BIOLOGICAL OVERVIEW

The function of hunchback is central to the establishment of an anterior-posterior gradient of gene activity in the transition from unfertilized egg to developing zygote. As its name suggests, hunchback has a special role in the development of the trunk (thorax) of the fly.

Maternal HB mRNA, is intitially distributed evenly throughout the egg. Nanos, whose mRNA is localized to the posterior pole of mature oocyte, functions to inhibit Hunchback: the Nanos protein inactivates HB mRNA, preventing its translation in the posterior. Thus Nanos, through its inhibition of HB translation, establishes a concentration gradient of maternally derived HB protein complementary to the gradient of Nanos protein (Pelegri, 1994). It is not Nanos itself that binds to the Nanos response elements of HB mRNA, but rather another protein, Pumilio, that apparently recruits Nanos into a multiprotein-RNA complex (Murata, 1995).

After fertilization, maternally derived Hunchback is supplanted by a zygotic HB transcript. Transcription is driven by Bicoid in the anterior. Bicoid is arrayed in an anterior to posterior gradient, and activates hunchback expression along this gradient, giving rise to an anterior-posterior Hunchback zygotic gradient.

Hunchback acts both to activate anterior gap gene function as a co-activator with Bicoid, and to shift the effective morphogenetic activity of Bicoid toward the posterior, thus extending the effective range of Bicoid (Simpson-Brose, 1994). Hunchback can operate both as a transcription activator or repressor, and as such determines the placement of both anterior and posterior gap genes. Hunchback's main role is as a repressor of posterior gap gene expression in the anterior. Krüppel expression in the middle of the embryo is regulated by HB. knirps and giant are expressed in the posterior, but these genes are repressed in the anterior by Hunchback.

Enhancer of zeste( E[z]) is required to maintain transcriptional repression of knirps and giant once repression has been initiated by Hunchback. A role for Polycomb group genes in the regulation of gap genes is a fairly recent idea; it is now apparent that Hunchback and E(z) act together at the same cis-acting sequences to establish repression in the knirps promoter (Pelegri, 1994).

Hunchback activity in the posterior is regulated by Tailless and Huckebein (Margolis, 1995). hunchback acts like a gap gene in the posterior. Mutants evince fused 7th and 8th segments [Images] (Tautz, 1987). Perhaps Hunchback acts as a cofactor with Krüppel and Knirps. It has been demonstrated that HB can associate with these gap gene products and that their interaction results in gene repression (Sauer, 1995).

Regulation of the Tribolium homologues of caudal and hunchback in Drosophila: evidence for maternal gradient systems in a short germ embryo

While the Bcd gradient has served as a model system in understanding pattern formation in Drosophila, it is suspected that this is not the case in more ancestral insects. The long-germ mode of development as found in Drosophila is probably an adaptation to its particularly rapid embryogenesis. The ancestral type of embryogenesis in insects and arthropods is the short germ type. In these embryos, the germ rudiment forms at the posterior ventral side of the egg. In extreme cases like the grasshopper, it may be restricted to only a few percent of the total egg length - which makes it difficult to imagine how an anteriorly localized BCD mRNA could determine pattern formation at the posterior end of the egg. Moreover, classical experiments have only yielded evidence for a posteriorly localized organizing activity. Therefore, bcd could be considered a late addition during insect evolution and its pivotal function during embryogenesis could be restricted to higher dipterans. This paper is concerned with early pattern formation of the flour beetle Tribolium castaneum. Tribolium is a typical example for short germ embryogenesis, representing the ancestral type of embryogenesis in insects, albeit not in its extreme form, like the grasshopper (see Tribolium early embryonic development). In contrast to Drosophila, only cephalic and thoracic segments, but not abdominal segments, are determined during the blastoderm stage. Furthermore, the most anterior 20% of the Tribolium blastoderm cells form an extra-embryonic membrane, the serosa. This structure is not found in this form in higher Dipterans like Drosophila, but is again an ancestral feature of insect embryogenesis. Prior to gastrulation, most blastoderm cells move from anterior and dorsal positions towards the posterior ventral region where they form the embryo proper. This germ rudiment then continues to grow from its posterior end to form a germ band which eventually encompasses all abdominal segments (Wolff, 1998).

Thus, in short germ embryos, the germ rudiment forms at the posterior ventral side of the egg, while the anterior-dorsal region becomes the extra-embryonic serosa. It is difficult to see how in these embryos an anterior gradient like that of Bicoid protein in Drosophila could be directly involved in patterning of the germ rudiment. Moreover, since it has not yet been possible to recover a bicoid homolog from any species outside the diptera, it has been speculated that the anterior Bicoid gradient could be a late addition during insect evolution. This question was addressed by analyzing the regulation of potential target genes of bicoid in the short germ embryo of Tribolium castaneum. Homologs of caudal and hunchback from Tribolium are regulated by Drosophila bicoid. In Drosophila, maternal Caudal mRNA is translationally repressed by Bicoid. Tribolium Caudal RNA is also translationally repressed by Bicoid, when it is transferred into Drosophila embryos under a maternal promoter. This strongly suggests that a functional bicoid homolog must exist in Tribolium. The second target gene, hunchback, is transcriptionally activated by Bicoid in Drosophila. Transfer of the regulatory region of Tribolium hunchback into Drosophila also results in regulation by early maternal factors, including Bicoid, but in a pattern that is more reminiscent of Tribolium hunchback expression, namely in two early blastoderm domains. Using enhancer mapping constructs and footprinting, it has been shown that Caudal activates the posterior of these domains via a specific promoter. These experiments suggest that a major event in the evolutionary transition from short to long germ embryogenesis was the switch from activation of the hunchback gap domain by Caudal to direct activation by Bicoid. This regulatory switch can explain how this domain shifted from a posterior location in short germ embryos to its anterior position in long germ insects, and it also suggests how an anterior gradient can pattern the germ rudiment in short germ embryos, i.e. by regulating the expression of caudal (Wolff, 1998).

The key to understanding the qualitative switch that took place in insect evolution is believed to lie in the more anterior serosa expression domain of Tribolium hb. Reporter gene data suggest that this domain may already be activated by Bcd in Tribolium. To explain the switch in the regulation of the more posterior gap domain of hb expression, one can envision an intermediate state, where the serosa domain and the embryonic (gap) domain have fused into a single domain. To achieve this, the evolution of a few additional Bcd binding sites in the hb upstream region would have been sufficient. In this intermediate stage both Bcd and Cad could have acted as activators on the gap domain of hb. Subsequent loss of Cad regulation would then have moved the posterior boundary of this combined domain towards the anterior. It is noted that the Tribolium hb gene has three known promoters, one of which appears to be specialized for mediating Cad regulation. In Drosophila, only two promoters are present, neither of which has a known responsiveness to Cad. Thus, in all likelihood, the Cad dependent promoter and its associated enhancer was lost. Since no other enhancer activity has been found for later expression patterns of hb in the cad dependent fragment, the loss of this region could have been a single step. Intriguingly, a combined serosa and gap domain is still evident in the lower dipteran Clogmia. In this fly, hb is expressed in a large anterior domain, from which at later stages also the serosa is recruited (Rohr, personal communication to Wolff, 1998). This mechanism, the modification of the way gap genes sense maternal positional information while this information itself remains constant, can explain how the blastoderm fate map changed during evolution of short germ insects to insects with long germ embryos. Moreover, it represents an intriguing example for the importance of regulatory adaptation during the evolution of developmental processes (Wolff, 1998).

Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS

In addition to its early regulatory functions during segmentation, Hunchback is also expressed in the developing nervous system (see Lateral views of Drosophila CNS). One possible CNS regulatory target for Hb is the POU gene pdm-1. Hb regulates pdm-1 expression at the cellular blastoderm stage (Lloyd, 1991; Cockerill, 1993), and may play a similar role in the CNS. Since Hb and Castor bind similar promoter target sequences, an exploration was carried out of the embryonic distribution of the three proteins using polyclonal antibodies. It is suggested that Hb and Cas act in a cooperative, non-overlapping manner to control POU gene expression during Drosophila CNS development. By silencing pdm expression in early and late NB sublineages, Hb and Cas establish three pan-CNS compartments whose cellular constituents are marked by the expression of either Hb, Pdm, or Cas. During the initial S1 and S2 waves of NB delaminations, Pdm-1 is expressed in most, if not all, neuroectoderm cells. However, no Pdm-1 is detected in fully delaminated NBs and during stage 9 only a small subset of ventral cord GMCs express detectable levels. At this time, Hb expression is detected in all fully delaminated NBs and in many of their GMCs but not in neuroectoderm cells. Starting at late stage 9, Hb immunoreactivity is progressively lost from NBs; by late stage 10 only a small subset of ventral cord NBs express Hb. However, Hb is detected in many GMC and in their progeny generated during the first rounds of GMC production. These early sublineages reside predominantly along the inner/dorsal surfaces of the developing ganglia. The reduction in Hb NB expression coincides with the activation of Pdm-1 NB expression; by late stage 10, Pdm-1 is detected in many cephalic lobe (see Views of cephalic lobe neuroblasts) and ventral cord NBs and in GMCs. Similar to the dynamics of Hb expression, Pdm-1 NB expression is transient. However, many GMCs and their progeny arising from the Pdm-expressing NBs maintain high levels of Pdm-1. (Kambadur, 1998).

Recombineering Hunchback identifies two conserved domains required to maintain neuroblast competence and specify early-born neuronal identity

The Hunchback/Ikaros family of zinc-finger transcription factors is essential for specifying the anterior/posterior body axis in insects, the fate of early-born pioneer neurons in Drosophila, and for retinal and immune development in mammals. Hunchback/Ikaros proteins can directly activate or repress target gene transcription during early insect development, but their mode of action during neural development is unknown. This study used recombineering to generate a series of Hunchback domain deletion variants and assay their function during neurogenesis in the absence of endogenous Hunchback. Previous studies have shown that Hunchback can specify early-born neuronal identity and maintain 'young' neural progenitor (neuroblast) competence. Two conserved domains required for Hunchback-mediated transcriptional repression were identified; transcriptional repression is necessary and sufficient to induce early-born neuronal identity and maintain neuroblast competence. pdm2 was identified as a direct target gene that must be repressed to maintain competence, but additional genes must also be repressed. It is proposed that Hunchback maintains early neuroblast competence by silencing a suite of late-expressed genes (Tran, 2010).

Hb acts as an activator and repressor of gene expression in the CNS, but only its transcriptional repressor function is essential for maintaining neuroblast competence and specifying early-born neuronal identity. Two repression domains within the Hb protein were identified: the Mi2-binding D domain and the dimerization (DMZ) domain (Tran, 2010).

How do the D and DMZ domains repress gene expression? It is interesting to note that the D and DMZ domains are not dedicated repression domains, such as the one found in Engrailed. Instead, both are known to mediate protein-protein interactions. The DMZ allows Hb dimerization, leading to the proposal that high Hb levels promote dimerization and thus transcriptional repression (Papatsenko, 2008). For example, at cellular blastoderm stages, high levels of Hb in the anterior of the embryo are required to repress Kr, whereas low Hb levels activate Kr, and mutations in the DMZ lead to an anterior expansion of the Kr expression domain (Hulskamp, 1994). Yet it remains unknown how Hb dimerization leads to gene repression. The D domain is also involved in protein-protein interactions. The region of Hb containing the D domain is known to bind the chromatin regulator Mi2, and this interaction promotes epigenetic silencing of the Hb target gene Ubx during early embryonic patterning. The current results suggest that the D and DMZ domains could act in distinct processes that are both required for transcriptional repression, or that they could act in a common pathway such as dimerization-dependent recruitment of Mi2 and/or other repressor proteins to the D domain (Tran, 2010).

Hb proteins lacking the D or DMZ domain have very similar phenotypes in the CNS. Although both the D and DMZ domains appear to be required for Hb-mediated transcriptional repression, they do not have identical functions. Overexpression of Hb^δD leads to the specification of two U5 neurons at the expense of the U4 cell identity, whereas overexpression of Hb^δDMZ results in normal U4 and U5 identities. Perhaps Hb^δDMZ retains some ability to repress cas expression, allowing the production of the Cas^- U4 identity. Alternatively, Hb might use the D and DMZ domains to repress different target genes. Currently, it is not possible to distinguish between these models owing to the limited number of known Hb direct target genes (Tran, 2010).

Both Hb and the related mammalian protein Ik have major roles as transcriptional repressors, but are also weak transcriptional activators. How does Hb activate gene expression within the CNS? It was not possible to identify a discrete activation domain despite the fact that the systematic deletion series covered the entire protein. It can be ruled out that the activation domain maps to the D region, as it does in the closely related Ik protein, because the Hb^δD protein has no effect on Kr transcriptional activation or the specification of U3 neuronal identity. The presence of a single activation domain within the A, B, B', E or DMZ domains can also be ruled out for the same reason. Mechanisms for Hb-mediated transcriptional activation consistent with these data are: (1) Hb activates transcription indirectly by blocking DNA binding of a repressor; (2) Hb has multiple activation domains; or (3) the Hb activation domain is tightly linked to an essential domain, such as the DBD. In any case, VP16::Hb experiments, together with repression domain deletion experiments, show that Hb-mediated transcriptional repression, not transcriptional activation, is essential for maintaining neuroblast competence and specifying early-born neuronal identity (Tran, 2010).

What are the Hb-repressed target genes that are involved in extending neuroblast competence? One negatively regulated target is pdm, as co-expression of Pdm with wild-type Hb failed to extend neuroblast competence. However, overexpression of VP16::Hb in a pdm mutant background (lacking both pdm1 and pdm2) was incapable of extending neuroblast competence, showing that Hb must repress multiple genes to extend competence. In the future, further characterization of Hb function in the CNS will require genomic analyses, such as chromatin immunoprecipitation to identify Hb binding sites within the genome, or TU-tagging experiments to identify all the genes regulated by Hb within the CNS. Such comparative analyses might help to elucidate the complex gene interactions involved in regulating neuroblast competence (Tran, 2010).

Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo

Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. This study presents evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5' knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. Different models are presented for 'enhancer synergy,' whereby two enhancers with overlapping activities produce authentic patterns of gene expression (Perry, 2011).

Candidate gap enhancers were identified using ChIP-chip data. Specifically, clustered binding sites for maternal and gap proteins were identified within 100 kb of every gap gene. This survey identified each of the known enhancers, as well as putative shadow enhancers. For example, a potential distal shadow enhancer was identified for hb, located 4.5-kb upstream of the proximal transcription start site (designated 'P2' in earlier literature) and upstream of the later-acting distal promoter (designated 'P1') (Perry, 2011).

A 400-bp genomic DNA fragment from this newly identified region was attached to a lacZ reporter gene and expressed in transgenic embryos. The resulting hb/lacZ fusion gene exhibits localized expression in anterior regions of the embryo similar to that seen for the endogenous gene and 'classic' enhancer identified over 20 y ago. The classic proximal and distal shadow enhancers exhibit similar responses to increasing Bicoid copy number (Perry, 2011).

ChIP-chip data also identified potential pairs of enhancers for Kr and kni. There are two distinct clusters of transcription factor binding sites upstream of Kr. The previously identified Kr 'CD2' enhancer contains the proximal enhancer but also part of the distal binding cluster. Subsequent lacZ fusion assays identified each ChIP-chip peak and underlying binding sites as separable proximal and distal enhancers. Similarly, more refined limits were determined for the kni intronic enhancer, in addition to the previously identified 5' distal enhancer. Both the distal Kr enhancer and the intronic kni enhancer produce somewhat broader patterns of expression than the endogenous gene. Additional gap enhancers were also identified for giant, including an additional distal enhancer located ~35-kb downstream within a neighboring gene (Perry, 2011).

The survey of gap and maternal binding clusters was extended to include the so-called 'head' and 'terminal' gap genes, critical for the differentiation of head structures and the nonsegmented termini of early embryos. Additional enhancers were identified for empty-spiracles (ems), huckebein (hkb), and forkhead (fkh). More refined limits were also determined for the previously identified ocelliless/orthodenticle (oc/otd) intronic enhancer. For simplicity, the two enhancers regulating a given gap gene will be identified as proximal and distal, based on their relative locations to the transcription start site (Perry, 2011).

BAC recombineering, phiC31-targeted genome integration, and quantitative in situ hybridization assays were used to determine the contributions of the proximal and distal enhancers to the hb expression pattern. BACs containing ~20 kb of genomic DNA encompassing the hb gene and flanking sequences were integrated into the same position in the Drosophila genome. The hb transcription unit was replaced with the yellow gene, which permits quantitative detection of nascent transcripts using an intronic hybridization probe. The modified BAC retains the complete hb 5' and 3' UTRs. Additional BACs were created by inactivating the proximal or distal enhancers by substituting critical regulatory elements with 'random' DNA sequences (Perry, 2011).

BAC transgenes lacking either the distal or proximal enhancer continue to produce localized patterns of transcription in anterior regions of transgenic embryos in response to the Bicoid gradient. However, the patterns are not as faithful compared with the BAC transgene containing both enhancers. Embryos were double-labeled to detect both yellow and hb nascent transcripts. During nuclear cleavage cycle (cc) 13, a substantial fraction of nuclei (14%) expressing hb nascent transcripts lack yellow transcription upon removal of the shadow enhancer. An even higher fraction of nuclei (24%) lack yellow transcription when the proximal enhancer is removed. Control transgenic embryos containing both enhancers exhibit more uniform patterns of transcription, whereby only an average of ~3% of nuclei fail to match the endogenous pattern of transcription (Perry, 2011).

The pairwise Wilcoxon rank sum test (also called the Mann-Whitney u test) was used to determine the significance of the apparent variation in gene expression resulting from the removal of either the proximal or distal enhancer. Control embryos containing the hb BAC transgene with both enhancers exhibit some variation in the number of nuclei that lack yellow nascent transcripts. Despite this variation, the statistical analyses indicate that the loss of either the proximal or distal enhancer results in a significant change in yellow transcription patterns compared with the control BAC transgene (Perry, 2011).

The preceding analyses suggest that multiple enhancers produce more uniform patterns of de novo transcription than individual proximal or distal enhancers. Additional studies were done to determine whether multiple enhancers also help produce authentic spatial limits of transcription (Perry, 2011).

The expression of hb normally diminishes at the anterior pole of cc13 to 14 embryos. This loss in expression has been attributed to attenuation of Bcd activity by Torso RTK signaling. However, the proximal enhancer fails to recapitulate this loss. In contrast, the distal enhancer is inactive at the anterior pole, and the two enhancers together produce a pattern that is similar to endogenous expression, including reduced expression at the pole (Perry, 2011).

To examine the relative contributions of the proximal and distal enhancers in this repression, yellow nascent transcripts were measured in transgenic embryos expressing BAC reporter genes containing one or both hb enhancers. Particular efforts focused on the early phases of cc14, when repression of endogenous hb transcripts is clearly evident. For the transgene lacking the proximal, classic enhancer, but containing the newly identified distal enhancer, a median of 6% (std 6%) of nuclei exhibit expression of yellow nascent transcripts but lack expression of the endogenous gene. In contrast, a median of 24% (std 11%) of nuclei displays a similar discordance upon removal of the distal enhancer. In control embryos, 16% (std 11%) of nuclei express yellow but lack hb nascent transcripts. It should be noted that the BAC transgene lacking the proximal enhancer exhibits 'super-repression' because of reduced activation at the anterior pole (Perry, 2011).

Kr/lacZ and kni/lacZ fusion genes containing either one or two enhancers were inserted into the same position in the Drosophila genome. Transgenic embryos were double-labeled to detect the expression of the transgene (lacZ) as well as the endogenous gap gene (Perry, 2011).

The kni proximal (intronic) enhancer alone produces an abnormally broad pattern of expression, especially in posterior regions. In contrast, the kni distal (5') enhancer produces erratic lacZ activation within nearly normal spatial limits. An essentially normal pattern of lacZ transcription is observed when both enhancers are combined in a common transgene (intronic enhancer 5' and distal enhancer 3' of lacZ). It appears that lacZ transcription is slightly broader than the endogenous pattern, but considerably narrower than the pattern observed for the intronic enhancer alone, and not statistically different from the expression limits of the distal enhancer alone. There is no significant narrowing of the Kr/lacZ expression pattern when both the distal and proximal enhancers are combined within the same transgene. Perhaps additional Kr regulatory elements are required for the type of narrowing observed for the kni intronic enhancer. Alternately, all of these transgenes use the eve basal promoter and it is possible that promoter-specific interactions are important for establishing the normal limits of the Kr expression pattern (Perry, 2011).

As discussed earlier, long-range repressors bound to the distal hb enhancer might inhibit the activities of the proximal enhancer at the anterior pole of precellular embryos. The distal kni enhancer might function in a similar manner to sharpen the expression limits of the intronic enhancer. The spatial limits of gap gene-expression patterns have been shown to depend on cross-repressive interactions. The kni intronic enhancer might lack critical gap repression elements because it produces an abnormally broad expression pattern. Indeed, whole-genome ChIP assays identify more putative Tailless binding sites in the distal vs. intronic enhancer. These Tailless repression elements might function in a dominant fashion to restrict the limits of the intronic enhancer (Perry, 2011).

The modest anterior expansion of the expression pattern driven by the kni intronic enhancer is more difficult to explain because this boundary is probably formed by the Hb repressor, which is not known to function in a long-range and dominant manner. If the action of short-range repressors is also affected by stochastic processes (e.g., binding of the repressor to enhancer or looping of a bound enhancer to promoter), perhaps having two enhancers might improve the chances of maintaining proper repression (Perry, 2011).

This study has presented evidence that the robust and tightly defined patterns of gap gene expression do not arise from the unique action of individual enhancers. Rather, these patterns depend on multiple and separable enhancers with similar, but slightly distinct regulatory activities. This enhancer synergy produces more homogeneous patterns of transcriptional activity, as well as more faithful spatial limits of expression (Perry, 2011).

The enhancer synergy documented in this study is somewhat distinct from the proposed role of the shadow enhancer regulating snail expression in the presumptive mesoderm. The dual regulation of snail by the proximal and distal (shadow) enhancers was shown to ensure homogenous and reproducible expression in embryo after embryo in large populations of embryos, even when they are subject to increases in temperature. In contrast, dual regulation of hb expression by proximal and distal enhancers appears to ensure homogenous activation in response to limiting amounts of the Bicoid gradient. They are used as an obligatory patterning mechanism rather than buffering environmental changes. Despite these apparent differences, it is possible that dominant repression is also used as a mechanism of synergy for the regulation of snail expression. The distal enhancer contains repressor elements (e.g., Huckebein) that inhibit the expression of the proximal enhancer at the termini (Perry, 2011).

Different mechanisms can be envisioned to account for enhancer synergy. Perhaps the simplest is that there are fewer inactive nuclei within a given gap expression domain because of the diminished failure rate of successful enhancer-promoter interactions with two enhancers rather than one. If the rates at which enhancers fail to activate transcription are completely independent, then one would expect the combined action of two enhancers to yield a multiplicative reduction in how often a given cell fails to express the gene within a given window of time. This sort of synergy does not require any direct physical or cooperative interactions between the enhancers. Nonetheless, the effect can be significant (as seen for hb). For example, two enhancers, each with a 10% uncorrelated failure rate, may together be expected to have a 1% failure rate, a 10-fold reduction. For genes that produce strong bursts of mRNA expression, this change in frequency of transcription may have a dramatic effect on the variation of total mRNA levels (Perry, 2011).

A second but critical potential mechanism of enhancer synergy concerns long-range, dominant repression. Repressors (such as Tailless) bound to one enhancer are sufficient to restrict the spatial limits of the other enhancer. There is no need for long-range repressor elements to appear in both enhancers to achieve normal spatial limits of gene expression. It has been suggested that long-range repressors, such as Hairy, mediate the assembly of positioned nucleosomes at the core promoter. Such repressive nucleosomes should block productive enhancer-promoter interactions, even for enhancers lacking repressor sites (Perry, 2011).

Regardless of the detailed molecular mechanisms, the combined action of multiple enhancers helps explain why an individual enhancer sometimes fails to recapitulate an authentic expression pattern when taken from its native context. Enhancers that produce abnormal patterns of expression (e.g., kni intronic enhancer) can nonetheless contribute to homogeneous and robust patterns of gene expression in conjunction with the additional enhancers contained within the endogenous locus (Perry, 2011).

Precision of hunchback expression in the Drosophila embryo

Activation of the gap gene hunchback (hb) by the maternal Bicoid gradient is one of the most intensively studied gene regulatory interactions in animal development. Most efforts to understand this process have focused on the classical Bicoid target enhancer located immediately upstream of the P2 promoter. However, hb is also regulated by a recently identified distal shadow enhancer as well as a neglected 'stripe' enhancer, which mediates expression in both central and posterior regions of cellularizing embryos. This study employed BAC transgenesis and quantitative imaging methods to investigate the individual contributions of these different enhancers to the dynamic hb expression pattern. These studies reveal that the stripe enhancer is crucial for establishing the definitive border of the anterior Hb expression pattern, just beyond the initial border delineated by Bicoid. Removal of this enhancer impairs dynamic expansion of hb expression and results in variable cuticular defects in the mesothorax (T2) due to abnormal patterns of segmentation gene expression. The stripe enhancer is subject to extensive regulation by gap repressors, including Kruppel, Knirps, and Hb itself. It is proposed that this repression helps ensure precision of the anterior Hb border in response to variations in the Bicoid gradient (Perry, 2012).

hunchback (hb) is the premier gap gene of the segmentation regulatory network. It coordinates the expression of other gap genes, including Kruppel (Kr), knirps (kni), and giant (gt) in central and posterior regions of cellularizing embryos. The gap genes encode transcriptional repressors that delineate the borders of pair-rule stripes of gene expression. hb is activated in the anterior half of the precellular embryo, within 20-30 min after the establishment of the Bicoid gradient during nuclear cleavage cycles 9 and 10 (~90 min following fertilization). This initial hb mRNA transcription pattern exhibits a reasonably sharp on/off border within the presumptive thorax. This border depends on cooperative interactions of Bicoid monomers bound to linked sites in the proximal ('classical') enhancer. However, past studies and recent computational modeling suggest that Bicoid cooperativity is not sufficient to account for this precision in hb expression (Perry, 2012).

The hb locus contains two promoters, P2 and P1, and three enhancers. The 'classical' proximal enhance and distal shadow enhancer mediate activation in response to the Bicoid gradient. Expression is also regulated by a third enhancer, the 'stripe' enhancer, which is located over 5 kb upstream of P2. Each of these enhancers was separately attached to a lacZ reporter gene and expressed in transgenic embryos. As shown previously, the Bicoid target enhancers mediate expression in anterior regions of nuclear cleavage cycle (cc) 12-13 embryos, whereas the stripe enhancer mediates two stripes of gene expression at later stages, during cc14. The anterior stripe is located immediately posterior to the initial hb border established by the proximal and distal Bicoid target enhancers (Perry, 2012).

BAC transgenesis was used to determine the contribution of the stripe enhancer to the complex hb expression pattern. For some of the experiments, the hb transcription unit was replaced with the yellow (y) reporter gene, which contains a large intron permitting quantitative detection of nascent transcripts. The resulting BAC mimics the endogenous expression pattern, including augmented expression at the Hb border. However, removal of the stripe enhancer from an otherwise intact y-BAC transgene leads to diminished expression at this border and in posterior regions (Perry, 2012).

The functional impact of removing the stripe enhancer was investigated by genetic complementation assays. A BAC transgene containing 44 kb of genomic DNA encompassing the entire hb locus and flanking regulatory DNAs fully complements deficiency homozygotes carrying a newly created deletion that cleanly removes the hb transcription unit. The resulting adults are fully viable, fertile, and indistinguishable from normal strains. Embryos obtained from these adults exhibit a normal Hb protein gradient, including a sharp border located between eve stripes 2 and 3 (Perry, 2012).

The Hb BAC transgene lacking the stripe enhancer fails to complement hb⁻/hb⁻ mutant embryos due to the absence of the posterior hb expression pattern, which results in the fusion of the seventh and eighth abdominal segments. In addition, the anterior Hb domain lacks the sharp 'stripe' at its posterior limit, resulting in an anterior expansion of Even-skipped (Eve) stripe 3 because the Hb repressor directly specifies this border. There is also a corresponding shift in the position of Engrailed (En) stripe 5, which is regulated by Eve stripe 3. The narrowing of En stripes 4 and 5, due to the anterior shift of stripe 5, correlates with patterning defects in the mesothorax (Perry, 2012).

Quantitative measurements indicate significant alterations of the anterior Hb expression pattern upon removal of the stripe enhancer. There is an anterior shift at the midpoint of the mature pattern, spanning two to three cell diameters. This boundary normally occurs at 47.2% egg length (EL; measured from the anterior pole). In contrast, removal of the stripe enhancer shifts the boundary to 45.6% EL. The border also exhibits a significant diminishment in slope. Normally, there is a decrease in Hb protein concentration of 20% over 1% EL. Removal of the stripe enhancer diminishes this drop in concentration, with a reduction of just 10% over 1% EL. The most obvious qualitative change in the distribution of Hb protein is seen in regions where there are rapidly diminishing levels of the Bicoid gradient. Normally, the transition from maximum to minimal Hb levels occurs over a region of 10% EL (43%-53% EL). Removal of the stripe enhancer causes a significant expansion of this transition, to 26% EL (27%-53% EL). It is therefore concluded that the stripe enhancer is essential for shaping the definitive Hb border (Perry, 2012).

The preceding studies suggest that the proximal and distal Bicoid target enhancers are not sufficient to establish the definitive Hb border at the onset of segmentation during cc14. Instead, the initial border undergoes a dynamic posterior expansion encompassing several cell diameters due to the action of the stripe enhancer. This enhancer is similar to the eve stripe 3+7 enhancer. Both enhancers mediate two stripes, one in central regions and the other in the posterior abdomen, and the two sets of stripes extensively overlap. Previous studies provide a comprehensive model for the specification of eve stripes 3 and 7, whereby the Hb repressor establishes the anterior border of stripe 3 and the posterior border of stripe 7 while the Kni repressor establishes the posterior border of stripe 3 and anterior border of stripe 7. Whole-genome chromatin immunoprecipitation (ChIP) binding assays and binding site analysis identify numerous Hb and Kni binding sites in the hb stripe enhancer, along with several Kr sites (Perry, 2012).

Site-directed mutagenesis was used to examine the function of gap binding sites in the hb stripe enhancer. Since the full-length, 1.4 kb enhancer contains too many binding sites for systematic mutagenesis, a 718 bp DNA fragment was identified that mediates weak but consistent expression of both stripes, particularly the posterior stripe. Mutagenesis of all ten Hb binding sites in this minimal enhancer resulted in a striking anterior expansion of the expression pattern. This observation suggests that the Hb repressor establishes the anterior border of the central stripe, as seen for eve stripe 3. There is no significant change in the posterior border of the central stripe or the anterior border of the posterior stripe, and repression persists in the presumptive abdomen (Perry, 2012).

Mutagenesis of the Kni binding sites resulted in expanded expression in the presumptive abdomen, similar to that seen for the eve 3+7 enhancer. More extensive depression was observed upon mutagenesis of both the Kni and Kr binding sites. These results suggest that the Kr and Kni repressors establish the posterior border of the central Hb stripe and the anterior border of the posterior stripe. This depressed pattern is virtually identical to the late hb expression pattern observed in Kr¹;kni¹⁰ double mutants. The reliance on Kr could explain why the Hb central stripe is shifted anterior of eve stripe 3, which is regulated solely by Kni (Perry, 2012).

The dynamic regulation of the zygotic Hb expression pattern can be explained by the combinatorial action of the proximal, shadow, and stripe enhancers. The proximal and distal shadow enhancers mediate activation of hb transcription in response to the Bicoid gradient in anterior regions of cc10-13 embryos. The initial border of hb transcription is rather sharp, but the protein that is synthesized from this early pattern is distributed in a broad and shallow gradient, extending from 30% to 50% EL. During cc14 the stripe enhancer mediates transcription in a domain that extends just beyond the initial hb border. Gap repressors, including Hb itself, restrict this second wave of zygotic hb transcription to the region when there are rapidly diminishing levels of the Bicoid gradient, in a stripe that encompasses 44%-47% EL. The protein produced from the stripe enhancer is distributed in a sharp and steep gradient in the anterior thorax. It has been previously suggested that the steep Hb protein gradient is a direct readout of the broad Bicoid gradient. However, the current studies indicate that this is not the case. It is the combination of the Bicoid target enhancers and the hb stripe enhancer that produces the definitive pattern (Perry, 2012).

It has been proposed that Hb positive autofeedback is an important feature of the dynamic expression pattern. However, the mutagenesis of the hb stripe enhancer is consistent with past studies suggesting that Hb primarily functions as a repressor. The only clear-cut example of positive regulation is seen for the eve stripe 2 enhancer. Mutagenesis of the lone Hb-3 binding site results in diminished expression from a minimal enhancer. It was suggested that Hb somehow facilitates neighboring Bicoid activator sites, and attempts were made to determine whether a similar mechanism might apply to the proximal Bicoid target enhancer. The two Hb binding sites contained in this enhancer were mutagenized, but the resulting fusion gene mediates an expression pattern that is indistinguishable from the normal enhancer). It is therefore likely that the reduction of the central hb stripe in hb⁻/hb⁻ embryos is the indirect consequence of expanded expression of other gap repressors, particularly Kr and Kni (Perry, 2012).

The hb stripe enhancer mediates expression in a central domain spanning 44%-47% EL, which coincides with the region exhibiting population variation in the distribution of the Bicoid gradient. Despite this variability, the definitive Hb border was shown to be relatively constant among different embryos. Previous studies suggest that the Kr and Kni repressors function in a partially redundant fashion to ensure the reliability of this border. This paper has presented evidence for direct interactions of these repressors with the hb stripe enhancer, and suggest that a major function of the enhancer is to 'dampen' the variable Bicoid gradient. Indeed, removal of this enhancer from an otherwise normal Hb BAC transgene results in variable patterning defects in the mesothorax, possibly reflecting increased noise in the Hb border (Perry, 2012).

Specification of neuronal subtypes by different levels of Hunchback

During the development of the central nervous system, neural progenitors generate an enormous number of distinct types of neuron and glial cells by asymmetric division. Intrinsic genetic programs define the combinations of transcription factors that determine the fate of each cell, but the precise mechanisms by which all these factors are integrated at the level of individual cells are poorly understood. This study analyzed the specification of the neurons in the ventral nerve cord of Drosophila that express Crustacean cardioactive peptide (CCAP). There are two types of CCAP neurons: interneurons and efferent neurons. Both were found to be specified during the Hunchback temporal window of neuroblast 3-5, but are not sibling cells. Further, this temporal window generates two ganglion mother cells that give rise to four neurons, which can be identified by the expression of empty spiracles. The expression of Hunchback in the neuroblast increases over time, and evidence is provided that the absolute levels of Hunchback expression specify the two different CCAP neuronal fates (Moris-Sanz, 2014).

This study analyzed how CCAP-expressing neurons are specified. Evidence was obtained that both the the efferent subset of CCAP neurons (CCAP-ENs) and interneuron subset (CCAP-INs) of all embryonic segments are generated by NB3-5. The results also indicate that CCAP neurons are generated in the Hb temporal window, are not sibling cells and that the CCAP-ENs are generated first followed by the CCAP-INs. Although the Hb temporal window in NB3-5 generates two GMCs that can be distinguished by the expression of Pdm in GMC1, Pdm does not seem to play any role in the specification of these neurons, as no phenotype was observed in pdm mutants (Moris-Sanz, 2014).

These findings raised the question of how these two neuronal fates are generated, and the results that are presented in this study suggest that different levels of Hb expression specify them. The evidence for this is as follows. First, Hb expression in NB3-5 increases over time from stage 9 to early stage 11, then its expression quickly fades, coinciding with the reported expression of Svp, which is known to close the Hb temporal window. During this time window, NB3-5 divides twice and generates four neurons. Second, overexpression of high levels of Hb using a pan-NB driver extends the IN fate. Third, in an hb hypomorphic condition CCAP-INs are lost or converted into ENs, as monitored by the expression of Dac and the presence of axons that exit the ganglion (Moris-Sanz, 2014).

This mechanism for generating distinct neuronal fates is different from that proposed for subdividing the Cas temporal window in NB5-6, which involves two sequential feed-forward loops and several genes to define the fates of four cells (Ap1-4) that are sequentially generated and form the Apterous (Ap) cluster of neurons. However, the mechanism that was proposed is very similar to the role that the grh gene plays in the Ap cluster, since Grh expression increases gradually over time from Ap1 to Ap4, and overexpression of Grh converts all four Ap neurons into Ap4 (Moris-Sanz, 2014).

In addition to the different levels of Hb expression observed in NB3-5, it was found that CCAP-ENs and CCAP-INs express low and high levels of Hb, respectively, and overexpression of Hb in postmitotic cells convert the ENs into INs. These observations raise the question of how a high level of Hb expression in the NB leads to a high level of expression in the neuron. A recent analysis of the hb regulatory region revealed a specific postmitotic enhancer, so it would be tempting to propose that this enhancer is only activated in neurons that are generated by a NB expressing a high level of Hb. However, no expression of this enhancer was detected in any of the CCAP neurons, and overexpression of Hb in the NB did not lead to activation of the enhancer in neurons. Therefore, further work is needed to identify the mechanism by which only a subset of the neurons generated in the Hb temporal window expresses a high level of Hb and how this is translated into different neuronal fates (Moris-Sanz, 2014).

CCAP-INs express a high level of Hb and do not express Dac, and upon Hb overexpression the expression of Dac is lost in many, although not all, cells. This could place dac as a direct target of Hb. Analysis of dac cis-regulatory domains indicates the presence of a 5.8 kb domain in the first intron that, when placed in a Gal4 vector, was sufficient to drive GFP expression in vivo in many neurons of late embryos . A preliminary analysis of the sequence of this domain suggests the presence of conserved regions and putative Hb binding sites. Further analysis will be required to confirm the presence and elucidate the function of such sequences (Moris-Sanz, 2014).

Ikaros (or Ikzf1), a mouse ortholog of Hb, is expressed in all early retinal progenitor cells (RPCs) of the developing retina. Its expression in RPCs is necessary and sufficient to confer the competence to generate early-born neurons. These and other observations suggest that, as in the Drosophila CNS, cell-intrinsic mechanisms act in the RPC to control temporal competence. Ikaros is expressed in the early RPCs that give rise to several cell types, namely horizontal, amacrine and gangion cells; however, it is unclear whether distinct levels of Ikaros expression are responsible for the production of these different cell types (Moris-Sanz, 2014).

In the early embryo, different concentrations of Hb seem to elicit different cellular responses. At low concentrations, Hb monomers function as activators, whereas at high concentrations they form dimers that either repress transcription or block activation. Analysis of the Hb protein has led to the identification of two conserved domains: a DNA-binding domain and a dimerization domain. More recently, it has been shown that, in CNS development, Hb repressor function is required to maintain early NB competence and to specify early-born neuronal identity. These results are compatible with the evidence presented in this study that it is the absolute level of Hb in a NB that determines whether it is expressed in the postmitotic progeny and so specifies the different neuronal subtypes (Moris-Sanz, 2014).

The Hunchback temporal transcription factor establishes, but is not required to maintain, early-born neuronal identity

Drosophila and mammalian neural progenitors typically generate a diverse family of neurons in a stereotyped order. Neuronal diversity can be generated by the sequential expression of temporal transcription factors. In Drosophila, neural progenitors (neuroblasts) sequentially express the temporal transcription factors Hunchback (Hb), Kruppel, Pdm, and Castor. Hb is necessary and sufficient to specify early-born neuronal identity in multiple lineages, and is maintained in the post-mitotic neurons produced during each neuroblast expression window. Surprisingly, nothing is currently known about whether Hb acts in neuroblasts or post-mitotic neurons (or both) to specify first-born neuronal identity. This study selectively removed Hb from post-mitotic neurons, and assayed the well-characterized NB7-1 and NB1-1 lineages for defects in neuronal identity and function. Loss of Hb from embryonic and larval post-mitotic neurons did not affect neuronal identity. Furthermore, removing Hb from post-mitotic neurons throughout the entire CNS has no effect on larval locomotor velocity, a sensitive assay for motor neuron and pre-motor neuron function. It is concluded that Hb functions in progenitors (neuroblasts/GMCs) to establish heritable neuronal identity that is maintained by a Hb-independent mechanism. It is suggested that Hb acts in neuroblasts to establish an epigenetic state that is permanently maintained in early-born neurons (Hirono, 2017).

This study showed that the temporal transcription factor Hb, despite being continuously expressed in the U1 motor neuron, is not required to maintain U1 neuronal identity. It is concluded that Hb acts transiently in the neuroblast, GMC, or new-born neuron to establish the U1 neuronal identity, and that this identity is subsequently maintained by a Hb-independent mechanism. This conclusion is also supported by the observation that Hb, like many temporal transcription factors, are re-used in other cell types or tissues to specify different cell fates, showing that cellular context shapes the response to Hb. It is likely that progenitors and post-mitotic neurons provide different contexts for Hb action; the role of Hb in early-born post-mitotic neurons has yet to be defined. For example, in the neuroblast/GMC progenitors, Hb confers temporal identity, in the early embryo Hb specifies anterior-posterior identity, and in adult male neurons Hb confers male-specific morphology. Similar findings are observed for other embryonic temporal transcription factors such as Kr, Pdm, and Castor (Hirono, 2017).

Interestingly, attempts to remove Hb from the entire NB7-1 lineage using en-gal4 UAS-hbRNAi resulted in residual Hb protein in NB7-1 and a weaker phenotype than complete genetic removal of Hb from the NB7-1 lineage. For example, both hb RNAi and hb null mutants resulted in U1 motor neurons that de-repressed zfh2 but only genetic hb null mutants result in absence of early-born Eve+ neurons. This suggests that the Hb protein present in the NB7-1 following hb RNAi is sufficient to produce long-lasting expression of Eve in the U1 motor neuron (Hirono, 2017).

It is concluded that Hb has no detectable function in post-mitotic U1 neurons. Might this lack of phenotype be due to low levels of residual Hb protein in neurons? Although this cannot be formally ruled out, there are several reasons to discount this possibility. First, Hb protein was stained for, and find most U1 neurons have no detectable Hb protein compared to background. Second, RNAi knockdown of Hb in neuroblasts produces a strong phenotype which would not be expected if very low levels of Hb are functional. Finally, Hb protein is not expected to persist following loss of hb RNA, as it has been shown that Hb protein in the CNS has a very short half life. These experiments co-stained neuroblasts and their progeny for Hb protein and active hb transcription (nuclear intron signal), and found that few or no cells had Hb protein but not hb transcription (Hirono, 2017).

The conclusion that Hb has no function in post-mitotic neurons is buttressed by previous findings that late-born Hb-negative neurons are unaffected by forced Hb misexpression. It is hypothesized that temporal transcription factors alter the epigenetic state of neuroblasts which is inherited by their progeny neurons. Thus, early-born neurons do not need Hb to maintain early-born identity, and are also unresponsive to forced expression of other temporal transcription factors; similarly, late-born neurons are unresponsive to forced expression of early temporal transcription factors. This model is supported by findings that Hb acts transiently at the cellular blastoderm stage together with the chromatin remodeler Mi-2 to permanently silence the Ubx gene. It is also supported by the observation that some temporal transcription factors are only transiently expressed in progenitors and new-born neurons, such as Pdm in embryonic lineagesor Eyeless, Sloppy paired, Dichaete, and Tailless in larval optic lobe lineages. In these cases, the temporal transcription factor must act transiently in the neuroblast or GMC to confer long-lasting neuronal identity. The current findings raise the possibility that all temporal transcription factors are required transiently in progenitors to specify permanent temporal identity, despite many of these factors being maintained in post-mitotic neurons. If these findings can be extended to other temporal transcription factors, it would highlight the differences between spatial or temporal patterning genes (required transiently in progenitors) and terminal selector genes (required permanently in post-mitotic neurons). It would also highlight the importance of properly linking spatial/temporal patterning to terminal selector gene expression, an important area for future investigation (Hirono, 2017).

The possibility can be ruled out that Hb is required in post-mitotic neurons for aspects of neuronal function that were not assayed. In fact, post-embryonic expression of Hb is required for proper Fruitless + male neurons morphogenesis; following hb RNAi these neurons are transformed to a female-like morphology. Although no striking axon or dendrite changes in the U neurons following hb RNAi, a slight decrease was seen in neuronal projections in connectives. Although Hb is not required to maintain dorsal axon projections in embryonic or larval U1 motor neurons, it may be required for proper ion channel or neurotransmitter production. Furthermore, mammalian post-mitotic neurons can be reprogrammed to another neuronal identity for a short time after their birth. Temporal transcription factors like Hb may stabilize neuronal identity to prevent such transformations; in this case, loss of neuronal Hb would only show a strong phenotype upon misexpression of a 'reprogramming factor', such as a later temporal transcription factor or a terminal selector gene for a different neural subtype (Hirono, 2017).

Perhaps the strongest evidence available against a Hb function in post-mitotic neurons is the finding that elimination of Hb protein from all post-mitotic neurons has no larval locomotor phenotype. Similar experiments driving pan-neuronal expression of neuronal silencers or activators leads to larval paralysis. Thus, it is highly unlikely that loss of Hb alters early-born interneuron or motor neuron neurotransmitter phenotypes or membrane properties. In the future, it would be interesting to use transcriptional profiling to compare Hb+ and Hb- early-born neurons -- the results suggest that there would be little transcriptional effect from removing Hb from post-mitotic neurons (Hirono, 2017).

It is concluded that Hb functions in progenitors (neuroblasts/GMCs) to establish heritable neuronal identity that is maintained by a Hb-independent mechanism. It is suggested that Hb acts in neuroblasts to establish an epigenetic state that is permanently maintained in early-born neurons (Hirono, 2017).

Bicoid-dependent activation of the target gene Hunchback requires a two-motif sequence code in a specific basal promoter

In complex genetic loci, individual enhancers interact most often with specific basal promoters. This study investigated the activation of the Bicoid target gene hunchback (hb), which contains two basal promoters (P1 and P2). Early in embryogenesis, P1 is silent, while P2 is strongly activated. In vivo deletion of P2 does not cause activation of P1, suggesting that P2 contains intrinsic sequence motifs required for activation. This study shows that a two-motif code (a Zelda binding site plus TATA) is required and sufficient for P2 activation. Zelda sites are present in the promoters of many embryonically expressed genes, but the combination of Zelda plus TATA does not seem to be a general code for early activation or Bicoid-specific activation per se. Because Zelda sites are also found in Bicoid-dependent enhancers, it is proposed that simultaneous binding to both enhancers and promoters independently synchronizes chromatin accessibility and facilitates correct enhancer-promoter interactions (Ling, 2019).

The promoter deletion and insertion experiments in this paper show that Bcd-dependent promoter usage at the hb locus is controlled by intrinsic DNA sequences that lie in the interval between ~51 and +69 with respect to the P2 TSS. Two sequence motifs (a strong Zld site plus TATATAAA) are critical for the efficient activation of the P2 promoter, and inserting them together into the inactive P1 promoter is sufficient to convert it to a partially active Bcd-dependent promoter. Because deletion of the P2 promoter does not result in the activation of the normally inactive P1 promoter, these motifs appear to function by actively and specifically promoting transcription, and there is little competition between P2 and P1 for Bcd-dependent activation (Ling, 2019).

Understanding P2 regulation is complicated by the Zld site's position immediately downstream of the Bcd-dependent Prox enhancer and by both the enhancer and the promoter being contained in a contiguous 390 bp fragment. One specific issue is whether the Zld site upstream of the TATATAAA sequence should be considered part of the Prox enhancer or part of the P2 promoter. Three considerations suggest that it is an integral part of the promoter. First, the Zld site extends from position ~41 to ~35 bp with respect to the hb TSS and only 5 bp upstream of the TATA sequence at position ~30. Second, the Prox enhancer deletion experiments suggest that the Zld site is required for strong activation by the Dist enhancer. Third, a study showed that at least 55 developmentally regulated promoters in Drosophila contain consensus Zld motifs that form a meta-peak ~50 bp upstream of the TSS. Altogether, it is proposed that Zld binding sites should be considered core promoter motifs for a subset of genes that are activated during the mid-blastula transition in the Drosophila (Ling, 2019).

Because Zld may function as a pioneer factor, its binding to the P2 promoter might loosen chromatin by displacing nucleosomes. Such a mechanism has been proposed for Zld sites in enhancer elements. In particular, the hb gene contains Zld sites in both its Bcd-dependent enhancers and in the P2 promoter. It is proposed that binding Zld generates an open chromatin configuration at both types of elements, which would synchronize the binding of Bcd to the enhancers and the binding of TFIID and other basal transcription factors to the P2 promoter. Because of the prevalence of Zld sites in the enhancers and promoters of embryonically expressed genes, this is likely to be a general mechanism that facilitates correct pairings between enhancers and promoters (Ling, 2019).

The P1 promoter does not contain either a strong Zld motif or a canonical TATA sequence, and it is in a closed chromatin configuration when Bcd-dependent activation of P2 occurs. This suggests that P1 is immune to Bcd-dependent activation but when placed adjacent to either the Prox or the Dist-Short enhancer or inserted into the position of P2 in the dual reporter, this promoter is efficiently activated. One explanation is that all three of these experiments position strong Zld sites in the enhancers within 100 bp of the P1 promoter. It is possible that these sites help organize a region of accessible chromatin that spreads into the adjacent P1 promoter, facilitating its activation, even in the absence of a canonical TATA box. To test this, the distance between the nearest Zld site and the P1 promoter was increased to more than 300 bp (Dist-P1), which resulted in the abolishment of expression. Perhaps this distance places the promoter beyond the range of spreading chromatin mediated by the Zld sites. In the endogenous hb gene, the P1 promoter is positioned more than 1 kb downstream of the nearest Zld site in the Dist enhancer and is inactive at this time (Ling, 2019).

Insertion of the 5' half of the P2 core, which contains the Zld site, the TATATAAA sequence, and the InrInr, causes significant activation of P1, but this activation is only about half that seen when the 120 bp P2 core sequence is inserted intact into the P1 position. This suggests that motifs downstream of the TSS are required for generating the transcription rates mediated by P2 in its normal position. Even the activation by the 120 bp P2 core sequence is less than two-thirds of that seen when the P2 is in its original position. It is possible that the Zld site in the Prox enhancer, which is not included in either P2 insertion experiment, augments P2 expression or that sequences between the Dist enhancer and P1 contribute to a region of compacted chromatin that represses the ability to activate at this stage. Future experiments will be required to test these hypotheses (Ling, 2019).

Several published studies suggested that promoters containing specific sequence motifs might attract interactions with enhancers bound by specific proteins, and it is tempting to speculate that the two-motif code discovered in this study is a common feature of promoters activated by Bcd-bound enhancers. This does not seem to be the case. For example, of the 24 embryonic promoters that contain Zld sites and TATA boxes mentioned earlier, only one is activated by a Bcd-dependent enhancer. To test this idea more rigorously, a survey was conducted of 25 well-annotated Bcd-dependent target promoters. About half of these target genes (11, including hb) were previously classified as pre-mid-blastula transition (MBT) genes because they rank among the first zygotically activated genes. Of these, seven contain TATA in their promoter sequences, and two of these contain Zld sites within 100 bp upstream of the TSS. A third promoter contains a single Zld site at ~90 but no TATA. The other 14 Bcd target genes are activated slightly later and were classified as mid-blastula transition zygotic (MBT-Zyg) or mid-blastula transition maternal (MBT-Mat) genes. Of these, only two have TATA-containing promoters, and only one of these also contains a canonical Zld site close to the TATA box. In summary, these results suggest a bias toward having TATA sequences in the promoters of the earliest expressed Bcd target genes, but they do not support the idea that Zld sites or TATA elements (or the combination of both) mediate Bcd-dependent activation per se (Ling, 2019).

Previous studies suggested that the Prox and Dist hb enhancers work together to maximize expression levels of hb. The data presented in this paper substantially extend these studies. First, the data show that both enhancers make productive interactions with P2. Deleting the Prox enhancer alone in the context of the dual reporter caused a 44% reduction in P2 expression, and deleting both the Prox and the Dist enhancers virtually abolished expression, confirming that the Dist enhancer can contribute significantly in the absence of the Prox enhancer. In vivo, deletion of the Prox enhancer causes a strong reduction in hb expression, causing lethality and the loss of two thoracic segments from the larval cuticle. Thus, the amount of hb produced by the Dist enhancer alone is insufficient to provide in vivo hb function. In contrast to previous studies, no significant effect was detected on P2 expression levels when the Dist enhancer was deleted from the reporter gene. Furthermore, deleting the Dist enhancer in vivo did not lead to a mutant phenotype, suggesting that it is dispensable for development under normal laboratory conditions. Thus, the Prox enhancer is critical for hb function, and although the Dist enhancer can interact with P2 to some degree, the level produced by this enhancer alone cannot replace that normally provided by the Prox enhancer (Ling, 2019).

Finally, the results show that the Zld site and TATATAAA each contribute quantitatively to the level of transcription driven by the P2 promoter. Furthermore, attempts to convert P1 into a Bcd-responsive promoter resulted in many output levels. Constructs carrying the Zld+TATA code were expressed at higher levels than those containing a single Zld or TATA site. In addition, a construct carrying the TATATAAA motif and the double initiator was expressed at higher levels than one carrying a simple TATA sequence and an Inr. Finally, changing the spacing between the Zld site and the TATA motif strongly affected expression levels. Altogether, these experiments suggest that basal promoter sequences can play critical roles in precisely determining levels of transcription, in addition to mediating specific enhancer-promoter interactions (Ling, 2019).

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 τ_n (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 (k_elo/k_ini~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 k_ini. 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 τ_n (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).

Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription

For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, this study showed that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid's partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate (Fernandes, 2022).

Recently, synthetic approaches have been used to understand how the details of gene regulation emerge from the plethora of binding sites for transcription factors buried in genomes. In developmental systems, these approaches are starting to help us unravel the evolution of gene regulatory modules. In many cases, using high-throughput analysis of systematically mutagenized regulatory sequences, expression was measured through synthesis of easily detectable fluorescent proteins, RNA sequencing or antibody or FISH staining on fixed samples. Even though these approaches allowed screening for a high number of mutated sequences with a very high resolution (single nucleotide level), the output measurements remained global and it was hard to capture the temporal dynamics of the transcription process itself. In addition, because effects of single mutations are frequently compensated by redundant sequences, it remained often difficult from these studies to highlight the mechanistic roles of the TF they bind to. This work combined the MS2 tagging system, which allows for a detailed measurement of the transcription process dynamics at high temporal resolution, with an orthogonal synthetic approach focusing on a few cis-regulatory elements with the aim of reconstructing from elementary blocks most features of hb regulation by Bcd. The number and placement of TF BS in the MS2 reporters are not identical to those found on the endogenous hb promoter and the number of combinations tested was very limited when compared to the high throughput approaches mentioned above. Nevertheless, this synthetic approach combined with quantitative analyses and modeling sheds light on the mechanistic steps of transcription dynamics (polymerase firing rate, bursting, licensing to be ON/OFF) involving each of the three TFs considered (Bcd, Hb, and Zld). Based on this knowledge from synthetic reporters and the known differences between them, an equilibrium model of transcription regulation was built that agrees with the data from the hb-P2 reporter expression (Fernandes, 2022).

Expression from the Bcd-only synthetic reporters indicate that increasing the number of Bcd BS from 6 to 9 shifts the transcription pattern boundary position toward the posterior region. This is expected as an array with more BS will be occupied faster with the required amount of Bcd molecules. Increasing the number of Bcd BS from 6 to 9 also strongly increases the steepness of the boundary indicating that cooperativity of binding, or more explicitly a longer time to unbind as supported by our model fitting, is likely to be at work in this system. In contrast, adding three more BS to the 9 Bcd BS has very limited impact, indicating that either Bcd molecules bound to the more distal BS may be too far from the TSS to efficiently activate transcription or that the system is saturated with a binding site array occupied with 9 Bcd molecules. In the anterior with excess Bcd, the fraction of time when the loci are active at steady state also increases when adding 3 Bcd BS from B6 to B9. By assuming a model of transcription activation by Bcd proteins bound to target sites, the activation rate increases by much greater fold (~4.5 times) than the number of BS (1.5-2 times) suggesting a synergistic effect in transcription activation by Bcd (Fernandes, 2022).

The burstiness of the Bcd-only reporters in regions with saturating amounts of Bcd, led us to build a model in two steps. The first step of this model accounts for the binding/unbinding of Bcd molecules to the BS arrays. It is directly related to the positioning and the steepness of the expression boundary and thus to the measurement of positional information. The second step of this model accounts for the dialog between the bound Bcd molecules and the transcription machinery. It is directly related to the fluctuation of the MS2 signals including the number of firing RNAP at a given time (intensity of the signal) and bursting (frequency and length of the signal). Interestingly, while the first step of the process is achieved with an extreme precision (10% EL), the second step reflects the stochastic nature of transcription and is much noisier. This model therefore also helps to understand and reconcile this apparent contradiction in the Bcd system (Fernandes, 2022).

As predicted by an original theoretical model, 9 Bcd BS in a synthetic reporter appear sufficient to reproduce experimentally almost entirely the spatial features of the early hb expression pattern i.e. measurements of positional information. This is unexpected as the hb-P2 promoter is supposed to only carry 6 Bcd BS and leaves open the possibility that the number of Bcd BS in the hb promoter might be higher. Alternatively, it is also possible that even though containing 9 Bcd BS, the B9 reporter can only be bound simultaneously by less than 9 Bcd molecules. This possibility must be considered if for instance, the binding of a Bcd molecule to one site prevents by the binding of another Bcd molecule to another close by site (direct competition or steric hindrance). Even though this possibility cannot be excluded, it is thought to be unlikely for several reasons: (1) some of the Bcd binding sites in the hb-P2 promoter are also very close to each other and the design of the synthetic constructs was made by multimerizing a series of 3 Bcd binding sites with a similar spacing as found for the closest sites in the hb-P2 promoter; (ii) the binding of Bcd or other homeodomain containing proteins to two BS is generally increased by cooperativity when the sites are close to each other (as close as two base pairs for the paired homeodomain) compared to binding without cooperativity when they are separated by five base pairs or more (Fernandes, 2022).

Importantly, even though it is not really known if the B9 and the hb-P2 promoter contain the same number of effective Bcd BS, the B9 reporter which solely contains Bcd BS recapitulates most spatial features of the hb-P2 reporter, clearly arguing that Bcd on its own brings most of the spatial (positional) information to the process. Interestingly, the B9 reporter is however much slower (2-fold) to reach the final boundary position than the hb-P2 reporter. This suggested that other maternally provided TFs binding to the hb-P2 promoter contribute to fast dynamics of the hb pattern establishment. Among these TFs, this study focused on two known maternal partners of Bcd: Hb which acts in synergy with Bcd and Zld, the major regulator of early zygotic transcription in fruit fly. Interestingly, adding Zld or Hb sites next to the Bcd BS array reduces the time for the pattern to reach steady state and modifies the promoter activity in different ways: binding of Zld facilitates the recruitment of Bcd at low concentration, making transcription more sensitive to Bcd and initiate faster while the binding of Hb affects strongly both the activation/deactivation kinetics of transcription (burstiness) and the RNAP firing rate. Thus, these two partners of Bcd contribute differently to Bcd-dependent transcription. Consistent with an activation process in two steps as proposed in this model, Zld will contribute to the first step favoring the precise and rapid measurements of positional information by Bcd without bringing itself positional information. Meanwhile, Hb will mostly act through the second step by increasing the level of transcription through a reduction of its burstiness and an increase in the polymerase firing rate. Interestingly, both Hb and Zld binding to the Bcd-dependent promoter allow speeding-up the establishment of the boundary, a property that Bcd alone is not able to achieve. Of note, the hb-P2 and Z2B6 reporters contain the same number of BS for Bcd and Zld but they have also very different boundary positions and mean onset time of transcription T0 following mitosis when Bcd is limiting. This is likely due to the fact that the two Zld BS in the hb-P2 promoter are not fully functional: one of the Zld BS is a weak BS while the other Zld BS has the sequence of a strong BS but is located too close from the TATA Box (5 bp) to provide full activity (Fernandes, 2022).

Zld functions as a pioneer factor by potentiating chromatin accessibility, transcription factor binding and gene expression of the targeted promoter. Zld has recently been shown to bind nucleosomal DNA and proposed to help establish or maintain cis-regulatory sequences in an open chromatin state ready for transcriptional activation. In addition, Zld is distributed in nuclear hubs or microenvironments of high concentration. Interestingly, Bcd has been shown to be also distributed in hubs even at low concentration in the posterior of the embryo. These Bcd hubs are Zld-dependent and harbor a high fraction of slow moving Bcd molecules, presumably bound to DNA. Both properties of Zld, binding to nucleosomal DNA and/or the capacity to form hubs with increased local concentration of TFs can contribute to reducing the time required for the promoter to be occupied by enough Bcd molecules for activation. In contrast to Zld, knowledge on the mechanistic properties of the Hb protein in the transcription activation process is much more elusive. Hb synergizes with Bcd in the early embryo and the two TF contribute differently to the response with Bcd providing positional and Hb temporal information to the system. Hb also contributes to the determination of neuronal identity later during development. Interestingly, Hb is one of the first expressed members of a cascade of temporal TFs essential to determine the temporal identity of embryonic neurons in neural stem cells (neuroblasts) of the ventral nerve cord. In this system, the diversity of neuronal cell-types is determined by the combined activity of TFs specifying the temporal identity of the neuron and spatial patterning TFs, often homeotic proteins, specifying its segmental identity. How spatial and temporal transcription factors mechanistically cooperate for the expression of their target genes in this system is not known. The current work indicates that Hb is not able to activate transcription on its own but that it strongly increases RNAP firing probability and burst length of a locus licensed to be ON. Whether this capacity will be used in the ventral nerve cord and shared with other temporal TFs would be interesting to investigate (Fernandes, 2022).

The Bcd-only synthetic reporters also provided an opportunity to scrutinize the effect of Bcd concentration on the positioning of the expression domain boundaries. This question has been investigated with endogenous hb in the past, always giving a smaller shift than expected given the decay length of 20% EL for the Bcd protein gradient and arguing against the possibility that positional information in this system could solely be dependent on Bcd concentration. When comparing the transcription patterns of the B9 reporter in Bcd-2X flies and Bcd-1X flies, a shift was detected of ~10.5 ± 1% EL of the boundary position. This shift revealed a gradient of Bcd activity with an exponential decay length of ~15 ± 1.4% EL (~75 μm), significantly smaller than the value observed directly (20% EL, ~ 100 μm) with immuno-staining for the Bcd protein gradient but closer from the value of 16.4% EL obtained with immuno-staining for Bcd of the Bcd-GFP gradient. Given the discrepancies of previous studies concerning the measurements of the Bcd protein gradient decay length, this work calls for a better quantification to determine how close the decay length of the Bcd protein gradient is from the decay length of the Bcd activity gradient uncovered here. This work opens the possibility that the effective decay length of 15% EL corresponds to a population of 'active' or 'effective' Bcd distributed in steeper gradient than the Bcd protein gradient observed by immunodetection which would include all Bcd molecules. Bcd molecules have been shown to be heterogenous in intranuclear motility, age and spatial distributions but to date, it is not known which population of Bcd can access the target gene and activate transcription. The existence of two (or more) Bicoid populations with different mobilities obviously raises the question of the underlying gradient for each of them. Also, the dense Bcd hubs persist even in the posterior region where the Bcd concentration is low. As the total Bcd concentration decreases along the AP axis, these hubs accumulate Bcd with increasing proportion in the posterior, resulting in a steeper gradient of free-diffusing Bcd molecules outside the hubs. At last, the gradient of newly translated Bcd was also found to be steeper than the global gradient. Finally and most importantly, reducing by half the Bcd concentration in the embryo induced a similar shift in the position of the hb-P2 reporter boundary as that of the Bcd-only reporters. This further argues that this gradient of Bcd activity is the principal and direct source of positional information for hb expression (Fernandes, 2022).

The effective Bcd gradient found here rekindles the debate on how a steep hb pattern can be formed in the early nuclear cycles. With the previous value of λ=20% EL for the decay length of the Bcd protein gradient, the Hill coefficient inferred from the fraction of loci's active time at steady state PSpot is ~6.9, beyond the theoretical limit of the equilibrium model of Bcd interacting with six target BS of the hb promoter. This led to hypotheses of energy expenditure in Bcd binding and unbinding to the sites, out-of-equilibrium transcription activation, hb promoters containing more than 6 Bcd sites or additional sources of positional information to overcome this limit. The effective decay length λeff ~15% EL, found here with a Bcd-only reporter but also hb-P2, corresponds to a Hill coefficient of ~5.2, just below the physical limit of an equilibrium model of concentration sensing with 6 Bcd BS alone. Of note, a smaller decay length also means that the effective Bcd concentration decreases faster along the AP axis. In the Berg & Purcell limit (Biophys. J., 1977), the time length to achieve the measurement error of 10% at hb-P2 expression boundary with λ=15% EL is ~2.1 times longer than with λ=20% EL. This points again to the trade-off between reproducibility and steepness of the hb expression pattern and reinforces the importance of Hb and Zelda in speeding-up the process (Fernandes, 2022).

Hunchback activates Bicoid in Pair1 neurons to regulate synapse number and locomotor circuit function

Neural circuit function underlies cognition, sensation, and behavior. Proper circuit assembly depends on the identity of the neurons in the circuit (gene expression, morphology, synapse targeting, and biophysical properties). Neuronal identity is established by spatial and temporal patterning mechanisms, but little is known about how these mechanisms drive circuit formation in postmitotic neurons. Temporal patterning involves the sequential expression of transcription factors (TFs) in neural progenitors to diversify neuronal identity, in part through the initial expression of homeodomain TF combinations. This study addresses the role of the Drosophila temporal TF Hunchback and the homeodomain TF Bicoid in the assembly of the Pair1 (SEZ_DN1) descending neuron locomotor circuit, which promotes larval pausing and head casting. Both Hunchback and Bicoid are expressed in larval Pair1 neurons, Hunchback activates Bicoid in Pair1 (opposite of their embryonic relationship), and the loss of Hunchback function or Bicoid function from Pair1 leads to ectopic presynapse numbers in Pair1 axons and an increase in Pair1-induced pausing behavior. These phenotypes are highly specific, as the loss of Bicoid or Hunchback has no effect on Pair1 neurotransmitter identity, dendrite morphology, or axonal morphology. Importantly, the loss of Hunchback or Bicoid in Pair1 leads to the addition of new circuit partners that may underlie the exaggerated locomotor pausing behavior. These data are the first to show a role for Bicoid outside of embryonic patterning and the first to demonstrate a cell-autonomous role for Hunchback and Bicoid in interneuron synapse targeting and locomotor behavior (Lee, 2022).

Neural circuit formation underlies the generation of behavior, and aberrant neural circuit development has been associated with many neural disorders, such as autism and attention deficit hyperactivity disorder. It is widely accepted that circuit formation requires the assembly of precise interconnectivity between diverse neuron subtypes. Although the mechanisms for generating molecularly and morphologically distinct neurons are well studied, little is known about how these developmental mechanisms regulate 'higher-order' neuronal properties such as pre- and post-synapse numbers or circuit partner choice (Lee, 2022).

In Drosophila, neuronal identity is specified by the combination of spatial and temporal transcription factors (TFs) acting on neuronal stem cells (neuroblasts in Drosophila). Spatial patterning creates molecularly distinct neuroblasts, followed by each neuroblast sequentially expressing a series of temporal TFs: Hunchback > Kruppel Temporal TFs are known to specify axon and dendrite morphology and targeting as well as behavior. For example, in neuroblast 7-1, the best characterized lineage in the embryo, the zinc-finger temporal TF Hunchback promotes expression of the homeodomain TF even-skipped that is required for proper motor neuron morphology and connectivity; and the combination of Kruppel and Pdm temporal TFs promotes expression of the homeodomain TF Nkx6 (FlyBase: HGTX) that is required for proper ventral projecting motor neuron morphology and connectivity. In both cases, transient temporal TF expression activates a homeodomain TF that persists in the postmitotic neuron to determine neuron morphology and neuromuscular connectivity. Similarly, work from the Hobert lab in C. elegans supports a model in which each of the 302 neurons is specified by a unique combination of homeodomain TFs. Overall, from worms to flies to mammals, temporal TFs activate homeodomain TFs to specify molecular and morphological neuronal identity (Lee, 2022).

Although homeodomain TFs are well known to specify these early aspects of motor neuron identity, their role in specifying later aspects of neuronal identity such as synapse number, position, and connectivity remains poorly understood. To address this question, the Pair1 (SEZ_DN1) locomotor circuit in Drosophila was used. Pair1 is a GABAergic interneuron with ipsilateral dendrites and contralateral descending axonal projections. The moonwalker descending neurons (MDN) provide inputs to Pair1, and Pair1 sends outputs to A27h neurons in the ventral nerve cord (VNC). When optogenetically activated, the Pair1 neurons induce a pause in forward locomotion and increase in head casting, in part by inhibiting the A27h neurons, which drive forward locomotion. Importantly, it was previously reported that the temporal TF Hunchback and the homeodomain TF Bicoid are expressed in Pair1 neurons throughout life, providing candidates to study the transcriptional regulation of Pair1 neuronal identity and connectivity (Lee, 2022).

Hunchback is the first temporal TF to be expressed in the Drosophila embryo and acts transiently to generate early born neurons. In the embryonic CNS, Hunchback is not required to maintain neuronal identity, although it is required to maintain proper dendrite morphology of the mAL interneuron in adult males. Bicoid is a homeodomain TF; however, its expression and function outside the early embryo had not been reported until recent work from this lab. Bicoid is well known to form an anterior-posterior morphogen gradient that directly activates hunchback to properly pattern the anterior-posterior body axis.26 Although the role of Hunchback in temporal patterning is conserved in mammals, Bicoid is found only in higher dipteran insects, making it an interesting contributor to insect evolution. This study tested the model that the temporal TF Hunchback activates the homeodomain TF Bicoid (opposite of their early embryo relationship) and whether Hunchback and Bicoid play a role in Pair1 neurotransmitter expression, neuron morphology, synapse number, circuit function, and behavior. The data support the emerging model that temporal TFs drive expression of homeodomain TFs that maintain distinct aspects of neuronal identity including synapse number/position, connectivity, and behavior (Lee, 2022).

The results show that Hunchback activates Bicoid in postmitotic Pair1 neurons, where it regulates specific and important aspects of neuronal identity-synapse number, synapse density, and connectivity. When Hunchback or Bicoid levels are decreased, synapse density is increased, with a corresponding disruption of the function of the Pair1 locomotor neural circuit. This work demonstrates a novel role for Hunchback and Bicoid-functioning postmitotically to regulate synapse number and to ensure proper circuit function. Importantly, this work also reproduces a phenotype previously seen in C. elegans-a single homeobox gene (unc-4) specifically regulates synaptic connectivity but not other aspects of neuronal identity. Interestingly, unc-4 expression is also regulated by a nonhomeodomain TF, suggesting that this regulatory pathway may be conserved between species to specify highly specific aspects of neuronal identity (Lee, 2022).

Unlike most early born neurons in the VNC that only transiently express Hunchback, and Bicoid which is only expressed in the first few hours of embryogenesis, the Pair1 neuron maintains both Hunchback and Bicoid expression into the adult. This suggests that a Pair1-specific regulatory mechanism may be leading to the persistent Hunchback and Bicoid expression and function. Given that the Pair1 neuron persists into adulthood, still expresses Hunchback and functions within a similar locomotor neural circuit, it is hypothesized that Hunchback and Bicoid expressions may be required in Pair1 neurons throughout life for the maintenance of the Pair1 locomotor neural circuit (Lee, 2022).

Surprisingly, Bicoid protein expression in larval Pair1 neurons was often detected in one or more spherical puncta located in the cytoplasm; this was observed with two independent Bicoid antibodies and a third FLAG-tagged Bicoid protein and was abolished by Bicoid RNAi. Given that Bicoid contains highly disordered regions with an abundance of glutamine and glycine, the spherical puncta may represent a phase-separation condensate, perhaps to keep nuclear Bicoid levels low. Interesting, Bicoid does not form spherical puncta outside of the larvae. Further investigation is needed to understand nature of the Bicoid cytoplasmic puncta, but these studies have the potential to elucidate a novel role for phase-separation in mature neurons (Lee, 2022).

Previous work showed that Bicoid activates hunchback in the early embryo. This study is the first to demonstrate the reverse that Hunchback can promotes Bicoid expression in vivo. Hunchback may regulate Bicoid directly or indirectly; supporting the former possibility are the findings that Hunchback protein binds two distinct regions at the 3' and 5' end of the bicoid locus. Alternatively, Hunchback may act indirectly by promoting Bicoid phase separation in larval neurons. Regardless, this finding supports the initial hypothesis that temporal TFs, like Hunchback, can activate homeodomain TFs, like Bicoid, to specify some or all aspects of neuronal identity. Other morphogens have been previously associated with establishing properties of neuronal identity, further suggesting that early developmental TFs may be important regulators of neuronal identity, connectivity, and circuit function in general (Lee, 2022).

Hunchback and Bicoid had no detectable role in regulating dendrite morphology, axon morphology, nor GABA expression, key aspects of Pair1 neuronal identity. However, both Hunchback and Bicoid are required for maintaining synapse number and functional connectivity of the Pair1 neuron. Trans-Tango experiments show that reduced Hunchback levels resulted in the addition of new synaptic partners of Pair1, although it cannot be excluded that these may be normal partners that are too weak to see in controls. Although the novel neuronal partners were not formally identified, the Drosophila larvae TEM volume was used to speculate that Pair1 could be synapsing with the A27h neurons located in the thoracic region. Given that A27h neurons are involved in forward locomotion, additional thoracic A27h neurons synapsing onto, and therefore being inhibited by Pair1 activation, could explain the increased pausing phenotype observed when Hunchback in knocked down in Pair1. Alternatively, abdominal A27h neurons could be forming more synapses with Pair1 in the posterior axonal regions (Lee, 2022).

Interestingly, it appears that Bicoid is not the only homeodomain TF functioning downstream of Hunchback in Pair1. When Hunchback is knocked down in Pair1, pausing speed is increased, head casting is increased, and recovery speeds are decreased. However, Bicoid knockdown only replicated the decreased recovery speed phenotype; this suggests that another homeodomain TF may be functioning downstream of Hunchback to regulate pausing speed and head casting. The data presented in this study begin to support this hypothesis, but additional work is needed to identify other homeodomain TFs functioning downstream of Hunchback (Lee, 2022).

This work is the first to demonstrate a role for Hunchback and Bicoid in postmitotic neurons to regulate synapse number, connectivity, and circuit function. These results raise the question of which is the more ancestral function of these two TFs: in segmentation, temporal patterning in neuroblasts, or postmitotic neuronal circuit maintenance (Lee, 2022)?

GENE STRUCTURE

There are two transcripts, P1 (maternal & zygotic) and P2 (zygotic), initiated from different promoters. The P2 site of initiation is within the DNA coding for the large intron of the maternal transcript. There is a small zygotic intron which shares a common acceptor site with the maternal intron (Tautz, 1987).

Bases in 5' UTR - 221 for P1 and 152 for P2

Exons - two

PROTEIN STRUCTURE

Amino Acids - 758

Structural Domains

Hunchback and Krüppel are homologous; they share four zinc finger domains. Hunchback has a higher molecular weight than Krüppel, because of an additional two zinc fingers at its C-terminal end (Tautz, 1987). 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).

Castor, a transcription factor with similar DNA binding specificity to that of Hb, contains a centrally located Zn-finger domain made-up of four consecutive C2-H2C2-H2 repeats. The second C2-H2 of each repeat closely resembles fingers of the Xenopus TFIIIA C2-H2 class. Flanking this repeat are motifs that may constitute either transcription transactivation or repression domains. UV induced protein-DNA cross-linking in vivo studies reveal that Cas binds genomic DNA. To determine if Cas is a sequence-specific DNA-binding protein, the cyclic amplification of selected targets protocol was used. After six rounds of selection/amplification, sequencing of cloned fragments revealed that all had at least one sequence motif in common and some contained two core recognition sequences. DNA fragments containing one site homologous to the consensus site produce a single prominent Cas-DNA gel-shift; a fragment with two, generates two complexes. Addition of Cas-specific antisera causes a super-shift of the Cas-DNA complex. A search of known transcription factor DNA-binding sites shows that the Cas recognition sequence is almost identical to that of the Drosophila Zn-finger protein Hunchback. The Cas consensus matches 9 out of 10 bp for the reported Hb sites. To determine if Cas binds Hb sites, gel-shift experiments using DNA fragments were carried out with exact sequence matches to Hb targets. Cas does indeed bind to these sites. The sequence-specificity of Cas-DNA binding to Hb recognition sites was further tested by competition assays and base-pair substitutions. Taken together, these experiments demonstrate that Cas can bind to the same DNA sites as Hb, raising the possibility that it modulates transcriptional activities of genes also regulated by Hb. Secondary structure predictions of the Cas finger domain indicate that only the first and third of its TFIIIA-like fingers contain alpha-helices. Interestingly, optimal alignment of Cas and Hb fingers reveals that the first and third a-helices of Cas share the highest homology with the corresponding a-helices of Hb (33% identity for the first and 27% for the third). Although speculative, their shared DNA-binding preferences may be due in part to the shared residues found in these predicted reading heads. Outside of their Zn-fingers, Hb and Cas show no obvious sequence similarities (Kambadur, 1998).

date revised: 2 January 2023 

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