (KLFs) represent a family of 15 different zinc finger proteins of the C(2)H(2) type that are involved in vertebrate development and which control cell proliferation, growth and differentiation. Structural-functional considerations have segregated KLF6 and KLF7 into a phylogenetically distinct group. This study reports the identification of Luna, the Drosophila progenitor of the mammalian KLF6/KLF7 group. This conclusion is based on the near sequence identity, as well as the comparable location of the DNA-binding domains and nuclear localization signals of the insect and mammalian proteins. The homology extends to the composition and function of the amino-terminal segment of Luna which, similarly to the mammalian counterparts, stimulates transcription in a reporter gene assay. Preliminary in vivo evidence is presented of Luna involvement in embryonic development and cell differentiation. First, luna RNA interference and luna overexpression during early Drosophila embryogenesis leads to developmental arrest at different embryonic stages. Second, targeted perturbation of luna expression in the forming compound eye interferes with terminal cell differentiation, but not cell specification. It is therefore proposed that Luna is a novel transcriptional determinant of Drosophila development (De Graeve, 2003).