hunchback: Biological Overview | Evolutionary Homologs | Regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

Gene name - hunchback

Synonyms -

Cytological map position - 85A3-B1

Function - transcription factor

Keywords - morphogen - anterior-posterior axis and gap gene

Symbol - hb

FlyBase ID:FBgn0001180

Genetic map position - 3-48.3

Classification - zinc finger protein (C2H2)

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene | HomoloGene | UniGene

Recent literature
Bothma, J. P., Garcia, H. G., Ng, S., Perry, M. W., Gregor, T. and Levine, M. (2015). Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo. Elife 4. PubMed ID: 26267217
Metazoan genes are embedded in a rich milieu of regulatory information that often includes multiple enhancers possessing overlapping activities. This study employed quantitative live imaging methods to assess the function of pairs of primary and shadow enhancers in the regulation of key patterning genes - knirps, hunchback, and snail - in developing Drosophila embryos. The knirps enhancers exhibit additive, sometimes even super-additive activities, consistent with classical gene fusion studies. In contrast, the hunchback enhancers function sub-additively in anterior regions containing saturating levels of the Bicoid activator, but function additively in regions where there are diminishing levels of the Bicoid gradient. Strikingly sub-additive behavior is also observed for snail, whereby removal of the proximal enhancer causes a significant increase in gene expression. Quantitative modeling of enhancer-promoter interactions suggests that weakly active enhancers function additively while strong enhancers behave sub-additively due to competition with the target promoter (Bothma, 2015).

Kozlov, K., Gursky, V. V., Kulakovskiy, I. V., Dymova, A. and Samsonova, M. (2015). Analysis of functional importance of binding sites in the Drosophila gap gene network model. BMC Genomics 16 Suppl 13: S7. PubMed ID: 26694511

The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. This study elaborates a two-layer model for the Drosophila gap gene network and includes in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. The new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. The functional importance of binding sites was quantified by calculating their impact on gene expression in the model, and how these impacts correlate across all sites were calculated under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains (Kozlov, 2015).

Liu, J., Xiao, Y., Zhang, T. and Ma, J. (2016). Time to move on: modeling transcription dynamics during an embryonic transition away from maternal control. Fly (Austin): [Epub ahead of print]. PubMed ID: 27172244
A recent study investigated the regulation of hunchback (hb) transcription dynamics in Drosophila embryos. The results suggest that shutdown of hb transcription at early nuclear cycle (nc) 14 is an event associated with the global changes taking place during the mid-blastula transition (MBT). This study developed a simple model of hb transcription dynamics during this transition time. With kinetic parameters estimated from published experimental data, the model describes the dynamical processes of hb gene transcription and hb mRNA accumulation. With two steps, transcription onset upon exiting the previous mitosis followed by a sudden impact that blocks gene activation, the model recapitulates the observed dynamics of hb transcription during the nc14 interphase. The timing of gene inactivation is essential, as its alterations lead to changes in both hb transcription dynamics and hb mRNA levels. This model provides a clear dynamical picture of hb transcription regulation as one of the many, actively regulated events concurrently taking place during the MBT.

Spirov, A. V., Myasnikova, E. M. and Holloway, D. M. (2016). Sequential construction of a model for modular gene expression control, applied to spatial patterning of the Drosophila gene hunchback. J Bioinform Comput Biol 14: 1641005. PubMed ID: 27122317
Gene network simulations are increasingly used to quantify mutual gene regulation in biological tissues. These are generally based on linear interactions between single-entity regulatory and target genes. Biological genes, by contrast, commonly have multiple, partially independent, cis-regulatory modules (CRMs) for regulator binding, and can produce variant transcription and translation products. This study presents a modeling framework to address some of the gene regulatory dynamics implied by this biological complexity. Spatial patterning of the hunchback (hb) gene in Drosophila development involves control by three CRMs producing two distinct mRNA transcripts. This example was used to develop a differential equations model for transcription which takes into account the cis-regulatory architecture of the gene. Potential regulatory interactions are screened by a genetic algorithms (GAs) approach and compared to biological expression data.
Desponds, J., Tran, H., Ferraro, T., Lucas, T., Perez Romero, C., Guillou, A., Fradin, C., Coppey, M., Dostatni, N. and Walczak, A. M. (2016). Precision of readout at the hunchback gene: Analyzing short transcription time traces in living fly embryos. PLoS Comput Biol 12(12): e1005256. PubMed ID: 27942043
The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives us a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows quantification of the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. An analysis approach based on tailor made autocorrelation functions was developed that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, signatures of bursty transcription initiation from the hunchback promoter were identified. The precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis was show to be low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos.
Torres-Oliva, M., Schneider, J., Wiegleb, G., Kaufholz, F. and Posnien, N. (2018). Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity. PLoS Genet 14(1): e1007180. PubMed ID: 29360820
Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. This study obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. The expression profiles of all genes expressed during eye-antennal disc development was revealed, and temporally co-expressed genes was revealed by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, the transcriptome dataset was combined with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, this study shows that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. It was confirmed that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, functional data is provided indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, the expression data was combined with a de novo Hb motif search to reveal stage specific putative target genes of which a significant number was found to be expressed in carpet cells.
Vincent, B. J., Staller, M. V., Lopez-Rivera, F., Bragdon, M. D. J., Pym, E. C. G., Biette, K. M., Wunderlich, Z., Harden, T. T., Estrada, J. and DePace, A. H. (2018). Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos. PLoS Genet 14(9): e1007644. PubMed ID: 30192762
Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. This study investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. Evidence was also found that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. These results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. How counter-repression may influence eve stripe 2 regulation and evolution is discussed.
Averbukh, I., Lai, S. L., Doe, C. Q. and Barkai, N. (2018). A repressor-decay timer for robust temporal patterning in embryonic Drosophila neuroblast lineages. Elife 7. PubMed ID: 30526852
Biological timers synchronize patterning processes during embryonic development. In the Drosophila embryo, neural progenitors (neuroblasts; NBs) produce a sequence of unique neurons whose identities depend on the sequential expression of temporal transcription factors (TTFs), including Hb, Kr, Pdm and Cas. The stereotypy and precision of NB lineages indicate reproducible TTF timer progression. This study combines theory and experiments to define the timer mechanism. The TTF timer is commonly described as a relay of activators, but its regulatory circuit is also consistent with a repressor-decay timer, where TTF expression begins when its repressor decays. Theory shows that repressor-decay timers are more robust to parameter variations than activator-relay timers. This motivated an experimental comparison of the relative importance of the relay and decay interactions in-vivo. Comparing WT and mutant NBs at high temporal resolution, this study show that the TTF sequence progresses primarily by repressor-decay. It is suggested that need for robust performance shapes the evolutionary-selected designs of biological circuits.
Sen, S. Q., Chanchani, S., Southall, T. D. and Doe, C. Q. (2019). Neuroblast-specific open chromatin allows the temporal transcription factor, Hunchback, to bind neuroblast-specific loci. Elife 8. PubMed ID: 30694180
Spatial and temporal cues are required to specify neuronal diversity, but how these cues are integrated in neural progenitors remains unknown. Drosophila progenitors (neuroblasts) are a good model: they are individually identifiable with relevant spatial and temporal transcription factors known. This study tested whether spatial/temporal factors act independently or sequentially in neuroblasts. Targeted DamID was used to identify genomic binding sites of the Hunchback temporal factor in two neuroblasts (NB5-6 and NB7-4) that make different progeny. Hunchback targets were different in each neuroblast, ruling out the independent specification model. Moreover, each neuroblast had distinct open chromatin domains, which correlated with differential Hb-bound loci in each neuroblast. Importantly, the Gsb/Pax3 spatial factor, expressed in NB5-6 but not NB7-4, had genomic binding sites correlated with open chromatin in NB5-6, but not NB7-4. These data support a model in which early-acting spatial factors like Gsb establish neuroblast-specific open chromatin domains, leading to neuroblast-specific temporal factor binding and the production of different neurons in each neuroblast lineage.

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).

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).

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 HbδD leads to the specification of two U5 neurons at the expense of the U4 cell identity, whereas overexpression of HbδDMZ results in normal U4 and U5 identities. Perhaps HbδDMZ 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 HbδD 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).

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).


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).

cDNA clone length - 3.2 kb for P1 and 2.9 kb for P2

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

Exons - two


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).

hunchback: Biological Overview | Evolutionary Homologs | Regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References
date revised: 2 December 2018 

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