Gene name - huckebein
Cytological map position - 82A2-6
Function - transcription factor
Keyword(s) - gap gene
Symbol - hkb
Genetic map position - 3-[47.1]
Classification - Zinc finger
Cellular location - nuclear
|Recent literature||Momen-Roknabadi, A., Di Talia, S. and Wieschaus, E. (2016). Transcriptional timers regulating mitosis in early Drosophila embryos. Cell Rep 16: 2793-2801. PubMed ID: 27626650
The development of an embryo requires precise spatiotemporal regulation of cellular processes. During Drosophila gastrulation, a precise temporal pattern of cell division is encoded through transcriptional regulation of cdc25string in 25 distinct mitotic domains. Using a genetic screen, it was demonstrated that the same transcription factors that regulate the spatial pattern of cdc25string transcription encode its temporal activation. buttonhead and empty spiracles were identified as the major activators of cdc25string expression in mitotic domain 2. The effect of these activators is balanced through repression by hairy, sloppy paired 1, and huckebein. Within the mitotic domain, temporal precision of mitosis is robust and unaffected by changing dosage of rate-limiting transcriptional factors. However, precision can be disrupted by altering the levels of the two activators or two repressors. It is proposed that the additive and balanced action of activators and repressors is a general strategy for precise temporal regulation of cellular transitions during development.
|Ho, E. K., Oatman, H. R., McFann, S. E., Yang, L., Johnson, H. E., Shvartsman, S. Y. and Toettcher, J. E. (2023). Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo. bioRxiv. PubMed ID: 36945584
Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. This study used optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. High or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. It was further shown that an all-or-none stimulus can be 'blurred' through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, this study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.
tailless and huckebein are known as terminal gap genes, due to their expression in both anterior and posterior ends (or terminals) of the egg. huckebein's efforts to regulate transcription are both positive and negative. As a repressor, HKB assures that the formation of mesoderm (by ventral invagination of the presumptive mesoderm) does not spread to the two poles of the egg. In hkb mutants, invagination is not normal, spreading both to the anterior and posterior beyond the usual limits.
The positive regulatory action of HKB results in the formation of proper endoderm, developed from invaginating gut primordia at either end of the egg. Gut invagination doesn't proceed normally in hkb mutants. Germ cells usually migrate through the gut to take up a position in the mesoderm. As a gap gene, hkb is also involved in regulating wingless and engrailed in the head, assuring proper subdivision of head somite compartments [Images].
Engrailed and Huckebein are essential for development of serotonin neurons in the Drosophila CNS. en and hkb coexpress uniquely in the serotonin neurons and in neuroblast 7-3 (NB7-3). In the grasshopper, the analogous serotonin neurons originate from the first ganglion mother cell produced from NB7-3. The corresponding NB7-3 in Drosophila can be identified by its time of birth, size, and relative position within each hemisegment. The serotonin neurons can be identified during late embryogenesis by the appearance of DOPA decarboxylase (DDC) immunoreactivity. The DDC enzyme catalyzes the last step in the biosynthesis of serotonin and dopamine and can be used as a marker for both cell types. In the ventral ganglion there are three anatomically distinguished types of DDC immunoreactive cells per segment, a pair of ventrolateral serotonin cells (VL), a single midline dopamine cell (M) and the dorsal lateral (DL) dopamine cells. en and hkb are coexpressed in the VL cells but not the DL or M cells. The high selectivity of coexpression of these two gene products suggests that their combined activities may be important for the development of NB7-3 progeny. Serotonin neuron differentiation is abnormal in en and hkb mutants. Although neither mutant shows a complete loss of DDC immunoreactive serotonin cells, the few escaper serotonin neurons may be due to low levels of functional hkb gene product in a hypomorphic allele. Since NB 7-3 appears normal in hkb mutants, the effect of hkb on development of the serotonin cell lineage must be at a later stage of development, either at division of the neuroblast or ganglion mother cells or on the identity of the GMC progeny (Lundell, 1996). For more information on serotonin and dopamine neurons see Islet and Zn finger homeodomain 1).
The Groucho corepressor mediates negative transcriptional regulation in association with various DNA-binding proteins in diverse developmental contexts. Groucho has been implicated in Drosophila embryonic terminal patterning: it is required to confine tailless and huckebein terminal gap gene expression to the pole regions of the embryo. An additional requirement for Groucho in this developmental process has been revealed by establishing that Groucho mediates repressor activity of the Huckebein protein. Putative Huckebein target genes are derepressed in embryos lacking maternal groucho activity and biochemical experiments demonstrate that Huckebein physically interacts with Groucho. Using an in vivo repression assay, a functional repressor domain in Huckebein that has been identified contains an FRPW tetrapeptide, similar to the WRPW Groucho-recruitment domain found in Hairy-related repressor proteins. Mutations in Huckebeins FRPW motif abolish Groucho binding and in vivo repression activity, indicating that binding of Groucho through the FRPW motif is required for the repressor function of Huckebein. Thus Groucho-repression regulates sequential aspects of terminal patterning in Drosophila (Goldstein, 1999).
One proposed Hkb target is the snail (sna) gene, which is transcribed in the ventral-most portion of the embryo. sna expression is thought to be excluded from the posterior pole by hkb activity. Accordingly, sna and hkb expression domains abut in cellularizing wild-type embryos, whereas sna expression extends to, and includes, the posterior pole of hkb mutant embryos. In torD embryos, hkb expression expands towards the center of the embryo and the sna domain correspondingly retracts. By contrast, in gromat- embryos, the expression of sna does not respect the sna posterior border and spreads to the pole, overlapping extensively with the hkb expression domain. The expression of the T-related gene brachyenteron (byn; also called Trg) also seems to be repressed by Hkb. byn is not expressed at the most posterior region of wild-type (or torD) embryos, whereas it extends throughout the posterior cap of hkb mutant embryos, consistent with hkb setting the posterior limit of byn expression. However, it is found that byn is ectopically expressed at the posterior tip of gromat- embryos. Together, these results suggest that gro is, directly or indirectly, necessary for hkb repressor functions (Goldstein, 1999).
To establish whether Hkb can function as a repressor, a HairyHkb chimera was constructed by replacing the C terminus of Hairy with Hkbs N-terminal 195 amino acids (lacking the Hkb Sp1-like zinc-finger DNA-binding domain). When expressed under the regulation of the hb promoter, the HairyHkb chimera causes effective repression of Sxl (normally a target of Hairy) in the anterior region of female embryos. Furthermore, this repression also causes female-specific lethality, probably due to the role of Sxl in dosage compensation. These results indicate that Hkb contains a potent repression domain within its N-terminal 195 aminoacids (Goldstein, 1999).
Hkb also behaves genetically as a positive regulator of forkhead (fkh) and serpent (srp) expression. In hkb mutant embryos, the posterior fkh domain is smaller than in wild-type embryos and srp expression at the poles is not initiated. Perhaps Hkb functions as an activator of fkh and srp expression that, when associated with Gro, represses other target genes. Arguing against this possibility, there is no direct evidence that Hkb contains an activation domain. For example, it does not promote activation of reporter genes when introduced into yeast cells. Additionally, the Hairy Hkb chimera containing the N-terminal 195 residues of Hkb does not cause activation of Sxl in male embryos, whereas this does occur in a Hairy fusion containing the viral VP16 activation domain. These results suggest that Hkb regulates fkh and srp transcription indirectly, possibly by repressing a repressor of these genes (Goldstein, 1999 and references).
This study describes the morphological and genetic analysis of the Drosophila mutant gürtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebeingörtelchen (hkbgurt) mutant embryos. It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. These analyses of the visceral phenotype of hkbgurt mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion (Wolfstetter, 2009).
During gastrulation the visceral mesoderm comes in close contact with endodermal cell layers of the prospective midgut. This contact is maintained during embryogenesis and finally both tissues arrange to form the larval midgut which consists of an inner epithelial layer surrounded by a web of visceral muscles. During midgut formation, guidance of migrating endodermal cells by the flanking visceral mesoderm is an important step, as is the mesenchymal-epithelial transition of the endodermal cells. Although endoderm differentiation has been extensively studied in various mutants exhibiting defective visceral mesoderm development, less has been reported concerning visceral morphogenesis in the absence of the underlying endoderm. With the identification of the visceral mutation gürtelchen as a novel allele of the endodermal transcription factor huckebein it was possible to study these influences in detail. hkbgurt mutant embryos display severe circular muscle outgrowth defects and exhibit a nearly complete loss of both midgut rudiments. In contrast to this observation somatic muscle development, visceral cell type determination, visceral muscle fusion and even initial myotube stretching is unaffected in hkbgurt mutant embryos indicating that an endodermal influence on visceral development is limited to the later process of myotube outgrowth. Moreover, morphological analysis of tailless and brachyenteron mutant embryos clearly demonstrates that these influences are based on endoderm specification, whereas proper development of ectodermal derived hindgut structures is not needed per se for visceral myotube stretching but probably limits visceral constriction formation. However, due to the accompanying loss of the longitudinal muscle primordia in the analysed mutant embryos it is not possible to access the role of the longitudinal muscles in the process of midgut constriction formation (Wolfstetter, 2009).
Morphological analysis of integrin mutant embryos has unveiled the importance of attachment of visceral muscles to the underlying endoderm. Loss of the integrin subunits αPS1 and αPS3 results in delayed endoderm migration whereas the lack of αPS2 leads to irregularities in the visceral mesoderm. More striking results were obtained by analysing double mutants of βν and βPS integrins that nearly phenocopy the visceral defects observed in hkbgurt embryos. Since integrins are cellular receptors for molecules of the extracellular matrix (ECM) the interactions between endoderm and visceral mesoderm that lead to myotube outgrowth and elongation require functional molecules and their receptors. From the visceral muscle side Dystroglycan is a potential candidate for such a receptor because it provides a direct link to the cytoskeletal rearrangements which probably enable myotube outgrowth and elongation (Wolfstetter, 2009).
Prior to the influence of the endoderm on the outgrowth process of visceral myotubes the somatic mesoderm is utilized as an external influence for proper visceral myogenesis. In this case, the ligand Jelly belly (Jeb) is secreted from somatic muscle precursor cells and together with the receptor tyrosine kinase ALK leads to the activation of signalling pathways responsible for visceral founder cell determination. Consequently, mutations in jeb or its cognate receptor ALK exhibit remnants of visceral mesoderm consisting solely of fusion competent myoblasts. These 'default state' visceral myoblasts are able to contribute partially to somatic muscles and expression of other visceral fusion competent myoblast determination genes appears correct. In contrast, the fate of all visceral trunk mesoderm cells can be converted to founder cells by the overexpression of Jeb raising an unanswered question of the pool of FCMs for the migrating longitudinal precursors (Wolfstetter, 2009).
Longitudinal founder cells are derived from the posterior end of the mesoderm and move in front of the posterior midgut primordium. For proper migration both tissues need guidance provided by the visceral bands that arise from the trunk mesoderm. Analyses of crocodile-lacZ and 48Y-Gal4 expression in hkbgurt mutant embryos reveal regular expression patterns indicating that longitudinal muscle migration neither depends on endoderm formation nor a possible template function provided by endodermal cells. Therefore, migration of longitudinal muscle and endodermal cells seems to be completely independent. It has been reported that longitudinal precursor cells migrate as syncytia and therefore display successive fusion with trunk mesoderm cells. Since bagpipe-lacZ labels all visceral trunk mesodermal cells including the FCMs that are proposed to fuse with migrating longitudinal precursors, it is concluded that circular and longitudinal myoblast fusions in huckebeingürtelchen mutant embryos are successful (Wolfstetter, 2009).
hkbgurt mutant embryos display regular visceral cell type determination and bagpipe-lacZ expression fails to identify any unfused visceral myoblasts, suggesting proper visceral myoblast fusion. Some stretching of fused circular muscles was observed even in the absence of endoderm formation. Since these processes take place in a regular spatiotemporal pattern the subsequent outgrowth and therefore the enclosure of the midgut by syncytial myotubes is blocked suggesting a main influence from the adjacent endoderm to serve as a substrate for the process of visceral muscle outgrowth. The earlier step of initial myotube stretching occurs during or directly after myoblast fusion. Stretching of circular muscles also occurs in the absence of myoblast fusion as suggested by previous studies on fusion mutants that display proper endoderm differentiation. If myoblast fusion and endoderm contact can be uncoupled from the initial stretching process this initial stretching seems therefore an inherent capacity of the visceral founder cells itself. This holds true for the analogous stretching process of somatic muscle FCs (Wolfstetter, 2009).
In conclusion, the isolation of hkbgurt in a screen for visceral muscle mutants highlights an important role of the endoderm in the later stages of visceral muscle development. Further experiments should shed light on the mechanisms regulating this process (Wolfstetter, 2009).
Exons - two
Bases in 3' UTR - 518
Huckebein encodes a triple zinc finger protein containing a glutamine rich activation domain and an alanine rich repressor domain (Bronner, 1994).
date revised: 15 August 2023
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