bicoid

DEVELOPMENTAL BIOLOGY

Oogenesis and Embryonic expression

Localization of maternally provided RNAs during oogenesis is required for formation of the antero-posterior axis of the Drosophila embryo. This paper describes a subcellular structure in nurse cells and oocytes that may function as an intracellular compartment for assembly and transport of maternal products involved in RNA localization. This structure, termed a "sponge body," consists of ER-like cisternae, embedded in an amorphous electron-dense mass. It lacks a surrounding membrane and is frequently associated with mitochondria. Sponge bodies are not identical to the Golgi complexes. It is suggested that the sponge bodies are homologous to the mitochondrial cloud in Xenopus oocytes, a granulo-fibrillar structure that contains RNAs involved in patterning of the embryo. Exuperantia protein, the earliest factor known to be required for the localization of Bicoid mRNA to the anterior pole of the Drosophila oocyte, is highly enriched in the sponge bodies but not an essential structural component of these. RNA staining indicates that sponge bodies contain RNA. However, neither the intensity of this staining nor the accumulation of Exuperantia in the sponge bodies is dependent on the amount of Bicoid mRNA present in the ovaries. Sponge bodies surround nuage, a possible polar granule precursor. Microtubules and microfilaments are not present in sponge bodies, although transport of the sponge bodies through the cells is implied by their presence in cytoplasmic bridges. It is proposed that the sponge bodies are structures that are involved in localization of mRNAs in Drosophila oocytes by means of the assembly and transport of included molecules or associated structures (Wilsch-Bräuninger, 1997).

Earliest zygotic translation of the Bicoid mRNA is detected immediately after fertilization and egg deposition (stage 1). The anterior-posterior gradient of Bicoid is immediately apparent. The level increases slightly until onset of cellularization and decreases more rapidly during gastrulation [Images]. Traces are still apparent at the end of germ band elongation (Driever, 1988). In embryos from females with a mutation in bicoid, head and thorax are lacking and replaced by a posterior telson (Fröhnhofer, 1986).

Different combinations of gap repressors for common stripes in Anopheles and Drosophila embryos

Embryos lacking both maternal and zygotic hb display a reduction and an anterior shift of ems and btd expression at the blastoderm stage. Thus, it has been proposed that head-specific ems expression at the blastoderm stage requires synergistic activation by bcd and hb. However, no hb consensus site could be detected within the 304 bp enhancer element. It cannot be excluded that hb binding sites exist in the ems enhancer outside this element. However, the results suggest that hb plays a relatively minor role in ems expression control in the head and brain (Hartmann, 2002).

Drosophila segmentation is governed by a well-defined gene regulation network. The evolution of this network was investigated by examining the expression profiles of a complete set of segmentation genes in the early embryos of the mosquito, Anopheles gambiae. There are numerous differences in the expression profiles as compared with Drosophila. The germline determinant Oskar is expressed in both the anterior and posterior poles of Anopheles embryos but is strictly localized within the posterior plasm of Drosophila. The gap genes hunchback and giant display inverted patterns of expression in posterior regions of Anopheles embryos, while tailless exhibits an expanded pattern as compared with Drosophila. These observations suggest that the segmentation network has undergone considerable evolutionary change in the dipterans and that similar patterns of pair-rule gene expression can be obtained with different combinations of gap repressors. The evolution of separate stripe enhancers in the eve loci of different dipterans is discussed (Goltsev, 2004).

Anopheles lacks bicoid and contains a lone Hox3 gene that is more closely related to zen and specifically expressed in the serosa. How is hunchback activated in the presumptive head and thorax in Anopheles? The homeobox gene orthodenticle can substitute for bicoid in Tribolium. However, orthodenticle does not appear to be maternally expressed in Anopheles, but instead, staining is strictly zygotic and restricted to anterior regions, similar to the pattern seen in Drosophila. Sequential patterns of orthodenticle, giant, and hunchback expression are established by differential threshold readouts of the Bicoid gradient in Drosophila. It is possible that an unknown maternal regulatory gradient emanating from the anterior pole is responsible for producing similar patterns of expression in Anopheles. It is proposed that this unknown regulatory factor may be localized to the anterior pole by Oskar. Oskar coordinates the assembly of polar granules and is essential for the localization of Nanos in the posterior plasm. It might also localize one or more unknown determinants in anterior regions of Anopheles embryos (Goltsev, 2004).

The eve stripe 2 enhancer is the most thoroughly characterized enhancer in the segmentation gene network. It can be activated throughout the anterior half of the embryo by Bicoid and Hunchback, but the Giant and Kruppel repressors delimit the pattern and establish the anterior and posterior stripe borders, respectively. Removal of the Giant repressor sites within the stripe 2 enhancer in cis or removal of the repressor in trans causes an anterior expansion of the stripe 2 pattern. However, ectopic expression does not extend to the anterior pole, suggesting that an additional anterior repressor regulates the stripe 2 enhancer. Recent studies identified Sloppy-paired as the likely anterior repressor. The limits of the giant and Kruppel expression patterns seen in Anopheles suggest that they might define the eve stripe 2 borders, just as in Drosophila. However, at the critical time when eve stripe 2 is formed in Anopheles, the giant staining pattern extends to the anterior pole, while the corresponding Drosophila gene is repressed in these regions. It is therefore possible that Giant is sufficient to form the anterior border in Anopheles and that repression by Sloppy-paired represents an innovation in Drosophila (Goltsev, 2004).

Why do some enhancers generate two stripes, while others direct just one? Consider the eve stripe 2 and stripe 3/7 enhancers in Drosophila. The stripe 3/7 enhancer is activated by ubiquitous activators, including dSTAT, and the two stripes are 'carved out' by the localized Hunchback and Knirps repressors. Knirps establishes the posterior border of stripe 3 and anterior border of stripe 7, while Hunchback establishes the anterior border of stripe 3 and posterior border of stripe 7. The stripe 2 enhancer directs just a single stripe due to the localized distribution of the stripe 2 activators, particularly Bicoid. In principle, a ubiquitous activator would cause the stripe 2 enhancer to direct two stripes, stripes 2 and 5. Opposing Giant and Kruppel repressor gradients would carve out the borders of the two stripes, similar to the way in which Hunchback and Knirps regulate the stripe 3/7 and stripe 4/6 enhancers. Presumably, the eve stripe 5 enhancer directs a single stripe of expression because it is regulated by a localized activator, possibly Caudal (Goltsev, 2004).

Stability and nuclear dynamics of the Bicoid morphogen gradient

Patterning in multicellular organisms results from spatial gradients in morphogen concentration, but the dynamics of these gradients remain largely unexplored. This study characterized, through in vivo optical imaging, the development and stability of the Bicoid morphogen gradient in Drosophila embryos that express a Bicoid-eGFP fusion protein. The gradient is established rapidly (1 hr after fertilization), with nuclear Bicoid concentration rising and falling during mitosis. Interphase levels result from a rapid equilibrium between Bicoid uptake and removal. Initial interphase concentration in nuclei in successive cycles is constant (+-10%), demonstrating a form of gradient stability, but it subsequently decays by approximately 30%. Both direct photobleaching measurements and indirect estimates of Bicoid-eGFP diffusion constants (D <=1 microm2/s) provide a consistent picture of Bicoid transport on short (min) time scales but challenge traditional models of long-range gradient formation. A new model is presented emphasizing the possible role of nuclear dynamics in shaping and scaling the gradient (Gregor, 2007a).

The principal results provide the following foundation for any mechanistic model for the formation or read out of the Bcd gradient:

• 1. The gradient is approximately (but not necessarily exactly) an exponential decay in intranuclear (and cytoplasmic) concentration that is established rapidly (less than 90 min).
• 2. Bcd diffuses relatively slowly (D = 0.3 microm²/s) in the cortical cytoplasm containing the nuclei.
• 3. The Bcd gradient is stable over nuclear cycles 10-14, when the number of nuclei is growing by a factor of two with each division and Bcd is concentrated and released from nuclei in a dynamic process. In particular, the initial postinterphase concentration in nuclei in successive cycles is constant to at least 10% but decays by approximately 30% during the subsequent interphase period.
• 4. Bcd is not simply trapped in nuclei; rather, it is in dynamic equilibrium between influx and efflux with the cytoplasm and, possibly, intranuclear degradation.
• 5. The total (bound + free) concentration of Bcd in the cortical cytoplasm is distinctly higher than in the yolk.
• 6. The spatial shape of the Bcd gradient (the length constant for an exponential decay) scales with embryo length over a factor of five range in lengths in different dipteran species.

Earlier work established that molecular motion in the embryo is described well by the diffusion equation on the time (1 hr) and space scales of relevance for morphogenesis. The present work shows that diffusion is an equally good description of Bicoid transport on the scale of minutes and microns. The difficulty is that the relevant diffusion constants differ by more than an order of magnitude. To explain the observation that the Bcd gradient reaches a nuclear steady state very quickly the larger diffusion constants are needed, but the dynamics of transport into and out of the nuclei are consistent with the smaller diffusion constant, which were also measured directly. New experiments will be required to decide which of these is correct (Gregor, 2007a).

The most dramatic qualitative feature that is see in watching the development of the embryos expressing Bcd-GFP is the filling and emptying of the nuclei. Quantitatively, this results in a startling juxtaposition of dynamics and stability. Thus, although Bcd concentrations vary in time over a factor of four during the course of a mitotic cycle, the nuclear concentration near the start of interphase is reproducible with 10% accuracy from cycle to cycle. Although the number of nuclei is changing by a factor of 16 from cycle 10 to cycle 14, the total number of Bcd molecules that are localized in nuclei changes hardly at all. At the present level of understanding, both these examples of stability in the presence of change seem to be the result of cancellation among several independent processes, which is implausible. One way of summarizing the problem is that the simplest model looks like it works, but this is only because many parameters have been adjusted to make it work, leaving the simplest model as an effective description of the dynamics after the mechanisms responsible for this adjustment have done their job. This layer of mechanisms remains to be discovered (Gregor, 2007a).

The Bicoid gradient is shaped independently of nuclei

Morphogen gradients provide embryos with positional information, yet how they form is not understood. Binding of the morphogen to receptors could affect the formation of the morphogen gradient, in particular if the number of morphogen binding sites changes with time. For morphogens that function as transcription factors, the final distribution can be heavily influenced by the number of nuclear binding sites. This study has addressed the role of the increasing number of nuclei during the formation of the Bicoid gradient in embryos of Drosophila melanogaster. Deletion of a short stretch of sequence in Bicoid impairs its nuclear accumulation. This effect is due to a approximately 4-fold decrease in nuclear import rate and a approximately 2-fold reduction in nuclear residence time compared with the wild-type protein. Surprisingly, the shape of the resulting anterior-posterior gradient as well as the centre-surface distribution are indistinguishable from those of the normal gradient. This suggests that nuclei do not shape the Bicoid gradient but instead function solely during its interpretation (Grimm, 2010).

This paper has addressed the role of nuclear import and retention on the formation of the Bicoid gradient by generating a mutant form of Bicoid that does not accumulate within nuclei. Dual-colour imaging of mutant and wt Bicoid within the same embryo were used to reduce the variability due to timing in cross-embryo observations. The principal finding is that nuclei do not affect the anterior-posterior or the centre-to-surface distribution of the protein. A failure to detect an effect makes it unlikely that nuclei represent a major site of Bicoid degradation, or that they play a significant role in shaping or scaling the morphogen gradient. Instead, nuclei appear to be passive sensors of the Bicoid gradient. This is a surprising finding given that the transcription factor Bicoid accumulates in nuclei, the number of which increases by three orders of magnitude in less than ~2 hours from fertilisation to cycle 14. Unfertilised eggs provide an alternative means of testing a role of nuclei in the formation of the Bicoid gradient. Previous observations were, however, inconclusive, possibly reflecting the difficulties associated with staging and imaging the Bicoid gradient in unfertilised eggs (Grimm, 2010).

One interpretation of these results is that the gradient reaches its steady state before cycle 10, at a stage when the number of nuclei is still low (~750 nuclei) and therefore trapping of Bicoid by nuclei would not affect the shape of the gradient. However, the subsequent increase in nuclear density that occurs between cycles 10 and 14 might still have been predicted to alter the distribution of Bicoid if trapping or degradation became more efficient as the number of nuclei increased with each cycle. Such an effect has been observed for the Torso-dependent gradient of activated MAPK, which is activated at the two poles of the egg and sharpens with each round of mitosis in the embryo (Coppey et al., 2008). Whether such a sharpening would also be expected for the Bicoid gradient depends on its mobility and its lifetime and whether its distribution in the final cleavage stages continues to depend on movement from an anterior source. Using previously measured parameters and an assumed lifetime significantly longer than that of activated MAPK, it has been calculated that the shape of the Bicoid gradient would be refractory to the number of nuclei and it is argued that the late distribution of Bicoid protein at cycle 14 reflects local synthesis from a graded mRNA source. Distinguishing these and other models will require more accurate measurements of the relevant parameters, but the exclusion of nuclear import, retention and degradation in shaping the gradient greatly simplifies such an analysis (Grimm, 2010).

Fundamental origins and limits for scaling a maternal morphogen gradient

Tissue expansion and patterning are integral to development; however, it is unknown quantitatively how a mother accumulates molecular resources to invest in the future of instructing robust embryonic patterning. A model, Tissue Expansion-Modulated Maternal Morphogen Scaling [TEM(3)S], was developed to study scaled anterior-posterior patterning in Drosophila embryos. Using both ovaries and embryos, a core quantity was measred of the model, the scaling power of the Bicoid (Bcd) morphogen gradient's amplitude nA. Model-derived predictions about Bcd gradient and patterning properties were also evaluate directly. The results show that scaling of the Bcd gradient in the embryo originates from, and is constrained fundamentally by, a dynamic relationship between maternal tissue expansion and bcd gene copy number expansion in the ovary. This delicate connection between the two transitioning stages of a life cycle, stemming from a finite value of nA~3, underscores a key feature of developmental systems depicted by TEM(3)S (He, 2015).

Mechanisms of gap gene expression canalization in the Drosophila blastoderm

Extensive variation in early gap gene expression in the Drosophila blastoderm is reduced over time because of gap gene cross regulation. This phenomenon is a manifestation of canalization, the ability of an organism to produce a consistent phenotype despite variations in genotype or environment. The canalization of gap gene expression can be understood as arising from the actions of attractors in the gap gene dynamical system. In order to better understand the processes of developmental robustness and canalization in the early Drosophila embryo, this study investigated the dynamical effects of varying spatial profiles of Bicoid protein concentration on the formation of the expression border of the gap gene hunchback. At several positions on the anterior-posterior axis of the embryo, attractors and their basins of attraction were analyzed in a dynamical model describing expression of four gap genes with the Bicoid concentration profile accounted as a given input in the model equations. This model was tested against a family of Bicoid gradients obtained from individual embryos. These gradients were normalized by two independent methods, which are based on distinct biological hypotheses and provide different magnitudes for Bicoid spatial variability. It was shown how the border formation is dictated by the biological initial conditions (the concentration gradient of maternal Hunchback protein) being attracted to specific attracting sets in a local vicinity of the border. Different types of these attracting sets (point attractors or one dimensional attracting manifolds) define several possible mechanisms of border formation. The hunchback border formation is associated with intersection of the spatial gradient of the maternal Hunchback protein and a boundary between the attraction basins of two different point attractors. How the positional variability for hunchback is related to the corresponding variability of the basin boundaries was demonstrated. The observed reduction in variability of the hunchback gene expression can be accounted for by specific geometrical properties of the basin boundaries. The mechanisms of gap gene expression canalization in early Drosophila embryos were clarified. These mechanisms were specified in the case of hunchback in well defined terms of the dynamical system theory (Gursky, 2011).

This study presents the dynamical analysis of the simplified model of the gap gene network on the ensemble of early Drosophila embryos. The main goal was to decode the mechanistic basis of the gap gene border formation and stability under the Bcd morphogen variance. The hb border formation mechanisms were described in terms of attracting sets and their attraction basins calculated in the nuclei surrounding the border position (Gursky, 2011).

The results reveal that the border formation can be associated with the event of intersection between a boundary separating the attraction basins of two di®erent point attractors and the initial Hb profile presenting the input from the maternally expressed hb gene. Attracting sets of another type, the unstable manifolds of saddle equilibria, actively participate in the adjustment of the border position. They do so by attracting the solution trajectories in the nuclei surrounding this position. The model predicts that these attracting manifolds can be involved in the border formation for some Bcd profiles (Gursky, 2011).

The hb border correctly forms in the model by the onset of gastrulation for all individual Bcd profiles. For about a half of these profiles, however, the Kr and Gt patterns in the solutions exhibit defects in the anterior part of the spatial domain (solution classes II and III). It turns out that the hb border formation mechanism involving the attracting manifolds is mostly associated with these cases. This may lead to the conclusion about restricted applicability of this mechanism in the case of hb expression. However, this mechanism exists and plays an important role for the gap domain borders in a posterior part of the embryo, where the domains form and vary in time under the control of an unstable manifold. To analyze canalization for the posterior borders, the variation for external inputs from Cad and Tll should be taken into account, where these transcription factors are among the key regulators, and a modified model should be considered including an input from the terminal gene huckebein (Gursky, 2011).

As previously reported, the model exhibits a significant filtration (canalization) of the Bcd positional variability at the level of hb border formation. The results show how this filtration stems from the stable behavior of the attraction basin boundaries. Has been shown that the mutual regulatory repression between the gap genes accounts for the observed variance reduction, thus presenting a buffering mechanism for canalization. This buffering mechanism was translated to the level of attractors and their attraction basins. As the hb border position is well encoded by the intersection between the initial Hb profile and corresponding attraction basin boundaries, the stability of hb border predicted by the model can be explained by inspecting the geometrical properties of these attraction basins (Gursky, 2011).

From this inspection, the following two mechanisms responsible for the observed robustness can be elucidated. First, the initial Hb profile is a monotonously decreasing function of A/P position, while the basin boundary to be crossed is a monotonously increasing one, i.e., these curves have opposite dependencies on the A/P position. This purely geometrical fact evidently prescribes a smaller variation of the intersection point when the basin boundary is changing due to the variance of Bcd concentration, as opposed to the case if the curves would jointly rise or jointly fall along the A/P axis (Gursky, 2011).

The second mechanism is associated with the specific nonlinear form of the response curve. The gap gene cross regulation of hb bends the response line exhibited in absence of this regulation. This bending e®ectively reduces the Hb positional variance by about half. In terms of attractors, this bending is controlled by the fact that a basin boundary responsible for the hb border formation does not change monotonously, but oscillate in the state space with the changing Bcd profile (Gursky, 2011).

The results show that the full range of the hb positional variance is broken down into two almost equal parts, the anterior and posterior ones. These parts are associated with two families of the Bcd individual profiles (Family I and Family II, respectively) and two di®erent mechanisms of hb border formation. The Bcd profiles from Family I lead to the hb border formation as a switch from a hb/ON attractor in a hb-expressing nucleus to a hb/OFF attractor in a hb-nonexpressing nucleus, while for Family II the border forms with the help of an attracting invariant manifold in a hb-nonexpressing nucleus. Since the difference between the two families is in the amplitude of the Bcd profiles, it is concluded that Bcd profiles of high amplitude canalize by a dynamical mechanism different from those of lower amplitude. Each dynamical mechanism provides only half of the full variance for the hb border, but in two adjacent spatial domains. Therefore, the change of the dynamical mechanism that happens with rising Bcd amplitude e®ectively doubles the variance (Gursky, 2011).

The hb border positions from the more posterior range are placed posterior to the spatial position of a bifurcation annihilating attractor A3. This bifurcation position delimits the anterior and posterior dynamical regimes in the model. Therefore, the Bcd profiles from the second family shift the hb border to the posterior dynamical regime, which is characterized by an active role of an attracting invariant manifold in the pattern formation (Gursky, 2011).

The results indicate that the posterior range of hb positional variation is almost equal to the anterior one only due to smaller variation of the Bcd profiles in Family II compared to Family I. This suggests that the solutions in the anterior and posterior dynamical regimes have quite different sensitivity rates to variation of the Bcd concentration. For Family I, the standard deviation for the hb border position is 2.6 times less than for the Bcd threshold position, while it is only 1.4 times less in the case of Family II. This difference can be explained by an observation that Bcd profiles of higher amplitude correspond to the linear part of the response curve, and this is a consequence of specific regulatory interactions in the gap gene circuit as explained further (Gursky, 2011).

The model was used (1) to study the canalization mechanisms based on the assessment that the model provides one of the best spatio-temporal precision for the description of gap gene expression. This model is an approximation to a more general model of gene regulation, which should be grounded on the statistical-mechanical formalism. One possible limitation is the linear approximation for the argument of the nonlinear regulation function g. The canalization mechanisms described in terms of attractors and attraction basins generally depend on the structure of the model that predicts these attracting states. Therefore, an important direction for future investigations should be verification of the proposed mechanisms in a phase space of a more general model (Gursky, 2011).

The nonlinear nature of the Bcd readout by the gap gene circuit is clearly represented in a specific nonlinear form of the response curve showing the Bcd dependence of the hb border position in the model. The nonlinear part of the curve can be explained by the regulatory actions on hb from the other gap genes. In particular, a regulatory analysis in the full model revealed that the regulatory interactions between hb, gt, and Kr underlie the folding part of the response curve. The gap gene cross-regulation also participate in the linear parts of the response curve by tuning the incline of these parts (Gursky, 2011).

It was previously pointed out that the gt and Kr expression borders in the anterior part of the A/P axis show large variation in the model in response to Bcd variation because the model is missing some regulators in this part. For example, these gt and Kr borders are absent in the solutions from class III. This fact raises doubts on the specific folding part that the response curve exhibits in the middle range of the Bcd concentration values. On the other hand, the folding part exists only for the Bcd profiles associated with the solutions from class I, with all expression borders formed correctly, which means that an essential portion of the artificial variation of the gt and Kr borders can be excluded from the consideration without affecting the folding form of the curve (Gursky, 2011).

The model was investigated on the ensemble of Bcd profiles normalized by the alternative method, which provided lower Bcd variance. One used this method as an artificial limit case, in which the ensemble possessing minimal Bcd variance was dealt with, and it was applied for the crosschecking purposes (Gursky, 2011).

No essential discrepancy was found in the mechanisms of hb border formation and canalization for the two normalization methods. A distinct bifurcation structure in the model with the new parameter values does not lead to changes in the solutions during the biologically important time. The model preserves an attracting invariant manifold related to the posterior dynamical regime. The same border formation mechanisms appear except the one associated with the attractor/manifold transition. It is important that, even though the second family of Bcd profiles does not appear in the alternative normalization case, the invariant manifolds still play their role in adjusting the border position. The model also demonstrates an essentially nonlinear response curve for the hb border. Therefore, the conclusions formulated above are robust with respect to the choice of the normalization method, and, in more general terms, they should be valid for different estimates of the actual Bcd variance (Gursky, 2011).

This correspondence can be explained by the fact that the parameters A and l obtained for the alternatively normalized Bcd profiles form a subset in similar parameters obtained in the case of the basic normalization method. Roughly speaking, the alternatively normalized Bcd profiles can be associated with Family I. In particular, this means that the Bcd data rescaled according to the alternative algorithm support the conclusion formulated above about different dynamical mechanisms of canalization for Bcd profiles of different amplitude (Gursky, 2011).

There is an important issue concerning the comparison of the Bcd variance filtration rates. The calculations reveal that, for the basic normalization method, the Hb positional variation of 1.3%EL in the model output follows from the Bcd positional variation of 4.5%EL, thus implying that more than 70% of the positional variance has been filtrated. The same calculations for the alternative normalization method give the filtration rate of approximately 60%. Therefore, the filtration still happens in the model even if Bcd profiles are normalize according to the precisionist hypothesis. This result is quite expected since the reported dynamical mechanisms underlying the processing of the Bcd variation in the model are valid irrespective of the absolute variation range. Whatever actual variation the Bcd morphogen exhibits, the nonlinear model response translates it to a smaller variation of the target gene patterns (Gursky, 2011).

It is concluded that the formation of hb border is coded by the intersection between the maternal Hb gradient and a boundary between attraction basins in the gap gene dynamical system. Small positional variance for hb border can be explained by the geometrical properties of this basin boundary and its nonmonotonic dependence on the Bcd concentration. Main features of the phase portraits underlying the canalization mechanisms do not depend on the normalization method for Bcd (Gursky, 2011).

Adaptation of the length scale and amplitude of the Bicoid gradient profile to achieve robust patterning in abnormally large Drosophila melanogaster embryos

The formation of patterns that are proportional to the size of the embryo is an intriguing but poorly understood feature of development. Molecular mechanisms controlling such proportionality, or scaling, can be probed through quantitative interrogations of the properties of morphogen gradients that instruct patterning. Recent studies of the Drosophila morphogen gradient Bicoid (Bcd), which is required for anterior-posterior (AP) patterning in the early embryo, have uncovered two distinct ways of scaling. Whereas between-species scaling is achieved by adjusting the exponential shape characteristic of the Bcd gradient profile, namely, its length scale or length constant (lambda), within-species scaling is achieved through adjusting the profile's amplitude, namely, the Bcd concentration at the anterior (B0). This study reports a case in which Drosophila melanogaster embryos exhibit Bcd gradient properties uncharacteristic of their size. The embryos under investigation were from a pair of inbred lines that had been artificially selected for egg size extremes. B0 in the large embryos is uncharacteristically low but lambda is abnormally extended. Although the large embryos have more total bcd mRNA than their smaller counterparts, as expected, its distribution is unusually broad. The large and small embryos develop gene expression patterns exhibiting boundaries that are proportional to their respective lengths. These results suggest that the large-egg inbred line has acquired compensating properties that counteract the extreme length of the embryos to maintain Bcd gradient properties necessary for robust patterning. This study documents a case of within-species Bcd scaling achieved through adjusting the gradient profile's exponential shape characteristic, illustrating at a molecular level how a developmental system can follow distinct operational paths towards the goal of robust and scaled patterning (Cheung, 2014).

Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo

Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. This study used optogenetic manipulation to switch off Bicoid-dependent transcription in the early Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. Bicoid transcriptional activity was found to be dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of Bicoid decoding (Huang, 2017).

Cortical movement of Bicoid in early Drosophila embryos is actin- and microtubule-dependent and disagrees with the SDD diffusion model

The SDD model (synthesis, diffusion, degradation) proposes that the bicoid (bcd) mRNA is located at the anterior pole of the embryo at all times and serves a source for translation of the Bicoid protein, coupled with diffusion and uniform degradation throughout the embryo. The ARTS model (active RNA transport, synthesis) proposes the mRNA is transported at the cortex along microtubules to form a mRNA gradient which serves as template for the production of Bcd, hence little Bcd movement is involved. Bcd was found to move along the cortex and not in a broad front towards the posterior as the SDD model predicted. Embryos were subjected to hypoxia where the mRNA remained strictly located at the tip at all times, while the protein was allowed to move freely, thus conforming to an ideal experimental setup to test the SDD model. Unexpectedly, Bcd still moved along the cortex. Moreover, cortical Bcd movement was sparse, even under longer hypoxic conditions. Hypoxic embryos treated with drugs compromising microtubule and actin function affected Bcd cortical movement and stability. Vinblastine treatment allowed the simulation of an ideal SDD model whereby the protein moved throughout the embryo in a broad front. In unfertilized embryos, the Bcd protein followed the mRNA which itself was transported into the interior of the embryo utilizing a hitherto undiscovered microtubular network. These data suggest that the Bcd gradient formation is probably more complex than previously anticipated (Cai, 2017).

Effects of Mutation or Deletion

Embryos from mothers with either fewer or greater than the normal two copies of bicoid show initial alterations in the expression of the gap, segmentation and segment polarity genes, as well as changes in early morphological markers. In the absence of a fate map repair system, one would predict that these initial changes would result in drastic changes in the shape and size of larval and adult structures. However, these embryos develop into relatively normal larvae and adults. This indicates a certain plasticity in Drosophila embryonic development along the anterior-posterior axis. Embryos laid by mothers with six copies of bcd have reduced viability, indicating a threshold for repairing anterior-posterior mispatterning. The cephalic furrow (CF) is displaced posteriorly in embryos generated for 6bcd females. Embryos that form the CF more posterior to the mean die at almost twice the frequence of the population of embryos that form the CF anterior to the mean position. Embryos of 4bcd animals either have one missing or a pair of fused denticle belts. Cell death plays a major role in correcting expanded regions of the fate map. There is a concomitant decrease of cell death in compressed regions of the fate map. Compression of the fate map does not appear to be repaired by the induction of new cell divisions. In addition, some tissues are more sensitive to fate map compression than others. 6bcd embryos display increased levels of cell death in the expanded presumptive head region, while 1bcd embryos have increased cell death in the abdominal segments (Namba, 1997).

I>bicoid-independent formation of thoracic segments in Drosophila

The maternal determinant Bicoid (Bcd) represents the paradigm of a morphogen that provides positional information for pattern formation. However, since bicoid seems to be a recently acquired gene in flies, the question has been raised as to how embryonic patterning is achieved in organisms with more ancestral modes of development. Because the phylogenetically conserved Hunchback (Hb) protein acts as a morphogen in abdominal patterning, it was asked which functions of Bcd could be performed by Hb. By reestablishing a proposed ancient regulatory circuitry in which maternal Hb controls zygotic hunchback expression, it has been shown that Hb is able to form thoracic segments in the absence of Bcd (Wimmer, 2000).

A functional hb transgene has been generated that is missing all P2 promoter sequences and relies solely on the P1 promoter (hbP1only). hbP1only constructs do not respond to bcd and do not mediate gene expression in the anterior cap domain. Therefore, hbP1only uncouples the direct link between the Bcd and Hb morphogen systems. Zygotic hb mutants derived from heterozygous parents do not develop labial or thoracic structures, and they also show a fusion of abdominal segments A7 and A8. When one copy of the hbP1only transgene is provided zygotically (by the father) to a hb mutant embryo, it rescues the posterior phenotype, and A7 and A8 developed normally. The labial/thoracic phenotype is not rescued. However, when hbP1only is provided as one copy by the mother to a hb mutant embryo, the posterior and part of the anterior phenotype are rescued. These embryos exhibit normal labial and prothoracic (T1) segments, and only lack meso- and meta-thoracic segments (T2 and T3). The anterior rescue is due to the maternal contribution of hbP1only because sibling embryos that do not inherit the hbP1only construct zygotically also exhibit the partial anterior (but not the posterior) rescue. This indicates that restoring high levels of maternal hb expression (i.e., two copies: one wild type plus one copy of hbP1only) is sufficient to rescue the labial and prothoracic segments in the zygotic hb mutant progeny. Therefore, the lack of zygotic hb leads only to the loss of T2 and T3 and to the fusion of A7 and A8, whereas the previously reported zygotic hb phenotype represents a combination of a haploinsufficient maternal plus a zygotic phenotype (Wimmer, 2000).

The loss of zygotic hb activity affects regions of the embryo that correspond to the two late stripes of zygotic hb expression: The A7-A8 fusion corresponds to the posterior stripe, whereas the loss of T2 and T3 corresponds to the PS4 stripe, which starts as a fairly wide domain covering the anlagen of T2 and T3. This correlation between the zygotic hb phenotype and the late stripe expression pattern led to a reconsideration of the importance of the early bcd-dependent anterior cap domain. Under some conditions, hbP1only (maternal hb contribution plus stripe expression) might suffice for normal segmentation of head and thorax, making superfluous the bcd-dependent anterior cap domain. Hence, the hb PS4 stripe is activated without bcd-dependent hb expression. This stripe is repressed by the knirps abdominal gap-gene product and is activated by high levels of Hb itself, either directly or indirectly (through repression of kni). Embryos that lack the bcd-dependent hb cap domain have been generated that contain an increased maternal hb contribution (to four copies) and kni reduced to one copy. These embryos display a range of partially rescued hb phenotypes, including some embryos with a full set of head and thoracic segments. Thus, bcd-dependent hb expression can in principle be dispensable for embryonic segmentation, and the only critical anterior domain of zygotic hb expression appears to be the PS4 stripe, with the bcd-dependent cap domain serving to activate this stripe. This role is likely achieved by the maternal hb contribution in species where zygotic hb is not under the control of bcd or where a bcd homolog might not exist (Wimmer, 2000).

The rescue of T2 and T3 structures by bcd-independent hb expression raises the question of whether these structures could develop in a completely bcd-independent manner. Embryos derived from bcd mutant mothers develop ectopic tail structures that replace head and thorax and exhibit a disruption of some abdominal segments. Although previous work has shown that, in the absence of bcd, high levels of maternal hb can rescue a normal abdomen and some thoracic structures, no complete thoracic segments can be induced. A bcd-independent source for high levels of zygotic hb expression was introduced into a bcd mutant background. By establishing this artificial zygotic Hb gradient, two notable results were obtained, with variable expressivity: (1) about 20% of the embryos exhibit rescued T2 and T3 segments. The maintenance of high Hb levels that lead to the rescue of thoracic segments is likely due to the activation of the hb stripe element because the hbP1AB reporter is activated as a stripe where T2 and T3 form. (2) Most of the ectopic tail structures that are anteriorly duplicated in bcd mutants are suppressed, suggesting further redundancy between Hb and Bcd. However, Hb and Bcd must act at different levels in suppressing these tail structures, which depend on the activity of the caudal (cad) gene: Bcd acts by repressing cad mRNA translation, whereas Hb does not but might instead interfere with Cad protein function. This bcd-independent suppression of cad function might be important in organisms where the Cad gradient only forms late and represents another variation as to how cad activity is suppressed at the anterior of the embryo (Wimmer, 2000).

Different species use various strategies for repression of Cad function: In Drosophila, translational repression of CAD mRNA involves the Bcd homeoprotein, whereas in Caenorhabditis elegans repression involves the KH-domain protein MEX-3. In vertebrates, a mutually antagonistic relation between otd-like and cad-like genes has been proposed to reflect an ancestral system to pattern the anteroposterior axis of the embryo. In arthropods, ancestral head determinants are probably encoded by otd-like genes as well. Thus, in the beetle Tribolium, where no bcd homologs but Bcd-like activities have been found, these activities are probably also covered by Otd or KH-domain proteins. This is consistent with the Otd-like DNA binding specificity of Bcd, which is atypical for a factor encoded by a gene duplication in the Hox cluster. This change in specificity was probably crucial for Bcd to acquire its key role in anterior patterning, because it allowed Bcd to function both as an RNA binding protein and as an Otd-like transcription factor. In this respect, it is not surprising that the zinc-finger protein Hb cannot completely replace Bcd in the head region. Even the highest levels of Hb obtained in these experiments were not able to induce head formation in the absence of Bcd. However, Hb is required for the posterior head region (maxillary and labial segment) and supports anterior head development by synergizing with Bcd. It will be interesting to see whether such a synergism can also take place between Hb and other more ancestral head determinants (Wimmer, 2000).

These results indicate that the two morphogenetic systems, Bcd and Hb, do not need to be directly linked. Hence, the direct regulation of hb by Bcd might represent a recent evolutionary addition to the insect body plan. In Drosophila, the abundance of bcd-dependent hb expression eventually renders superfluous the maternal hb contribution, which is widespread within arthropods. Consistent with the idea that the bcd-dependent hb expression represents a recent evolutionary acquisition, the P2 promoter contains only activator sites that allow the direct response to a specific threshold level of a morphogen. This might be a unique situation, given that most other developmentally regulated promoters contain, in addition to activator sites, repressor elements for setting the exact borders of gene expression. By tinkering with the rather plastic mechanisms of early development, the ontogeny of Drosophila could be changed toward an inferred ancestral state where maternal Hb controls zygotic hb. This change could be brought about by altering patterns and levels of gene expression; this presents the most likely variation on which evolutionary processes are based (Wimmer, 2000).

Two new roles for the Drosophila AP patterning system in early morphogenesis

Although gastrulation is regarded as the stage during Drosophila development when the AP patterning system first influences morphological processes, transcription is already regulated in complex patterns by cycle 10. How soon this transcriptional complexity produces spatial differences in morphology has been unclear. Two new processes are described that establish visible morphological inhomogeneities before the onset of gastrulation. The first of these is the regulation of syncytial nuclear densities in the anterior end of the egg and represents the first zygotically driven AP asymmetry in the embryo. The second process is the generation of a fine-scale pattern in the actin/myosin array during cellularization. Three domains of different yolk stalk diameters as well as depths of cellularization along the AP axis have been found. These domains are established under the control of the AP patterning system and require bicoid activity. The anterior-most domain is a region of large yolk stalk diameters and corresponds to the region of decreased nuclear densities observed during syncytial stages. The middle domain shows smaller yolk stalk diameters and more rapid cellularization. Its establishment requires wild-type paired activity and thus indirectly requires bicoid. It occurs in a region of the embryo that ultimately gives rise to the cephalic furrow and may account for the effect of paired on that structure during gastrulation. These results therefore suggest a link between cytoskeletal organization during cellularization and subsequent morphogenetic processes of gastrulation (Blankenship, 2001).

To assay the degree of uniformity along the AP axis during cellularization, the furrow canals of wild-type embryos were visualized by staining for Myosin. By mid-cellularization, three domains of differing diameters of yolk stalks could be observed. The first domain is centered around the anterior pole of the embryo, and possesses the largest diameters. The second domain, immediately posterior to the first anterior domain, has the smallest diameters. This domain is centered around the location where the cephalic furrow will eventually form. The third domain lies posterior to this pre-CF domain and has furrow canals of intermediate diameter (Blankenship, 2001).

These three domains also differ in the depth of the cellularization front. The anterior domain, which has large yolk stalk diameters, is the shallowest part of the embryo in its depth of cellularization. By the end of cellularization, the most anterior part of this domain can possess a depth of cellularization (15-20 µm) half that of the rest of the embryo (~35 µm on average). The region posterior to the pre-CF domain has an intermediate depth, whereas the pre-CF domain, which has the smallest yolk stalk diameters, has the greatest depth of cellularization. The regions of differing yolk stalk diameters correspond to the regions of varying depth during cellularization (Blankenship, 2001).

To test whether these cellularization phenomena are due simply to the position or geometry of the embryo (e.g. the curvature of the anterior part of the egg), or if these phenomena are being specified and positioned by the AP patterning system, the three domains were sought in embryos that lacked all positional information along this axis. In embryos derived from females triply mutant for bicoid nanos torso-like, yolk stalk diameters are uniform, even though other aspects of cellularization occur normally. The shallow cellularization front of the anterior domain, as well as the greater depth of the pre-CF, is also lost. Thus, these phenomena require the activity of the AP patterning system for their morphogenesis (Blankenship, 2001).

These cellularization phenomena respond to specific levels of the bicoid gradient. Embryos that carry six copies of bicoid produce more Bicoid protein, shifting any given concentration of Bicoid to a position posterior to where it would be in a wild-type embryo. In embryos carrying six copies of bicoid, the pre-CF domain of small yolk stalk diameters and greater depth is shifted posteriorly, while the anterior domain of large yolk stalk diameters expands to cover close to half of the embryo. bicoid appears to be the important factor specifying these phenomena, since embryos derived from females mutant for bicoid lack the anterior and pre-CF domains (Blankenship, 2001).

Since the pre-CF domain is a relatively narrow domain, it seemed unlikely that the broad Bicoid gradient would be directly specifying the pre-CF domain. Several pair-rule genes are expressed in approximately the right time and place to be mediating the formation of the pre-CF domain. While Even-skipped (Eve) is expressed in the posterior half of the pre-CF domain, Prd expression is directly centered on the pre-CF domain. Prd expression also has an unusual feature. When Prd expression is first detected during cycle 13, it is observed in a single, gap gene-like domain. Thus, Prd is expressed before the pre-CF domain is formed, and in the right location in the embryo. Moreover, the early expression of Prd in a single stripe, rather than in the stereotypical seven-stripe pattern, suggests that it could specify the pre-CF domain independent of other factors (Blankenship, 2001).

Various patterning mutants were examined for a disruption of the pre-CF domain. prd homozygotes show a normal anterior domain of larger yolks, but there is very little difference in the yolk stalk diameters between the pre-CF domain and the posterior domain. The relatively subtle appearance of the pre-CF domain in different genetic backgrounds required the scoring of embryos in a blind test. On a classification scale of 0-5, with 0 indicating a complete absence of the pre-CF domain, prd homozygotes scored a 1.2, while their non-homozygous siblings scored a 3.3. These classifications are significant (P <0.001, n=61). Thus, prd is already active in the regulation of morphogenesis during the process of cellularization (Blankenship, 2001).

The pre-CF domain is first visible when the cellularization front has reached about 25% of its depth. However, the larger yolk stalks of the anterior domain are visible throughout cellularization. The density of nuclei in the anterior is reduced by about 30% from the nuclear density that is found in the rest of the embryo. This anterior domain of lower nuclear density is centered around the anterior pole and extends to approximately two to three nuclei in front of the first stripe of Eve, or ~70% EL. Judging from the staining patterns of Eve and Prd, this is the same area of the embryo in which the anterior domain of large yolk stalk diameters meets the pre-CF domain. During cellularization the anterior nuclear domain is consistently observed in OreR embryos, with an average decrease in nuclear density of 27.6%. In this sample, the values for individual embryos ranged from 34% to 20%. This domain of lower nuclear densities is maintained and stays constant throughout the process of cellularization, and can still be observed in gastrulating embryos. This greater spacing of nuclei in the anterior would necessarily lead to a cellularization network with larger diameters. Additionally, by mid-cellularization, when a pre-CF domain is already visible, a slight clustering of nuclei in this region occurs (Blankenship, 2001).

Since the anterior domain of lower nuclear densities is present at the start of cycle 14, it was asked when this AP asymmetry arises. While both the sphere of nuclei that migrate to the surface of the embryo during cycle 9, and the cortical nuclei of cycle 10 are uniform along the AP axis, the first sign of increased nuclear spacing in the anterior can be observed in cycle 11 embryos. By cycle 12, a pronounced AP asymmetry is observed, which is maintained through the subsequent mitosis to the start of cellularization. The anterior domain of lower nuclear densities is also observable in living embryos. Nuclear counts on GFP-histone embryos demonstrate a similar ~30% reduction in nuclear density in vivo. These observations on living embryos also reveal that, except for when the nuclei are undergoing mitosis and the associated yolk contractions, the anterior nuclear domain is present throughout the rest of the cell cycle (Blankenship, 2001).

Before cycle 14, cortical nuclei are associated with large actin caps. In embryos without actin caps, the regular spacing of nuclei is disrupted. One possible mechanism for the formation of the anterior domain of lower nuclear densities would use the regulation of the size of these actin caps. To see if asymmetries in actin cap size occur along the AP axis, wild-type embryos were stained with fluorescently labeled phalloidin to visualize F-actin. Measurements show that anterior actin caps are larger than caps in the rest of the embryo in cycle 11 and cycle 12, but not during cycle 10. This difference, although small, is highly reproducible from embryo to embryo (Blankenship, 2001).

The exceptionally early appearance of the AP asymmetries in nuclear distributions led to an examination of whether the anterior domain of lower nuclear densities is specified by the AP patterning system. In embryos derived from bicoid nanos torso-like mutant females, nuclear spacing is uniform along the AP axis. In addition, the anterior nuclear domain can be expanded posteriorly in embryos carrying six copies of bicoid. Additionally, embryos from bicoid females are uniform in their nuclear densities, arguing that the anterior domain of lower nuclear densities is set up by the Bicoid gradient. Finally, the larger diameters of the actin caps in the anterior do not occur in embryos from bicoid nanos torso-like females (Blankenship, 2001).

Since zygotic transcription only starts around cycle 10, the early appearance of the anterior domain by cycle 11 raised the question of whether zygotic transcription is required for this domain's formation. It is possible that bicoid might be regulating nuclear spacing through a post-transcriptional mechanism. To assay the effect of transcription on nuclear spacing, embryos were injected with alpha-amanitin to block RNA polymerase II. Consistent with earlier studies, such embryos develop to cycle 14 with normal gross morphology. However, when these embryos were examined with nuclear stains, they showed a total absence of asymmetric nuclear distributions. Embryos injected with alpha-amanitin fail to form the anterior domain of lower nuclear densities. It is concluded that bicoid regulates the zygotic transcription to bring about the formation of the anterior domain of lower nuclear densities (Blankenship, 2001).

Two genes necessary for cephalic furrow formation have been identified. Embryos mutant for either eve or buttonhead (btd) lack cephalic furrows. Both eve and btd have a defect in the earliest phase of CF formation. At no point during development are the stereotypical cell shape changes of shortening along the apical-basal axis and widening of the cell observed in these mutants. Mutations in prd also cause a disruption in CF formation. At the onset of gastrulation, the cephalic furrow does not form, nor do initiator cells undergo their characteristic cell shape change. Because there is an absolute correlation between initiator cell behavior and the middle nucleus of the first stripe of Eve in wild-type embryos, the location where initiator cells should form in prd embryos can still be identified. In prd embryos these Eve-marked cells are indistinguishable from their neighbors at stages during gastrulation when the ventral furrow has formed and the CF would normally be visible in wild-type embryos. However, by mid-germband extension (GBE), prd embryos have formed a regular, CF-like invagination. This late fold is thought to arises through the same mechanisms that govern normal CF formation, but these processes are delayed relative to the development of wild-type embryos (in the stage 7 prd embryo there are no cell shape changes in the region where the CF would form, while the stage 6 wild-type embryo has an obvious CF). Initiator cells form in wild-type embryos at stage 6, at the onset of gastrulation. Imaging of prd embryos reveals initiator cell activities beginning at stage 7. At this stage, wild-type embryos have already begun to deform the yolk sack with a basal bulge of the epithelium. At the beginning of stage 8, or germband extension, wild-type embryos have a furrow that is many cells deep, while prd embryos have just begun to deform the yolk sack. It is only by mid-GBE that most, but not all, prd embryos have a regular CF stretching around the entire circumference of the embryo (Blankenship, 2001).

The abnormalities in CF formation observed at the beginning of gastrulation in prd embryos are superficially similar to those observed in embryos mutant for eve or btd. In the absence of the activity of eve, btd or prd, the cell shape changes that occur in the row of cells that initiate CF formation at the beginning of gastrulation do not occur. In contrast to eve or btd, however, early activities of prd during cellularization have been identified that may account for the later differences observed in CF formation. Consistent with this view the analysis indicates that prd embryos often recover and form a fairly regular CF by mid-germband extension, unlike the severe disruption of the CF in eve and btd embryos. While the start of gastrulation occurs normally in prd embryos, CF formation and initiator cell behavior is delayed. It is proposed that this delay is due not to a function of prd during gastrulation in the specification of initiator cells, as has been proposed proposed for eve and btd function. These latter mutants completely block initiator cell and CF formation. Instead, prd function is necessary for the formation of the pre-CF domain, and it is suggested that prd functions in CF formation only through this disruption of the pre-CF domain. The recovery of the CF observed in prd embryos may be a reflection that cellularization throughout the embryo has finally reached the point that the pre-CF domain reaches at the very beginning of gastrulation, and so CF formation, although delayed, may be correctly initiated. The advanced rate of cellularization in the pre-CF domain may reflect a required premature closing of the base of the initiator cells so that cell volumes may be maintained during the severe cell shape changes of gastrulation, or that cytoskeletal components involved in cellularization must be freed for initiator cell movements. Because little deformation, or loss of volume, occurs in the presumptive initiator cells of early prd embryos, the latter of these two possibilities is favored. Thus, the subtle regulation of one stage of development can have profound effects upon a later, seemingly discrete, process of development (Blankenship, 2001).

The cellularization front that arises during early cycle 14 is rich in actin and myosin, and is thought to provide a contractile force that orients and drives the process of cellularization. This network, as well as cellularization itself, proceeds through a two-phase process. The first phase is a basal movement of the cellularization front towards the interior of the embryo. During this phase, the furrow canals stay constant in their small size, and the actin/myosin array has a hexagonal shape. The second phase is a lateral movement of the cellularization front, which creates a pinching off at the bases of cells in the newly forming cell sheet. The morphology of the cellularization front is not uniform along the AP axis. The pre-CF domain is distinguished from other regions of the embryo by its small yolk stalk diameters and greater depth of cellularization (Blankenship, 2001).

These results suggest the following model for the generation of AP asymmetries during cellularization. In the pre-CF domain, the Bicoid gradient directs the correct localization of the early gap gene-like expression domain of prd, which, in turn, directs a greater local contraction of the cellularization network. When cellularization is in its first phase of inward directed movement, the greater contraction of the pre-CF domain leads to a greater advance inwards of the cellularization front, thus creating the greater depth of the pre-CF domain. Then, when cellularization shifts to the second phase of a lateral, pinching-off movement, the greater contractility of the pre-CF domain leads to a greater widening of the furrow canals, which creates the smaller yolk stalk diameters observed in the pre-CF domain. The creation of this domain of advanced cellularization may be necessary for the initiator cell shape change required for cephalic furrow formation (Blankenship, 2001).

In certain respects, the anterior domain of large yolk stalk diameters and shallow cellularization appears to be the opposite of the pre-CF domain, and thus might be produced by a downregulation of the same contractile mechanisms that are proposed to operate in the pre-CF domain. However, the formation of the larger yolk stalk diameters in the anterior domain clearly involves a different mechanism. The anterior cellularization domain is pre-figured by an anterior domain of lower nuclear densities, while the pre-CF domain initiates in a region where nuclear densities are uniform. The greater spacing of nuclei in the anterior necessarily causes the formation of actin/myosin arrays of greater diameter during cellularization. A model for the formation of the anterior domain is favored in which the bicoid gradient, by cycle 11, has regulated the transcription of a set of zygotic genes, which in turn regulate the size of the actin caps overlaying the nuclei. This regulation of the actin caps results in larger caps in the anterior that necessitates a greater spacing of nuclei. By cycle 14, when cellularization is initiated, the greater spacing of nuclei dictates the generation of a cellularization network in the anterior in which the hexagonal components are larger in diameter. The initiation of large hexagons would thus lead to larger yolk stalks. It may be that the larger hexagons of the cellularization network contract less efficiently, thus generating the shallower depth of cellularization that is observed for the anterior domain (Blankenship, 2001).

The concept of a mid-blastula transition (MBT) has generally referred to a time when the genome of an embryo begins to exert an influence on development, presumably through zygotic transcription. The best-defined MBT is for Xenopus, where the MBT was characterized by a cessation of synchronous mitotic cycles, the start of zygotic transcription, and a change in the morphology of blastula cells (i.e., the acquisition of cell motility). Various aspects of this characterization have since been called into question. There is low level zygotic transcription before the MBT, and it appears as though cell motility may not be a function of zygotic transcription, but of slower mitosis. Although these discrepancies call into question the usefulness of the MBT as a concept, the idea of an MBT remains an attractive model for explaining the changing morphology of the Drosophila embryo (Blankenship, 2001).

In flies, the MBT has traditionally been discussed in terms of cycle 14 development. It is at this point that the fly embryo ceases its synchronous syncytial divisions and several morphogenetic processes require zygotic transcription for their genesis. At a superficial level, the first observable defects in embryos deficient for zygotic transcription is at cycle 14, when alpha-amanitin-injected embryos show defects in the processes of lipid droplet clearing and cellularization. However, as in Xenopus, defining a precise MBT presents some difficulties. Although cycle 14 marks a major increase in transcriptional efficiency, the start of zygotic transcription occurs in different nuclear cycles for different genes. In general, for the early acting genes involved in patterning, transcription begins around cycle 10, although some genes are transcribed as early as cycle 8, and mutations in specific segmentation genes show subtle disruption as early as cycle 10. What is striking in the results reported for Bicoid and Prd is the correspondence between spatial patterns of expression and regional alterations in morphology. These results show that the genome of the embryo is active in the spatial regulation of morphogenesis at a much earlier time than previously described. The genome directs reproducible asymmetries in morphology by cycle 11. The generation of an anterior domain of lower nuclear densities argues against a single discrete MBT at cycle 14. These results further suggest a more refined view of the Drosophila MBT in which there is a gradual shifting in the guidance of development from maternal to zygotic gene products, one that stretches from as early as cycle 8 until the cessation of mitosis and formation of a cellularized embryo at cycle 14 (Blankenship, 2001).

Maternal expression relaxes constraint on innovation of the anterior determinant

The origin of evolutionary novelty is believed to involve both positive selection and relaxed developmental constraint. In flies, the redesign of anterior patterning during embryogenesis is a major developmental innovation and the rapidly evolving Hox gene bicoid plays a critical role. This study reports evidence for relaxation of selective constraint acting on bicoid as a result of its maternal pattern of gene expression. Evolutionary theory predicts 2-fold greater sequence diversity for maternal effect genes than for zygotically expressed genes, because natural selection is only half as effective acting on autosomal genes expressed in one sex as it is on genes expressed in both sexes. Single individuals have been sampled from ten populations of Drosophila melanogaster and nine populations of D. simulans for polymorphism in the tandem gene duplicates bcd, which is maternally expressed, and zerknüllt, which is zygotically expressed. In both species, the ratio of bcd to zen nucleotide diversity was found to be two or more in the coding regions but one in the noncoding regions, providing the first quantitative support for the theoretical prediction of relaxed selective constraint on maternal-effect genes resulting from sex-limited expression. These results suggest that the accelerated rate of evolution observed for bcd is owing, at least partly, to variation generated by relaxed selective constraint (Barker, 2005).

Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila

Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is a particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. A subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. Evidence is provided that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation (Werz, 2005).

Although this study largely focus on the maternal effect mutants bicoid and oskar, it is likely that the principles uncovered are of broader significance. Segmentation mutants acting downstream of bicoid and oskar, including mutants of gap genes (Krüppel, knirps), pair-rule genes (odd, fushi-tarazu) and segment polarity genes (wg, hedgehog, engrailed) induce expression of hid. These mutants are characterized by loss of larval tissue. As in the case of bicoid and oskar, hid expression is upregulated during stage 9 of embryogenesis in the regions of the mutant embryos that are later deleted in the larvae. In addition, hid mutants rescue the cuticle phenotype of armadillo mutants. Finally, hid expression accompanied by TUNEL-positive cell death was found in dorsal and Toll^10b mutants, which cause dorsalizing and ventralizing phenotypes, respectively, along the dorsoventral axis of Drosophila embryos. Thus, these data support the notion that upregulation of hid appears to be a common trigger for a caspase-dependent cell death program in mis-specified cells of patterning mutants (Werz, 2005).

Furthermore, mutations affecting imaginal disc development result in loss of the adult appendage due to inappropriate cell death. It is currently being determined whether these mutants also require hid expression to develop the final phenotypes. Moreover, many gene disruptions in mice result in inappropriate cell death in the tissue that requires the function of the disrupted gene, suggesting that similar mechanisms might exist in mammalian development. Finally, cell death may be an important contributing factor to human congenital birth defects. Thus, an understanding of the underlying mechanisms is of general interest (Werz, 2005).

Interestingly, not all segment polarity mutants analyzed induce hid expression and cell death. Embryos mutant for patched, which encodes the hedgehog receptor, were not found to express hid and do not exhibit increased cell death, although hedgehog mutants both upregulate hid and contain increased amounts of cell death. The reasons for these differences are not known, but partial redundancy might account for lack of hid expression in patched mutants. The Drosophila genome encodes another patched homolog, patched-related, which might provide the survival requirement for mis-specified cells in patched mutants (Werz, 2005).

Mis-specified cells in bicoid and oskar mutants induce expression of hid. No increased reaper or grim expression was observed in these mutants. However, expression of reaper has been reported in crumbs mutants, which affect epithelial integrity. X-ray-treated embryos also preferentially respond by upregulation of reaper, rather than hid. Although crumbs mutants were not analyzed for hid expression, it appears that cells contain several developmental checkpoints, which activate different cell death-inducing regulators depending on the type of abnormal cellular development (Werz, 2005 and references therein).

Mis-specified cells can survive if an alternative survival pathway is provided. The example presented here is the acron into telson transformation in bicoid mutants, which is mediated by the torso signaling pathway. Although the cells giving rise to telson structures at the anterior tip are mis-specified based on Abd-B-labeling experiments, they survive because they receive a survival signal from the torso signaling system. In this case, transformation rather than cell death is favored. It has been shown that activation of the Ras/Mapk pathway protects cells from hid-induced apoptosis, both by transcriptional repression of hid and by phosphorylation of Hid protein by Mapk. Because Torso, which encodes a receptor tyrosine kinase (RTK), is known to activate Ras and Mapk, tests were performed to see whether manipulation of active Mapk levels using a gain-of-function allele, Mapk^Sem, can suppress hid expression and cell death in bicoid mutants. However, this was found not to be the case. Thus, torso appears to protect mis-specified cells independently of Mapk activation (Werz, 2005).

The hid bicoid double mutant analysis reveals that the transformation of anterior into posterior identity expands beyond the telson, and that this expansion undergoes hid-induced cell death in bicoid single mutants. The rescued cells secrete larval cuticle elements, suggesting that mis-specified cells have the developmental capacity to terminally differentiate. However, in hid⁺ background, they instead die, presumably because equivalent survival signals are lacking. It is proposed that mis-specified cells undergo cell death if no alternative survival pathway is provided to protect them (Werz, 2005).

An alternative survival mechanism might also operate in other developmental mutants where transformation rather than cell death occurs. Mutations in the sev RTK and its ligand boss result in transformation of the R7 photoreceptor cell into a non-neuronal cone cell. Survival of this cell could be mediated by the Drosophila Egf receptor (Egfr), another RTK, which is required to maintain cell survival in the developing eye disc. Accordingly, activation of the Ras/Mapk pathway by Egfr would inhibit hid expression and support survival of the presumptive R7 photoreceptor cell. This interpretation is also consistent with observations that egfr^- clones are small and undergo cell death, and that this death can be suppressed in hid mutants. Thus, transformation of the R7 photoreceptor to a cone cell rather than R7 cell death in sev and boss mutants could occur because of survival signaling by the Egfr (Werz, 2005).

The hid bicoid double mutant analysis suggests that mis-specified cells can continue to develop and differentiate. Yet, they die. Presumably, this cell death protects the organism from potentially dangerous cells. For example, it is conceivable that in mammals, surviving mis-specified cells might lie dormant in the host organism for years. During this time, they might acquire additional genetic alterations that could drive the progressive transformation of these cells into malignant cancer. In wild-type embryos, mis-specification probably occurs in cells in isolation, and elimination of these cells does not interfere with development and survival of the organism. Only in extreme situations, such as the patterning mutants analyzed in this study, is the mis-specification caused by aberrant development so severe that the affected organism dies (Werz, 2005).

The cause of mis-specification in each segmentation mutant is different. Usually, the expression of other segmentation genes is shifted and expanded, resulting in flattened gradients. Yet, irrespective of the cause of mis-specification, most of these mutants have in common that they induce hid expression. It is currently unknown how the mis-specified fate of cells is recognized, and how hid expression is induced. One possibility might be that the protein gradients established by bicoid⁺ and oskar⁺, as well as other segmentation genes are used as readout for proper cellular specification. The steepness of protein gradients as a means to determine life or death decisions has recently been proposed. Such a model would imply that cells are able to determine their position in a graded field and compare this readout with their neighbors. Because in bicoid and oskar mutants these gradients do not form, the concentration difference between neighboring cells would be zero. If the concentration difference between two neighboring cells is below a crucial threshold, they induce the expression of hid and undergo cell death. This model could also explain embryonic pattern repair, which was described in embryos that express six copies of the bicoid gene. In these embryos, the head and thorax primordia are expanded because of the presence of six copies of bicoid. However, this expansion is corrected for by induction of cell death, and relatively normal larvae develop. In this case, the Bicoid protein gradient does form, but would be flatter compared with wild type. Thus, the concentration difference between neighboring cells would be below a critical threshold, sufficient to induce hid-dependent cell death. However, it is largely unknown how cells compare their position in a graded field with those of their neighbors. It has been proposed that short-range cell interactions mediated via the cell-surface proteins Capricious and Tartan provide cues that support cell survival during wing development. Cells unable to participate in these interactions are eliminated by cell death. It is unclear, however, whether short-range interactions are sufficient to explain the cell death phenotype in bicoid and oskar mutants (Werz, 2005).

Irrespective of the underlying mechanism for sensing mis-specification, the current results highlight the role of an active gene-directed process that removes mis-specified cells during development. However, if a survival mechanism is provided, mis-specified cells can survive and adopt a different fate. In wild-type embryos, mis-specification probably occurs in cells in isolation, and hence is difficult to study. However, in bicoid and oskar mutants, large regions of neighboring cells are mis-specified and undergo cell death simultaneously, providing a unique opportunity to clarify the signals that initiate cell death in situations where cells are developmentally mis-specified (Werz, 2005).

Genome-wide measurement of spatial expression in patterning mutants of Drosophila melanogaster

Patterning in the Drosophila melanogaster embryo is affected by multiple maternal factors, but the effect of these factors on spatial gene expression has not been systematically analyzed. This study characterized the effect of the maternal factors Zelda, Hunchback and Bicoid by cryosectioning wildtype and mutant blastoderm stage embryos and sequencing mRNA from each slice. The resulting atlas of spatial gene expression highlights the intersecting roles of these factors in regulating spatial patterns, and serves as a resource for researchers studying spatial patterning in the early embryo. A large number of genes was identified with both expected and unexpected patterning changes, and through integrated analysis of transcription factor binding data common themes were identified in genes with complex dependence on these transcription factors (Combs, 2017).

Abdu, U., Bar D. and Schüpbach T. (2006). spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila. Development 133: 1477-1484. PubMed Citation: 16540510

Abe, N., Dror, I., Yang, L., Slattery, M., Zhou, T., Bussemaker, H. J., Rohs, R. and Mann, R. S. (2015). Deconvolving the recognition of DNA shape from sequence. Cell 161(2): 307-318. PubMed ID: 25843630

Adhikary, R., Tan, Y. X., Liu, J., Zimmermann, J., Holcomb, M., Yvellez, C., Dawson, P. E. and Romesberg, F. E. (2017). Conformational heterogeneity and DNA recognition by the morphogen Bicoid. Biochemistry [Epub ahead of print]. PubMed ID: 28547993

Ali-Murthy, Z. and Kornberg, T. B. (2016). Bicoid gradient formation and function in the Drosophila pre-syncytial blastoderm. Elife 5: e13222. PubMed ID: 26883601

Andrioli, L. P. M., et al. (2002). Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms. Development 129: 4931-4940. 12397102

Arn, E. A., Cha, B. J., Theurkauf, W. E. and Macdonald, P. M. (2003). Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins. Dev. Cell 4: 41-51. 12530962

Baird-Titusa, J. M., et al. (2005). The solution structure of the native K50 Bicoid homeodomain bound to the consensus TAATCC DNA-binding site. J. Molec. Biol. 356(5): 1137-51. 16406070

Barker, M. S., Demuth, J. P. and Wade, M. J. (2005). Maternal expression relaxes constraint on innovation of the anterior determinant, bicoid. Plos Genetics 1: e57. 16299585

Baum, B., Li, W. and Perrimon, N. (2000). A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast. Curr. Biol. 10: 964-973. PubMed Citation: 10985383

Berleth, T., et al. (1988). The role of localized bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J 7: 1749-56. PubMed Citation: 2901954

Bellaiche, Y., et al. (1996). Neither the homeodomain nor the activation domain of Bicoid is specifically required for its down-regulation by the Torso receptor tyrosine kinase cascade. Development 122, 3499-3508. PubMed Citation: 8951065

Blankenship, J. T. and Wieschaus, E. (2001). Two new roles for the Drosophila AP patterning system in early morphogenesis. Development 128: 5129-5138. 11748148

Boring, L. Weir, M. and Schubiger, G. (1993). Egg ligation alters the Bcd protein gradient and segmentation gene expression in embryos of Drosophila. Mech Dev 42: 97-111. PubMed Citation: 8369226

Brown, S., et al. (2001). A strategy for mapping bicoid on the phylogenetic tree Curr. Bio. 11: R43-R44. 11231138

Bullock, S. L. and Ish-Horowicz, D. (2001). Conserved signals and machinery for RNA transport in Drosophila oogenesis and embryogenesis. Nature 414(6864): 611-6. 11740552

Cai, X., Akber, M., Spirov, A. and Baumgartner, S. (2017). Cortical movement of Bicoid in early Drosophila embryos is actin- and microtubule-dependent and disagrees with the SDD diffusion model. PLoS One 12(10): e0185443. PubMed ID: 28973031

Campione, M., et al. (1999). The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. Development 126(6): 1225-1234. PubMed Citation: 10021341

Carr, A. and Biggin, M. D. (1999). A comparison of in vivo and in vitro DNA-binding specificities suggests a new model for homeoprotein DNA binding in Drosophila embryos. EMBO J. 18(6): 1598-1608. PubMed Citation: 10075930

Chen, B., et al. (2000). Completion of meiosis in Drosophila oocytes requires transcriptional control by Grauzone, a new zinc finger protein. Development 127: 1243-1251. PubMed Citation: 10683177

Chen, H., Xu, Z., Mei, C., Yu, D., Small, S. (2012). A system of repressor gradients spatially organizes the boundaries of Bicoid-dependent target genes. Cell 149(3): 618-29. PubMed Citation: 22541432

Cheung, D., Miles, C., Kreitman, M. and Ma, J. (2011). Scaling of the Bicoid morphogen gradient by a volume-dependent production rate. Development 138(13): 2741-9. PubMed Citation: 21613328

Cheung, D., Miles, C., Kreitman, M. and Ma, J. (2014). Adaptation of the length scale and amplitude of the Bicoid gradient profile to achieve robust patterning in abnormally large Drosophila melanogaster embryos. Development 141: 124-135. PubMed ID: 24284208

Cho, P. F., et al. (2005). A new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP. Cell 121(3): 411-23. 15882623

Cheung, D. and Ma, J. (2015). Probing the impact of temperature on molecular events in a developmental system. Sci Rep 5: 13124. PubMed ID: 26286011

Clegg, N. J., et al. (1997). maelstrom is required for an early step in the establishment of Drosophila oocyte polarity: posterior localization of grk mRNA. Development 124(22): 4661-4671. PubMed Citation: 9409682

Combs, P. A. and Eisen, M. B. (2017). Genome-wide measurement of spatial expression in patterning mutants of Drosophila melanogaster. F1000Res 6: 41. PubMed ID: 28299188

Corado, M. S., et al. (2002). The activity of the Drosophila morphogenetic protein Bicoid is inhibited by a domain located outside its homeodomain. Development 129: 1669-1680. 11923203

Crawford, M. J., et al. (1997). Human and murine PTX1/Ptx1 gene maps to the region for Treacher Collins syndrome. Mamm. Genome 8(11): 841-5. PubMed Citation: 9337397

Driever, W. and Nüsslein-Volhard, C. (1988). A gradient of bicoid protein in Drosophila embryos. Cell 54: 83-93. PubMed Citation: 3383244

Driever, W. et al. (1989). Rescue of bicoid mutant Drosophila embryos by bicoid fusion proteins containing heterologous activating sequences. Nature 342(6246): 149-54. PubMed Citation: 2509940

Dubin-Bar D., et al. (2008). The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis. BMC Cell Biol. 9: 51. PubMed Citation: 18796167

Dubin-Bar, D., Bitan, A., Bakhrat, A., Amsalem, S. and Abdu, U. (2011). Drosophila javelin-like encodes a novel microtubule-associated protein and is required for mRNA localization during oogenesis. Development 138(21): 4661-71. PubMed Citation: 21989913

Dubnau, J. and Struhl, G. (1996). RNA recognition and translational regulation of a homeodomain protein. Nature 379: 694-699. PubMed Citation: 8602214

Epps, J. L. and Tanda, S. (1998). The Drosophila semushi mutation blocks nuclear import of bicoid during embryogenesis. Curr. Biol. 8: 1277-1280. 9822580

Fahmy, K., Akber, M., Cai, X., Koul, A., Hayder, A. and Baumgartner, S. (2014). αTubulin 67C and Ncd are essential for establishing a cortical microtubular network and formation of the Bicoid mRNA gradient in Drosophila. PLoS One 9: e112053. PubMed ID: 25390693

Fauvarque, M.O., Zuber, V and Dura, J. M. (1995). Regulation of polyhomeotic transcription may involve local changes in chromatin activity in Drosophila. Mech Dev 52: 343-355. PubMed Citation: 8541220

Fernandes, G., Tran, H., Andrieu, M., Diaw, Y., Perez Romero, C., Fradin, C., Coppey, M., Walczak, A. M. and Dostatni, N. (2022). Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription. Elife 11. PubMed ID: 35363606

Ferrandon, D., et al. (1994). Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell 79: 1221-1232. PubMed Citation: 8001156

Ferrandon, D., et al. (1997). RNA-RNA interaction is required for the formation of specific bicoid mRNA 3' UTR-STAUFEN ribonucleoprotein particles. EMBO J. 16: 1751-58. PubMed Citation: 9130719

Finkelstein, R. and Perrimon, N. (1990). The orthodenticle gene is regulated by bicoid and torso and specifies Drosophila head development. Nature 346: 485-8. PubMed Citation: 1974036

Frigerio, G., et al. (1986). Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell 47: 735-46. PubMed Citation: 2877746

Frigerio, G., Burri, M., Bopp, D., Baumgartner, S. and Noll, M. (1986). Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell 47: 735-746. PubMed Citation: 2877746

Fröhnhofer, H.G. and Nüsslein-Volhard, C. (1986). Organization of anterior pattern in the Drosophila embryo by the maternal gene bicoid. Nature 324: 120-125

Fu, D. and Ma, J. (2005). Interplay between positive and negative activities that influence the role of Bicoid in transcription. Nucleic Acids Res. 33(13): 3985-93. 16030350

Gage, P. J. and Camper, S. A. (1997). Pituitary homeobox 2, a novel member of the bicoid-related family of homeobox genes, is a potential regulator of anterior structure formation. Hum. Mol. Genet. 6(3): 457-64

Gamberi, C., Peterson, D. S., He, L. and Gottlieb, E. (2002). An anterior function for the Drosophila posterior determinant Pumilio. Development 129: 2699-2710. 12015297

Gao, Q., Wang, Y. and Finkelstein, R. (1996). orthodenticle regulation during embryonic head development in Drosophila. Mech. Dev. 56: 3-15

Gao, Q. and Finkelstein, R. (1998). Targeting gene expression to the head: the Drosophila orthodenticle gene is a direct target of the Bicoid morphogen. Development 125(21): 4185-4193. 9753673

Garcia, H. G., Tikhonov, M., Lin, A. and Gregor, T. (2013). Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning. Curr Biol 23: 2140-2145. PubMed ID: 24139738

Gavis, E. R. and Lehmann, R. (1994). Translational regulation of nanos by RNA localization. Nature 369: 315-8

Gillespie, D. E., and Berg, C. A. (1995). Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases. Genes Dev 9: 2495-2508

Goltsev, Y., et al. (2004). Different combinations of gap repressors for common stripes in Anopheles and Drosophila embryos. Dev. Biol. 275: 435-446. 15501229

Goriely, A., et al. (1996). A functional homologue of goosecoid in Drosophila. Development 122: 1641-1650

Gottlieb, E. (1992). The 3' untranslated region of localized maternal messages contains a conserved motif involved in mRNA localization. Proc Natl Acad Sci 89: 7164-8

Gregor, T., Wieschaus, E. F., McGregor, A. P., Bialek, W. and Tank, D. W. (2007). Stability and nuclear dynamics of the Bicoid morphogen gradient. Cell 130: 141-152. PubMed Citation: 17632061

Gregor, T., Tank, D. W., Wieschaus, E. F. and Bialek. W. (2007). Probing the limits to positional information. Cell 130(1): 153-64. Medline abstract: 17632062

Grimm, O. and Wieschaus, E. (2010). The Bicoid gradient is shaped independently of nuclei. Development 137(17): 2857-62. PubMed Citation: 20699297

Grossniklaus, U., Cadigan, K. M. and Gehring, W. J. (1994). Three maternal coordinate systems cooperate in the patterning of the Drosophila head. Development 120: 3155-3171. PubMed Citation: 7720559

Guichet, A., Peri, F. and Roth, S. (2001). Stable anterior anchoring of the oocyte nucleus is required to establish dorsoventral polarity of the Drosophila egg. Dev. Bio. 237: 93-106. 11518508

Gursky, V. V., Jaeger, J., Kozlov, K. N., Reinitz, J. and Samsonov, A. M. (2004). Pattern formation and nuclear divisions are uncoupled in Drosophila segmentation: Comparison of spatially discrete and continuous models. Physica D 197: 286-302. Full text: Gursky, 2004

Gursky, V. V., Panok, L., Myasnikova, E. M., Manu, Samsonova, M. G., Reinitz, J. and Samsonov, A. M. (2011). Mechanisms of gap gene expression canalization in the Drosophila blastoderm. BMC Syst. Biol. 5(1): 118. PubMed Citation: 21794172

Häcker, U., et al. (1995). The Drosophila fork head domain protein crocodile is required for the establishment of head structures. EMBO J 14: 5306-5317

Hanes, S. D., et al. (1994). Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen. Mol Cell Biol 14: 3364-75

Hannon, C. E., Blythe, S. A. and Wieschaus, E. F. (2017). Concentration dependent chromatin states induced by the Bicoid morphogen gradient. Elife 6. PubMed ID: 28891464

Hartmann, B., Reichert, H. and Walldorf, U. (2001). Interaction of gap genes in the Drosophila head: tailless regulates expression of empty spiracles in early embryonic patterning and brain development. Mech. Dev. 109(2): 161-72. 11731230

He, F., et al. (2008). Probing intrinsic properties of a robust morphogen gradient in Drosophila. Dev Cell. 15(4): 558-67. PubMed Citation: 18854140

He, F., Ren, J., Wang, W. and Ma, J. (2011). A multiscale investigation of bicoid-dependent transcriptional events in Drosophila embryos. PLoS One 6(4): e19122. PubMed Citation: 21544208

He, F., Wei, C., Wu, H., Cheung, D., Jiao, R. and Ma, J. (2015). Fundamental origins and limits for scaling a maternal morphogen gradient. Nat Commun 6: 6679. PubMed ID: 25809405

Hoch, M., Seifert, E. and Jaeckle, H. (1991). Gene expression mediated by cis-acting sequences of the Krüppel gene in response to the Drosophila morphogens bicoid and hunchback. EMBO J. 10:2267-2278. PubMed Citation: 2065664

Horner, V. L., et al. (2006). The Drosophila calcipressin Sarah is required for several aspects of egg activation. Curr. Biol. 16(14): 1441-6. Medline abstract: 16860744

Huang, A., Amourda, C., Zhang, S., Tolwinski, N. S. and Saunders, T. E. (2017). Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo. Elife 6. PubMed ID: 28691901

Ip, Y. T., Levine, M. and Small, S. J. (1992). The bicoid and dorsal morphogens use a similar strategy to make stripes in the Drosophila embryo. J Cell Sci Suppl 16: 33-8. PubMed Citation: 1297650

Jack, T. and McGinnis, W. (1990). Establishment of the Deformed expression stripe requires the combinatorial action of coordinate, gap and pair-rule proteins. EMBO J 9: 1187-98. PubMed Citation: 2323337

Jaeger, J., et al. (2004a). Dynamic control of positional information in the early Drosophila embryo. Nature 430: 368-371. 15254541

Jaeger, J., et al. (2004b). Dynamical analysis of regulatory interactions in the gap gene system of Drosophila melanogaster. Genetics 167: 1721-1737. 15342511

Janody, F., et al. (2000a). Phosphorylation of Bicoid on MAP-kinase sites: contribution to its interaction with the torso pathway. Development 127: 279-289. PubMed Citation: 10603346

Janody, F. Reischl, J. and Dostatni, N. (2000b). Persistence of Hunchback in the terminal region of the Drosophila blastoderm embryo impairs anterior development. Development 127: 1573-1582. 10725234

Janody, F., et al. (2001). Two distinct domains of Bicoid mediate its transcriptional downregulation by the Torso pathway. Development 128: 2281-2290. 11493547

Januschke, J., et al. (2002). Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation. Current Biology 12: 1971-1981. 12477385

Jaeger, J., et al. (2004), Dynamical analysis of regulatory interactions in the gap gene system of Drosophila melanogaster. Genetics 67(4): 1721-37. 15342511

Juge, F., Zaessinger, S., Temme, C., Wahle, E., Simonelig, M. (2002). Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila. EMBO J. 21(23): 6603-13. 12456666

Jung, C., Schnepf, M., Bandilla, P., Unnerstall, U. and Gaul, U. (2019). High sensitivity measurement of transcription factor-DNA binding affinities by competitive titration using fluorescence microscopy. J Vis Exp(144). PubMed ID: 30799844

Jung, C., Bandilla, P., von Reutern, M., Schnepf, M., Rieder, S., Unnerstall, U. and Gaul, U. (2018). True equilibrium measurement of transcription factor-DNA binding affinities using automated polarization microscopy. Nat Commun 9(1): 1605. PubMed ID: 29686282 ue): W56-62. PubMed ID: 23703209

Kennerdell, J. R. Yamaguchi, S. and Carthew, R. W. (2002). RNAi is activated during Drosophila oocyte maturation in a manner dependent on aubergine and spindle-E. Genes Dev. 16: 1884-1889. 12154120

Kerszberg, M. and Changeux, J. P. (1994). A model for reading morphogenetic gradients: autocatalysis and competition at the gene level. Proc. Natl. Acad. Sci. 91: 5823-7. PubMed Citation: 8016073

Khuc Trong, P., Doerflinger, H., Dunkel, J., St Johnston, D. and Goldstein, R.E. (2015) Cortical microtubule nucleation can organise the cytoskeleton of Drosophila oocytes to define the anteroposterior axis. Elife 4: e06088. PubMed ID: 26406117Kim, Y. K., Furic, L., DesGroseillers, L. and Maquat, L. E. (2005). Mammalian Staufen1 recruits Upf1 to specific mRNA 3'UTRs so as to elicit mRNA decay. Cell 120: 195-208. PubMed Citation: 15680326

Kioussi, C., et al. (2002). Identification of a Wnt/Dvl/ß-Catenin --> Pitx2 pathway mediating cell-type-specific proliferation during development. Cell 111: 673-685. 12464179

La Rosee, A., et al. (1997). Mechanism and Bicoid-dependent control of hairy stripe 7 expression in the posterior region of the Drosophila embryo. EMBO J. 16(14): 4403-4411

Lamonerie, T., et al. (1996). Ptx1, a bicoid-related homeo box transcription factor involved in transcription of the pro-opiomelanocortin gene. Genes Dev. 10: 1284-1295

Lanctot, C., Lamolet, B. and Drouin, J. (1997). The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm. Development 124(14): 2807-2817

Lanctot, C., Gauthier, Y. and Drouin, J. (1999a). Pituitary homeobox 1 (Ptx1) is differentially expressed during pituitary development. Endocrinology 140(3): 1416-22

Lanctot, C. and Moreau, A., et al. (1999b). Hindlimb patterning and mandible development require the Ptx1 gene. Development 126: 1805-1810

Lane, M. E. and Kalderon, D. (1994). RNA localization along the anteroposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization. Genes Dev 8: 2986-2995

Lebrecht, D., et al (2005). Bicoid cooperative DNA binding is critical for embryonic patterning in Drosophila. Proc. Natl. Acad. Sci. 102(37): 13176-81. 16150708

Lee, K. M., Linskens, A. M. and Doe, C. Q. (2022). Hunchback activates Bicoid in Pair1 neurons to regulate synapse number and locomotor circuit function. Curr Biol 32(11): 2430-2441.e2433. PubMed ID: 35512697

Lemke, S., et al. (2010). Maternal activation of gap genes in the hover fly Episyrphus. Development 137(10): 1709-19. PubMed Citation: 20430746

Li, J., Sagendorf, J. M., Chiu, T. P., Pasi, M., Perez, A. and Rohs, R. (2017). Expanding the repertoire of DNA shape features for genome-scale studies of transcription factor binding. Nucleic Acids Res 45(22): 12877-12887. PubMed ID: 29165643

Liaw, G.-J. and Lengyel, J.A. (1992). Control of tailless expression by bicoid, dorsal and synergistically interacting terminal system regulation elements. Mech. Dev. 40: 47-61. PubMed Citation: 8443106

Liaw, G. J. and Lengyel, J. A. (1993). Control of tailless expression by bicoid, dorsal and synergistically interacting terminal system regulatory elements. Mech Dev 40: 47-61. PubMed Citation: 8443106

Lieberfarb, M. E., et al. (1996). Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development. Development 122: 579-588. PubMed Citation: 8625809

Liu, J. and Ma, J. (2013). Dampened regulates the activating potency of Bicoid and the embryonic patterning outcome in Drosophila. Nat Commun 4: 2968. PubMed ID: 24336107

Liu, J. and Ma, J. (2015). Modulation of temporal dynamics of gene transcription by activator potency in the Drosophila embryo. Development 142(21):3781-90. PubMed ID: 26395487

Liu, W. and Niranjan, M. (2011). The role of regulated mRNA stability in establishing bicoid morphogen gradient in Drosophila embryonic development. PLoS One 6(9): e24896. PubMed Citation: 21949782

Llamusi, B., Bargiela, A., Fernandez-Costa, J. M., Garcia-Lopez, A., Klima, R., Feiguin, F. and Artero, R. (2013). Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model. Dis Model Mech 6(1): 184-196. PubMed ID: 23118342

Logan, M. and Tabin, C. J. (1999). Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity. Science 283(5408): 1736-9. PubMed Citation: 10073939

Lucas, T., Ferraro, T., Roelens, B., De Las Heras Chanes, J., Walczak, A. M., Coppey, M. and Dostatni, N. (2013). Live imaging of bicoid-dependent transcription in Drosophila embryos. Curr Biol 23: 2135-2139. PubMed ID: 24139736

Luk, S. K., et al. (1994). Components acting in localization of bicoid mRNA are conserved among Drosophila species. Genetics 137: 521-530. 8070663

Ma, X, et al., et al. (1996). The Drosophila morphogenetic protein Bicoid binds DNA cooperatively. Development 122: 1195-1206

Macdonald, P. M., et al. (1993). RNA regulatory element BLE1 directs the early steps of bicoid mRNA localization. Development 118: 1233-43

Macdonald, P. M., Leask, A. and Kerr, K. (1995). exl protein specifically binds BLE1, a bicoid mRNA localization element, and is required for one phase of its activity. Proc. Natl. Acad. Sci. 92: 10787-10791. 7479884

Mancebo, R., et al. (2001). BSF binds specifically to the bicoid mRNA 3' untranslated region and contributes to stabilization of bicoid mRNA. Mol. Cell. Bio. 21: 3462-3471. 11313472

Martinez, C., Rest, J. S., Kim, A. R., Ludwig, M., Kreitman, M., White, K. and Reinitz, J. (2014). Ancestral resurrection of the Drosophila S2E enhancer reveals accessible evolutionary paths through compensatory change. Mol Biol Evol 31(4): 903-16 PubMed ID: 24408913

Meignin, C. and Davis, I. (2008). UAP56 RNA helicase is required for axis specification and cytoplasmic mRNA localization in Drosophila. Dev. Biol. 315(1): 89-98. PubMed Citation: 18237727

Micklem, D. R., et al. (2000). Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation. EMBO J. 19: 1366-1377

Mir, M., Reimer, A., Haines, J. E., Li, X. Y., Stadler, M., Garcia, H., Eisen, M. B. and Darzacq, X. (2017). Dense Bicoid hubs accentuate binding along the morphogen gradient. Genes Dev 31(17): 1784-1794. PubMed ID: 28982761

Moon, W. and Hazelrigg, T. (2004). The Drosophila microtubule-associated protein Mini spindles is required for cytoplasmic microtubules in oogenesis. Curr. Biol. 14: 1957-1961. Medline abstract: 15530399

Nakamura, A., Amikura, R., Hanyu, K. and Kobayashi, S. (2001). Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development 128(17): 3233-42. 11546740

Namba, R., et al. (1997). Drosophila embryonic pattern repair: how embryos respond to bicoid dosage alteration Development 124: 1393-1403. 9118810

Niessing, D., et al. (1999). Sequence interval within the PEST motif of Bicoid is important for translational repression of Caudal mRNA in the anterior region of the Drosophila embryo. EMBO J. 18(7): 1966-1973. 10202159

Niessing, D., et al. (2000). Homeodomain position 54 specifies transcriptional versus translational control by Bicoid. Molec. Cell 5: 395-401. 10882080

Niessing, D., Blanke, S. and Jäckle, H. (2002). Bicoid associates with the 5'-cap-bound complex of caudal mRNA and represses translation. Genes Dev. 16: 2576-2582. 12368268

Ochoa-Espinosa, A., Yucel, G., Kaplan, L., Pare, A., Pura, N., Oberstein, A., Papatsenko, D. and Small S. (2005). The role of binding site cluster strength in Bicoid-dependent patterning in Drosophila. Proc. Natl. Acad. Sci. 102(14): 4960-5. 15793007

Ochoa-Espinosa, A., Yu, D., Tsirigos, A., Struffi, P. and Small, S. (2009). Anterior-posterior positional information in the absence of a strong Bicoid gradient. Proc. Natl. Acad. Sci. 106(10): 3823-8. PubMed Citation: 19237583

Olivas, W. and Parker, R. (2000). The Puf3 protein is a transcript specific regulator of mRNA degradation in yeast. EMBO J. 19: 6602-6611. 11101532

Page, A. W. and Orr-Weaver, T. L. (1996). The Drosophila genes grauzone and cortex are necessary for proper female meiosis. J. Cell Science 109: 1707-1715. PubMed Citation: 8832393

Park, J. M., et al. (2001). Drosophila mediator complex is broadly utilized by diverse gene-specific transcription factors at different types of core promoters. Mol. Cell. Bio. 21: 2312-2323. 11259581

Papatsenko, D., Goltsev, Y. and Levine, M. (2009). Organization of developmental enhancers in the Drosophila embryo. Nucl. Acids Res. 37: 5665-5677. PubMed Citation: 19651877

Papatsenko, D. and Levine, M. (2011). The Drosophila gap gene network is composed of two parallel toggle switches. PLoS One 6(7): e21145. PubMed Citation: 21747931

Payre, F., Crozatier, M. and Vincent, A. (1994). Direct control of transcription of the Drosophila morphogen bicoid by the serendipity delta zinc finger protein, as revealed by in vivo analysis of a finger swap. Genes Dev. 8: 2718-28. PubMed Citation: 7958928

Payre, F., et al. (1997). Two types of zinc fingers are required for dimerization of the Serendipity delta transcriptional activator. Mol. Cell. Biol. 17(6): 3137-45. PubMed Citation: 9154812

Perez Ipina, E. and Ponce Dawson, S. (2017). The effect of reactions on the formation and readout of the gradient of Bicoid. T. Phys Biol. 14(1):016002. PubMed ID: 28054512

Perkins, T. J., Jaeger, J., Reinitz, J. and Glass, L. (2006). Reverse engineering the gap gene network of Drosophila melanogaster. PLoS Comput. Biol. 2(5): e51. 16710449

Perrin, L., Romby, P., Laurenti, P., Berenger, H., Kallenbach, S., Bourbon, H. M. and Pradel, J. (1999). The Drosophila modifier of variegation modulo gene product binds specific RNA sequences at the nucleolus and interacts with DNA and chromatin in a phosphorylation-dependent manner. J. Biol. Chem. 274: 6315-6323. 10037720

Perry, M. W., Bothma, J. P., Luu, R. D. and Levine, M. (2012). Precision of hunchback expression in the Drosophila embryo. Curr Biol 22: 2247-2252. PubMed ID: 23122844

Pignoni, F., Steingrimsson, E. and Lengyel, J.A. (1992). bicoid and the terminal system activate tailless expression in the early Drosophila embryo. Development 115: 239--251

Pokrywka, N. J. and Stephenson, E. C. (1995). Microtubules are a general component of mRNA localization systems in Drosophila oocytes. Dev. Biol. 167: 363-70

Pokrywka, N. J., Fishbein, L. and Frederick, J. (2000). New phenotypes associated with the swallow gene of Drosophila: evidence for a general role in oocyte cytoskeletal organization. Dev. Genes Evol. 210: 426-435

Pokrywka, N. J., Meng, L., Debiec, K. and Stephenson, E. C. (2004). Identification of hypomorphic and null alleles of swallow via molecular and phenotypic analyses. Dev. Genes Evol. 214: 185-192. PubMed Citation: 15029497

Ramos, A., et al. (2000). RNA recognition by a Staufen double-stranded RNA-binding domain. EMBO J. 19: 997-1009\

Riechmann, V. and Ephrussi, A. (2004). Par-1 regulates bicoid mRNA localisation by phosphorylating Exuperantia. Development 131: 5897-5907. 15539486

Rivera-Pomar, R. and Jäckle, H. (1996a). From gradients to stripes in Drosophila embryogenesis: Filling in the gaps. Trends Genet 12: 478-483. 8973159

Rivera-Pomar, R., et al. (1996b). RNA binding and translational suppression by bicoid. Nature 379, 746-749

Ronchi, E., et al. (1993). Down-regulation of the Drosophila morphogen bicoid by the torso receptor-mediated signal transduction cascade. Cell 74: 347-55

Roth, S., et al (1995). cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila. Cell 81: 967-978

Rube, H. T., Rastogi, C., Kribelbauer, J. F. and Bussemaker, H. J. (2018). A unified approach for quantifying and interpreting DNA shape readout by transcription factors. Mol Syst Biol 14(2): e7902. PubMed ID: 29472273

Ruez, C., Payre, F. and Vincent, A. (1998). Transcriptional control of Drosophila bicoid by Serendipity delta: cooperative binding sites, promoter context, and co-evolution. Mech. Dev. 78(1-2): 125-134

Ryu, Y. H. and Macdonald, P. M. (2015). RNA sequences required for the noncoding function of oskar RNA also mediate regulation of Oskar protein expression by Bicoid Stability Factor. Dev Biol 407(2): 211-223. PubMed ID: 26433064

Salles, F. J., et al. (1994). Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266: 1996-1999

Sanchez, L. and Thieffry, D. (2001). A logical analysis of the gap gene system. J. Theor. Biol. 211: 115-141. 11419955

Sauer, F., Hansen, S. K. and Tjian, R. (1995a). Multiple TAFIIs directing synergistic activation of transcription. Science 270(5243): 1783-8

Sauer, F., Hansen, S. K. and Tjian, R. (1995b). DNA template and activator-coactivator requirements for transcriptional synergism by Drosophila bicoid. Science 270: 1825-1828

Sauer, F., Hansen, S. K. and Tjian, R. (1996). Multiple TAFIIs directing synergistic activation of transcription. Science 270: 1783-1788

Schaeffer, V., et al. (1999). Bicoid functions without its TATA-binding protein-associated factor interaction domains. Proc. Natl. Acad. Sci. 96(8): 4461-6

Schaeffer, V., et al. (2000). High Bicoid levels render the terminal system dispensable for Drosophila head development. Development 127: 3993-3999. PubMed Citation: 10952897

Schnepf, M., von Reutern, M., Ludwig, C., Jung, C. and Gaul, U. (2020). Transcription factor binding affinities and DNA shape readout. iScience 23(11): 101694. PubMed ID: 33163946

Schnorrer, F., Bohmann, K. and Nüsslein-Volhard, C. (2000). The molecular motor dynein is involved in targeting Swallow and bicoid mRNA to the anterior pole of Drosophila oocytes. Nat. Cell Biol. 2: 185-190. 10783235

Schnorrer, F., Luschnig, S., Koch, I. and Nüsslein-Volhard, C. (2002). γ-Tubulin37C and γ-tubulin ring complex protein 75 are essential for bicoid rna localization during Drosophila oogenesis. Developmental Cell 3: 685-696. 12431375

Serano, T. L. and Cohen, R. S. (1995). A small predicted stem-loop structure mediates oocyte localization of Drosophila K10 mRNA. Development 121: 3809-3818

Shang, J., Luo, Y. and Clayton, D. A. (1997). Backfoot is a novel homeobox gene expressed in the mesenchyme of developing hind limb. Dev. Dyn. 209(2): 242-53

Simpson-Brose, M., Treisman, J. and Desplan, C. (1994) Synergy between the hunchback and bicoid morphogens is required for anterior patterning in Drosophila. Cell 78: 855-865

Small, S., Blair, A. and Levine, M. (1992). Regulation of even-skipped stripe 2 in the Drosophila embryo. EMBO J 11: 4047-57

Smidt, M. P., et al. (1997). A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc. Natl. Acad. Sci. 94(24): 13305-13310

Snee, M. J., Arn, E. A., Bullock, S. L. and Macdonald, P. M. (2005). Recognition of the bcd mRNA localization signal in Drosophila embryos and ovaries. Mol Cell Biol. 25(4): 1501-10. 15684399

Spirov, A., Fahmy, K., Schneider, M., Frei, E., Noll, M. and Baumgartner, S. (2009). Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient. Development 136(4): 605-14. PubMed Citation: 19168676

Stauber, M., Jackle, H. and Schmidt-Ott, U. (1999). The anterior determinant bicoid of Drosophila is a derived Hox class 3 gene. Proc. Natl. Acad. Sci. 96(7): 3786-9

Stauber, M., Taubert, H. and Schmidt-Ott, U. (2000). Function of bicoid and hunchback homologs in the basal cyclorrhaphan fly Megaselia (Phoridae). Proc. Natl. Acad. Sci. 97: 10844-10849

Stauber, M., Prell, A. and Schmidt-Ott, U. (2002). A single Hox3 gene with composite bicoid and zerknüllt expression characteristics in non-Cyclorrhaphan flies. Proc. Natl. Acad. Sci. 99: 274-279. 11773616

Surdej, P. and Jacobs-Lorena, M. (1998). Developmental regulation of Bicoid mRNA stability is mediated by the first 43 nucleotides of the 3' untranslated region. Mol. Cell. Biol. 18(5): 2892-2900

Szeto, D. P., et al. (1999). Role of the bicoid-related homeodomain factor pitx1 in specifying hindlimb morphogenesis and pituitary development. Genes Dev. 13(4): 484-94

Takeo, S., Tsuda, M., Akahori, S., Matsuo, T. and Aigaki, T. (2006). The calcineurin regulator Sra plays an essential role in female meiosis in Drosophila. Curr. Biol. 16(14): 1435-40. Medline abstract: 16860743

Theurkauf, W. E. and Hazelrigg, T. I. (1998). In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization pathway. Development 125(18): 3655-3666

Torigoi, E., et al. (2000). Chip interacts with diverse homeodomain proteins and potentiates Bicoid activity in vivo. Proc. Natl. Acad. Sci. 97: 2686-2691

Treier, M., et al. (2001). Hedgehog signaling is required for pituitary gland development. Development 128: 377-386. 11152636

Tremblay, J. J., Lanctot, C. and Drouin, J. (1998). The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol. Endocrinol. 12(3): 428-41

Trovisco, V., Belaya, K., Nashchekin, D., Irion, U., Sirinakis, G., Butler, R., Lee, J. J., Gavis, E. R. and St Johnston, D. (2016). bicoid mRNA localises to the Drosophila oocyte anterior by random Dynein-mediated transport and anchoring. Elife 5: e17537. PubMed ID: 27791980

Tsai, C, and Gergen, J. P. (1994). Gap gene properties of the pair-rule gene runt during Drosophila segmentation. Development 120: 1671-1683

Vorbruggen, G., et al. (1997). Embryonic expression and characterization of a Ptx1 homolog in Drosophila. Mech. Dev. 68(1-2): 139-47

Walldorf, U. and Gehring, W. J. (1992). empty spiracles, a gap gene containing a homeobox involved in Drosophila head development. EMBO J 11: 2247-59

Wang, S. and Hazelrigg, T. (1994). Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369: 400-03. 7910952

Weil, T. T., Forrest, K. M. and Gavis, E. R. (2006). Localization of bicoid mRNA in late oocytes is maintained by continual active transport. Dev. Cell. 11(2): 251-62. Medline abstract: 16890164

Weil, T. T., Parton, R., Davis, I. and Gavis, E. R. (2008). Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition. Curr. Biol. 18: 1055-1061. PubMed Citation: 18639459

Weil, T. T., et al. (2010). Distinguishing direct from indirect roles for bicoid mRNA localization factors. Development 137(1): 169-76. PubMed Citation: 20023172

Werz, C., et al. (2005). Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila. Development 132(24): 5343-52. 16280349

Wharton, R. P. and Struhl, G. (1991). RNA regulatory elements mediate control of Drosophila body pattern by the posterior morphogen nanos. Cell 67: 955-67

Wilhelm, J. E., et al. (2000). Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes. J. Cell Biol. 148: 427-440

Wilsch-Bräuninger, M., Schwarz, H. and Nüsslein-Volhard, C. (1997). A sponge-like structure involved in the association and transport of maternal products during Drosophila oogenesis. J. Cell Biol. 139: 817-829

Wimmer, E. A., et al., (1995). Trans- and cis-acting requirements for blastodermal expression of the head gap gene buttonhead. Mech. Dev. 53: 235-245

Wimmer, E. A., et al. (2000). bicoid-independent formation of thoracic segments in Drosophila. Science 287: 2476-2479

Wolff, C., et al. (1998). Regulation of the Tribolium homologues of caudal and hunchback in Drosophila: evidence for maternal gradient systems in a short germ embryo. Development 125(18): 3645-3654

Wu, H., Manu, Jiao, R. and Ma, J. (2015). Temporal and spatial dynamics of scaling-specific features of a gene regulatory network in Drosophila. Nat Commun 6: 10031. PubMed ID: 26644070

Xie, J. and Hu, G. H. (2016). Hydrodynamic modeling of Bicoid morphogen gradient formation in Drosophila embryo. Biomech Model Mechanobiol 15(6):1765-1773. PubMed ID: 27193268

Yoshioka, H., et al. (1998). Pitx2, a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry. Cell 94(3): 299-305

Zabet, N. R. and Adryan, B. (2014). Estimating binding properties of transcription factors from genome-wide binding profiles. Nucleic Acids Res 43(1):84-94. PubMed ID: 25432957

Zhao, C., et al. (2000). Target selectivity of Bicoid is dependent on nonconsensus site recognition and protein-protein interaction. Mol. Cell. Bio. 29: 8112-8123

Zhao, C., Fu, D., Dave, V., Ma, J. (2003). A composite motif of the Drosophila morphogenetic protein bicoid critical to transcription control J. Biol. Chem. 278: 43901-43909. 12939280

Zhou, T., Yang, L., Lu, Y., Dror, I., Dantas Machado, A. C., Ghane, T., Di Felice, R. and Rohs, R. (2013). DNAshape: a method for the high-throughput prediction of DNA structural features on a genomic scale. Nucleic Acids Res 41(Web Server issue):W56-62. PubMed ID: 23703209

Zhu, W., Foehr, M., Jaynes, J. B. and Hanes, S. D. (2001). Drosophila SAP18, a member of the Sin3/Rpd3 histone deacetylase complex, interacts with Bicoid and inhibits its activity. Dev. Genes Evol. 211: 109-117. Medline abstract: 11455422

Zimyanin, V. L., Belaya, K., Pecreaux, J., Gilchrist, M. J., Clark, A., Davis, I. and St Johnston, D. (2008). In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization. Cell 134: 843-853. PubMed Citation: 18775316

date revised: 23 April 2021

 

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

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