Krüppel: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - Krüppel

Synonyms -

Cytological map position - 60F3

Function - transcription factor

Keywords - gap gene - temporal determinant in embryonic CNS development

Symbol - Kr

FlyBase ID:FBgn0001325

Genetic map position - 2-107.6

Classification - zinc finger

Cellular location - nuclear

NCBI link: Entrez Gene
Kr orthologs: Biolitmine
Recent literature
Mishra, A. K., Bargmann, B. O., Tsachaki, M., Fritsch, C. and Sprecher, S. G. (2016). Functional genomics identifies regulators of the phototransduction machinery in the Drosophila larval eye and adult ocelli. Dev Biol [Epub ahead of print]. PubMed ID: 26769100
Sensory perception of light is mediated by specialized Photoreceptor neurons (PRs) in the eye. During development all PRs are genetically determined to express a specific Rhodopsin (Rh) gene and genes mediating a functional phototransduction pathway. While the genetic and molecular mechanisms of PR development is well described in the adult compound eye, it remains unclear how the expression of Rhodopsins and the phototransduction cascade is regulated in other visual organs in Drosophila, such as the larval eye and adult ocelli. Using transcriptome analysis of larval PR-subtypes and ocellar PRs this study identified and studied new regulators required during PR differentiation or necessary for the expression of specific signaling molecules of the functional phototransduction pathway. The transcription factor Kruppel (Kr) is enriched in the larval eye and controls PR differentiation by promoting Rh5 and Rh6 expression. Camta, Lola, Dve and Hazy were also identified as key genes acting during ocellar PR differentiation. These transcriptional regulators control gene expression of the phototransduction cascade in both larval eye and adult ocelli. The results show that PR cell type-specific transcriptome profiling is a powerful tool to identify key transcriptional regulators involved during several aspects of PR development and differentiation. The findings greatly contribute to the understanding of how combinatorial action of key transcriptional regulators control PR development and the regulation of a functional phototransduction pathway in both larval eye and adult ocelli.

El-Sherif, E. and Levine, M. (2016). Shadow enhancers mediate dynamic shifts of gap gene expression in the Drosophila embryo. Curr Biol [Epub ahead of print]. PubMed ID: 27112292
Drosophila patterning genes often contain pairs of primary and shadow enhancers that possess overlapping activities. It has been suggested that this regulatory "redundancy" helps ensure reliable activation of gene expression under stressful conditions such as increases in temperature. There is also evidence that shadow enhancers help produce sharp on/off boundaries of gene expression in response to small changes in the levels of regulatory factors, such as the maternal Bicoid gradient. This study uses live-imaging methods to visualize the temporal dynamics of the gap genes Kruppel and knirps, which are essential for the patterning of the thorax and abdomen, respectively. Previous analyses of fixed embryos suggested anterior shifts of the Kruppel and knirps expression patterns. Using computational visualization methods, the precise temporal dynamics of these shifts was revealed which leads to the suggestion that shadow enhancers are crucial for this process. The study also discusses potential mechanisms for enhancer dominance, whereby one enhancer represses the other to foster temporal dynamics.

Dresch, J. M., Zellers, R. G., Bork, D. K. and Drewell, R. A. (2016). Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome. Gene Regul Syst Bio 10: 21-33. PubMed ID: 27330274
Relatively little is known about the sequence-specific binding preferences of many transcription factors (TFs), especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. This study used a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. The results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. The ability of different models to identify the known Kruppel TF binding sites in cis-regulatory modules (CRMs) was directly compared, and a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development.
Surkova, S., Sokolkova, A., Kozlov, K., Nuzhdin, S. V. and Samsonova, M. (2019). Quantitative analysis reveals genotype- and domain- specific differences between mRNA and protein expression of segmentation genes in Drosophila. Dev Biol. PubMed ID: 30629954
This study has characterized differences between mRNA and protein expression of Drosophila segmentation genes at the level of individual gene expression domains. Quantitative imaging data was obtained on expression of gap genes gt and hb and pair-rule gene eve for Drosophila wild type embryos, Kr null mutants and Kr+/Kr- heterozygotes. To compare mRNA and protein expression several criteria were used including difference in amplitude and positions of expression domains, pattern shape and positional variability. For a number of gene expression domains examples are shown where protein expression does not repeat mRNA expression even after a temporal delay. Time delays were calculated between eve pattern formation at the level of mRNA and protein for wild type embryos, Kr mutants and Kr+/Kr- heterozygotes. In wild type embryos, the amplitudes of eve stripes 3 and 7 do not differ significantly at the level of mRNA, however, stripe 3 is higher than stripe 7 at the protein level. It was further shown that hb mRNA and protein expression in both anterior and posterior domains significantly differs at specific time points. The formation of hb PS4 stripe at the mRNA level proceeds five times faster than at the level of protein. With regard to spatial expression, the offset between posterior gt mRNA and protein domains is much larger in Kr mutants than in wild type embryos and heterozygotes. Finally, differences were analyzed in positional variability of eve stripe 7 expression in Kr mutants and Kr+/Kr- heterozygotes at the level of mRNA and protein. These results enable further perspectives to uncover mechanisms underlying discrepancies between mRNA and protein expression in early embryo.
Scholes, C., Biette, K. M., Harden, T. T. and DePace, A. H. (2019). Signal integration by shadow enhancers and enhancer duplications varies across the Drosophila embryo. Cell Rep 26(9): 2407-2418.e2405. PubMed ID: 30811990
Transcription of developmental genes is controlled by multiple enhancers. Frequently, more than one enhancer can activate transcription from the same promoter in the same cells. How is regulatory information from multiple enhancers combined to determine the overall expression output? This study measured nascent transcription driven by a pair of Krüppel shadow enhancers, each enhancer of the pair separately, and each duplicated, using live imaging in Drosophila embryos. This set of constructs allows quantification of the input-output function describing signal integration by two enhancers. Signal integration performed by these shadow enhancers and duplications varies across the expression pattern, implying that how their activities are combined depends on the transcriptional regulators bound to the enhancers in different parts of the embryo. Characterizing signal integration by multiple enhancers is a critical step in developing conceptual and computational models of gene expression at the locus level, where multiple enhancers control transcription together.
Surkova, S., Sokolkova, A., Kozlov, K., Nuzhdin, S. V. and Samsonova, M. (2019). Quantitative analysis reveals genotype- and domain- specific differences between mRNA and protein expression of segmentation genes in Drosophila. Dev Biol 448(1): 48-58. PubMed ID: 30629954
In many biological systems gene expression at mRNA and protein levels is not identical. This study characterizes differences between mRNA and protein expression of Drosophila segmentation genes at the level of individual gene expression domains. Quantitative imaging data was obtained on expression of gap genes gt and hb and pair-rule gene eve for Drosophila wild type embryos, Kr null mutants and Kr+/Kr- heterozygotes. To compare mRNA and protein expression, several criteria were used, including difference in amplitude and positions of expression domains, pattern shape and positional variability. For a number of gene expression domains, examples are shown where protein expression does not repeat mRNA expression even after a temporal delay. Time delays were calculated between eve pattern formation at the level of mRNA and protein for wild type embryos, Kr mutants and Kr+Kr- heterozygotes. In wild type embryos, the amplitudes of eve stripes 3 and 7 do not differ significantly at the level of mRNA, however, stripe 3 is higher than stripe 7 at the protein level. It was further shown that hb mRNA and protein expression in both anterior and posterior domains significantly differs at specific time points. The formation of hb PS4 stripe at the mRNA level proceeds five times faster than at the level of protein. With regard to spatial expression, the offset between posterior gt mRNA and protein domains is much larger in Kr mutants than in wild type embryos and heterozygotes. Finally, differences were analyzed in positional variability of eve stripe 7 expression in Kr mutants and Kr+/Kr- heterozygotes at the level of mRNA and protein. These results enable further perspectives to uncover mechanisms underlying discrepancies between mRNA and protein expression in early embryo.
Waymack, R., Gad, M. and Wunderlich, Z. (2021). Molecular competition can shape enhancer activity in the Drosophila embryo. iScience 24(9): 103034. PubMed ID: 34568782
Transgenic reporters allow the measurement of regulatory DNA activity in vivo and consequently have long been useful tools for studying enhancers. Despite their utility, few studies have investigated the effects these reporters may have on the expression of other genes. Understanding these effects is required to accurately interpret reporter data and characterize gene regulatory mechanisms. By measuring the expression of Kruppel (Kr) enhancer reporters in live Drosophila embryos, it was found that reporters inhibit one another's expression and that of a nearby endogenous gene. Using synthetic transcription factor (TF) binding site arrays, evidence is presented that competition for TFs is partially responsible for the observed transcriptional inhibition. A simple thermodynamic model was developed that predicts competition of the measured magnitude specifically when TF binding is restricted to distinct nuclear subregions. These findings underline an unexpected role of the non-homogenous nature of the nucleus in regulating gene expression.
Duk, M. A., Gursky, V. V., Samsonova, M. G. and Surkova, S. Y. (2021). Application of Domain- and Genotype-Specific Models to Infer Post-Transcriptional Regulation of Segmentation Gene Expression in Drosophila. Life (Basel) 11(11). PubMed ID: 34833107
Unlike transcriptional regulation, the post-transcriptional mechanisms underlying zygotic segmentation gene expression in early Drosophila embryo have been insufficiently investigated. Condition-specific post-transcriptional regulation plays an important role in the development of many organisms. A recent study revealed the domain- and genotype-specific differences between mRNA and the protein expression of Drosophila hb, gt, and eve genes in cleavage cycle 14A. This study used this dataset and the dynamic mathematical model to recapitulate protein expression from the corresponding mRNA patterns. The condition-specific nonuniformity in parameter values is further interpreted in terms of possible post-transcriptional modifications. For hb expression in wild-type embryos, the results predict the position-specific differences in protein production. The protein synthesis rate parameter is significantly higher in hb anterior domain compared to the posterior domain. The parameter sets describing Gt protein dynamics in wild-type embryos and Kr mutants are genotype-specific. The spatial discrepancy between gt mRNA and protein posterior expression in Kr mutants is well reproduced by the whole axis model, thus rejecting the involvement of post-transcriptional mechanisms. These models fail to describe the full dynamics of eve expression, presumably due to its complex shape and the variable time delays between mRNA and protein patterns, which likely require a more complex model. Overall, this modeling approach enables the prediction of regulatory scenarios underlying the condition-specific differences between mRNA and protein expression in early embryo.


Krüppel is a gap gene. The term gap comes from the effect of mutation: gap genes cause the loss of central segments from the embryo, thus creating gaps in the developing structure.

The domain of early Krüppel expression is in the center of the embryo. Gap genes like knirps, giant and Krüppel are among the earliest genes expressed during development. They subdivide the embryo along the anterior/posterior axis, creating a framework for the subsequent expression of pair-rule genes. In turn, pair-rule genes are expressed in seven symmetrical stripes under the control of gap genes and a number of genes expressed in the egg before fertilization, such as bicoid, hunchback and nanos.

Krüppel represses transcription of other genes, not unlike the subduing effects adults might have on an otherwise noisy adolescent party. It represents one of the earliest calls to reason, assuring that development remains an orderly process.

After gastrulation the party is only just beginning. One example of the part Kr plays in tissue differentiation will be given. In each Malpighian tubule of Drosophila, one cell is singled out, the tip cell, whose function during embryogenesis is to promote cell division in its neighbours. The tip cell arises by division of a tip mother cell, which is selected from a cluster of equivalent cells, each expressing Krüppel in each tubule primordium. Each cluster is marked out by the expression of proneural genes, and the selection of a single cell from each group involves lateral inhibition, mediated by the neurogenic genes. Here achaete is responsible for tip cell allocation, but Kr acts as the selector gene, responsible for tip cell fate. The tip cell directs the growth of the Malpighian tubules and organizes the mitotic response and migration of the other cells forming each tubule (Hoch, 1994). Therefore Krüppel is responsible for cell fate in the Malpighian tubules, a role quite distinct from Krüppel's role as a gap gene.

The available in vivo evidence suggests that Kruppel acts as a transcriptional repressor; however, conclusive in vivo evidence demonstrating that Krüppel can additionally function as an activator of gene expression has only recently been found. Krüppel binds to the consensus sequence AAAAC/GGGGTTAA (Rosenberg, 1986 and Pankratz, 1989). The zinc finger domain of the Kr protein is framed by two evolutionarily conserved transrepressor domains [an N-terminal TR1 (transrepressor domain 1) and C64 (C-terminal repressor domain)] and a single, weak transactivator domain (TA1). C64 was initially identified when transferred to the DNA-binding domain of the yeast transcriptional activator GAL4: all three transacting domains, TR1, TA1 and C64, have been shown to confer their activities to the bacterial LacI protein. The two independent and transferable repressor domains of Kruppel have been shown to control expression of the pair-rule gene hairy; the minimal cis-acting element of hairy stripe7 mediates either Kruppel-dependent activation or repression in different regions of the blastoderm embryo (La Rosee-Borggreve, 1999).

In Drosophila cultured cells, TA1 alone is incapable of acting as a weak transactivator domain. TA1 is however, activation-competent in the presence of the adjacent stretch of 51 amino acid residues of the Kr protein. This 51 amino acid region contains sequence motifs similar to those observed in the transactivation domains of CTF/NF1, Sp1 and Pit1, but since this sequence alone fails to mediate gene activation, it is referred to as the co-activating domain (CAD). Combined TA1 and CAD causes reporter gene activation even in the presence of TR1. Together, these two domains override the TR1-dependent transrepression activity. In contrast, when TA1 and CAD are directly fused with the C64 repressor domain of Kr, reporter expression is nullified. Therefore, the opposite regulatory activities of the TA1/CAD and C64 domains are extinguished when fused. Thus, it appears necessary that, as in the full-size Krüppel protein, these domains are separated in order to exert opposite regulatory functions on transcription (La Rosee-Borggreve, 1999).

The hairy stripe7 enhancer element, decodes the activity of three activators: the maternal homeodomain proteins Caudal and Bicoid, and the zinc finger protein Kruppel. Caudal and Krüppel activities are necessary, and sufficient, to activate h7-mediated lacZ reporter gene (h7-lacZ) expression but Bicoid activity is additionally required to achieve wildtype expression levels. Absence of Kr activity not only significantly reduces the level of h7-dependent reporter gene activation in the posterior region of the embryo, but also results in the appearance of a second and novel expression domain in a position corresponding to the highest levels of Krüppel in wildtype blastoderm embryos. Thus, h7 not only mediates gene expression in response to low levels of Krüppel in the posterior region of the blastoderm embryo, but it simultaneously prevents reporter gene expression at high concentrations in the central region of the embryo. To determine the ability of Kr to directly interact with the h7 element, DNaseI footprinting experiments were performed using bacterially produced Kr and subfragments of the h7 element. The h7 element has been shown to contain five in vitro Krüppel binding sites; this opens the possibility that Krüppel may act through multiple binding sites within the h7 element (La Rosee-Borggreve, 1999).

The C-terminal region of Krüppel that encompasses the predominant repressor domain is not essential for activation, but is required to fully suppress h7-mediated transcription in response to high levels of Krüppel activity. This domain contains an interaction motif for dCtBP, a homologue of the human co-repressor CtBP. dCtBP activity is, however, dispensable for Krüppel-mediated repression in the embryo since Krüppel-mediated repression functions in the absence of dCtBP (La Rosee-Borggreve, 1999).

In vitro experiments have shown that C64 provides a homodimerization surface that permits Krüppel homodimer formation at high protein concentrations. The homodimer acts exclusively as a transcriptional repressor, whereas the Krüppel monomer has been shown to function as a transcriptional activator both in vitro and in Drosophila tissue culture assays (Sauer, 1993). Based on the in vitro results, it has been proposed that Krüppel acts as a transcriptional repressor in the central region of the blastoderm embryo and may function as an activator of target genes outside the central region where the concentration of Krüppel gradually decreases (Sauer, 1993). The h7-mediated expression pattern in KrV mutant embryos is consistent with this proposal: the lack of the C-terminus, and hence the dimerization domain, does not affect Krüppel's ability to co-activate h7-mediated gene expression in a position of low Krüppel concentration in the embryo, but rather, strongly reduces its repressor function at high concentrations (La Rosee-Borggreve, 1999).

Gray and Levine (1996) proposed two models to explain Krüppel-mediated repression. One model suggests that Krüppel possesses two separate activities, one interfering with enhancer-bound activators by quenching, and the other directly inhibiting transcription by interacting with components of the basal transcription machinery. The second model proposes that Krüppel recruits a repressor complex that only functions locally. Some aspects of the results presented here fit with the first model, others with the second. For example, TR1 could be the repressor domain that acts through quenching. In this case, TR1 would interfere with Bicoid-dependent activation mediated by h7 in the central region of the embryo, but not with Caudal-dependent activation, which is predominant in the posterior region of the embryo. This assignment is consistent with the finding that repression of h7-mediated gene expression is strongly reduced in the central region of the KrV mutant embryo but no effect is observed in the posterior region of the embryo when Krüppel lacking the C-terminal region is expressed throughout the embryo (La Rosee-Borggreve, 1999).

Alternatively or additionally, C64 could act either by blocking activation via inhibiting basal transcription, or it may interfere with, and thereby extinguish, both Caudal and Bicoid activities directly. Direct inhibition of the basal transcription machinery would be consistent with in vitro data showing that C64 prevents transcription by interacting with the general transcription factor TFIIEbeta (Sauer, 1995a). This proposal would, however, be consistent with the recent finding that the C-terminal repression region of Krüppel inhibits certain activators (Hanna-Rose, 1997) only if the subset of affected activators would target TFIIEbeta to exert their function. The second model which explains transcriptional repression via a repressive complex formation is consistent with the observation that the C-terminal domain enables Kr to form heterodimer complexes with other transcription factors such as Knirps (Sauer, 1995a). A further possibility is that the C-terminal domain could serve to recruit more general co-repressors such as Groucho or CtBP to template DNA. A CtBP-binding motif has indeed been noted in the C- terminal repressor region of Krüppel (Nibu, 1998). The Drosophila homolog, dCtBP, has been shown to interact in vitro with the gap gene product Knirps and gene-dosage interaction studies with dCtBP and knirps mutants have suggested that Knirps-dCtBP interactions are also able to occur in vivo (Nibu, 1998). The recruitment of dCtBP by short-range repressors, such as Knirps and Krüppel, may theoretically be able to alter the chromatin structure, its status of acetylation or the presence of transcriptional activators bound to a nearby site within the enhancer. Nevertheless, the weakest known knirps mutant, knirps14F, which lacks the dCtBP-interaction motif, develops an almost normal abdominal segment pattern with the exception that the abdominal segment 4 is consistently missing. This suggests that dCtBP may possibly be important for some specific but not all aspects of Knirps-dependent repressor function. The results shown here indicate that dCtBP is neither required for Krüppel-dependent repression of h7-mediated activation in the central region of the embryo, nor for Knirps-dependent repression of the expression domain in the posterior region of the embryo. Furthermore, dCtBP is also not required for repression of this expression domain in response to ubiquitously expressed Krüppel (La Rosee-Borggreve, 1999 and references therein).

The results shown here describe a previously missing piece of information surrounding Krüppel function; namely, that Krüppel possesses both activator and repressor function in vivo. The switch between activator and repressor functions is dependent on the concentration of Krüppel protein and is mediated by the C-terminus. The precise mechanism by which this mode of switching is regulated and potential cofactors of Krüppel are still unknown and need to be addressed by future studies (La Rosee-Borggreve, 1999).

Drosophila Polycomb complexes restrict neuroblast competence to generate motoneurons

Similar to mammalian neural progenitors, Drosophila neuroblasts progressively lose competence to make early-born neurons. In neuroblast 7-1 (NB7-1), Kruppel (Kr) specifies the third-born U3 motoneuron and Kr misexpression induces ectopic U3 cells. However, competence to generate U3 cells is limited to early divisions, when the Eve+ U motoneurons are produced, and competence is lost when NB7-1 transitions to making interneurons. This study found that Polycomb repressor complexes (PRCs) are necessary and sufficient to restrict competence in NB7-1. PRC loss of function extends the ability of Kr to induce U3 fates and PRC gain of function causes precocious loss of competence to make motoneurons. PRCs also restrict competence to make HB9+ Islet+ motoneurons in another neuroblast that undergoes a motoneuron-to-interneuron transition, NB3-1. In contrast to the regulation of motoneuron competence, PRC activity does not affect the production of Eve+ interneurons by NB3-3, HB9+ Islet+ interneurons by NB7-3, or Dbx+ interneurons by multiple neuroblasts. These findings support a model in which PRCs establish motoneuron-specific competence windows in neuroblasts that transition from motoneuron to interneuron production (Touma, 2012).

This study used multiple genetic approaches to investigate the timing and specificity of competence restriction by PRCs in Drosophila neuroblasts. The data show that PRCs establish motoneuron competence windows in two distinct neuroblast lineages, regulating the production of both Eve+ and HB9+ Islet+ motoneurons. This provides a mechanistic explanation for the loss of competence that has been previously described in NB7-1 and NB3-1. The experiments manipulating the timing of Pdm and Cas expression show that this mechanism is not limited to fate specification by Kr but is involved in establishing a broad motoneuron competence window. Consistent with this model, there appears to be little restriction of competence in a lineage that produces exclusively interneurons (NB3-3) and, correspondingly, PRC activity does not affect the ability of Kr to alter interneuron fates in this lineage. In addition, whereas Ph gain-of-function is sufficient to inhibit production of HB9+ Islet+ motoneurons by NB3-1, the production of HB9+ Islet+ interneurons by NB7-3 and of Dbx+ interneurons by multiple neuroblasts are unaffected (Touma, 2012).

The initial screen revealed a requirement for a subset of PRC1 and PRC2 genes in the regulation of competence. Lack of a statistically significant phenotype for other genes might be due to dosage: all embryos are heterozygous for the mutant allele and there is maternal contribution of Polycomb group and Trithorax group transcripts. Subsequent studies primarily used the Su(z)123 (null allele) and phd401, ph-p602 (ph-d401 is hypomorphic, ph-p602 is null) mutants. Su(z)12 is a component of PRC2 and Ph is a component of PRC1, allowing assessment the roles of each PRC complex. Su(z)12 loss-of- function extended competence to the end of the NB7-1 lineage. Su(z)12 is an essential co-factor of the E(z) H3K27 methyltransferase and levels of Su(z)12 activity correlate with the extent of H3K27 methylation at target genes. This suggests that the degree of competence restriction is determined by the levels of H3K27 methylation at genes required for motoneuron production. Progressive restriction of competence was still observed in the ph-d401, ph-d602 mutants, which was likely to be due to residual Ph activity. However, competence in these mutants is not completely lost until nearly twice the number of neuroblast divisions have occurred than are normally associated with loss of competence (nine divisions in ph mutants versus five in wild type). It is hypothesized that PRC-induced chromatin modifications accumulate over multiple neuroblast divisions and must reach some threshold for inhibiting motoneuron fates, similar to the accumulation of H3K27 trimethylation at the Neurog1 locus during competence restriction in mammalian cortical progenitors. Without testing additional Polycomb group and Trithorax group genes as homozygous mutants and generating maternal nulls (which in some cases might not survive to the relevant stages of neurogenesis), precisely which Polycomb group proteins are necessary for the restriction of competence cannot be precisely identifed. The core components of PRC1 and PRC2 are likely to be ubiquitously and constitutively expressed throughout neurogenesis, and this has been confirmed for Pc and Ph. However, cell type-specific PRC complexes and developmentally regulated changes in PRC composition have been described previously, suggesting that PRC1 or PRC2 co-factors might regulate the timing of competence restriction. It will be interesting to test the role of co-factors that are known or predicted to recruit PRC2 to specific genes, such as the PhoRC complex, Pipsqueak and Grainy head (Touma, 2012).

The sequential generation of motoneurons followed by interneurons has been observed during nervous system development of many insects. Clonal analysis of Drosophila neuroblasts suggests that motoneurons are always produced first, as demonstrated for NB7-1 and NB3-1, although precise birth order data are lacking for most other lineages. In the mammalian spinal cord, motoneurons and interneurons are produced from spatially segregated populations of progenitors that develop along the dorsal-ventral axis of the neural tube. Drosophila lacks this spatial segregation of motoneuroncommitted or interneuron-committed progenitors. Instead, temporal changes allow single progenitors to produce mixed lineages. PRCs appear to work in parallel to the temporal identity transcription factors by establishing competence windows in which temporal identity factors can specify motoneuron fates. Competence windows might represent a 'quality control' mechanism in which PRCs reinforce the timing of fate specification, similar to the role proposed for miRNAs during Drosophila development. Competence windows might also allow temporal identity factors to be 'redeployed' at later divisions. The majority of neuroblasts express Kr and Cas a second time and this study has confirmed that NB7-1 re-expresses Kr when interneurons are being produced. The function of Kr during later neuroblast divisions remains to be determined. If PRC activity alone were responsible for blocking a Kr-specified motoneuron late in the NB7-1 lineage, at least one ectopic U3 might be expected in ph-d, ph-p hemizygous or Su(z)12 homozygous mutant embryos. However, no altered U motoneuron fates were observed in such mutants. There are at least two potential explanations for this result. First, residual PRC activity in these mutants might allow sufficient changes in chromatin states to block endogenous Kr from specifying a motoneuron. This possibility is supported by data showing a dosage-sensitive relationship between Kr and PRC levels in specifying U3 fates, and the eventual loss of competence in ph mutants subjected to heat shock-induced pulses of Kr. Alternatively, there might be an additional transcription factor (or factors) that specifies interneuron fates in the NB7-1 lineage. This interneuron fate determinant could have a dominant effect, such that even when PRC activity is reduced, interneuron fates (or an Eve- 'hybrid' fate) prevail. Conversion to an Eve+ motoneuron might therefore only occur in a combined PRC loss-of-function and Kr gain-of-function background (Touma, 2012).

In both NB7-1 and NB3-1, later-born motoneuron fates are preferentially inhibited in Ph gain-of-function experiments, supporting a link between the number of neuroblast divisions and the restriction of motoneuron competence. The timing of competence restriction might also be regulated by the temporal identity factors themselves. Previous studies of competence in NB7-1 and NB3-1 have shown that constitutive expression of Hb can maintain neuroblasts in a fully competent state. In addition, precocious Pdm expression can inhibit Kr expression and block U3 fates in NB7-1 and RP3 fates in NB3-1. How Hb or Pdm might interact with Polycomb or Trithorax complexes during the regulation of competence remains to be determined (Touma, 2012).

In an attempt to identify PRC target genes that affect competence, NB7-1 fates were analyzed in embryos with wor-GAL4 driving expression of Kr in combination with the following candidates: the anterior-posterior patterning Hox genes Ultrabithorax, abdominal A, Antennapedia and Abdominal B, the nervous system-expressed Hox gene BarH1, the neuroblast fate determinant gooseberry, and the cell cycle regulator Cyclin A. No extension of competence was detected when these PRC targets are coordinately overexpressed with Kr. It would be technically very challenging and beyond the scope of this work to identify direct PRC targets in NB7-1 or NB3-1. However, clues are provided by previous studies that identified PRC targets in Drosophila embryos). One interesting set of PRC targets is a group of genes involved in motoneuron formation or function: eve, islet, HB9, Nkx6 (HGTX - FlyBase), zfh1 and Lim3. All motoneurons that innervate dorsal muscles express Eve, most motoneurons that innervate ventral muscles express some combination of Lim3, Islet, HB9 and Nkx6, and all somatic motoneurons express Zfh1. None of these genes is sufficient to confer motoneuron fates on their own, and some (eve, HB9, islet) are also expressed in subsets of interneurons. It is possible that PRCs silence the transcription of multiple genes that establish motoneuron fate 'combinatorial codes.' Relevant PRC target genes might be coordinately regulated by the temporal identity transcription factors (as suggested by the ability of high levels of Kr to partially overcome competence restriction) or transcription of these targets might depend on indirect interactions (Touma, 2012).

In mammalian embryonic stem cells, PRCs maintain pluripotency by inhibiting transcription of developmental pathway genes. These genes contain 'bivalent' histone modifications, with PRC-associated H3K27 methylation and Trithorax-associated H3K4 methylation keeping developmental regulators silenced but poised for activation. During differentiation of embryonic stem cells into neural progenitors, neural development genes lose PRC-associated modifications but retain H3K4 methylation, resulting in increased transcription. Although PRC silencing maintains pluripotency in embryonic stem cells, PRCs are likely to have an additional role in restricting fate potential once a progenitor becomes lineage committed. This was recently demonstrated for mouse embryonic endoderm progenitors, which undergo a fate choice for liver or pancreas development. The regulatory elements of liver and pancreas genes have distinct chromatin patterns prior to commitment to either lineage, and EZH2 [an ortholog of Drosophila E(z)] promotes liver development by restricting the expression of pancreatic genes. Similar chromatin 'prepatterns' might exist for motoneuron and interneuron genes in newly formed Drosophila neuroblasts, with subsequent PRC activity selectively silencing motoneuron genes in NB7-1 and NB3-1. PRC activity has also been shown to regulate the timing of terminal differentiation in mouse epidermal progenitors and the transition from neurogenesis to astrogenesis in mouse cortical progenitors. The identification of a related mechanism in Drosophila neuroblasts suggests that temporal restriction of fate potential is a common function of PRCs. Drosophila embryonic neuroblasts will provide a useful system for addressing several outstanding questions regarding PRC regulation of fate potential, including how PRCs are recruited to target genes, the composition of the relevant silencing complexes, and how PRC activity is temporally regulated (Touma, 2012).

Neuronal cell fate diversification controlled by sub-temporal action of Kruppel

During Drosophila CNS development, neuroblasts express a programmed cascade of five temporal transcription factors that govern the identity of cells generated at different time-points. However, these five temporal genes fall short of accounting for the many distinct cell types generated in large lineages. This study finds that the late temporal gene castor sub-divides its large window in neuroblast 5-6 by simultaneously activating two cell fate determination cascades and a sub-temporal regulatory program. The sub-temporal program acts both upon itself and upon the determination cascades to diversify the castor window. Surprisingly, the early temporal gene Kruppel acts as one of the sub-temporal genes within the late castor window. Intriguingly, while the temporal gene castor activates the two determination cascades and the sub-temporal program, spatial cues controlling cell fate in the latter part of the 5-6 lineage exclusively act upon the determination cascades (Stratmann, 2016).

This study, along with previous work, found that the temporal gene cascade results in the expression of Cas in the latter part of NB5-6T. cas acts together with spatial input, provided by Antp, hth, exd and lbe to activate col in the NB. col in turn activates ap and eya in the early postmitotic cells, which represents a transient and generic Ap cluster cell fate. col subsequently acts in a feedforward loop of col>ap/eya>dimm>Nplp1 to determine Tv1 cell fate. However, in addition to col, cas activates five other genes, including the last temporal gene grh, and the sub-temporal genes sqz, nab, svp and, as shown in this study, Kr. These five genes engage in a postmitotic cross-regulatory interplay, unique to each of the three cell types, which results in the propagation of the col>ap/eya>dimm>Nplp1 terminal selector cascade exclusively in Tv1, and the ap/eya/dac>dimm/BMP>FMRFa cascade in Tv4, while the Tv2/3 cells acquire a non-peptidergic interneuron identity. The role of Kr is to suppress the sub-temporal gene svp, in order to safeguard the expression of col and dimm, and thereby ensures the propagation of the col>ap/eya>dimm>Nplp1 terminal selector cascade, crucial for specification of the Tv1 cells. The other four genes (grh, sqz, nab, svp) each have unique roles, and act as sub-temporal micromanagers to ensure high fidelity and precision in the sub-division of the cas temporal window (Stratmann, 2016).

The temporal gene cas plays a pivotal role in the specification process of the different Ap cluster cells due to its activator role on a number of downstream regulators; col, a terminal selector in Tv1 specification, the sub-temporal genes sqz, nab, svp and Kr, as well as the temporal gene grh. Strikingly, cas thus activates both of the two terminal selector feedforward loops (FFLs), and the genes required to refine both FFLs (Stratmann, 2016).

cas activates Kr and svp, but how is Kr expression then restricted to only Tv1 and svp expression to Tv2/3? For Kr, restricted expression of sqz, nab and svp in Tv2-Tv4, all of which suppress Kr, can explain the confined expression pattern of Kr to Tv1. The gradually restricted expression of svp in Tv2-3 is in turn explained by Kr repressing svp in Tv1, and by grh repressing svp in Tv4. However, because grh misexpression is not sufficient to repress svp, it is tempting to speculate that there exists a similar factor to Kr, being exclusively expressed in the Tv4 cell, acting to suppress svp expression in a highly confined manner to ensure FMRFa/Tv4 specification (Stratmann, 2016).

Besides its activation by cas, col activation requires additional spatial information, provided by lbe, Antp, hth and exd, which subsequently initializes the generic Ap cluster program, by activating ap and eya. In contrast, cas alone activates grh and the sub-temporal factors, which are then important for the cell diversification, whether by activating or repressing each other's actions, or the FFLs, or partake in the FFL (grh), in order to allocate the correct cell fate to the four Ap cluster neurons. Remarkably, the four spatial inputs (lbe, Antp, hth and exd) act only on col, while the temporal input (cas) acts both on col, as well as the temporal and sub-temporal factors (sqz, nab, svp, Kr and grh). It is tempting to speculate that this may point to a general role for spatial versus temporal cues, and may be logically explained by the fact that spatial cues generally do not display the highly selective temporal expression profile necessary for sub-temporal cell diversification (Stratmann, 2016).

An unexpected finding in this study pertains to the dual role of Kr, first acting early in the canonical temporal cascade and subsequently late in the sub-temporal cascade, to ensure the specification of the Tv1 cell. The main role of Kr in Tv1 cells is to suppress svp, hence allowing for the maintenance of Col, which itself is critical for the propagation of the terminal FFL, fundamental for Tv1 cell fate. Interestingly, dual expression of Kr, first in the neuroblast and subsequently in neurons, was previously observed in NB3-3, but the functional role of the second Kr expression pulse was not addressed. svp itself also displays a dual expression and function, being expressed early in many NB lineages to suppress hb, then being re-expressed in several lineages, and in NB5-6T it acts to suppress col and dimm. With regards to postmitotic activity, another example of a temporal gene acting postmitotically applies to the role of the last temporal gene, grh, which is necessary and sufficient for FMRFa expression in Tv4 cells, and can trigger ectopic FMRFa in Ap neurons when misexpressed postmitotically. Yet in contrast to Kr, grh does not experience a dual expression profile. Hence, with several examples of dual (Kr and svp) and progenitor versus postmitotic roles of temporal genes (Kr and grh), it is tempting to speculate that this type of temporal multi-tasking may indeed be a common feature for many temporal genes, both in Drosophila and in higher organisms (Stratmann, 2016).

Diverse spatial expression patterns emerge from unified kinetics of transcriptional bursting

How transcriptional bursting relates to gene regulation is a central question that has persisted for more than a decade. This study measure nascent transcriptional activity in early Drosophila embryos and characterize the variability in absolute activity levels across expression boundaries. Boundary formation follows a common transcription principle: a single control parameter determines the distribution of transcriptional activity, regardless of gene identity, boundary position, or enhancer-promoter architecture. The underlying bursting kinetics were inferred, and the key regulatory parameter was identified as the fraction of time a gene is in a transcriptionally active state. Unexpectedly, both the rate of polymerase initiation and the switching rates are tightly constrained across all expression levels, predicting synchronous patterning outcomes at all positions in the embryo. These results point to a shared simplicity underlying the apparently complex transcriptional processes of early embryonic patterning and indicate a path to general rules in transcriptional regulation (Zoller, 2018).

A multitude of processes influence eukaryotic transcription rates. It is not clear which events might be more likely than others to determine the kinetics of bursting-either globally or in a gene specific manner, nor is it known how bursting kinetics compare across endogenous genes over a range of expression levels. Quantitative bursting measurements reveal that all gap gene (hunchback, knirps, Kruppel and giant) expression boundaries arise from the same underlying kinetics regardless of the differences in regulatory elements. Thus, from the complex combination of diverse interactions specific to each gene emerges a simple, common strategy for transcriptional regulation (Zoller, 2018).

The recognition of shared regulation surfaced only upon development of a highly precise single-molecule method of quantification. Conclusions about bursting depend heavily upon understanding sources and extent of measurement error and minimizing variability from extrinsic sources. Extrinsic processes, such as cell growth and division, DNA duplication, and mRNA transport and decay, can significantly affect the apparent variability between cells and thus also bursting rates. These effects were minimized by measuring transcription at nascent sites in an endogenous system with synchronized cell divisions. Moreover, explicit quantification of measurement error resulted in a noise model that significantly constrained the inference framework. All these approaches are generally applicable to enable precise quantification in any system (Zoller, 2018).

The fundamental mean-cumulant relationships uncovered in this study demonstrate that a single-parameter distribution globally determines transcriptional activity. Employing the telegraph model, this study found that the modulation of mean occupancy (η) predicts mean mRNA synthesis rates comparable with previous measurements and reproduces the distribution of nascent activity, whereas kini and τn (see Terminology and Parameterization of Transcription Rates) are conserved. The global behavior observed is surprising, given that bursting is generally believed to be gene and promoter specific. Multiple factors and processes, including enhancer-promoter interactions, chromatin context, nucleosome occupancy, Pol II pausing, and transcription factor interactions, all impinge on bursting rates. It remains to be determined whether the same processes are modulated in the same manner or, conversely, whether different regulatory strategies have converged to generate identical transcriptional activity across genes (Zoller, 2018).

These observations raise the question of whether the common transcriptional bursting kinetics carry a functional advantage. In early embryos, the precise positioning of cell fates requires minimizing variability between nuclei, which is achieved by a combination of long mRNA lifetimes permitting accumulation and spatial averaging through the syncytial cytoplasm. In principle, modulating kini (Pol II initiation rate) at a constitutive promoter would generate the theoretical minimal (Poisson) transcriptional noise at all levels. The fact that neither constitutive activity (η≤0.85) nor Pol II saturation (kelo/kini~215 bp >;> Pol II footprint) is ever observed suggests that some constraint prohibits this system from maintaining a continuous active state and/or it is not straightforward to alter kini. Instead, a constant switching correlation time suggests that this value is important in facilitating robust patterning. It is proposed that both expression timing and noise minimization jointly constrain switching rates (Zoller, 2018).

The mechanistic origins of the conserved parameters are unknown. One possibility is that protein-DNA affinities have been individually selected to confer the switching rates that were observe. However, it is unclear how transient transcription factor interactions, usually on the order of seconds, could generate bursts on the order of minutes. Another possibility is that the fast transcription factor binding kinetics are masked by the slower dynamics of common general factors involved in the transcription process. In fact, recent evidence suggests that mediator and TATA-binding protein binding, as well as the core promoter and its shape, play a key role in bursting. Alternatively, processes of potentially even slower dynamics, such as long-range enhancer-promoter interactions, chromatin modification, or Pol II pausing, may determine common bursting kinetics (Zoller, 2018).

The observed constancy of τn (switching correlation time; see Terminology and Parameterization of Transcription Rates) will guide further modeling and identification of the molecular mechanisms. This constancy is connected to the binomial noise level. Extensions of the two-state model must provide similar filtering of the binomial noise, which will restrict the possible class of models. For example, two particular extensions of the two-state model were tested. One possibility is a three-state model consisting of a two-step reversible activation. Alternatively, a model with an additional noise term, such as input noise stemming from input transcription factor diffusion, could explain dual modulation of switching rates observed under the two-state model. However, distinguishing these models will require live imaging (Zoller, 2018).

The common transcriptional parameters of the gap genes highlight a form of complexity reduction: despite the variety of upstream regulatory elements, all expression boundaries result from similar bursting kinetics. Whether this signature results from an underlying molecular simplicity has yet to be determined. Regardless of the mechanistic means by which these similarities are achieved, the convergence suggests the general constraints that limit the range of permitted bursting rates and/or minimize transcription variability. The unexpected conservation of the initiation rate and the correlation time might indicate a path to general rules in transcriptional regulation. It is now possible to inquire about the breadth of these generalities and whether they apply to the same gene expressed in different cell types, to the transcriptome as a whole, or even across organisms. Indeed, it appears plausible that other classes of genes share similarly constrained bursting kinetics. The methods utilized in this study are applicable in a variety of systems and permit the discovery of the molecular mechanism(s) conferring unified transcription kinetics (Zoller, 2018).

A novel temporal identity window generates alternating Eve(+)/Nkx6(+) motor neuron subtypes in a single progenitor lineage

Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. In vertebrates, these mechanisms generate 'cardinal classes' of neurons that share a transcription factor identity and common morphology. In Drosophila, two cardinal classes are Even-skipped (Eve)(+) motor neurons projecting to dorsal longitudinal muscles, and Nkx6(+) motor neurons projecting to ventral oblique muscles. Cross-repressive interactions prevent stable double-positive motor neurons. The Drosophila neuroblast 7-1 (NB7-1) lineage uses a temporal transcription factor cascade to generate five distinct Eve(+) motor neurons; the origin and development of Nkx6(+) motor neurons remains unclear. This study used a neuroblast specific Gal4 line, sparse labelling and molecular markers to identify an Nkx6(+) VO motor neuron produced by the NB7-1 lineage. Lineage analysis to birth-date the VO motor neuron to the Kr(+) Pdm(+) neuroblast temporal identity window. Gain- and loss-of-function strategies to test the role of Kr(+) Pdm(+) temporal identity and the Nkx6 transcription factor in specifying VO neuron identity. Lineage analysis identifies an Nkx6(+) neuron born from the Kr(+) Pdm(+) temporal identity window in the NB7-1 lineage, resulting in alternation of cardinal motor neuron subtypes within this lineage (Eve>Nkx6>Eve). Co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage - the first evidence that this TTF combination specifies neuronal identity. Moreover, the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for motor neuron targeting to ventral oblique muscles, thereby revealing a molecular specification pathway from temporal patterning to cardinal transcription factor expression to motor neuron target selection. Thus this study shows that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm > Nkx6 pathway is necessary and sufficient to promote VO motor neuron targeting to the correct ventral muscle group (Seroka, 2020).

Neural diversity from flies to mice arises from two major developmental mechanisms. First, neural progenitors acquire a unique and heritable spatial identity based on their position along the rostrocaudal or dorsoventral body axes. Second, temporal patterning based on neuronal birth-order results in individual progenitors producing a diverse array of neurons and glia. Temporal patterning is best characterized in Drosophila; neural progenitors (neuroblasts) located in the ventral nerve cord, central brain, and optic lobes all undergo temporal patterning, in which the neuroblast sequentially expresses a cascade of TTFs that specify distinct neuronal identities. Although all neuroblasts undergo temporal patterning, the TTFs are different in each region of the brain. Similar mechanisms are used in the mammalian cortex, retina, and spinal cord, although many TTFs remain to be identified (Seroka, 2020).

A major open question is how transient expression of TTFs like Kr and Pdm lead to long-lasting specification of molecular and morphological neuronal diversity. Good candidates for integrating spatial and temporal cues to consolidate motor neuron identity are homeodomain transcription factors expressed in post-mitotic motor neurons. In vertebrates, dorsoventral domains of the spinal cord are partitioned into 12 distinct cardinal classes of neurons - each characterized by development from a common progenitor domain, expression of unique homeodomain transcription factors with cross-repressive interactions to stabilize boundaries, and generating neurons with common morphology. This nomenclature is adapted to define Eve+ and Nkx6+ (Flybase: HGTX) motor neurons as two 'cardinal classes' of motor neurons: each class expresses a homeodomain transcription factor (Eve or Nkx6) with cross-repressive interactions, and each class consists of motor neurons with related neuronal morphology (Eve+ motor neurons project to dorsal and lateral longitudinal muscles; Nkx6+ motor neurons project to ventral muscle groups) (Seroka, 2020).

The Drosophila neuroblast 7-1 (NB7-1) is arguably the best characterized system for understanding TTF expression and function. Similar to most other ventral nerve cord neuroblasts, NB7-1 expresses the canonical TTF cascade Hb-Kr-Pdm-Cas with each TTF inherited by the GMCs born during an expression window, and transiently maintained in the two post-mitotic neurons produced by each GMC. The TTF cascade generates diversity among the five Eve+ U1-U5 motor neuron progeny of NB7-1: Hb specifies U1 and U2, Kr specifies U3, Pdm specifies U4, and Pdm/Cas together specify U5. Identifying TTF target genes, including transcription factors and cell surface molecules, will provide a comprehensive view of how developmental determinants direct neuronal morphology and synaptic partner choices (Seroka, 2020).

It has long been thought that the cardinal classes of motor neurons derive from distinct progenitors; Eve+ motor neurons derive from NB7-1, NB1-1, and NB4-2 whereas Hb9+ or Nkx6+ motor neurons derive from NB3-1 and others. However, DiI labeling of NB7-1 identified a potentially unknown motor neuron innervating ventral muscles, which is distinct from dorsal and lateral longitudinal muscles targeted by the Eve+ motor neurons. The observed ventral projection in this lineage could reflect transient exuberant outgrowths that are lost during larval life, or they could be due to an uncharacterized motor neuron that forms stable synapses with ventral muscles (Seroka, 2020).

This study shows that a newly discovered Kr/Pdm TTF window generates an Nkx6+ Eve-motor neuron, born between U3 and U4 in the NB7-1 lineage, that projects to ventral oblique (VO) muscles. It was also shown that overexpression of Kr/Pdm together, or Nkx6 alone, generates ectopic VO motor neurons based on molecular marker expression. Finally, this study demonstrates that Nkx6 is required for proper motor neuron axon targeting to ventral oblique muscles. These results establish a genetic pathway from TTFs (Kr/Pdm), to a cardinal motor neuron transcription factor (Nkx6) to motor axonal targeting. Also the unexpected discovery was made that a single progenitor can alternate production of different cardinal motor neuron classes (Seroka, 2020).

Kr/Pdm co-expression has been detected in several neuroblast lineages, but until now there has not been evidence that this TTF combination could specify neuronal identity. Previous work showed that the Kr/Pdm window generates a Kr/Pdm GMC, and this study shows that this GMC generates an Nkx6+ ventral-projecting motor neuron. It is unknown whether Kr/Pdm directly or indirectly activate nkx6 expression. The nkx6 gene lies in a 45kb region devoid of genes, and there are only a few, sparse predicted Kr or Pdm binding sites in this genomic expanse. How do Kr and Pdm together specify one fate (Nkx6+ VO motor neuron) whereas Kr or Pdm alone specify completely different fates (Eve+ dorsal motor neurons)? It is likely that Kr/Pdm together activate a different suite of target genes than either alone. For example, Kr/Pdm together may directly activate nkx6 expression, whereas neither alone has that potential. The emergence of single cell transcriptome and ChIP studies will help to reveal how the combination of Kr/Pdm TTFs generates different cell fate output compared to Kr or Pdm alone (Seroka, 2020).

The production of an Nkx6+ VO motor neuron in Kr/Pdm window interrupts the sequential production of Eve+ dorsal motor neurons in the NB7-1 lineage, resulting in an Eve > Nkx6 > Eve alternation of cardinal motor neuron production within the lineage. This is unusual, as in most cases neurons with similar morphology or function are produced together in a lineage. In mammals, progenitors generate neurons first, followed by glia; no examples are known of neuron>glia>neuron production from a single lineage. Similarly, Drosophila central brain neuroblast lineages produce the mushroom body γ neurons, then α'/ β' neurons, and lastly α/β neurons, with no evidence for alternating or interspersed fates. In the abdominal NB3-3 lineage, the early-born cells are in a mechanosensitive circuit, whereas the late-born cells are in a proprioceptive circuit. The only possible example of interleaved production of two morphological classes of neurons is in the Drosophila lateral antennal lobe neuroblast lineage, which alternate between uniglomerular and multiglomerular (AMMC) projection neurons. The use of clonal and temporal labeling tools will be needed to examine additional lineages to determine the prevalence of lineages producing temporally interleaved neuronal subtypes as in the NB7-1 lineage (Seroka, 2020).

Overexpression of Kr/Pdm or Nkx6 can induce only 2-3 ectopic VO motor neurons within the NB7-1 lineage. Clearly not all neurons in the lineage are competent to respond to these transcription factors. Early-born temporal identities specified by Hb and Kr (U1-U3) are unaffected by Kr/Pdm or Nkx6 overexpression, which is similar to previous data showing that early temporal fates are not affected by overexpression of later TTFs in multiple lineages. It remains a puzzle why the Kr+ U3 neuron does not switch to a VO fate upon overexpression of Kr/Pdm. There may need to be an equal level of Kr and Pdm to specify VO fate, although this would not explain why Kr/Pdm overexpression converts the Pdm+ U4 motor neuron to a VO fate. Alternatively, there may be an early chromatin landscape that blocks access to relevant Pdm target loci (Seroka, 2020).

It is noted that the assay of VO neuronal identity was done in newly-hatched larvae. Although motor circuits are functional at this time, larvae grow for five more days. These is no data on whether the ectopic VO motor neurons are functional or are maintained through the life of the larvae. This would be an important question for the future (Seroka, 2020).

Nkx6 and Eve have cross-repressive interactions (Broihier, 2004), but with some limitations: early-born Eve+ motor neurons are not affected by Nkx6 overexpression. Wild type animals even show sporadic expression of Nkx6 in the Eve+ U2 motor neuron, but in these neurons it has no effect on Eve expression, nor does it promote targeting to ventral oblique muscles. There appears to be a mechanism to block endogenous or overexpressed Nkx6 function in the early lineage of neuroblasts producing Eve+ motor neurons. The mechanism 'protecting' early-born Eve+ neurons from Nkx6 repression of Eve is unknown. Early lineages may lack an Nkx6 cofactor; Nkx6 could act indirectly via an intermediate transcription factor missing in early lineages; the early TTFs Hb or Kr may block Nkx6 function; or the eve locus could be in a subnuclear domain inaccessible to Nkx6 (Seroka, 2020).

Nkx6 promotes motor neuron specification in both Drosophila and vertebrates. In Drosophila, loss of Nkx6 reduces ventral projecting motor neuron numbers and increases the number of Eve+ neurons, while overexpression increases ventral projecting motor neuron numbers at the expense of Eve+ neurons. In vertebrates the Nkx6 family members Nkx6.1/Nkx6.2 appear to play a broader role in motor neuron specification. Nkx6.1/Nkx6.2 show early expression throughout the pMN domain; mice mutant for both Nkx6 family members lack most somatic motor neurons; and Nkx6.1 overexpression in chick or zebrafish can induce ectopic motor neurons. It would be interesting to investigate whether vertebrate Nkx6.1/Nkx6.2 are required to suppress a specific motor neuron identity, similar to the antagonistic relationship between Nkx6 and Eve in Drosophila (Seroka, 2020).

Neuroblasts in all regions of the Drosophila CNS (brain, ventral nerve cord, optic lobe) use TTF cascades to generate neuronal diversity, yet less is known about TTF target genes. It is likely that TTFs induce expression of suites of transcription factors that persist in neurons and confer their identity. Examples may include the 'morphology transcription factors' that specify adult leg motor neuron dendrite projections, but in this case it remains unknown whether these transcription factors control all other aspects of adult motor neuron identity. It is possible that 'morphology transcription factors' are one module downstream of a broader regulatory tier similar to the terminal selector genes in C. elegans (Seroka, 2020).

This study has identified a linear pathway from Kr/Pdm to Nkx6 which specifies VO motor neuron identity. TTFs could act by two non-mutually exclusive mechanisms: inducing a stable combinatorial codes of transcription factors that consolidate neuronal identity, or by altering the chromatin landscape to have a heritable, long lasting effect on motor neuron gene expression. The observation that Nkx6 is maintained in the VO neuron after fading of Kr/Pdm expression supports the former mechanism. Identification of Kr/Pdm or Nkx6 target genes would give a more comprehensive understanding of TTF specification of neuronal identity (Seroka, 2020).

The results presented in this work lead to several interesting directions. Other embryonic VNC lineages exhibit a Kr/Pdm window; does this window generate neurons in these lineages? Are there common features to neurons born in the Kr/Pdm window? Furthermore, do ectopic VO neurons make functional presynapses with the ventral oblique muscles, and do they have the normal inputs to their dendritic postsynapses? In only a few cases has it been shown the TTF-induced neurons are functionally integrated into the appropriate circuits [30]. Kr and Pdm orthologs have been identified in vertebrates. Looking for dual expression of Kr and Pdm orthologs in vertebrates may reveal a role in specifying temporal identity, similar to evidence for Hb and Cas TTFs having vertebrate orthologs that specify temporal identity (Seroka, 2020).

Shadow enhancers can suppress input transcription factor noise through distinct regulatory logic

Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of organisms and are critical for robust developmental patterning. However, their mechanism of action is unknown. It is hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor (TF) inputs at the individual enhancers. By measuring the transcriptional dynamics of several Kruppel shadow enhancer configurations in live Drosophila embryos, this study showed that individual member enhancers act largely independently. TF fluctuations were found to be an appreciable source of noise that the shadow enhancer pair can better buffer than duplicated enhancers. The shadow enhancer pair is also uniquely able to maintain low levels of expression noise across a wide range of temperatures. A stochastic model demonstrated the separation of TF inputs is sufficient to explain these findings. These results suggest the widespread use of shadow enhancers is partially due to their noise suppressing ability (Waymack, 2020).

Given that shadow enhancers are common and necessary for robust gene expression, it is proposed that shadow enhancers may function to buffer the effects of fluctuations in the levels of key developmental TFs. This study has extensively characterized the noise associated with shadow enhancers critical for patterning the early Drosophila embryo. By either tracking biallelic transcription or simultaneously measuring input TF levels and transcription, the hypothesis was tested that shadow enhancers buffer noise through a separation of TF inputs to the individual member enhancers. The results show that TF fluctuations play a significant role in transcriptional noise and that a shadow enhancer pair is better able to buffer both extrinsic and intrinsic sources of noise than duplicated enhancers. Using a simple mathematical model, it was found that fluctuations in TF levels are required to reproduce the observed correlations between reporter activity and that the low noise driven by the shadow enhancer pair may be a natural consequence of the separation of TF inputs to the member enhancers. Lastly, this study showed that a shadow enhancer pair is uniquely able to buffer expression noise across a wide range of temperatures. Together, these results support the hypothesis that shadow enhancers buffer input TF noise to drive robust gene expression patterns during development (Waymack, 2020).

When measured in fixed embryos, the TFs used in Drosophila embryonic development show remarkably precise expression patterns, displaying errors smaller than the width of a single nucleus. It therefore was unclear whether fluctuations in these regulators play a significant role in transcriptional noise in the developing embryo. By measuring the temporal dynamics of the individual Kr enhancers, each of which is controlled by different transcriptional activators, this study shows that TF fluctuations do significantly contribute to the noise in transcriptional output of a single enhancer. Within a nucleus, expression controlled by the two different Kr enhancers is far less correlated than expression driven by two copies of the same enhancer, indicating that TF inputs, as opposed to more global factors, are the primary regulators of transcriptional bursting in this system. The current findings leave open the possibility that additional mechanisms, such as differences in 3D nuclear organization between different reporters, may also contribute to the differences in noise that is see (Waymack, 2020).

This study also showed that activity driven by the Kr shadow enhancer pair is less sensitive to levels of a single TF than is activity driven by an individual Kr enhancer. While prior work has shown that changes in TF levels precede changes in target transcription, the sensitivity of individual enhancers to changes in TF levels had not been previously quantified. The correlation between Bcd levels and activity of the distal enhancer is modest, and it is expected that this reflects both the influence of additional TF inputs and nuclear heterogeneity that causes the local Bcd levels available to the enhancer to differ from total nuclear levels. It is suspected that the correlation between the activity of the distal enhancer and Bcd levels in the microenvironment surrounding the enhancer is higher than what was possible to measure in this study. New and emerging technologies will likely allow for live measurements of multiple TF inputs at higher spatial resolution, enabling further insights into the dynamics of expression regulation (Waymack, 2020).

The finding that the Kr shadow enhancer pair is less sensitive to TF levels helps reconcile the finding that the individual Kr enhancers are influenced by fluctuations in input TFs with previous studies showing that endogenous Kr expression patterns are rather reproducible. Previous work has cited the role of spatial and temporal averaging, which buffers noisy nascent transcriptional dynamics to generate more precise expression levels. Shadow enhancers operate upstream of this averaging, driving less noisy nascent transcription than either single enhancers or enhancer duplications (Waymack, 2020).

A stochastic mathematical model of Kr enhancer dynamics and mRNA production was developed that recapitulates the main experimental results. This model is based on that by Bothma (2015), but it is expanded to include the dynamics of a TF that regulates each enhancer. A strong emphasis was placed on the simplicity of this model, for example by using a single abstract TF for each enhancer. This choice both avoids a combinatorial explosion of parameters and makes the model results and parameters easier to interpret. One of the most notable features of the model is that it recreates the differences in noise between shadow and duplicated enhancer constructs without any additional fitting, indicating that these differences in the model system are a direct result of the separation of input TFs to the proximal and distal enhancers (Waymack, 2020).

Future versions of this model can include refinements. For example, in the current model, the influence of repressive TFs was not included nor were the multiple modes of action used by activating TFs included. Future experiments and models can also be designed to identify the mechanism of enhancer non-additivity: changes in promoter-enhancer looping, saturation of the promoter, or other mechanisms (Waymack, 2020).

In the investigation of sources of noise, total noise was decomposed into extrinsic and intrinsic components. It has been shown that the activity of one reporter does not inhibit expression of another reporter, and therefore the calculations assumed no negative covariance between the reporters' expression output. In the current system, a small amount of negative covariance was found between the activity of two alleles in the same nucleus. For this reason, the measurements were called covariance and inter-allele noise. The negative covariance observed indicates that activity at one allele can sometimes interfere with activity at the other allele, suggesting competition for limited amounts of a factor necessary for reporter visualization. The two possible limiting factors are MCP-GFP or an endogenous factor required for transcription. If the MS2 coat protein-GFP (MCP-GFP) were limiting, it would be expectec to see the highest levels of negative covariance at the center of the embryo, where the highest number of transcripts are produced and bound by MCP-GFP. Since the fraction of nuclei with negative covariance is highest at the edges of the expression domain, the limiting resource is likely not MCP-GFP, but instead a spatially-patterned endogenous factor, like a TF (Waymack, 2020).

Currently, the field largely assumes that adding reporters does not appreciably affect expression of other genes. However, sequestering TFs within repetitive regions of DNA can impact gene expression, and a few case studies show that reporters can affect endogenous gene expression. If TF competition is responsible for the observed negative covariance between reporters, a closer examination of the effects of transgenic reporters on the endogenous system is warranted. In addition, TF competition may be a feature, not a bug, of developmental gene expression control, as modeling has indicated that molecular competition can decrease expression noise and correlate expression of multiple targets (Waymack, 2020).

There are likely several features of shadow enhancers selected by evolution outside of their noise-suppression capabilities. It has been shown that all shadow enhancers of shavenbaby, a developmental TF gene in Drosophila, drive expression patterns in tissues and times outside of their previously characterized domains in the larval cuticle (Preger-Ben Noon, 2018). This suggests that shadow enhancers, while seemingly redundant at one developmental stage, may play separate, non-redundant roles in other stages or tissues. Additionally, a recent study investigating shadow enhancer pairs associated with genes involved in Drosophila embryonic development found that CRISPR deletions of the individual enhancers result in different phenotypes, suggesting each plays a slightly different role in regulating gene expression (Dunipace, 2019). In several other cases, both members of a shadow enhancer pair are required for the precise expression pattern generated by the endogenous locus. These sharpened expression patterns achieved by a shadow enhancer pair may reflect enhancer dominance or other forms of enhancer-enhancer interaction and are likely another important function of shadow enhancers (Waymack, 2020).

In the case of Kr, the endogenous expression pattern is best recapitulated by the shadow enhancer pair, with the individual enhancers driving slightly more anterior or posterior patterns of expression. Additionally, the early embryonic Kr enhancers drive observable levels of expression in additional tissues and time points, but these expression patterns overlap those driven by additional, generally stronger, enhancers, suggesting that the primary role of the proximal and distal enhancers is in early embryonic patterning. Therefore, while it cannot be ruled out that the proximal and distal enhancers perform separate functions at later stages, it seems that their primary function, and evolutionary substrate, is controlling Kr expression pattern and noise levels during early embryonic development (Waymack, 2020).

This study has investigated the details of shadow enhancer function for a particular system, and it is expected that some key observations may generalize to many sets of shadow enhancers. Shadow enhancers seem to be a general feature of developmental systems, but the diversity among them has yet to be specifically addressed. While this study worked with a pair of shadow enhancers with clearly separated TF activators, shadow enhancers can come in much larger groups and with varying degrees of TF input separation between the individual enhancers. To discern how expression dynamics and noise driven by shadow enhancers depend on their degree of TF input separation, these characteristics are being investigated in additional sets of shadow enhancers with varying degrees of differential TF regulation. The current results combined with data gathered from additional shadow enhancers will inform fuller models of how developmental systems ensure precision and robustness (Waymack, 2020).


Bases in 5' UTR - 186

Exons - two

Bases in 3' UTR - 264


Amino Acids - 466

Structural Domains

Krüppel is a zinc finger protein with four tandomly repeated zinc finger domains (Rosenberg, 1986). A subset of zinc finger transcription factors contain amino acid sequences that resemble those of Krüppel. They are characterized by multiple zinc fingers containing the conserved sequence CX2CX3FX5LX2HX3H (X is any amino acid, and the cysteine and histidine residues are involved in the coordination of zinc) that are separated from each other by a highly conserved 7-amino acid inter-finger spacer, TGEKP(Y/F)X, often referred to as the H/C link.

Each 30-residue zinc finger motif folds to form an independent domain with a single zinc ion tetrahedrally coordinated beween an irregular, antiparallel, two stranded ß-sheet and a short alpha-helix. Each zinc finger of mouse Zif268 (which has three fingers) binds to DNA with the amino terminus of its helix angled down into the major groove. An important contact between the first of the two histidine zinc ligands and the phosphate backbone of the DNA contributes to fixing the orientation of the recognition helix. Although the two fingers of Drosophila Tramtrack interact with DNA in a way very similar to those of Zif268, there are important differences. Tramtrack has an additional amino-terminal ß-strand in the first of the three zinc fingers. The charge-relay zinc-histidine-phosphate contact of Zif268 is substituted by a tyrosine-phosphate contact. In addition, for TTK, the DNA is somewhat distorted with two 20 degree bends. This distortion is correlated with changes from the rather simple periodic pattern of amino base contacts seen in Zif268 and finger 2 of TTK (Klug, 1995 and references).

To identify biologically functional regions in the product of the Drosophila melanogaster gene Kruppel, the Kruppel homolog was cloned from Drosophila virilis. Both the previously identified amino (N)-terminal repression region and the DNA-binding region of the D. virilis Kruppel protein are greater than 96% identical to those of the D. melanogaster Kruppel protein, demonstrating a selective pressure to maintain the integrity of each region during 60 million to 80 million years of evolution. An additional region in the carboxyl (C) terminus of Kruppel that is most highly conserved was examined further. A 42-amino-acid stretch within the conserved C-terminal region also encodes a transferable repression domain. The short, C-terminal repression region is a composite of three subregions of distinct amino acid composition, each containing a high proportion of either basic, proline, or acidic residues. Mutagenesis experiments have demonstrated, unexpectedly, that the acidic residues contribute to repression function. Both the N-terminal and C-terminal repression regions were tested for the ability to affect transcription mediated by a variety of activator proteins. The N-terminal repression region is able to inhibit transcription in the presence of multiple activators. However, the C-terminal repression region inhibits transcription by only a subset of the activator proteins. The different activator specificities of the two regions suggest that they repress transcription by different mechanisms and may play distinct biological roles during Drosophila development (Hanna-Rose, 1997).

Krüppel: Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 4 April 2022

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.