Krüppel
Transcripts of Krüppel are first detected after the 11th nuclear cleavage in the syncytial blastoderm, in a central domain that is 45-55% of egg length. During the cellular blastoderm an additional site of expression appears in the polar cap. At the time of gastrulation, a third zone of expression appears in the anterior region of the embryo. After the beginning of gastrulation, the expression pattern becomes more complex.
Krüppel is expressed under separate control in many different tissues including the anterior domain, stomodeum, amnioserosa, Malphighian tubule precursors, CNS and muscle precursors (Hoch, 1990, 1991).
By the end of germ-band expression (6 hours), KR transcripts have accumulated from the posterior edge of the cephalic furrow throughout the thoracic and abdominal anlagen. During the extended germ band stage [Image], expression is mesodermal, neuroblastic and in Malpighian tubules as well (Knipple, 1985).
Kr begins to be expressed along the entire neuroectoderm during stage 8, when the first proneural clusters become resolved. This pattern of Kr neuroectodermal expression persists until late stage 11. The spatio-temporal pattern of neuroectodermal Kr expression is heterogeneous, that is, certain areas and cells accumulate higher levels of protein than others and this varies with time. Kr is also found in a large fraction of the ganglion mother cells and in their neuronal and glial progeny. Kr mutants exhibit strong neural aberrations that are not confined to the thoraic and anterior abdominal region but are also found in the gnathal and three last abdominal segments. Kr is coexpressed with engrailed in a subset of neurons and glia that include the medial-lateral cluster of en-expressing neurons and the dorsal channel glia cells. In Kr mutants, the medial-lateral cluster is either absent or fails to express en, but the dorsal channel glia cells are not affected. These medial-lateral cluster cells gives rise to serotoninergic neurons, and almost no neurons synthesizing serotonin remain in these mutant embryos. In Kr mutants, the number of gooseberry neural-expressing cells increases from 10% to 50%. Ectopic Kr expression leads to a strong reduction in gsb-n expressing neurons. Kr is also required for glial development. Kr is expressed in several glia types: in cell body glia cells, including medial components; in the exit glia cells and in the intersegmental nerve root glia media cells. While Kr is not expressed in longitudinal glia, it is found in the corresponding glioblast. In Kr mutants there are a reduced number of longitudinal glia and a reduction in cell body glial cells. Absence of Kr is required for correct specification of cell body glial cells; its absence leads to abnormal migration and eventually cell death (Romani, 1996).
To identify genes important in fat-cell metabolism and development, Drosophila stocks carrying an engineered transposable element that can reveal the presence of nearby enhancer elements were screened. Those "enhancer-trap lines" that contain transposable P elements integrated near fat-cell specific enhancer elements were identified by marker expression studies. Genes associated with these enhancers will provide information concerning fat-cell function and serve as target genes for studying fat-cell specific gene expression. The identification of enhancer-trap lines active in the developing fat cell should provide an entry point into the molecular and genetic analysis of early fat-cell development. Analysis of two lines has revealed that the transcription factors seven up (a steroid-hormone receptor) and Krüppel (a zinc-finger protein) are present in the fat body; these factors are likely to be involved in fat-cell gene expression. In two other lines, beta-galactosidase was detected in a subset of adepithelial cells that may be the precursors to the adult fat cell. In a single line, transgene activity is present in the progenitor cells of the embryonic fat body. The genes associated with these enhancer-trap lines may be involved in fat-cell development (Hoshizaki, 1995).
The development of the head and tail regions of the Drosophila embryo is dependent upon the localized polar activation of Torso (Tor), a receptor tyrosine kinase that is uniformly distributed in the membrane of the developing embryo. Trunk (Trk), the proposed ligand for Tor, is secreted as an inactive precursor into the perivitelline fluid that lies between the embryonic membrane and the vitelline membrane (VM), the inner layer of the eggshell. The spatial regulation of Trk processing is thought to be mediated by the secreted product of the torsolike (tsl) gene, which is expressed during oogenesis by a specialized population of follicle cells present at the two ends of the oocyte. Tsl protein has been shown to be specifically localized to the polar regions of the VM in laid eggs. Although Tsl can associate with nonpolar regions of the VM, the activity of polar-localized Tsl is enhanced, suggesting the existence of another spatially restricted factor acting in this pathway. The incorporation of Tsl into the VM provides a mechanism for the transfer of spatial information from the follicle cells to the developing embryo. Tsl represents the first example of an embryonic patterning determinant that is a component of the eggshell (Stevens, 2003).
Activation of Tsl may be spatially regulated through the action of gap genes. Spatial regulation of the expression of the gap genes, the first zygotic patterning genes to be expressed during embryogenesis, is determined by the activity of the three maternal pathways (anterior, posterior, and terminal) required for the development of the anterior-posterior axis of the embryo. In addition to localized maternal input, interactions between the gap gene products themselves led to further refinement of their expression domains. tll expression, for example, is specifically repressed in the segmented region of the embryo by central gap gene products such as Kr. Thus, depending on their relative levels of activity, Kr and Tll are both capable of suppressing one another's expression. This raises the possibility that centrally expressed Kr is responsible for the polar restriction of tll expression that is observed in the progeny of tsl mutant females expressing tsl from the germline. To address this question, the CBBtsl insertion was crossed into females that were mutant for all three anterior-posterior maternal pathways. Embryos produced by mothers triply mutant for bicoid (anterior), oskar (posterior), and tsl (terminal) lack all anterior-posterior patterning, express low levels of Kr uniformly along the anterior-posterior axis, and do not express tll at all. In contrast, the embryos produced by triply mutant females carrying CBBtsl did express tll in distinct polar domains, either at the anterior alone or at both poles. Consistent with this pattern of tll expression, Kr expression is specifically repressed in the corresponding polar domains. Further, these embryos also differentiate Filzkörper material, a posterior cuticular structure that requires terminal pathway activity, at one or both poles. Thus, although Tsl was distributed uniformly in their VMs, these embryos developed gap gene expression patterns and cuticular phenotypes consistent with polar activation of the Tor receptor. These findings suggest that the activity of Tsl is enhanced at, and perhaps restricted to, the polar regions of the VM; this finding implies that there is an as yet unidentified component of the terminal class pathway that is restricted to the poles and is required for the function of Tsl (Stevens, 2003).
The existence of another localized factor in this pathway indicates that there are at least four levels of control that ensure the polar restriction of tll expression during embryonic development. (1) First to act is the restriction of tsl expression to a specific subpopulation of follicle cells present at the poles of the oocyte. (2) Next is the stabilization of secreted Tsl protein at the poles of the VM and its incorporation into the eggshell in an active form. (3)The facilitation of Tsl function follows, through its proposed interaction with another localized factor. (4)The final layer of control is the exclusion of tll expression from nonpolar regions through the inhibitory effects of centrally expressed gap genes. Although it has long been assumed that the spatial restriction of tsl expression was the uniquely localized element in the terminal pathway, data is presented implying the existence of another factor that enhances the activity of Tsl specifically at the poles. The function of Tsl itself is unknown, and there are currently no candidate genes encoding proteins with the enzymatic activity to bring about the proposed processing of the Trk precursor. It is likely, therefore, that the identification of this factor will greatly enhance understanding of the mechanism by which the Tor ligand is formed (Stevens, 2003).
Different combinations of gap repressors for common stripes in Anopheles and Drosophila embryos
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).
In Drosophila, different levels of the Hunchback and Knirps gap repressor gradients define the limits of eve stripes 3, 4, 6, and 7, while Giant and Kruppel establish the borders of stripes 2 and 5. In situ hybridization probes were prepared for Anopheles orthologues of all four of these gap genes, as well as a fifth gap gene, tailless. hunchback displays a broad band of expression in the anterior half of the Anopheles embryo, encompassing both the presumptive head and thorax. This pattern is similar to that observed in Drosophila, although there are a few notable deviations: (1) there is no obvious maternal expression seen in early Anopheles embryos, whereas maternal hunchback mRNAs are strongly expressed throughout early Drosophila embryos; (2) there is a significant change in the posterior staining pattern. The Drosophila gene displays a strong posterior stripe of expression that is comparable in intensity to the anterior staining pattern. In Anopheles, this staining is significantly weaker than that of the anterior domain, and the posterior pattern is shifted anteriorly into the presumptive abdomen (Goltsev, 2004).
The Kruppel and knirps staining patterns are similar in Anopheles and Drosophila embryos. In both cases, the principal sites of expression are seen in the presumptive thorax and abdomen, respectively. However, the remaining two gap genes, giant and tailless, exhibit distinctive staining patterns. In Anopheles, giant exhibits a continuous band of staining in anterior regions, whereas the Drosophila gene is excluded from the anterior pole. Moreover, there is a prominent band of staining in the presumptive abdomen of Drosophila embryos that is not seen in Anopheles. Finally, tailless is expressed in a narrow stripe in the posterior pole of Drosophila embryos, whereas Anopheles embryos display a dynamic pattern that (transiently) extends throughout the presumptive abdomen (Goltsev, 2004).
The combinations of gap repressors that define the borders of eve stripes 2 to 7 are known in Drosophila. Stripes 2 and 5 are formed by the combination of Giant and Kruppel repressors, while distinctive borders for stripes 3, 4, 6, and 7 are established by the differential repression of the stripe 3/7 and stripe 4/6 enhancers in response to distinct concentrations of the Hunchback and Knirps repressor gradients. Double-staining assays provide immediate insights into the likely combination of gap repressors that are used for any given stripe. For example, the giant and Kruppel expression patterns abut the borders of eve stripes 2 and 5. Double-staining assays were done to determine the potential regulators of the Anopheles eve stripes. These experiments involved the use of digoxigenin-labeled hunchback, Kruppel, knirps, and giant hybridization probes along with an FITC-labeled eve probe. Different histochemical substrates were used to separately visualize the two patterns (Goltsev, 2004).
The anterior hunchback pattern extends through eve stripe 2 and approaches the anterior border of stripe 3. While the posterior pattern extends through stripes 6 and 7, this pattern is quite distinct from the posterior hunchback pattern seen in Drosophila, which abuts the posterior border of eve stripe 7. The anterior giant pattern extends from the anterior pole to eve stripe 2, while the posterior pattern abuts the posterior border of eve stripe 7. In Drosophila, the posterior giant pattern extends from eve stripe 5 to stripe 7. The Kruppel pattern may be somewhat narrower in Anopheles than Drosophila. It encompasses eve stripe 3 in Anopheles but includes both stripes 3 and 4 in Drosophila. Finally, knirps exhibits the same limits of expression in Anopheles as Drosophila. In both cases, the staining pattern extends from eve stripes 4 to 6. In Anopheles, the anterior knirps pattern straddles the anterior border of eve stripe 1. Some of the eve stripes are associated with the same combinations of gap repressors in flies and mosquitoes (e.g., stripes 2, 3, and possibly 4), whereas others show distinctive combinations of gap repressors (e.g., stripes 5, 6, and 7 (Goltsev, 2004).
There are numerous differences in the patterns of gap gene expression in Drosophila and Anopheles. In Drosophila, the posterior stripe of hunchback expression is the source of a repressor gradient that specifies the posterior borders of eve stripes 6 and 7. Anopheles exhibits a distinct posterior staining pattern, with expression extending through stripes 6 and 7. It is therefore unlikely that Hunchback regulates these stripes as seen in Drosophila. Instead, the location of the posterior hunchback pattern suggests that it regulates the posterior border of eve stripe 5 in Anopheles. In Drosophila, this border is formed by Giant, but in Anopheles, the posterior giant expression pattern is restricted to the posterior pole where it abuts stripe 7. Thus, a combination of Kruppel and Giant defines the eve stripe 5 borders in Drosophila, whereas Kruppel and Hunchback might be used in Anopheles (Goltsev, 2004).
In Drosophila, eve stripes 6 and 7 are regulated by different concentrations of Knirps and Hunchback. Low levels of Knirps define the anterior border of stripe 7, while higher levels are needed to repress eve stripe 6. Conversely, low levels of Hunchback establish the posterior border of eve stripe 6, while higher levels regulate stripe 7. The position of the knirps expression pattern is consistent with the possibility that it defines the anterior limits of stripes 6 and 7, just as in Drosophila. However, the posterior borders of these stripes are probably not regulated by Hunchback. The expanded pattern of tailless expression seen in Anopheles might permit it to establish the posterior border of eve stripe 6 and possibly stripe 7. An alternative candidate for the posterior stripe 7 border is giant, which is expressed in a tight domain within the posterior pole. Consistent with this possibility is the observation that the posterior giant pattern comes on relatively late, and the posterior stripe 7 border is the last to form among the seven eve stripes. The reversal of the posterior hunchback and giant expression patterns, along with the expanded tailless pattern, strongly suggests that different combinations of gap repressors are used to define eve stripes 5, 6, and 7 in Drosophila and Anopheles (Goltsev, 2004).
An implication of the preceding arguments is that each of the seven eve stripes is regulated by a separate enhancer in Anopheles. Only five enhancers regulate eve in Drosophila since four of the seven stripes (3, 4, 6, and 7) are regulated by just two enhancers (3/7 and 4/6) that respond to different concentrations of the opposing Hunchback and Knirps repressor gradients. The change in the posterior hunchback pattern virtually excludes the use of this strategy in Anopheles. Thus, stripes 3 and 7 are probably regulated by separate enhancers since different combinations of gap repressors appear to define the stripe borders. Similar arguments suggest that stripes 4 and 6 are also regulated by separate enhancers (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).
It is suggested that ancestral dipterans contained an eve locus with separate enhancers for every stripe. Anopheles eve might represent an approximation of this ancestral locus. The consolidation of enhancers that generate multiple stripes was made possible by cross-repression of gap gene pairs. In Drosophila, there are mutually repressive interactions between Hunchback and Knirps, as well as between Giant and Kruppel. The use of these interacting gap pairs along with ubiquitous activators permits the formation of two stripes from a single enhancer. It is possible to envision two ways in which mutual cross-repression of these gap genes helps to establish the precise patterns of pair-rule gene expression: (1) it ensures that there are zones free of repressor activity on both sides of Kruppel (for the Kruppel and Giant pair) and Knirps (for the Knirps and Hunchback pair) domains; (2) it protects the patterns of pair-rule gene expression from mutations that could potentially shift the domains of gap gene expression. For example, a mutation that could shift the expression of Kruppel would simultaneously shift the expression of Giant always leaving a repressor-free zone where Eve stripes would be established. Therefore, the evolution of the eve locus depends on the changes in the preceding tier of the segmentation network: refinement in gap gene cross-regulatory interactions (Goltsev, 2004).
Finally, it is easy to imagine that certain dipterans have a single enhancer for stripes 2 and 5, rather than the separate enhancers seen in Drosophila. Perhaps, the symmetric repression of Giant and Kruppel is a relatively recent occurrence, only now creating the opportunity for consolidated expression of stripes 2 and 5 (Goltsev, 2004).
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).
Temporal patterning is an important aspect of embryonic development, but the underlying molecular mechanisms are not well understood. Drosophila neuroblasts are an excellent model for studying temporal identity: they sequentially express four genes (hunchback -> Krüppel -> pdm1 -> castor) whose temporal regulation is essential for generating neuronal diversity. hunchback -> Krüppel timing is regulated transcriptionally and requires neuroblast cytokinesis, consistent with asymmetric partitioning of transcriptional regulators during neuroblast division or feedback signaling from the neuroblast progeny. Surprisingly, Krüppel -> pdm1 -> castor timing occurs normally in isolated or G2-arrested neuroblasts, and thus involves a neuroblast-intrinsic timer. Finally, Hunchback potently regulates the neuroblast temporal identity timer: prolonged Hunchback expression keeps the neuroblast 'young' for multiple divisions, and subsequent downregulation allows resumption of Krüppel -> pdm1 -> castor expression and the normal neuroblast lineage. It is concluded that two distinct 'timers' regulate neuroblast gene expression: a hunchback -> Krüppel timer requiring cytokinesis, and a Krüppel -> pdm1 -> castor timer which is cell cycle independent (Grosskortenhaus, 2004).
It is concluded that hb is regulated at the transcriptional level in neuroblasts, based on strong correlation with active transcription (intron probe) and protein levels (antibody probe). In addition, hb transcription in GMCs and differentiated neurons, but at this point it cannot be determine if the correlation between protein and transcription is as tight as in the neuroblasts. This does not rule out a role for posttranscriptional regulation, however, to ensure a very short half-life of both hb mRNA and protein. There are predicted miRNA binding sites in the hb 3'UTR and protein degradation (PEST) motifs in the Hb protein that may be necessary to restrict Hb protein to the early portion of neuroblast lineages. There is ample precedent for posttranscriptional regulation of hb in both Drosophila early embryos and C. elegans, but only for translational repression. In Drosophila, Nanos represses hb translation in the early embryo via binding to its 3'UTR. In C. elegans, the hb ortholog hbl-1 regulates temporal identity as part of the heterochronic pathway, and hbl-1 is a target of microRNA regulation through its 3'UTR. It is concluded that precise regulation of hb transcription, coupled with a short half-life of hb mRNA and protein, leads to the observed restriction of Hb protein to the initial cell cycles of neuroblast lineages. Identification of the hb cis-regulatory sequences necessary for proper hb CNS expression has been initiated, and it will be interesting to determine the associated factors that positively and negatively regulate hb transcription in neuroblasts (Grosskortenhaus, 2004).
Cell cycle-arrested neuroblasts maintain hb expression. However, a direct role of the cell cycle (e.g., counting S phases) or an indirect role (e.g., generation of a GMC which could signal back to the neuroblast) has not been distinguished. This study shows that hb transcription is maintained in pebble mutant neuroblasts, which lack cytokinesis but nevertheless go through repeated cell cycles including DNA replication, nuclear envelope breakdown, chromosome condensation, and spindle assembly. Thus, the timely downregulation of hb transcription requires cytokinesis. The requirement for cytokinesis is consistent with two quite different mechanisms: (1) feedback signaling from the GMC to the neuroblast to repress hb transcription, and (2) asymmetric partitioning of an hb transcriptional activator into the GMC to halt hb transcription (Grosskortenhaus, 2004).
Which mechanism is used has not yet been distinguished. Two candidate transcription factors have been tested for a role in hb regulation: Hb and Prospero. The Hb protein does not positively regulate its own transcription in the CNS -- neither hb mRNA nor protein is partitioned into the GMC during neuroblast cell division. The Prospero transcription factor is known to be partitioned into the GMC during neuroblast division, but Prospero protein is cytoplasmic in neuroblasts, and thus unlikely to positively activate hb transcription in this cell type. In addition, misexpression of Prospero in neuroblasts is unable to extend the window of hb transcription and prospero mutants have normal hb expression in neuroblasts, although there is reduced Hb protein in GMCs and neurons by stage 13 and beyond. Thus, Prospero may have a role in maintaining hb transcription in GMCs and neurons, consistent with its nuclear localization in these cell types, but it is not required for timing of hb transcription in neuroblasts (Grosskortenhaus, 2004).
To investigate the role of feedback signaling from the GMC, it would be ideal to do GMC ablations and assay for extended hb transcription in the parental neuroblast, but this experiment is technically very demanding, and even short GMC-neuroblast contact might be enough for the signaling to occur. Whether the feedback signal is mediated by the Notch pathway, which is active in all neuroblasts and GMCs examined to date was tested: blocking the pathway with a sanpodo mutant has no effect on the timing of hb -> Kr -> pdm1 -> cas neuroblast expression. The identification of trans-acting factors that associate with the hb cis-regulatory DNA may be the best approach to distinguish between feedback signaling and transcription factor partitioning mechanisms (Grosskortenhaus, 2004).
Previous work provided strong hints that global extrinsic signals are not required for timing neuroblast temporal identity transitions. (1) Neuroblast lineages are asynchronous, with later-forming neuroblasts expressing hb at the same time adjacent early-forming neuroblasts are expressing cas, making it unlikely that global extrinsic signals trigger gene expression transitions. (2) In vitro culture experiments reported differentiated neuronal clones containing nonoverlapping populations of Hb+, Pdm1+, and Cas+ neurons, consistent with a normal progression of gene expression in the parental neuroblast over time, although gene expression timing was not assayed in neuroblasts. These observations have been confirmed and extended. Isolated neuroblasts progress from Hb+ to Kr+ to triple negative (presumptive Pdm1+) to Cas+ over time in culture, and clones in which the GMCs expressed a later gene than the neuroblast (e.g., Hb+ or Kr+ neuroblasts never had Pdm1+ or Cas+ GMCs). Thus, Hb -> Kr -> Pdm1 -> Cas neuroblast gene expression timing occurs normally in isolated neuroblasts, demonstrating that lineage-extrinsic factors are not required for neuroblast temporal identity transitions. It is possible that extrinsic cues may still override or entrain an intrinsic program, however, which could be tested by heterochronic neuroblast transplants. In summary, in vitro and in vivo data show that timing of temporal identity transitions is regulated by a neuroblast lineage-intrinsic mechanism. For the latter genes in the cascade, it appears that the mechanism is actually intrinsic to the neuroblast itself (Grosskortenhaus, 2004).
All available data suggest that Kr and Cas timing are regulated at the transcriptional level. Kr mRNA and protein are both detected in neuroblasts during embryonic stage 10 and subsequently maintained in a subset of neurons. Similarly, cas mRNA and protein are both widely detected in neuroblasts only at stage 12, and maintained in a subset of late-born neurons. In the future, it will be important to do mRNA/protein double labels for Kr, pdm1, and cas to determine the extent to which mRNA/protein levels are correlated at the single cell level. Unfortunately, it is not easy to assay for active transcription of Kr or cas due to the lack of large introns (Grosskortenhaus, 2004).
Surprisingly, it was found that cell cycle-arrested neuroblasts that lack Hb still express Kr -> Pdm1 -> Cas with the same timing as in wild-type embryos. What mechanism might time Kr -> Pdm1 -> Cas expression in the absence of cell division? Extrinsic cues can be ruled out, because isolated neuroblasts still undergo normal Kr -> Pdm1 -> Cas gene expression timing. A change in nucleo-cytoplasmic ratio, known to time certain early embryonic events, can be ruled out, because wild-type neuroblasts increase their nucleo-cytoplasmic ratio over time, but G2-arrested neuroblasts decrease their nucleo-cytoplasmic ratio as they enlarge without dividing (Grosskortenhaus, 2004).
The most attractive model for Kr -> Pdm1 -> Cas in G2-arrested neuroblasts is a cascade of transcriptional regulation between Kr, Pdm1, and Cas. Misexpression studies have shown that each gene can activate expression of the next gene in the series, and repress the 'next + 1' gene, which could account for the sequential activation of each gene. If each transcription factor can also repress its activator, similar to the known ability of Cas to negatively regulate pdm1 expression, it could explain the sequential downregulation of each gene as well. Currently, all misexpression data are consistent with this simple model. However, analysis of hb and Kr mutants reveals additional complexity. hb mutants show relatively normal Kr -> Pdm1 -> Cas timing, and Kr mutants show relatively normal Pdm1 -> Cas timing. Thus, there must be at least one unidentified input that can activate Kr in the absence of Hb, and pdm1 in the absence of Kr. Regulation of Hb -> Kr -> Pdm1 -> Cas appears to be primarily at the transcriptional level, and thus identification of the relevant cis-regulatory DNA and associated transcription factors should provide insight into the 'timer' mechanism that controls sequential gene expression in neuroblasts (Grosskortenhaus, 2004).
Hb seems to have a special role in advancing the temporal identity timer. It is the only factor in the cascade whose downregulation requires cytokinesis, and as long as it is present (either because of cell cycle arrest or misexpression) the timer is unable to advance. Misexpression of Hb beyond its normal expression window leads to generation of extra early cell types and blocks Kr -> Pdm1 -> Cas progression. However, these experiments do not reveal whether Hb generates these early fates by overriding Kr -> Pdm1 -> Cas neuronal identity while the temporal timer is advancing or if it arrests progress of the temporal timer. The results show that continuous expression of Hb blocks the advancement of the temporal identity timer, keeping the neuroblast in a 'young' state that is fully capable of resuming its normal cell lineage following downregulation of Hb. The ability of Hb to keep the neuroblast in a 'young' multipotent state, despite repeated rounds of cell division, raises the interesting question of how Hb acts at the mechanistic level. Transcriptional targets of Hb in the CNS are so far unknown. A mammalian homolog, Ikaros, is associated with chromatin and remodeling proteins and Drosophila Hb is thought to regulate chromatin-mediated heritable expression of homeotic genes. Thus, Hb might modulate chromatin structure in neuroblasts to prevent expression of later temporal identity genes, or to maintain plasticity of gene expression necessary for maintaining the multipotent state of the neuroblast (Grosskortenhaus, 2004).
Cellular competence is an essential but poorly understood aspect of development. Is competence a general property that affects multiple signaling pathways (e.g., chromatin state), or is competence specific for each signaling pathway (e.g., availability of cofactors)? This study has found that (1) Drosophila neuroblast 7-1 (NB7-1) has a single early window of competence to respond to four different temporal identity genes (Hunchback, Krüppel, Pdm, and Castor); (2) each of these factors specifies distinct motor neuron identities within this competence window but not outside it, and (3) progressive restriction to respond to Hunchback and Krüppel occurs within this window. This work raises the possibility that multiple competence windows may allow the same factors to generate different cell types within the same lineage (Cleary, 2006).
To determine whether NB7-1 undergoes progressive restriction in competence to respond to Kr, similar to that observed for Hb, pulses of Kr were generated at progressively later points in the NB7-1 lineage. Both hsp70-Kr and hsp70-hb were used to allow precise comparison of the effects of both genes. Progressively later pulses of Hb produce a decreasing frequency of U1/U2 neurons. Similarly, progressively later Kr pulses generate decreasing frequencies of extra U3 at each subsequent stage, with the exception of the earliest portion of the lineage, where Hb is known to be dominant to Kr. Thus, NB7-1 shows progressive restriction in competence to respond to both Hb and Kr, and competence to respond to both Hb and Kr is lost at the same point in the lineage (after five divisions) (Cleary, 2006).
An independent method was used to measure the competence window in the NB7-1 lineage. prospero-gal4 was used to induce expression of Kr within the NB7-1 lineage from the fourth division onward. When one copy of UAS-Kr was used at 22°C, which provides relatively low levels of Kr, only five to six Eve+ U neurons were observed, mostly U1, U2, and three U3 neurons (91%), but also U1, U2, and four U3 neurons (9%). Thus, NB7-1 loses competence to respond to prolonged Kr expression after five to six cell divisions, similar to results from the Kr pulse experiments described above. Prolonged expression of Hb using the same conditions (prospero-gal4, one copy of UAS-Hb, 22°C) also results in just five to six Eve+ U neurons. It is concluded that NB7-1 has a single competence window for generating U1-U3 neurons in response to Hb and Kr (Cleary, 2006).
Next to be tested was whether the later-expressed temporal identity factors Pdm and Cas share the same early competence window with Kr, or if they have distinct competence windows. Pdm specifies the U4 neuronal identity, while Pdm/Cas together specify U5 neuronal identity. scabrous-gal4 was used to prolong Kr expression for a variable length of time within the NB7-1 lineage (two copies of UAS-Kr at 29°C), which delayed but did not prevent the sequential expression of Kr, Pdm, and Cas. This experiment allowed NB7-1 competence to be assayed when presented with Kr, Pdm, or Cas at different times in its lineage (Cleary, 2006).
It was found that the scabrous-gal4 UAS-Kr embryos always had a total number of seven to eight Eve+ U neurons, although ectopic U3 neurons ranged from two to six in number. Interestingly, hemisegments with only two ectopic U3 neurons typically had U4/U5 neurons; those with three ectopic U3 neurons had only a U4 neuron, and those with four or more ectopic U3 neurons lacked both U4/U5 neuronal fates. These data are interpreted in the following way: in segments where Kr declines the fastest (fewest ectopic U3 neurons), there is time for Pdm to induce U4 fate and Pdm/Cas to induce U5 fates prior to loss of competence; however, in segments where Kr lasts the longest, both Pdm and Cas expression occur after the competence window and no U4/U5 fates are produced. Taken together, this experiment allows several conclusions to be drawn: (1) prolonged Kr expression can partially extend the neuroblast competence window (from five to six divisions to seven to eight divisions); (2) competence to respond to Kr, Pdm, and Cas is simultaneously lost at the end of this competence window, suggesting that there is a single competence window for responding to multiple temporal identity factors, and (3) each temporal identity factor specifies different U1-U5 motor neuron identities within the competence window, but not outside it. It is currently an open question as to how prolonged expression of one factor (Kr or Hb) can extend the competence window to respond to three distinct factors (Kr, Pdm, and Cas) (Cleary, 2006).
The previous experiment showed that prolonging Kr expression (scabrous-gal4 UAS-Kr) in NB7-1 lineage can only partially extend neuroblast competence. Interestingly, similar experiments prolonging Hb expression (scabrous-gal4 UAS-hb) revealed that the neuroblast maintains full competence for as long as Hb is expressed, in some cases over 15 divisions, with normal U3-U5 fates appearing after Hb levels decline. Thus, extended Hb expression (but not extended Kr expression) can maintain the neuroblast in a young, fully competent state. This raised the possibility that down-regulation of Hb is required for loss of neuroblast competence; alternatively, Hb may be more potent than Kr in maintaining neuroblast competency (Cleary, 2006).
To distinguish these models, the effect was tested of high-level Hb or Kr expression beginning at the fourth neuroblast division (prospero-gal4, 2x UAS-hb or UAS-Kr, 29°C), which would allow Hb down-regulation and permit comparison of the efficacy of Hb versus Kr in extending neuroblast competence. Performing this experiment with Hb resulted in a partial extension of neuroblast competence and the production of an average of 9.1 Eve+ U neurons: U1-U3, 6.1 extra U1, and no U4/U5. Performing the experiment with Kr resulted in an almost identical phenotype of 9.8 Eve+ U neurons: U1/U2, 7.8 U3s, and no U4/U5. Thus, Hb and Kr appear equally efficient at extending neuroblast competence; this is supported by their equivalent effect when expressed under heat shock or lower level prospero-gal4 control (competence lost after five divisions). More importantly, a comparison of the scabrous-gal4 UAS-hb and prospero-gal4 UAS-hb experiments shows that Hb down-regulation is critical for loss of neuroblast competence. When Hb is maintained from the beginning of the lineage (scabrous-gal4 UAS-hb), competence persists for the length of Hb expression, in some cases over 15 divisions; when Hb down-regulation occurs followed by permanent Hb re-expression one division later (prospero-gal4 UAS-hb), then competence is lost after approximately nine divisions. It is concluded that down-regulation of Hb, but not Kr, initiates progressive restriction in neuroblast competence that is normally complete after five divisions (Cleary, 2006).
Thus far, how neuroblast competence changes over multiple rounds of cell division was investigated. Now, how competence changes during neuronal differentiation is considered. Kr was expressed in high levels in the newborn post-mitotic U1-U5 neurons (eve-gal4 UAS-Kr). In these embryos, Kr is first detected just as the U1-U5 neurons are born. Despite high levels of Kr protein, no change in U1-U5 fate was ever detected. Conversely, transient expression of Kr in NB7-1/GMCs can occasionally generate ectopic U3 neurons that do not maintain Kr expression, despite the ability of Kr to positively autoregulate within the CNS. Thus, mitotic progenitors but not post-mitotic neurons are competent to respond to Kr. Similar results have been observed for competence to respond to Hb (Cleary, 2006).
These experiments, combined with previous studies, allow four major conclusions to be drawned.
eyeless is expressed both early and late in Bolwig's organ cells, which serve as the larval photoreceptor. The ey expression in Bolwig's organ occurs during embryonic development at the end of stage 12. Krüppel expression can also be detected in all 12 Bolwig's organ precursors, but whether ey and Krüppel are coexpressed in all precursors is unknown. ey is down-regulated and absence during most phases of Bolwig's organ development, which includes morphogenetic movement and axonal growth, elongation and projection (Sheng, 1997).
During Drosophila embryogenesis, mesodermal cells are recruited to form a complex pattern of larval muscles. The formation of the
pattern is initiated by the segregation of a special class of founder myoblasts. Single founders fuse with neighbouring nonfounder
myoblasts to form the precursors of individual muscles. Founders and the muscles that they give rise to have specific patterns of gene
expression, and it has been suggested that it is the expression of these founder cell genes that determines individual muscle attributes
such as size, shape, insertion sites and innervation. The segmentation gene Krüppel is expressed in a subset of founders
and muscles. Krüppel protein is expressed in a variety of muscles, including two dorsal muscles (the dorsal acute muscle 1 and the dorsal oblique muscle 1), three lateral muscles (including the lateral longitudinal muscle 1 and the lateral transverse muslces 2 and 4), and four ventral muscles (ventral longitudinal muscle 3, ventral acute muscle2 and ventral oblique muscles 2 and 5). Krüppel regulates specific patterns of gene expression in these cells, specifically the homeodomain gene NK1, also known as S59. Krüppel is required for the acquisition of proper muscle identity.
Gain and loss of Krüppel expression in sibling founder cells is sufficient to switch these cells (and the muscles to which they
give rise) between alternative cell fates. Thus Kr is not responsible for myogenic differentiation but for the specific characteristics of individual muscles. Ubiquitous expression of Kr does not alter the pattern of NK1 expression in muscle progenitors and the onset of NK1 expression is normal in the absence of Kr (Ruiz-Gomez, 1997).
Terminal divisions of myogenic lineages in the Drosophila embryo generate sibling myoblasts that act as founders for
larval muscles or form precursors of adult muscles. The formation of individual muscle fibers is seeded by a special class of founder myoblasts that fuse with neighboring mesodermal cells to form the syncytial precursors of particular muscle. Alternative fates adopted by sibling myoblasts are
associated with distinct patterns of gene expression. During normal development (embryonic stage 11), two ventrally located progenitor cells divide once to produce three muscle founders and the precursor of an adult muscle (known as a persistent Twist cell because of its continued expression of twist). The more dorsal of the two progenitors divides, first giving rise to the founders of muscles VA1 and VA2, followed by the more ventral progenitors which produce the VA3 founder and the ventral adult persistent Twist precursor (VaP). As the progenitors divide, Numb is included in one of the two dorsal progenitors and in one of the two ventral progenitors. Thus the division of a muscle progenitor produces an unequal distribution of Numb between the founders: one contains Numb, the other does not. In numb mutants, some muscles are lost and others are transformed. For example VA1 and VaP are duplicated and VA2 and VA3 are lost. Genes expressed in the progenitor cell are
maintained in one sibling and repressed in the other. Krüppel, S59 and even skipped expression mark a subset of the developing muscles. In numb mutants the expression of Krüppel, S59 and even skipped is initiated normally but is lost from both founder cells after they are formed. Thus in numb mutants there are no muscles that express Kr, eve or S59. In contrast, when numb is ectopically expressed throughout the mesoderm, Kr, S59 and eve expression are maintained in both founders and in the muscle precursors to which they give rise. In these embryos, Kr, S59 and eve-expressing muscles are duplicated (Gomez, 1997).
The Drosophila central nervous system contains many types of neurons that are derived from a limited number of progenitors as evidenced in the ventral ganglion. The situation is much more complex in the developing brain. The main neuronal structures in the adult brain are generated in the larval neurogenesis, although the basic neuropil structures are already laid down during embryogenesis. The embryonic factors involved in adult neuron origin are largely unknown. To shed light on how brain cell diversity is achieved, a study was carried out of the early temporal and spatial cues involved in the specification of lateral horn leucokinin peptidergic neurons (LHLKs). The analysis revealed that these neurons have an embryonic origin. Their progenitor neuroblast were identified as Pcd6 in the Technau and Urbach terminology. Evidence was obtained that a temporal series involving the transcription factors Kr, Pdm, and Cas participates in the genesis of the LHLK lineage, the Castor window being the one in which the LHLKs neurons are generated. It was also shown that Notch signalling and Dimmed are involved in the specification of the LHLKs. It is concluded that serial homologies with the origin and factors involved in specification of the abdominal leucokinergic neurons (ABLKs) have been detected (Herrero, 2013).
Studies on neuroblast lineages in the developing ventral ganglia are numerous, but
investigations of which lineages are present in cerebral ganglia and which are not have only
just begun. Drosophila neurogenesis takes place at two stages: an embryonic stage, in
which larval functions are established, and a larval stage, in which neurons involved in adult
functions are added. Temporal genes regulating the postembryonic neuroblast lineages in the
central brain and in the optic lobes have been identified, but little is known of brain neuroblast embryonic lineages. LHLK neurons offer the possibility of studying the embryonic origins of brain neurons and comparing them to the
origins of other lineages including LK-expressing progeny. This study shows that LK-expressing
neurons from different segments of brain and abdomen not only share neuropeptide
expression but also cell number per hemisegment and neuronal cell appearance, characterized by
long axons full of varicosities, large superficially located somas, but lack of coexpression of any
small neurotransmitters. The results obtained above provide clues for
defining the serial homology between neuroblasts from the protocerebrum and from the
ventral ganglia, and for analyzing differences between the complex combinatorial code that
defines the fates of LK-expressing neurons (Herrero, 2013).
The results suggest that the canonical temporal gene cascade Hb-Kr-Pdm-Cas-Grh is active in
protocerebral neuroblasts as it is in thoracic and abdominal neuroblasts. Consequently, as in
the VNC, temporal factors in the brain also activate the next gene and repress the 'next plus
one' or the previous one. These factors, except for Hb and Kr, are weakly expressed in LHLK
neurons at the early first instar larva, but the most important clues concerning their temporal
implications are the effects of their loss and gain of function: LHLK specification is partially
inhibited in kr and pdm mutants, and completely blocked in cas mutant. Only the grh mutant
has no phenotypic effect on LHLKs, although its overexpression does have a phenotype,
indicating that the Cas window is
negatively regulated by Grh. On the other hand, svp is also involved in LHLK specification,
probably not via its relation to hb but because it is expressed in another phase after the Cas
window, as in many embryonic abdominal neuroblast lineages. Although the
temporal factors implicated in the origin of LHLKs fit the model accepted for other NB lineages
in the embryonic CNS, more studies are required to provide
precise information about the timing of temporal factor expression and about the specification
of the other progeny in the lineage and in other embryonic brain lineages (Herrero, 2013).
The results obtained in dimm overexpression experiments demonstrate the existence of other
neurons with potential LK fates in the Drosophila brain. In this situation it seems that
expression of the neuroendocrine differentiation gene dimm forces the 'almost' leucokinergic
neurons to complete their differentiation. There are analogies with the results obtained for
FMRFamide, where ectopic FMRFamide expression in Tv neurons is
only observed when dimm is misexpressed. dimm is essential for transforming the synaptic
vesicles of neurons into functional peptidergic vesicles. This study
demonstrates that other neurons in the brain have the LK fate determinants but not the
ability to adopt the neuropeptidergic cell fate. Interestingly, the ectopic LK neurons found in
dimm overexpression correspond to different brain segments, namely deutocerebrum,
tritocerebrum and protocerebrum. This could be pointing to serial homology in some brain
lineages. Further analysis is needed to probe the LK fate in these segments (Herrero, 2013).
The two LK-expressing cell types share two main characteristics: the ventral-lateral location
within their segments and their embryonic origin.
LHLK neurons arise from a lineage located dorsally and near to the optic primordium,
which corresponds to the protocerebral dorsal central lineage in Urbach (2003)
terminology, or the basolateral dorsal lineage in Pereanu (2004) terminology.
ABLKs arise from abdominal NB5-5, which is laterally located in the
VNC, both are lateral in their respective segments, arise during embryonic neurogenesis and
start expressing LK at the end of stage 17 (Herrero, 2013).
There are some differences in terms of temporal genes between LHLK and ABLK
lineages. The analysis suggests that Cas is the temporal factor window specifying LHLK fate,
whilea Cas/Grh temporal window has been proposed for ABLKs. There is
evidence that the Cas window is long in some NBs of the trunk, and Cas has
also been identified in postembryonic brain development.
In the light of these findings it is proposed that, as in trunk neuroblasts, the Cas time window in
the neuroblast Pcd6 lineage is extensive and the Cas inhibitory effect of Grh is delayed with
respect to the abdominal segments. As a result, the LHLKs can be generated before grh
expression; so that this factor is dispensable for the appearance of LHLKs. Hence, Grh effects
on LHLKs are only observed when grh is overexpressed (Herrero, 2013).
Of the 27 genes, 7 were not expressed in either of the two types of LK neurons and their loss of function had different effects on their phenotypes. Three of these genes expressed in the ABLKs were hkb, gsb and ind, whose NB expression is weak in the protocerebrum. The
expression of other two genes, also expressed in the ABLKs: unpg and runt, is sustained until the end of embryogenesis in the postmitotic cells. However
ABLKs are controlled by the pair rule gene runt and the homeodomain gene unpg. It has been reported that runt regulates the expression boundaries of segment polarity genes in the VNC but not in the procephalon, while unpg, together with otd, is involved in
the protocerebrum/deutocerebrum interface in the procephalic neuroctoderm.
Hence these different functions could explain the different expression (Herrero, 2013).
Finally, ap and klu show extended brain expression in neuroblasts (klu) and in postmitotic neurons (ap) in the brain; however their effects are not the same in LHLKs and ABLKs: Ap regulates LK expression in LHLKs, while Klu does it in ABLKs. Xiao (2012) has shown that Klu is necessary in the brain for the renewal maintenance of type II
neuroblasts, whereas VNC type I neuroblasts are probably not affected because other factors
provide this function. Thus Klu has different functions in the brain and the VNC.
In spite of these differences, ABLKs and LHLKs do share the presence or absence of
expression of 19 genes, among which are not only the aforementioned temporal genes and
postmitotic cofactors nab and sqz, but the segment polarity gene wg. Just as engrailed (en)
marks the posterior border segment, wg marks the anterior one,
as in the trunk segment, although less obviously. Four cephalic
segments have been describe: intercalary, antennal, ocular and labral, the last two being part of the protocerebrum. The wg, en, gsb-d and hh segment polarity genes and the ind, msh, vnd
columnar genes mark some of their boundary. The ocular segment contains the largest
number of neuroblasts (60), and it is the most difficult to study because of its complexity. However it is clear that the anterior region of this neuromere is
extended the most, with more than 25% of the wg-expressing neuroblasts at stage 11. On the
other hand, the en expressing region is very much smaller (only 10 NBs). The LHLKs, like the
ABLKs, belong to an anterior segment lineage. ABLK-progenitor neuroblast expresses ind but LHLKs cannot be assigned to a particular columnar neuroblast
because the ocular segment has almost no ind identity. It may be
concluded that the neuroblasts Pcd6 and NB5-5, from which the LK-expressing neurons arise in
an equivalent temporal embryonic window, are serially homologous, although several
individual characteristics distinguish their development. In some of the serially homologous
neuroblast lineages of the VNC, there are differences between thoracic and abdominal
neuromeres, and it is expected that such segment-specific differences
would be more pronounced between the brain and the VNC where the genetic backgrounds
are different, and the canonical orthogonal expression genes described in the VNC are mainly
not conserved in the protocerebral neuromeres. Clarification of the
progression of the Leucokinin-progenitor neuroblasts during brain development and
comparison with the situation in the trunk could help in an understanding of what makes the brain
different from the VNC (Herrero, 2013).
Ion channel gene expression can vary substantially
among neurons of a given type, even though
neuron-type-specific firing properties remain stable
and reproducible. The mechanisms that modulate
ion channel gene expression and stabilize neural
firing properties are unknown. In Drosophila, this study
demonstrates that loss of the Shal potassium channel
induces the compensatory rebalancing of ion channel
expression including, but not limited to, the
enhanced expression and function of Shaker and
slowpoke. Using genomic and network modeling
approaches combined with genetic and electrophysiological
assays, it was demonstrated that the transcription
factor Kruppel is necessary for the homeostatic
modulation of Shaker and slowpoke expression.
Remarkably, Kruppel induction is specific to the
loss of Shal, not being observed in five other potassium
channel mutants that cause enhanced neuronal
excitability. Thus, homeostatic signaling systems
responsible for rebalancing ion channel expression
can be selectively induced after the loss or impairment
of a specific ion channel (Parrish, 2014).
This study provides evidence that the cell fate regulator Kr
is a critical player in the compensatory control of potassium
channel gene expression. It is speculated that the induction of
Kr drives a pattern of gene expression, first used to establish
neuronal identity in the embryo and then, postembryonically, to
rebalance ion channel expression in the face of persistent or
acute perturbation of the Shal channel. Surprisingly, Kr is
induced after the loss of Shal, but not other potassium channel
gene mutations that have been shown to cause neural hyperexcitability.
It is concluded that Shal function is specifically coupled
to a homeostatic feedback system that includes the Kr-dependent
transcriptional response. As such, these data imply the
existence of discoverable 'rules' that define how individual neurons
will respond to mutations in ion channel genes.
Recent work underscores the possibility that the regulation of
ion channel expression can be conserved from Drosophila to
mammalian central neurons. In Drosophila, the translational
regulator Pumilio was shown to be necessary and sufficient for
the modulation of sodium channel transcription after persistent
changes to synaptic transmission in the CNS. More recent data indicate that Pumilio-2
regulates NaV1.6 translation in rat visual cortical pyramidal
neurons in a manner consistent with that observed in Drosophila. In mammalian neurons, Kr-like genes (KLF)
respond to neuronal activity and are studied
intensively in the context of axonal regeneration, but a role in ion channel expression or homeostatic rebalancing
has yet to be defined (Parrish, 2014).
Kr and its homologs are potent regulators of neuronal cell fate.
KLF4 and KLF5, in particular, have been shown to both maintain
and reprogram embryonic stem cell fate (Sur, 2009). This study has provided
evidence that Kr protein levels diminish to nearly undetectable
levels in the postembryonic CNS. Kr expression is then induced
to achieve potassium channel regulation. It is tempting to speculate
that the rebalancing of ion channel expression postembryonically
is a reinduction of the embryonic mechanisms that
initially specify neuronal active properties (Parrish, 2014).
All three thoracic and five anterior abdominal segments are deleted in Krüppel mutants, and replaced by a mirror-image duplication of the normal posterior abdomen (Preis, 1985).
During Drosophila embryogenesis the Malpighian
tubules evaginate from the hindgut anlage and in
a series of morphogenetic events form two pairs of long
narrow tubes, each pair emptying into the hindgut
through a single ureter. Some of the genes that are involved
in specifying the cell type of the tubules have
been described. Mutations of previously described
genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules.
Of those ten, four block tubule development at
early stages; four block later stages of development, and
two, rib and raw, alter the shape of the tubules without arresting specific
morphogenetic events. Three of the genes, sna, twi, and
trh, are known to encode transcription factors and are
therefore likely to be part of the network of genes that
dictate the Malpighian tubule pattern of gene expression (Jack, 1999).
The transcription factors encoded by the genes Kr and cut
are required for the development of the Malpighian tubules.
Kr is required for the Malpighian tubule expression
of many genes including cut, which controls a subset
of the genes that are expressed specifically in the Malpighian
tubules. Other transcription
factors downstream of Kr must control genes that are
specifically expressed in the tubules but that do not require
ct activity.
Genes downstream of these transcriptional regulators
encode the proteins that control and execute the morphogenetic
events that form the Malpighian tubules. Some
genes required for the morphogenesis of the tubules have
been identified. The control of cell proliferation in the
tubules is one aspect of
morphogenesis. In addition, wingless is required to establish
the appropriate number of tubules, and the genes rib and raw are required
for proper shaping of the tubules. Although the Kr transcription
factor activates cut in the Malpighian tubules, it
may act through other factors. However, no mutation examined,
regardless of the effect on Malpighian tubule development,
blocks the expression of Cut protein in the
tubules. Furthermore, a survey of deletions that together
cover 70% of the genome found no deletion that causes
the loss of ct activity in the tubules although one of the
deletions blocks morphogenesis completely (Jack, 1999).
Neural precursors often generate distinct cell types in a specific order, but the intrinsic or extrinsic cues regulating the timing of cell fate specification are poorly understood. Drosophila neuroblasts sequentially express the transcription factors Hunchback->Krüppel->Pdm->Castor, with differentiated progeny maintaining the transcription factor profile present at their birth. Hunchback is necessary and sufficient for first-born cell fates, whereas Krüppel is necessary and sufficient for second-born cell fates: this is observed in multiple lineages and is independent of the cell type involved. It is proposed that Hunchback and Krüppel control early-born temporal identity in neuroblast cell lineages (Isshiki, 2001).
To begin investigating birth order dependent cell fate specification in the Drosophila CNS, the morphological and molecular features that distinguish early- versus late-born neurons were investigated. The axon projections and cell positions of first-born neurons were examined from datasets of DiI-labeled neuroblast clones, and find that first-born neurons typically occupy the deepest (most internal) position in the clone and have the longest axon projections of any cell in the clone (see also Brody, 2000). Conversely, later-born neurons lie in more superficial positions (nearest the ventral epithelium) and have relatively short projections (Isshiki, 2001).
What genes might regulate these birth order-specific neuronal properties? It is known that deep layer neurons are Hb+, middle layer neurons are Pdm+, while superficial layer neurons are Cas+ (Kambadur, 1998). These findings have been confirmed in this study and Kr is identified as a new deep layer transcription factor. Kr is weakly detected in Hb+ neurons, and strongly Kr+ neurons define a deep layer that lies between the Hb+ and Pdm+ layers. Some neurons showing coexpression of Kr/Pdm and Pdm/Cas are also detected. Tests were made to see whether Hb, Kr, Pdm, and Cas are expressed in a temporal order within neuroblasts at the time each layer of neurons is being generated. Indeed, sequential, transient expression of Hb -> Kr -> Pdm -> Cas in neuroblasts was observed that is 'stabilized' in progeny born during each window of gene expression (Kambadur, 1998; Brody, 2000; Isshiki, 2001). In addition, Pdm is transiently expressed in a subset of newborn Hb+ neuroblasts and their first-born GMCs, probably due to persistence of Pdm from the neuroectoderm, but it is usually not maintained in their Hb+ neuronal progeny (Kambadur, 1998; Isshiki, 2001).
Interestingly, the temporal expression pattern of Hb, Kr, Pdm, and Cas within neuroblasts parallels the spatial pattern of these genes during segmentation. Hb, Kr, Pdm, and Cas are detected in progressively more posterior domains at cellular blastoderm, respectively. Thus, the spatial order of these genes during segmentation is the same as their temporal order in neuroblasts, raising the possibility of a conserved gene cassette used in both segmentation and neurogenesis (Isshiki, 2001).
Identified neuroblast lineages were assayed to test the hypothesis that transient Hb -> Kr -> Pdm -> Cas expression in neuroblasts is stably maintained in neuronal progeny born during each window of gene expression. Three model neuroblast lineages were characterized, an early forming neuroblast (7-1) and two late-forming neuroblasts (7-3 and 2-4), where specific neuronal progeny were tracked from birth to differentiation. Although early- and late-forming neuroblasts begin their cell lineages hours apart, all show the same sequential, transient Hb -> Kr -> Pdm -> Cas pattern of expression (Isshiki, 2001).
Neuroblast 2-4 sequentially expresses Hb/Kr, Kr, Kr/Pdm, Pdm, Pdm/Cas, and Cas. GMCs and neurons with most of these expression patterns can be detected in deep to superficial layers of the CNS, respectively, except Kr+/Pdm+ or Pdm+ GMCs were rarely observed. In addition, GMC-1 is transiently Pdm+. To track gene expression patterns at the level of identified neuronal/glial progeny, gene expression was examined in the neuroblast 7-3 and 7-1 lineages (Isshiki, 2001).
Neuroblast 7-3 produces only three GMCs: GMC-1 generates the EW1 interneuron and the GW motoneuron, GMC-2 produces the EW2 interneuron and an EW2 sibling that rapidly undergoes programmed cell death, and GMC-3 differentiates directly into the EW3 interneuron. These neurons are referred to here by the abbreviated names of 1/1G, 2, 3 (respectively) to reflect their birth order. Neuroblast 7-3 sequentially expresses Hb, Hb/Kr, Kr, Kr/Pdm, Pdm; it is never Cas+. In addition, the newborn neuroblasts 7-3 and GMC-1 are transiently Pdm+. GMC-1 is Hb+ Kr+ and generates the Hb+ Kr+ 1/1G neurons; GMC-2 is Kr+ and generates the Kr+ interneuron 2, and GMC-3 is Pdm+ and generates the transiently Pdm+ interneuron 3 (Isshiki, 2001).
Neuroblast 7-1 generates over 20 GMCs, but only the first five GMCs express the nuclear marker Even-skipped (Eve). Each of the five Eve+ GMCs produces one Eve+ motoneuron (named U1, U2, U3, U4, U5, based on birth order) and one Eve- sibling neuron which cannot be tracked. Neuroblast 7-1 sequentially expresses Hb/Kr, Kr, Kr/Pdm, Pdm, and Pdm/Cas. The Hb+ Kr+ neuroblast produces two Hb+ Kr+ GMCs which develop into Hb+ Kr+ U1 and U2 motoneurons; the Kr+ neuroblast generates a Kr+ GMC-3 which produces the Kr+ U3 motoneuron; and GMC-4 and GMC-5 are born during the Pdm+ and Pdm+ Cas+ phases of gene expression, respectively, and produce the Pdm+ U4 motoneuron and the Pdm+ Cas+ U5 motoneuron (Isshiki, 2001).
Three conclusions are drawn from this detailed gene expression analysis: (1) nearly all of the 30 known neuroblasts go through an invariant temporal pattern of Hb -> Kr -> Pdm -> Cas gene expression, including early-forming and late-forming neuroblasts; (2) Hb -> Kr -> Pdm -> Cas gene expression is transient in neuroblasts, but is maintained in differentiated neuronal progeny; (3) Hb -> Kr -> Pdm -> Cas gene expression is correlated with birth order and not a particular cell type. For example, Hb+ progeny are all early-born, but can differentiate as interneurons, motoneurons, or glia depending on their parental neuroblast (Isshiki, 2001).
In the wild-type neuroblast 7-3 lineage, the first-born GMC and its 1/1G neuronal progeny are Hb+ Kr+; later-born interneurons 2 and 3 do not express Hb. In addition, the molecular markers Zfh-1, Zn finger homeodomain 2 (Zfh-2), and the neurotransmitter corazonin label different subsets of these neurons. In embryos specifically lacking Hb in the CNS (hb CNS mutants, either a duplication of interneuron 2 at the expense of the first-born 1/1G neurons is observed (11%), consistent with a duplication of GMC-2 fate, or a specific loss of the first-born 1/1G neurons (89%) is observed (Isshiki, 2001).
In the wild-type neuroblast 7-3 lineage, the first-born GMC-1 and its 1/1G neuronal progeny are Hb+ Kr+, while the second-born GMC-2 and interneuron 2 are Hb- Kr+. In embryos lacking Kr CNS expression (Kr CNS mutants), the first-born 1/1G sibling neurons are variably affected: both can be missing (17%), 1G can be missing (73%), or both can be normal (10%); however, the second-born interneuron 2 is always missing (100%) and the third-born interneuron 3 is almost always normal (90%). Absence of interneuron 2 could be due to cell death or due to skipping of the GMC-2 fate. In contrast, when neuroblast 7-3 is forced to continuously express Kr ('UAS-Kr,'), there are extra cells in the lineage (4-8; average of 5.5), and all but two cells differentiate as GMC-2 derived interneuron 2; the two unaffected cells are the GMC-1 derived 1/1G neurons. It is concluded that Kr contributes to GMC-1 development (where it is expressed with Hb) and is essential for GMC-2 development (where it is expressed without Hb); that continuous Kr can transform all progeny except GMC-1 toward a GMC-2 fate, and that continuous Kr leads to an extension of the neuroblast cell lineage (Isshiki, 2001).
In the wild-type neuroblast 7-1 lineage, the first two GMCs and their U1/U2 motoneuron progeny are Hb+ Kr+, GMC-3 and its U3 motoneuron progeny are Kr+, and subsequent GMCs do not express Kr. In Kr CNS mutants, one of the U3/U4 motoneurons is frequently missing (73%), although all earlier- and later-born neurons develop normally. It is suspected that the missing neuron is the normally Kr+ U3, based on cell position, but markers to distinguish U3/U4 in Kr mutant embryos are not available. As in the 7-3 lineage, the Kr phenotype may arise through cell death or a skipping of the GMC-3 fate. In contrast, continuous expression of Kr in neuroblast 7-1 results in extra Eve+ neurons (8-14; average of 10.0) with all differentiating as U3 or U4 motoneurons, except the normal pair of early-born U1/U2 motoneurons. It is suspected all neurons have the normally Kr+ U3 fate, but markers are not available to distinguish U3/U4 fates. It is concluded that Kr is necessary for U3 motoneuron development, and that continuous Kr can transform most or all 7-1 progeny, except the first-born U1/U2 neurons, toward a U3 neuron fate (Isshiki, 2001).
Accurate temporal regulation of Hb, Kr, and Cas is critical for proper CNS development, so it is important to determine the mechanisms that regulate sequential gene expression in neuroblasts. Regulatory interactions between Hb, Kr, Pdm, and Cas can be detected using misexpression assays: overexpression of Hb activates Kr and represses Pdm and Cas; overexpression of Kr activates Pdm, represses Cas, but has no effect on Hb expression; and Pdm positively regulates Cas expression (Brody, 2000), leading to the model that each gene can activate the next gene in the pathway and repress the 'next plus one' gene. These interactions are not necessary for driving sequential gene expression, however, as it is observed that hb, Kr, or cas mutations have only subtle alterations in the remaining gene expression profiles, and loss of hb or Kr does not appear to affect the fate of cells born later in the lineage based on existing markers. Thus, an independent pathway must also drive the sequential expression of Hb -> Kr -> Pdm -> Cas in neuroblasts. This mechanism involves cell cycle progression, directly or indirectly, because newly formed neuroblasts remain Hb+ Kr+ if they are cell cycle-arrested before their first division. They rarely if ever make the transition to Kr+ Hb-, Pdm+, or Cas+ (Weigmann, 1995 and Cui, 1995). It is concluded that a cell cycle-dependent 'clock' is required to drive the transitions in Hb -> Kr -> Pdm -> Cas gene expression (Isshiki, 2001).
The data do not support a model in which global temporal cues trigger gene expression transitions simultaneously in all neuroblasts. Most of the 30 neuroblasts, including the earliest neuroblasts to form (e.g., 7-1 and 7-4) and some of the latest neuroblasts to form (e.g., 2-4 and 7-3), go through the same Hb -> Kr -> Pdm -> Cas gene expression cascade. Exceptions are neuroblasts 2-1, 3-3, 5-1, and 6-1 (which start with Kr, Pdm, or Cas). Thus, early-forming neuroblasts can generate Cas+ progeny at the same time that late-forming neuroblasts produce Hb+ progeny (Isshiki, 2001).
A model is favored in which the timing of Hb -> Kr -> Pdm -> Cas expression is regulated primarily by a cell cycle-dependent clock but also by regulation within the Hb -> Kr -> Pdm -> Cas pathway (Kambadur, 1998). Evidence for the latter mechanism is that misexpression studies show that Hb, Kr, Pdm, and Cas typically activate the next gene in the pathway and repress the 'next plus one' gene in the pathway; and that loss of function mutations can result in premature expression of later genes in the pathway and the skipping of GMC fates. Evidence for the cell cycle-dependent clock mechanism is that hb and Kr mutants have relatively subtle changes in hb, Kr, pdm, or cas expression or in later-born GMC fates; that cell cycle arrested neuroblasts remain Hb+ Kr+ and fail to make a transition to Hb- Kr+, Pdm+, or Cas+; and that when neuroblast 1-1 is cell cycle arrested for several hours prior to its first cell division and then triggered to divide, it will produce an Eve+ GMC-1 instead of a later-born Eve- GMC (Weigmann, 1995), highlighting the importance of the cell cycle progression rather than developmental time in regulating GMC identity (Isshiki, 2001).
All early developing neuroblasts, such as 7-1, produce two Hb+ GMCs before downregulating Hb expression, whereas many late developing neuroblasts, such as 7-3, produce just one Hb+ GMC. How do neuroblasts regulate the number of Hb+ GMCs produced? All neuroblasts could express Hb for the same length of time but have different cell cycle rates, or all neuroblasts may have the same cell cycle rate but vary the length of Hb expression (Isshiki, 2001).
Hb, Kr, and Cas are transiently expressed in neuroblasts but maintained in GMC and neural progeny (Pdm can be transient or stable in neuroblast progeny, depending on the lineage) (Kambadur, 1998; Brody, 2000; Isshiki, 2001). By inheriting and maintaining the gene expression profile of their parental neuroblasts, GMCs can 'memorize' their birth order. This seems a powerful and efficient way for stem cells to make a variety of fate-restricted progeny in invariant sequence. A similar mechanism may be used during vertebrate cortical and retinal development, where precursors transiently express genes that are maintained in a subset of differentiated progeny. It is currently unknown what distinguishes transient neuroblast expression from persistent GMC/neuronal expression (Isshiki, 2001).
Loss of Hb or Kr from early-born GMCs results in loss or transformation of neurons normally derived from these GMCs, but later-born neurons develop normally based on nuclear markers, neurotransmitter expression, and axon projections. Neuronal loss in hb and Kr mutants is likely due to multiple mechanisms: (1) Cell death. Necrotic Eve+ or Eg+ neurons can be detected in the 7-1 or 7-3 lineages of Kr CNS mutants, supporting a cell death model. Although necrotic Eve+ or Eg+ neurons in hb mutants have not been detected, more early 7-3 lineages are seen in which three Prospero+ GMCs are born than mature 7-3 lineages with progeny from all three GMCs, suggesting that cell death also occurs in hb mutants. (2) GMC skipping (i.e., the neuroblast skips a GMC fate without duplicating a later-born fate). In Kr mutants, neuroblast 7-3 typically produces two Prospero+ GMCs at the time GMC-1 and GMC-2 are normally born, consistent with a skip of GMC-2 fate. Similarly, hb mutants often produce only two Prospero+ GMCs in the 7-3 lineage, consistent with a skip of the GMC-1 fate. It is not possible to assay for GMC skipping in the 7-1 lineage. (3) GMC transformation. hb mutants clearly show transformation of GMC-1 to GMC-2 fate in the 7-3 lineage, based on the observed duplication of interneuron 2 at the expense of the 1/1G sibling neurons. This phenotype may arise if the endogenous Kr in GMC-1 is sufficient to induce GMC-2 fate in the absence of Hb. In Kr mutants, a similar transformation of GMC-2 into the GMC-3 fate is never observed, perhaps because the Kr- GMC-2 does not prematurely express Pdm. Thus, there is evidence that hb mutants show all three phenotypes, GMC death, skipping, and transformation, while Kr mutants show only GMC death and skipping phenotypes (Isshiki, 2001).
Misexpression of Hb or Kr can transform all GMCs toward a first-born or second-born fate, respectively. This is likely to be a transformation of GMC identity, rather than an increase in the proliferation of early-born GMCs, because the extra early-born neurons are produced at the expense of the later-born neurons in the lineage, and the extra cells are not reduced in size (extra embryonic divisions lead to reduced cell size). Interestingly, Kr misexpression fails to transform early-born Hb+ GMCs into the later-born Kr+ fate. Kr is induced early enough to affect GMC-1 in the 7-3 lineage because GMC transformations are seen in other lineages (e.g., 7-1) prior to the time GMC-1 is born in the 7-3 lineage, yet it has no effect. The model that first-born Hb+ fates are dominant over second-born Kr+ fates is favored. In the future, cell type-specific Hb misexpression studies could be used to determine precisely when birth order-specific cell fates become fixed: in neuroblasts, GMCs, or neurons? It would also be interesting to determine if a pulse of Hb expression midway through a neuroblast lineage is sufficient to induce first-born cell fates, and if so, does the temporal program resume or reset to the beginning of the lineage after the Hb pulse ends (Isshiki, 2001)?
hb, Kr, pdm, and cas are not the only genes controlling temporal identity in neuroblast lineages. Some markers for first-born fate occur normally in hb mutants, such as Eve expression in the 1-1 and 4-2 lineage. Moreover, Hb misexpression may not fully transform every cell in the 7-3 lineage to a first-born fate: a full transformation would produce equal numbers of interneuron 1/motoneuron 1G siblings, but typically only 2-3 motoneurons and 5-6 interneurons are seen, suggesting that only the first 2-3 GMCs are fully transformed to a first-born fate. Finally, in most lineages, there are GMCs produced after Cas expression ends; additional genes such as grainyhead (Brody, 2000) may specify the temporal identity of these GMCs (Isshiki, 2001).
Hb is expressed in virtually all first-born GMCs, and these can differentiate into motoneurons, interneurons, or glia, depending on the neuroblast lineage. Similarly, high level Kr is detected in virtually all second-born GMCs (i.e., the GMCs following the Hb+ GMCs), and they can differentiate into motoneurons, interneurons, or glia. Not only are Hb and Kr expressed in multiple cell types, but they are necessary and sufficient for the proper cell fate specification of motoneurons, interneurons, or glia, depending on the neuroblast lineage (Isshiki, 2001).
An extremely interesting question is how GMC 'temporal identity' (regulated by Hb and Kr) is coordinated with individual 'neuroblast identity' to achieve the proper sequence of cell types that characterizes each neuroblast lineage. The Hb protein, its putative mammalian ortholog Ikaros, and the mammalian Kr-related EKLF protein all associate with chromatin remodeling proteins, and both Hb and Kr are thought to regulate chromatin-mediated heritable expression of homeotic genes. Thus, Hb and Kr may modulate chromatin structure such that different subsets of genes are accessible for transcription in first-born versus second-born GMCs, with the palette of genes expressed by a first-born or second-born GMC, dependent on the neuroblast-specific transcription factors they inherit. In this manner, neuroblast identity might be integrated with GMC temporal identity to create the unique cell types characterizing each neuroblast lineage. This is conceptually similar to homeotic genes and tissue-specific genes working together to uniquely specify distinct cell types in each tissue at different anterior-posterior levels of the body axis (Isshiki, 2001).
When neuroblast 7-3 is forced to continuously express Hb or Kr, it generates an extended lineage of up to ten neurons instead of four neurons and one programmed cell death. The increase does not appear to be due to an extra round of cell division by the normally postmitotic neurons because the extra cells are produced at the expense of later-born cell types and because smaller cells are not seen (as expected, since extra cell divisions in the embryo lead to smaller cell size. It is proposed that Hb or Kr misexpression results in production of extra GMCs, and it is suggested that each neuroblast has an intrinsic mechanism for triggering quiescence that is related to successful transition from Hb and Kr expression to later genes in the hierarchy. These data also show that Hb and Kr can regulate features of neuroblast cell biology (cell cycle control) in addition to regulating GMC temporal identity (Isshiki, 2001).
The temporal gene expression in neuroblasts (early to late: Hb -> Kr -> Pdm -> Cas) mimics the major domains of gene expression at cellular blastoderm (anterior to posterior: Hb -> Kr -> Pdm -> Cas). Additional studies will be needed to discern common and distinct regulatory features between Hb, Kr, Pdm, and Cas expression during segmentation and neurogenesis. The expression of all genes at both stages of development raises the question of which function is ancestral. Hb is detected in the CNS of various arthropod, leech, and C. elegans embryos. In mammals, Hb-related genes of the Ikaros family are best known for regulating immune development, but some also show CNS expression. A mammalian Pdm homolog, SCIP/Oct-6, is expressed in specific cortical layers of the brain, and a mammalian Cas ortholog exists but has not been characterized. It will be interesting to determine whether genes regulating temporal identity in Drosophila neuroblasts have similar functions in the mammalian CNS or immune system (Isshiki, 2001).
The body wall musculature of the Drosophila larva consists of a stereotyped pattern of 30 muscles per abdominal hemisegment that are innervated by about 40 distinct motoneurons. Proper innervation by motoneurons is established during late embryogenesis. Guidance of motor axons to specific muscles requires appropriate pathfinding decisions as they follow their pathways within the central nervous system and on the surface of muscles. Once the appropriate targets are reached, stable synaptic contacts between motoneurons and muscles are formed. Recent studies have revealed a number of molecular components required for proper motor axon pathfinding and demonstrated specific roles in fasciculation/defasciculation events, a key process in the formation of discrete motoneuron pathways. The gene capricious (caps), which encodes a cell-surface protein, functions as a recognition molecule in motor axon guidance, regulating the formation of the selective connections between the SNb-derived motoneuron RP5 and muscle 12. Krüppel (Kr), best known as a segmentation gene of the gap class, functionally interacts with caps in establishing the proper axonal pathway of SNb including the RP5 axons. The results suggest that the transcription factor Krüppel participates in proper control of cell-surface molecules which are necessary for the SNb neurons to navigate in a caps-dependent manner within the array of the ventral longitudinal target muscles (Abrell, 2001).
Use has been made of a Kr gain-of-function mutation, termed Irregular facets (If), to identify modifiers of Kr activity during eye morphogenesis. One suppressor of ectopic Kr activity in the eye, the P-element insertion l(3)02937, was found to reside within the gene caps. caps encodes a CAM of the 'leucine-rich repeat' family and has been shown to be required for proper pathfinding and synapsing of the RP5 motoneuron with muscle 12. In addition to l(3)02937, a second P-element insertion, l(3)05121, was identified that resides in the first exon of the caps gene. Genetic analyses showed that the two P-element insertions failed to complement each other and the previously identified caps65.2 null-mutation, indicating their allelism to caps. Furthermore, precise excision of the P-element insertion l(3)05121 results in a reversion of the caps mutant phenotype to wildtype, indicating that the P-element insertion is the cause of the mutation. Moreover, the phenotype caused by the newly identified caps alleles is indistinguishable from the caps65.2 loss-of-function phenotype, suggesting that they represent either strong hypomorphic or lack-of-function caps mutations (Abrell, 2001).
While the innervation of muscles occurs during embryogenesis, previous studies on caps function have focussed on mutant defects that were observed in the motoneuronal pattern of third instar larvae, showing that Caps is necessary for proper pathfinding of the RP5 axons. RP5 axons are part of the SNb fascicle. During embryogenesis, the SNb enters the ventral muscle field between muscle 15 and 28 (choice point 'target entry'). Its axons pass the ventral oblique muscle field towards the ventral longitudinal muscle targets in the most internal muscle layer. Close to the second choice point of the SNb nerve, the RP3 axon separates from SNb to target the cleft between muscles 7 and 6. The SNb continues along muscle 14 and enters the ventral longitudinal muscle field. At a position close to muscle 30, the RP1 and RP4 axons separate to target muscle 13. The remaining RP5 bypasses muscle 13 to synapse with muscle 12. This muscle as well as the RP5 axons are characterized by caps expression both in the embryo and in larvae (Abrell, 2001). The early route of SNb pathfinding and the defasciculation patterns of RP1, RP3 and RP4 are not affected in caps homozygous larvae, whereas the pathway of RP5 is altered. RP5 loses its target specificity and synapses with both muscle 12 and 13 instead of muscle 12 only. Furthermore, overexpression of caps in all muscles causes the same phenotype. These results were taken to indicate that caps plays an important role in selective target recognition and synapse formation by the motoneuron RP5 (Abrell, 2001).
In order to see whether and to what extent caps affects neuromuscular connectivity in the embryo, the phenotypes of the different caps mutants were examined. Of a total of 111 homozygous caps65.2 mutant individuals examined, all embryos developed into normal looking larvae. However, the majority of the caps65.2 mutant larvae (62%) failed to hatch. Out of 81 hatched larvae examined, only two individuals survived to pupal stages and developed into adults. Similar results were obtained with homozygous capsl(3)02937 (64% unhatched larvae; 3% adults) and capsl(3)05121 (73% unhatched larvae; 3% adults) mutants. The two newly identified caps alleles are therefore similar in strength to caps65.2, previously shown to be a lack-of-function mutation. Moreover, these data indicate that caps functions primarily during embryogenesis and that previous studies on caps function in larvae have been performed with the fraction of mutants that develop into larvae (Abrell, 2001).
SNb development was examined in homozygous mutant embryos using monoclonal antibodies directed against the CAM FasII. In all three caps mutants, the SNb enters the ventral muscle field and the RP axons defasciculate normally. Furthermore, RP1, RP3 and RP4 properly synapse with their respective target muscles, whereas the RP5 axons stall, show enlarged growth cone-like structures and fail to contact the target muscle 12. Instead, the RP5 axons are split and found also in direct contact with the transversal nerve (TN), a link never observed in wildtype. These phenotypes suggest that pathfinding of RP5 axons during embryogenesis is retarded and in cases where the RP5 axons defasciculate and elongate, they show erratic targeting and synapsing, as has been described for third instar larvae (Abrell, 2001).
The specific SNb defect of the caps mutants on RP5 pathfinding and its failure to synapse exclusively with muscle 12 correlates with the observation that both RP5 neurons and their target muscle are characterized by caps expression both in embryos and larvae. In addition, overexpression of caps in all muscles causes a phenotype similar to loss-of-function mutations, suggesting that relative levels of Caps are important for pathfinding. Thus, both lack-of-function and gain-of-function studies indicate a specific role for caps in selective target recognition and synapse formation. The results show that this function of caps is required during embryogenesis and that the previously reported phenotype observed in third instar larvae represents only a weak phenotype common to escapers. In all other cases, impairment of caps activity causes the RP5 axons to stall immediately after defasciculation from the SNb fascicle. This observation indicates that caps is a critical component specifically required for RP5 pathfinding after the defasciculation of the RP5 axons from SNb has occurred (Abrell, 2001).
caps is expressed in both motoneuron RP5 and its synaptic target muscle 12 in third instar larvae. In view of the embryonic caps mutant phenotypes, it was asked whether caps is also expressed during early embryogenesis and whether Kr (which is expressed in neuroblasts and in a distinct subset of embryonic muscles) could be responsible for the control of caps expression (Abrell, 2001).
caps is indeed expressed in neuroblasts. However, this aspect of the caps expression pattern was not altered in Kr lack-of-function mutant embryos except in the central region which is distorted due to the earlier segmentation function of Kr. caps expression was monitored in Krres mutant embryos in which the early segmentation function of Kr is specifically rescued. No difference was observed in the caps expression patterns of Krres mutant and wildtype embryos and the Kr neural expression pattern was unchanged in caps mutant embryos, indicating that Kr and caps activities are independently controlled. Since caps acts as a dose-dependent modifier of ectopic Kr activity during eye formation, it was next asked whether later aspects of the expression patterns, caps expression in RP5 neurons and their target muscles and Kr expression in a subset of muscles along the SNb pathway, might reflect the need of the two gene activities for the proper guidance of SNb axons (Abrell, 2001).
Previous work has shown that in the absence of Kr activity, the SNb stalls at the second choice point and RP axons fail to defasciculate. This observation is consistent with the proposal that the two genes act in the same genetic pathway. In order to test this proposal, genetic interaction studies were performed using capsl(3)05121 and Krres mutant combinations, asking whether a reduction of Kr and caps activities causes defects in neuromuscular connectivity (Abrell, 2001).
Heterozygous Krres/+ or capsl(3)05121/+ single mutant embryos developed a normal motoneuron pattern. Each of the RP axons was properly connected to its target muscle as revealed by anti-FasII antibody staining. Thus, a reduction of either Kr or caps activity has no effect on motoneuron development and pathfinding. In contrast, double heterozygous Krres/+;capsl(3)05121/+ embryos, where the dosage of both genes was reduced at the same time, develop a specific SNb nerve phenotype without affecting the ISN and SNa. In about one-third of the cases, the SNb stop along ventral longitudinal muscles, ending with a large growth cone-like structure. In addition, properly defasciculated RP axons fail to continue along their normal paths; a portion of them elongate and stall either in a position very close to the TN or is directly connected to it. Double homozygous Krres;capsl(3)05121 mutants develop an even stronger phenotype; the SNb is absent in most of the double mutants analysed or does not extend beyond its second choice point close to muscle 28. In only few cases, the SNb stalls in the ventral muscle field as had been described for homozygous Krres embryos (Abrell, 2001).
These phenotypes were also obtained with similar frequencies in double homozygous Krres;caps65.2 mutants, indicating that the phenotype is not dependent on a particular caps allele. The results show in addition that the defects are stronger and more pronounced in double mutant embryos than those obtained with single mutant embryos (Abrell, 2001).
The failure to detect the SNb nerve in the majority of homozygous Krres;capsl(3)05121 mutant embryos correlates with a thickening of the ISN. In order to test whether SNb might have lost its identity due to a transformation into ISN identity, the SNb-derived RP neurons were labelled by virtue of the transgenic islH-tau-myc marker gene. The results show that the RP axons were present. However, the SNb fails to separate from the ISN or in the cases separation occurs, it stalls shortly after the defasciculation. These observations indicate that the SNb is not transformed into ISN identity and suggest that the SNb has lost the capability to respond to guidance cues such as CAMs and repellents. Moreover, the results indicate that Kr and caps activities cooperate in a synergistic fashion necessary for proper defasciculation of the SNb axons at the exit junction and for RP axon guidance in the ventral muscle field (Abrell, 2001).
Previous studies have shown that overexpression of caps in all neurons causes a specific misrouting or stalling of RP5 at the second choice point near muscle 30 in about one-third of the embryos. This effect of panneural caps expression is dependent on the extracellular domain of Caps, suggesting that Caps functions as a cell-adhesion component which participates in the guidance of the SNb at the specific choice point near muscle 30. In contrast, panmuscular expression of caps has no effect on SNb guidance and pathfinding, but severely interfers with synapsing of RP5 resulting in connections being formed not only with muscle 12, but also with the neighboring muscle 13 (Abrell, 2001).
In order to investigate whether misexpression of Kr can interfere with SNb formation, Kr was ectopically expressed in all motoneurons or muscles using the Gal4/UAS system. To achieve this, the ftzNG-Gal4 ('panmotoneuronal expression) and the 24B-Gal4 driver lines ('panmuscular expression') were used in combination with one and two copies of UAS-Kr transgenes. Panmotoneural Kr expression from one transgene in wildtype embryos results in a minor phenotype of the SNb, in which the distal RP axons fail to reach their target muscles and maintain growth cone-like structures at their ends. Panmotoneural Kr expression from two transgenes causes a stronger phenotype. In 36% of the cases, the most distal RP axons stalls and the RP5 axon does not innervate the target muscle 12, whereas in all other cases, the SNb stalls at the second choice point, a phenotype that is similar to the Krres homozygous mutant phenotype. This observation indicates that the Kr overexpression phenotype is dosage-dependent and that both the lack-of-function and gain-of-function effects of Kr interfere with SNb development. Since Kr is a cell-autonomous transcription factor, it is likely that it is required for and can interfere with the transcription of motoneuronal genes necessary for proper motoneuronalguidance (Abrell, 2001).
In the wildtype embryos, Kr is not only expressed in the nervous system but also in specific subsets of muscle founder cells and muscles. Relevant sites of Kr expression during the formation of neuromuscular connectivity are the ventral oblique muscles 14 and 16, the ventral acute muscle 27 and the ventral longitudinal muscles 6, 7 and 13. Heterozygous Krres mutant embryos develop a normal muscle pattern, whereas in homozygous Krres mutant embryos muscle 27 is transformed into a duplicated muscle 26. The other muscles that normally express Kr either appear to be normal or develop a variably altered morphology. Overexpression of four copies of Kr in all muscles leads to the reverse result, i.e. muscle 26 is transformed into a second muscle 27. Thus, Kr is necessary to determine muscle identity and can alter muscle fate upon ectopic expression in muscles that normally do not express the gene (Abrell, 2001).
Pan muscular expression of Kr in response to one or two copies of the 24B-driven Gal4/UAS-Kr cDNA transgenes does not disturb the muscle pattern. However, it has severe consequences for the formation and defasciculation of SNb. With one copy of Kr, the RP3 axon separates properly from SNb and succeeds in finding the cleft between target muscles 7 and 6, whereas the remaining RP axons fail to defasciculate. In the majority of cases, however, the SNb passes the ventral oblique muscles and enters the ventral longitudinal muscles normally, but it stalls at the second choice point and RP axons fail to defasciculate. Two copies of transgene-derived Kr expression in all cases cause stronger defects that the SNb stalled at the position where RP3 would normally defasciculate. The same Kr-dependent phenotypes are found in response to a different panmuscular Gal4 driver, namely the twi Gal4 line. Thus, in contrast to panmuscular expression of caps, panmuscular expression of Kr appears to interfere with a muscle-specific program that regulates defasciculation of the RP axons and/or the elongation of SNb after the second choice point has been reached. The finding is consistent with the argument that Kr determines the spectrum of molecules expressed in muscles that are used to transmit signals to other cells, namely cell-surface and secreted molecules (Abrell, 2001).
In order to test whether panmuscular Kr expression interferes with SNb guidance in a Kr- and caps-dependent manner, the phenotype was examined of double homozygous Krres; capsl(3)05121 mutant embryos that receive panmuscular Kr expression. Upon panmuscular Kr expression, the SNb is absent in 70% of the cases. In the other cases, the SNb had formed and separated from the ISN, but stalled in the region of the ventral longitudinal muscles. The observed phenotype is reminiscent of the phenotype obtained with Krres;capsl(3)05121 double homozygous mutants, but a higher proportion of axons defasciculate in the exit junction region. This observation indicates that panmuscular Kr expression can partially rescue the SNb phenotype of the double homozygous mutants. However, defasciculation from the stalled SNb occurs in an erratic manner, implying that axon guidance is still strongly impaired. The lack of a better rescue is likely to be due to the fact that Kr has not been expressed in its wildtype muscular pattern and/or that the correct levels and neuronal Kr activity are not provided through panmuscular expression. Nevertheless, the results are consistent with the proposal that Kr regulates a muscular programme which in turn regulates SNb axon guidance along the muscles. This proposal is supported by the notion that the phenotype is reminiscent of mutants affecting CAMs in motoneurons such as FasII (Abrell, 2001).
The similarity of motoneuronal phenotypes of Kr and CAM mutants, and the interaction between Kr and caps activity suggests that Kr might also interact genetically with additional CAMs which are necessary for proper path finding. This proposal was tested using mutant combinations of Krres and the loss-of-function mutant FasIIeb112. FasII is a more general CAM than Caps. It is expressed on all motoneurons during late embryonic stages and is necessary to maintain adhesion between the axons (Abrell, 2001).
Heterozygous Krres/+ or FasIIeb112/+ embryos develop a normal SNb pattern and all RPs are properly connected to their target muscles. In double heterozygous FasIIeb112/+;Krres/+ mutants, however, the SNb enters the ventral muscle field normally in all cases, but the nerve stops at the second choice point by forming a growth cone-like structure. No individual RP axons could be observed. This phenotype is very similar to the one observed with homozygous Krres mutant embryos, implying that the two gene activities cooperate to allow for proper SNb development (Abrell, 2001).
In addition to position, size and morphology, the innervation of muscles by specific motoneurons represents a diagnostic feature for the determination of muscle identity. Previous results have shown that Kr is expressed in a specific subset of muscle progenitors and is necessary for the acquisition of a specific muscle fate as shown by muscle transformations that occur in response to gain-of-function and lack-of-function Kr mutations. Muscle 27 is transformed into a duplicated muscle 26 in homozygous Kr mutant embryos whereas high level overexpression (four copies of Kr) in all muscles leads to the reverse transformation. The data suggest that Kr contributes not only to identifying characteristics of muscle 27 but also provides adhesion properties to other Kr-expressing muscles along the SNb pathway, i.e. muscles 14 and 16, the ventral acute muscle 27, and the ventral longitudinal muscles 6, 7 and 13. The genetic interactions between Kr and the CAMs FasII and Caps support the hypothesis and imply that the adhesive properties of motoneurons and/or muscles are established in such a way that a concomitant reduction in adhesion of Krres/+ ; caps/+ or Krres/+; FasIIeb112/+ mutants results in a situation which no longer provides sufficient information to allow accurate axonal pathfinding and innervation. This proposal is also consistent with the finding that the presence of one muscle in an otherwise muscle-depleted embryo can be sufficient for the defasciculation of nerve bundles (Abrell, 2001).
In muscles 6, 7 and 13, Kr is known to maintain the expression of a direct target gene, knockout (ko). ko mutant embryos display a Kr-like motoneuron phenotype, suggesting that the gene, which encodes a novel protein with unknown biochemical characteristics, plays a key role in SNb defasciculation and RP pathfinding by acting downstream of Kr. However, in contrast to Kr, ectopic misexpression of ko does not affect SNb branching and synaptic targeting of RP neurons, and no genetic interaction as observed between ko and caps could be found. It is therefore likely that Kr transmits its signal not only via ko, but also through other factors that are still to be identified. It was also found that in contrast to caps, ectopic panmotoneural expression of Kr causes defects similar to the Kr lack-of-function mutation, and a reduction of combined caps or FasII and Kr activities. It is therefore speculated that Kr activity can also directly interfere with the spectrum of CAMs in motoneurons, resulting in non-compatible cell-surface characteristics between axons and muscles, which in turn interfere with neuromuscular connectivity (Abrell, 2001).
The anterodorsal projection neuron lineage of Drosophila melanogaster produces 40 neuronal types in a stereotypic order. This study takes advantage of this complete lineage sequence to examine the role of known temporal fating factors, including Chinmo and the Hb/Kr/Pdm/Cas transcriptional cascade, within this diverse central brain lineage. Kr mutation affects the temporal fate of the neuroblast (NB) itself, causing a single fate to be skipped, whereas Chinmo null only elicits fate transformation of NB progeny without altering cell counts. Notably, Chinmo operates in two separate windows to prevent fate transformation (into the subsequent Chinmo-indenpendent fate) within each window. By contrast, Hb/Pdm/Cas play no detectable role, indicating that Kr either acts outside of the cascade identified in the ventral nerve cord or that redundancy exists at the level of fating factors. Therefore, hierarchical fating mechanisms operate within the lineage to generate neuronal diversity in an unprecedented fashion (Kao, 2012).
Knocking down Kr from the NB led to skipping of a single temporal fate during adPN neurogenesis. Removing Kr from specific GMCs further revealed that GMC, which normally makes the missing adPN, had precociously adopted the next temporal fate in the absence of Kr. These observations indicate that Kr regulates temporal fate transitions in the adPN NB and is continuously required in the GMC to suppress the next temporal fate. Despite no evidence for the involvement of Hb/Pdm/Cas, Kr's role in delaying fate transition in the Kr-positive GMC suggests an analogous role in an alternate temporal cascade that confers a specific temporal fate from a set of contiguous fates. Furthermore, loss of Kr exerted no detectable effect on the remaining cascade, reminiscent of the chain control in the sequential expression of Hb/Kr/Pdm/Cas (Kao, 2012).
Kr confers the VA7l fate in adPN lineage. Notably, the temporal fate that precedes VA7l fate defines a polyglomerular PN with a rather diffuse AL elaboration. It is challenging to definitely locate the embryonic-born polyglomerular adPN due to colabeling with a large number of uniglomerular siblings. To exclude Hb as the temporal factor that precedes Kr, whether the embryonic-born polyglomerular adPN exists and has properly differentiated in hb mutant NB clones was reexamined. A combination of two sparse GAL4 drivers that collectively label three adPNs, including the embryonic polyglomerular PN plus two earlier-born uniglomerular siblings, was used to identify NB clones generated near the beginning of the lineage and simultaneously to assess the pre-VA7l polyglomerular PN. The same three adPNs were observed in the wild-type, hb, as well as Kr mutant NB clones. These results strengthen the conclusion that Kr acts alone without Hb/Pdm/Cas to specify only one middle temporal fate in the protracted adPN lineage (Kao, 2012).
In contrast to Kr defining only one temporal fate, Chinmo acts in two windows to support eight temporal fates in the adPN lineage. The two windows are separated by only one Chinmo-independent adPN that happens to split two otherwise indistinguishable VM3-targeting adPNs. Interestingly, the fate transformation of the last two embryonic adPNs (transformed from the VM3[b] and DL4 types to larval-born D type) is similar to the chinmo-elicited fate transformation of larval-born adPNs (Kao, 2012).
Chinmo has previously been implicated in governing neuronal temporal identity in the MB lineage and one partially resolved neuronal lineage. This study observed a distinct pattern of Chinmo requirement in the adPN lineage. Notably, chinmo mutant neurons aberrantly adopt later temporal cell fates within their original lineages in all cases. Moreover, Chinmo governs multiple continuous fates in MB as well as in adPN lineages. Despite these similarities, detailed mechanisms of Chinmo actions are apparently distinct. In the MB lineages, reducing Chinmo expression elicits systematical early-to-late MB temporal fate transformations, and ectopic Chinmo can specify early MB fates in late siblings. By contrast, a partial reduction in Chinmo sometimes conferred hybrid adPN fate showing features of both the prospective cell fate and the chinmo-null default fate, rather than exhibiting the morphologies reminiscent of the fates in between. And ectopic Chinmo also failed to promote early fates in late-born adPNs, providing no evidence for dosage-dependent Chinmo-mediated fate determination in the adPN lineage. Therefore, both loss- and gain-of-function genetic mosaic studies suggest that Chinmo does not directly determine any temporal cell fate in adPN lineage, but rather it suppresses a later temporal fate in early siblings to allow further neuronal diversification. Further, mechanism(s) must exist to restrict the activities of Chinmo to specific windows, because ectopic Chinmo exerted no detectable effect on adPNs within the rest of the lineage. It is also not clear whether and how Chinmo directly diversifies neuron fate (Kao, 2012).
Unlike Kr that regulates temporal fate transition in the NB, Chinmo apparently acts in the offspring and potentially downstream of some NB transcriptional cascade to increase neuron diversity. This distinction is supported by the follwing: (1) postmitotic expression of transgenic Chinmo restored proper temporal cell fates in chinmo mutant adPNs, arguing that Chinmo acts in newborn neurons to regulate adPN temporal identity; (2) deleting chinmo from NB through the entire lineage did not affect overall temporal fate transitions, as evidenced by no change in total cell count or length of the lineage; and (3) ectopic expression of chinmo exerted no detectable effect on the NB temporal fate transitions. All these observations indicate that Chinmo acts in postmitotic neurons to refine temporal identity. Temporal patterning by the Kr-containing transcriptional cascade in the NB and via Chinmo in newborn neurons exemplifies a hierarchical mode of temporal cell-fate specification. Identifying additional genes controlling adPN temporal identity and determining their mechanisms of action by iterative use of the strategy used in this paper will allow elucidation of developmental mechanisms specifying the great diversity of neuron types in the complex brain (Kao, 2012).
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Krüppel:
Biological Overview
| Evolutionary Homologs
| Regulation
| Targets of Activity
| Protein Interactions
| Developmental Biology
| Effects of Mutation
date revised: 22 April 2021
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