BIOLOGICAL OVERVIEW

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 Huckebein’s 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 tor^D embryos, hkb expression expands towards the center of the embryo and the sna domain correspondingly retracts. By contrast, in gro^mat- 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 tor^D) 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 gro^mat- 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 Hairy^Hkb chimera was constructed by replacing the C terminus of Hairy with Hkb’s 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 Hairy^Hkb 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).

Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm

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 huckebein^görtelchen (hkb^gurt) 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 hkb^gurt 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. hkb^gurt 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 hkb^gurt 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 hkb^gurt 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 hkb^gurt 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 huckebein^gürtelchen mutant embryos are successful (Wolfstetter, 2009).

hkb^gurt 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 hkb^gurt 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).

Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo

Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. This study used optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient 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 were found to produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. It was further shown that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, this study provides a blueprint for using optogenetics to dissect developmental signal interpretation in space and time (Ho, 2023).

This study has dissected the regulation of the byn stripe by combining precise optogenetic inputs in space and time with live biosensors of target gene expression. Using ectopic activation of Ras on the ventral side of wild-type embryos, high- and low-amplitude OptoSOS inputs were defined that induce distinct byn transcriptional dynamics – a pulse of expression in early NC14 versus more sustained expression – that match its endogenous responses in the posterior terminus and stripe-forming region. These conditions were then used to characterize the tll and hkb inputs that explain these byn dynamics in space and time (Ho, 2023).

This approach yielded novel insights about both the temporal and spatial interpretation of ERK inputs to pattern the byn stripe. First, differences in signal amplitude are interpreted through the timing of tll and hkb expression. The onset of tll expression is always rapid, occurring as quickly as 4 min after signaling onset, whereas there is a dose-dependent delay in the onset of hkb expression. This delay in hkb expression is a function of Ras/ERK input amplitude, not of developmental time. These data are consistent with previous observations in OptoSOS embryos that hkb RNA only accumulates to high levels in response to blue light inputs over 30 min. They also broaden the conception of the thresholds for tll and hkb expression: tll and hkb can be induced by inputs of the same amplitude, but hkb requires that the signal persist for a longer time. If the amplitude is low enough, the signal must persist longer than the developmental window allows, and hkb is never expressed. Thus, cumulative dose of ERK input (amplitude integrated over time) appears to be the relevant feature sensed by the circuit, as has been proposed for the terminal pattern as well as other systems. Integration of signal over time has similarly been shown to be important for interpretation of several morphogen pathways including Hedgehog, Wnt, Nodal and BMP. The byn circuit then processes this input through the relative timing of tll and hkb, rather than simply their presence, to determine local byn expression (Ho, 2023).

This more nuanced understanding of byn regulation resolves a conundrum of the endogenous pattern: how can the transient pulse of expression of byn in the high-ERK, Hkb-positive domain be reconciled with the presence of its inhibitor? It is shown in this study that at the high light levels which produce a comparable pulse of byn transcription, hkb transcription is delayed relative to tll and this delay is also evident in the accumulation of their protein products. Thus, there is a temporal window in which only the positive regulator is present, allowing for a pulse of byn expression, before accumulation of the repressor. The sequential appearance of Tll and Hkb was hypothesized during the initial characterizations of posterior patterning but has only now been directly shown. It is interesting to note that Tll has been characterized as a transcriptional repressor, implying that there is an intermediate node between tll and byn. However, the identity of this node and how it affects the timing of byn activation and repression remain unknown (Ho, 2023).

Improved understanding of byn regulation also explains how a byn stripe can form in conditions where tll and hkb transcription have the same spatial domain. The current study revisits these results with improved tools, in particular endogenously tagged transcriptional reporters of tll and hkb that are able to clearly resolve differences in transcriptional dynamics that were obscured by enhancer-based reporter constructs.It was found that stimulus conditions that support sustained byn can also support hkb expression in NC14, but under these conditions hkb expression is largely absent from earlier nuclear cycles. The co-expression of sustained byn with hkb under low light differs from the wild-type pattern, where the byn stripe forms in a region only expressing tll. Presumably the endogenous ERK gradient induces tll expression at even lower activity levels than optogenetic inputs. It is noted that the shortened bursting duration of sustained byn at the ectopic position (~25 min) compared with the endogenous stripe (~45 min) suggests that the late-appearing hkb under low light does ultimately repress byn in late NC14. It is also possible that the network dynamics reported in this study provide robustness to the byn circuit, allowing it to produce different outputs for even a narrow range of input strengths (Ho, 2023).

This study reveals that the tll-hkb-byn circuit can be classified as an incoherent feedforward loop with rapid activation and delayed repression, a circuit with well-characterized pulse-generation and stripe-forming properties. A unique feature of this circuit however is that the delay in hkb expression is dose-dependent, meaning that differences in signal amplitude are converted to differences in hkb dynamics and thus different byn responses (i.e. transient if hkb onset is fast, sustained if hkb onset is slow). Interestingly, similar dose-dependent delays in transcriptional onset were recently shown for Dorsal and BMP signaling targets. What is the mechanism underlying this delay in hkb onset? The dose-dependence of tll and hkb has been a longstanding open question even without the complexity of temporal dynamics. ERK signaling activates transcription of both tll and hkb through relief of the same repressor, Cic, and it is unclear why these genes would require different doses of ERK signaling. The experiments rule out a few possible explanations. Developmental time does not appear to be crucial, given that the delay in hkb transcription is observed regardless of when light is applied and both the tll and hkb loci are known to be accessible early. It is also possible to rule out interactions with other components of the anterior-posterior patterning machinery given that this study was able to produce an ectopic byn stripe rotated 90° from its endogenous counterpart. One intriguing possibility, supported by previous ChIP-seq results, is that Cic leaves the enhancers of hkb more slowly than those of tll. It is also possible that signaling-dependent chromatin changes are involved. These models will be tested in future studies (Ho, 2023).

The second major finding is that the boundary of a uniform OptoSOS input is blurred in space downstream of Ras to produce two domains from a single input – a transient byn domain within the high-ERK illuminated region and a sustained domain in the low-ERK unilluminated region. These non-local effects of a local Ras input are most likely mediated by diffusion of active intracellular components, a well-established contributor to developmental patterning in the syncytial Drosophila embryo. It remains unknown whether the endogenous terminal dpERK gradient is produced from a similar gradient of active Torso receptors, or is due to the combination of a discrete domain of Torso activity at the poles and cytoplasmic diffusion of downstream components. If the latter model is correct, the developmental rescue by an all-or-none OptoSOS input may not be an example of a simple input replacing the function of a complex one, but rather a good approximation of endogenous activation in the terminal system. A number of systems once thought to depend strictly on input concentration have similarly been shown to depend on an unexpectedly simple form of the input (Ho, 2023).

Several limitations of the optogenetic system reveal opportunities for future investigation. These experiments were performed at an ectopic position in the embryo where position-specific gene expression may influence ERK interpretation differently than at the poles. For example, the gap gene knirps has been shown to repress tll in the center of the embryo, and it was observed that the total domain of tll and byn expression was smaller under low light. Because of these positional differences in ERK sensitivity, it is not possible to make absolute comparisons about input and output strengths with the endogenous terminal pattern. In the future it will be interesting to investigate this circuit in embryos lacking other sources of positional information, preventing localized gap gene expression . Also, it is possible that the methods left some transcriptional bursts undetected, and it is not possible to distinguish whether an upper bound of ~75% transcriptionally active nuclei represents true transcriptional heterogeneity or an experimental limit of detection. These limitations could be overcome by future studies using techniques that simultaneously label the target DNA locus and measure transcription in live embryos, or advances in high-quality volumetric imaging and machine-learning approaches. Finally, many questions remain about the precise temporal relationships between ERK activation, gene transcription and protein accumulation. What is the relative influence of tll and hkb transcripts produced by early versus late nuclear cycles, and what is the delay between RNA production and protein accumulation? Combining transcriptional and protein reporters in the same embryo with mathematical models will allow these questions to be addressed (Ho, 2023).

Altogether, this work provides a blueprint for dissecting a developmental circuit with optogenetic tools to reveal new insights about network architecture. This study has manipulated amplitude, duration, timing and spatial pattern of the signal to understand the contributions of each factor to signal interpretation. This framework will be an effective strategy for dissecting other developmental circuits in the future (Ho, 2023).

GENE STRUCTURE

Exons - two

Bases in 3' UTR - 518

PROTEIN STRUCTURE

Amino Acids - 296

Structural Domains

Huckebein encodes a triple zinc finger protein containing a glutamine rich activation domain and an alanine rich repressor domain (Bronner, 1994).

date revised: 1 January 2024

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