In order to determine where mid and H15 fit in the genetic hierarchy controlling heart development, their expression was examined in several mutant backgrounds. The initiation of mid expression in the dorsal mesoderm in early stage 12 occurs after the expression of tin and pnr, as well as after the period of Wg signaling in the dorsal mesoderm, suggesting that mid and H15 are regulated downstream of the factors that confer cardiac fate. Indeed, the dorsal vessel expression of mid and H15 is completely lost in both wgcx4 and tinec40 mutant embryos, which fail to specify dorsal mesoderm. Embryos mutant for pnr have greatly decreased numbers of cardioblasts. Accordingly, mid and H15 expression is variably lost in pnrvx6 null mutant embryos, with most embryos completely lacking mid expression in the dorsal mesoderm. Ectopic expression of pnr throughout the mesoderm using the GAL4/UAS system is able to induce ectopic expression of mid and H15. These results indicate that the initiation of mid and H15 in the dorsal mesoderm is downstream of factors required for the specification of cardiac fate (Miskolczi-McCallum, 2005).
Segmentation of the Drosophila embryo is a classic paradigm for pattern formation during development. The Wnt-1 homolog Wingless (Wg) is a key player in the establishment of a segmentally reiterated pattern of cell type specification. The intrasegmental polarity of this pattern depends on the precise positioning of the Wg signaling source anterior to the Engrailed (En)/Hedgehog (Hh) domain. Proper polarity of epidermal segments requires an asymmetric response to the bidirectional Hh signal: wg is activated in cells anterior to the Hh signaling source and is restricted from cells posterior to this signaling source. This study reports that Midline (Mid) and H15, two highly related T box proteins representing the orthologs of zebrafish hrT and mouse Tbx20, are novel negative regulators of wg transcription and act to break the symmetry of Hh signaling. Loss of mid and H15 results in the symmetric outcome of Hh signaling: the establishment of wg domains anterior and posterior to the signaling source predominantly, but not exclusively, in odd-numbered segments. Accordingly, loss of mid and H15 produces defects that mimic a wg gain-of-function phenotype. Misexpression of mid represses wg and produces a weak/moderate wg loss-of-function phenocopy. Furthermore, it has been shown that loss of mid and H15 results in an anterior expansion of the expression of serrate (ser) in every segment, representing a second instance of target gene repression downstream of Hh signaling in the establishment of segment polarity. The data presented indicate that mid and H15 are important components in pattern formation in the ventral epidermis. In odd-numbered abdominal segments, Mid/H15 activity plays an important role in restricting the expression of Wg to a single domain (Buescher, 2004).
The larval cuticle of Drosophila is a model system for generating patterns from fields of cells. The ventral cuticle exhibits a segmentally reiterated array of six rows of unique denticles separated by areas of naked cuticle. These external structures reflect the cellular diversity within the underlying epidermis, and defective cuticle patterns are indicative of incorrectly specified cell fates. The secreted products of two segment polarity genes, wg and hh, are the key players that initiate progressive patterning events that ultimately result in epidermal differentiation at the single-cell level. Thus, patterning requires the tight regulation of the spatial limits of Wg and Hh expression. In early embryogenesis, pair-rule gene activity initiates the expression of Wg and Hh in adjacent stripes, with Wg just anterior to the Hh-expressing cells. After stage 9, reciprocal signaling between Wg- and Hh-expressing cells stabilizes their expression domains. Acting anisotropically, Hh signaling activates Wg anterior, but not posterior, to the Hh stripe. Finally, Wg expression becomes independent of Hh and is maintained through an autoregulatory feedback loop. Previous studies have led to the conclusion that Hh signaling is bidirectional because it maintains patched (ptc)-gene expression in narrow stripes anterior and posterior to the En/Hh stripe. The ptc gene product is a repressor of Wg expression, and maintenance of Wg expression at stage 9 requires the Hh-mediated derepression. However, despite the symmetry of Hh signaling, the outcome with respect to Wg expression is asymmetric and results in a single Wg stripe anterior to the En/Hh stripe. To rationalize the differential response of Wg to the Hh signal, a model has been put forward that subdivides each parasegment into two domains: the posterior half of the parasegment represents the wg-competent domain, and the anterior half is the en-competent domain. The wg-competent domain encompasses those cells that express Wg in a ptc mutant background. Additional studies have shown that wg competence requires the activity of the pair-rule/segment polarity genes sloppy-paired 1, 2 (slp1, 2), which are expressed in broad stripes anterior to the En/Hh stripe. It has been suggested that Slp permits the activation of Wg anterior to the En/Hh stripe by antagonizing one (or several) putative repressor(s) of Wg (Buescher, 2004 and references therein).
This study reports that Mid and H15 act to repress the Hh-dependent activation of Wg in the en-competent domain predominantly in odd-numbered segments. Furthermore, these results suggest that the Slp-mediated repression of Mid/H15 anterior to the En/Hh stripe is an important component of wg competence (Buescher, 2004).
Mutant alleles of mid (mid1, mid2), recessive embryonic-lethal zygotic mutations, were first identified in the classic screen for segmentation genes (Nüsslein-Volhard, 1984). An additional allele, midGA174, was isolated from a collection of EMS-induced mutations. mid mutant larvae are characterized by patches of naked cuticle in the ventral-most part of the abdominal denticle belts. In addition, a near-complete loss of denticle belts (or segmental halves of denticle belts) was occasionally observed. Both aspects of the phenotype are more pronounced in odd-numbered segment, whereas even-numbered segments show similar but milder defects. Genetic mapping (Nüsslein-Volhard, 1984) has placed mid at cytological position 25E (Buescher, 2004).
This region was examined for genes with expression patterns that suggest a role in segmentation. A P element insertion upstream of H15 (CG6604) displays a ß-gal expression pattern consistent with such a role (Griffin, 2000). Both H15 and an adjacent gene, CG6634, encode highly homologous T box proteins with essentially identical expression patterns ; thus, both genes represented candidates for mid. Deletion of H15 by X-ray resulted in a homozygous pharate adult lethal line (H15x4) with no appreciable cuticle phenotype. In contrast, sequencing of CG6634 DNA from homozygous mid1, mid2, and midGA174 embryos reveals nonsense mutations. The putative translation products of mid1 and midGA174 lack the DNA binding domain and are most probably nonfunctional. These data indicate that CG6634 encodes the mid gene. This conclusion is corroborated by the observation that ectopic expression of CG6634 in the mid1 mutant background rescues the cuticle defect. To determine if, in the absence of Mid function, H15 contributes to cuticle formation, early larvae lacking both copies of H15 and one copy of mid (H15x4/Df-GpdhA) were examined. A weak mid phenotype was observed. Removal of both copies of H15 and mid (hereafter referred to as mid/H15) results in a strong enhancement of the phenotype; the denticle rows 1-5 are frequently lost in odd-numbered segments, whereas even-numbered segments show milder defects. These findings suggest that Mid and H15 act redundantly to control denticle formation (Buescher, 2004).
The mid RNA expression pattern is typical of segment polarity genes. H15 RNA expression pattern is identical with the exception that expression levels up to stage 9 are significantly lower than mid expression levels. Fourteen stripes of mid expression are first detected in stage 5 embryos. Alternating stripes differ in width and intensity; even-numbered stripes are wider and show higher levels of expression. (Note that the first five mid stripes localize to the presumptive head and thoracic segments. Therefore, the even-numbered stripes 6, 8, 10, and 12 correspond to the stripes that are found in the odd-numbered abdominal segments 1, 3, 5, and 7.) During germband extension, expression becomes more uniform in consecutive stripes and is approximately equal in all stripes from stage 8 onward. mid-expressing stripes are maintained until the end of embryogenesis. However, mid expression occurs in distinct phases, during which it is regulated by different factors. Dynamic changes of the mid stripes with respect to width and location reflect the different regulatory inputs (Buescher, 2004).
The precise location of the mid stripes was first determined by double labeling wild-type embryos with a mid RNA in situ probe and an anti-Wg antibody. During stages 5-11, mid expression abuts the posterior side of the Wg stripe, but from late stage 11 onward, the mid and the Wg stripes are separated by two rows of cells that express neither gene. The Wg and mid stripes remain separated until the end of embryogenesis. To determine the posterior limits of mid expression, wild-type embryos were stained with the mid RNA in situ probe and an anti-En antibody. Up to stage 9, mid expression is found in the En/Hh cells, but even-numbered mid stripes extend farther to include two rows of cells just posterior to the En/Hh domain. Weaker mid expression is found posterior to the En/Hh domain in odd-numbered segments. This early expression of mid depends on pair-rule gene activity. By stage 10, the mid-expressing domain contracts to coincide with the En/Hh stripe. This stripe persists until early stage 11, after which mid expression in the En domain decays. The maintenance of mid expression during stages 9-11 within the En domain requires Wg function; in wg null mutant embryos (wgCX4), mid expression decays prematurely from stage 9 onward and is absent at stage 11. This dependence on Wg may provide a rationale for the narrowing of the mid stripe after initiation by the pair-rule genes because Wg signaling is not effective posterior to the En domain. During late stage 11, mid expression is reinitiated in a 2- to 3-cell-wide stripe posterior to the En stripe. This stripe persists until the end of embryogenesis. This late expression of mid is sensitive to Hh signaling; raising the level of Hh signaling (enGal4; UAS-hh) results in a posterior expansion of the mid stripe. It is noteworthy that this late expression of mid represents another example of an asymmetric outcome of bidirectional Hh signaling (Buescher, 2004).
In odd-numbered abdominal segments, and to a lesser extent in even-numbered segments, concomitant loss of both Mid and H15 results in an excess of naked cuticle similar to that caused by ectopic Wg expression. To investigate if loss of Mid/H15 affects the Wg expression pattern, double-mutant embryos were stained with a anti-Wg antibody or a wg RNA in situ probe. From stage 9 onward, ectopic 1-cell-wide Wg stripes were observed in odd-numbered abdominal segments and weak patches of ectopic Wg were occasionally found in even-numbered segments. The initially weak ectopic stripes subsequently increased in intensity so that by stage 13 high levels of ectopic Wg were found in odd-numbered segments and lower levels of ectopic Wg were seen in some even-numbered segments. Hence, the pair-rule-biased pattern of ectopic Wg expression reflects the defect observed in mid/H15 larval cuticles; namely, this defect is a gain of naked cuticle predominantly, but not exclusively, in odd-numbered segments. Ectopic Wg expression was also found in stage 9 mid single mutants, albeit less frequently and less robustly. It was also observed very rarely in H15 single mutants. The precise location of the ectopic Wg expression was determined by double staining mid/H15 mutant embryos with anti-Wg and anti-En antibodies. The ectopic Wg stripes abut the posterior side of the En stripes (within the domain of early mid expression, generating a pattern in which En-expressing cells are straddled by Wg-expressing cells (Buescher, 2004).
To assess the effects of mid gain-of-function on Wg expression, several Gal4 drivers were used to misexpress mid. The cuticle phenotype of the resulting larvae was examined; this phenotype represents a highly sensitive read-out of even small changes in the level of Wg signaling. The cuticle phenotype of ptcGal4;UAS-mid and scaGal4;UAS-mid larvae mimicked that of a late loss of wg function: in the posterior half of the larvae, nearly all naked cuticle was replaced with denticles. In the anterior half, extra denticles appeared predominantly in the ventral-most region. This phenotype appeared with 100% penetrance. In addition, ptcGal4;UAS-mid larvae (and to a lesser extent scaGal4;UAS-mid embryos) showed specific morphological defects characteristic of wg loss-of-function mutants -- reduced body size, strong segmental indentations, and malformation of the head. To confirm that the cuticle defects caused by ectopic Mid were due to decreased Wg expression, UAS-mid, UAS-wg, or both were expressed under the control of prdGal4, which is strongly expressed in the Wg domain in the even-numbered abdominal segments. Coexpression of wg with mid resulted in near-complete suppression of the fusion of alternating denticle belts as a result of misexpression of mid alone. The cuticle defects observed in ptcGal4;UAS-mid larvae were matched by altered levels of Wg protein: from stage 10 onward, Wg protein in the ventral ectoderm decayed, and at stage 13 it was nearly absent in the ventral epidermis. Hence, misexpression of mid is sufficient to antagonize Wg in its endogenous expression domain (Buescher, 2004).
The appearance of the ectopic Wg stripes in mid/H15 mutants coincides with the stabilization phase of the endogenous Wg expression by Hh signaling. The endogenous Wg expression (anterior to the En/Hh stripe) was not affected in either mid single or mid/H15 double mutants at any time, suggesting that Hh signaling is normal. To corroborate this conclusion, the expression of the hh gene, the distribution of Hh protein, and the effect of Hh signaling were examined on the expression of the ptc gene. ptcRNA expression in narrow stripes anterior and posterior to the En domain has been shown to be a read-out of bidirectional Hh signaling. No difference was observed in the hh and ptc expression patterns in wild-type and mid/H15 embryos. This demonstrates that the regulatory interactions that result in the maintenance of endogenous Wg at stage 9 and the autoregulation of Wg from stage 11 onward function normally in mid/H15 double mutants. This raises the possibility that Hh signaling may activate Wg expression in a symmetrical manner but that expression of Wg posterior to the En stripe is antagonized by Mid/H15. To investigate this possibility, the effects were studied of manipulating Hh signaling in a mid/H15 mutant background. When the level of Hh signaling was raised from within the endogenous Hh/En domain (enGal4;UAS-hh), the ectopic Wg expanded to a 2- to 3-cell-wide stripe, thus demonstrating that the level of Hh controls the spatial limits of ectopic Wg expression. Reciprocally, in hh/mid/H15 triple mutants, no ectopic Wg expression was observed. These data show that Hh signals symmetrically with respect to cells anterior and posterior to the En/Hh stripe and Mid and H15 are required to prevent posterior Wg expression, thus ensuring that Wg expression remains restricted to a single domain (Buescher, 2004).
Loss of mid/H15 leads to ectopic Wg expression in a single row of cells. Raising the level of Hh signaling (mid/H15;enGal4;UAS-hh) results in an expansion of the ectopic Wg stripe. To unmask the potential of cells to express Wg in the absense of Mid-mediated repression, mid1ptc9 double-mutant embryos in which Wg expression is largely independent of Hh were examined. In ptc mutant embryos, the expression domain of Wg broadens in the anterior direction, and ectopic En expression is induced de novo in cells anterior to these broadened domains. The anterior region of the segment between the ectopic En stripe and the next endogenous En stripe does not express Wg. Double labeling of ptc embryos with the mid RNA in situ probe and anti-En antibody showed that mid fills this region. Removal of Mid function caused all cells in odd-numbered segments to express either Wg or En (but not both) and resulted in a complete loss of the odd-numbered denticle belts (Buescher, 2004).
Close examination revealed some unexpected features: the wg transcription domain is slightly widened in mid1ptc9 embryos as compared to ptc single mutants; however, this occurs in all segments without any pair-rule bias. Moreover, consecutive wg RNA stripes form pairs (1-2, 3-4, and so on) that are separated by wider gaps from the subsequent pair. The region between two partners of each wg pair is entirely filled with En-expressing cells, suggesting a fusion of the ectopic and the endogenous En domains. Taken together, these data indicate that concomitant loss of mid and ptc results in defects that go beyond a simple change of Wg and En expression. This notion is supported by the observation that, as compared to even-numbered segments, odd-numbered segments in mid1ptc9 embryos are shorter by approximately two rows of cells. Future studies will show if mid plays a role in proper segment formation, which requires cell survival, cell division, and cell sorting (Buescher, 2004).
Earlier studies of the regulation of Wg expression showed that loss of ptc results in an anterior expansion of the wg transcription domain. It does not, however, result in ectopic Wg expression posterior to the En domain. To rationalize this asymmetric response of the wg promoter to bi-directional Hh signaling, it was proposed that each parasegment is divided into two domains: the posterior half of the parasegment represents the wg-competent domain and the anterior half is the en-compentent domain. Wg-competence requires the slp genes which are expressed in broad stripes anterior to the En/Hh stripe. This study has shown that loss of mid/H15 results in the ectopic expression of Wg within the en-competent domain (where Slp is absent). This prompted a study to examined any possible regulatory interactions between slp and mid/H15. Double staining of H15-lacZ embryos (which are viable and wild-type with respect to Wg expression) with a slp in situ probe and anti-ß-gal antibody showed that, at stage 9, slp and H15 (mid) are expressed in non-overlapping domains that are separated by one row of cells in the center of each segment. Within this central row, slp-positive and mid-positive cells intermingle with cells expressing neither gene (Buescher, 2004).
It is conceivable that the ectopic expression of Wg in mid/H15 mutant embryos could be a secondary effect brought about by a gain of Slp expression posterior to the En stripe. RNA in situ analysis revealed no change of the Slp expression pattern in mid/H15 embryos. Hence, in odd-numbered segments of wild-type embryos, the lack of wg competence in the cells immediately posterior to the En/Hh stripe is a consequence of Wg repression via Mid/H15 rather than of a lack of activation via Slp (Buescher, 2004).
Previous work has suggested that Slp permits the Hh-dependent activation of Wg anterior to the En/Hh stripe by antagonizing a repressor of Wg. Based on the data presented above, Mid/H15 appear to be such repressors. To determine if Slp is a negative regulator of mid expression, the effect of slp loss-of-function and slp misexpression was studied on the distribution of mid RNA. In slp mutant embryos the early mid expression is normal. However from early stage 9 onward the mid stripes broaden to approximately twice their normal width. Using mid-positive neuroblasts as a landmark (these remain unchanged in slp mutant embryos), it was possible to characterize the increase in mid expression as an anterior expansion. This aberrant mid expression pattern is unstable; from stage 11 onward mid decays in odd-numbered segments. Conversely, misexpression of slp in the ventral ectoderm from early stage 9 onward led to a complete loss of ectodermal mid expression. These data show that Slp functions as a repressor of mid expression. Taken together with the observation that misexpression of mid in otherwise wild-type embryos results in the loss of Wg expression, these results lead to the conclusion that the Slp-mediated repression of mid anterior to the En/Hh stripe is an important component of wg competence (Buescher, 2004).
As a further test of the relationship between slp and mid, the effect was compared of expressing mid and slp, alone or in combination, on Wg expression. Ectopic expression of mid results in a rapid and almost complete loss of Wg expression, whereas ectopic expression of slp results in weak ectopic expression of Wg posterior to the En/Hh stripe. This slp-induced phenotype resembles that of the loss of mid, except that ectopic Wg expression is weaker and appears randomly in even- and odd-numbered segments. The ectopic Wg expression is blocked when mid and slp are expressed together, suggesting that in this context mid acts downstream of slp. The Wg expression anterior to the En/Hh stripe still decays in UAS-mid/UAS-slp embryos, albeit more slowly and variably than in UAS-mid alone. This result may reflect that Wg expression is sensitive to the amounts of available Mid and Slp. It may also indicate that anterior to the En/Hh stripe, Slp function is required for more than just repression of mid and may possibly have independent activating functions. An analysis of slp1,slp2;mid/H15 quadruple mutants would be highly helpful in clarifying the relationship between slp genes and mid/H15. Unfortunately, the generation of such a quadruple mutant by genetic recombination is impossible because the slp deletion that removes these genes (Δ34B) is on a balancer chromosome that precludes recombination (Buescher, 2004).
The pair-rule modulation of the mid/H15 deletion phenotype results from a different requirement in alternate segments for mid/H15-mediated repression of Wg downstream of Hh signaling. However, in mid/H15 mutant larvae, even-numbered denticle belts also show some defects. This prompted analysis of whether mid/H15 plays a role in the expression of other regulators of late segmental patterning. After stage 11, Wg and Hh signaling regulate other target genes, resulting in the subdivision of each segment into smaller territories. In the posterior, adjacent to the En/Hh-expressing cells, a Rhomboid (Rho)-expressing domain is created. Rho processes membrane bound, inactive Spitz (Spi) to an active, secreted form. Competing Spi and Wg signaling control the decision between naked cuticle and denticle formation, with Spi activating denticle-type specification and Wg specifying naked cuticle. After stage 11, the mid stripe marks the anterior most rows of cells in every segment and colocalizes with rho. In situ hybridization of stage 13 mid/H15 mutants with a rho-specific probe reveals a reduction/absence of rho expression in odd-numbered abdominal segments, suggesting that the excess of naked cuticle in mid/H15 mutant larvae arises from the gain of Wg and the concomitant loss of Spi signaling. The expression of Serrate (Ser), which is normally restricted to cells posterior of Rho in abdominal segments because of repression by Hh signaling, was examined. The Ser domain expands into the Rho domain at the anterior end of the segment in embryos lacking mid, whereas Ser expression is lost completely in embryos with ectopic mid expression. This suggests that Mid acts downstream of Hh signaling to repress Ser in addition to Wg. The anterior expansion of Ser in every segment may contribute to the defects found in even-numbered segments, in which only variable weak ectopic Wg expression is detected (Buescher, 2004).
The data indicate that mid/H15 are negative regulators of Wg and Ser. In the ventral ectoderm of odd-numbered abdominal segments, mid/H15 act to break the symmetry of the Hh-dependent activation of Wg expression. It has been proposed that pair-rule gene activity leaves 'imprints' on all cells and that these imprints predispose cells to express either Wg or En. Slp was found to be such an 'imprint' anterior to the En/Hh stripe, where it predisposes cells to express Wg. The early, pair-rule gene-driven mid/H15 expression appears to be another such 'imprint' that predisposes cells posterior to the En/Hh stripe not to express Wg (Buescher, 2004).
These findings raise several questions. (1) Because the outcome of bidirectional Hh signaling is asymmetric in all abdominal segments, additional factors that prevent the inappropriate expression of Wg in even-numbered segments must exist. At present, such factors are not known. (2) Is the decreased Rho expression in alternating segments a direct consequence of a loss of activation by mid/H15 or a result of negatively acting, ectopic Wg expression? The latter explanation is favored because the ectopic pair-rule-biased expression of Wg corelates with the pair-rule-biased loss of Rho, and ectopic activation of the Wg pathway is sufficient to repress Rho expression posterior to the En/Hh stripe. (3) What is the molecular mechanism of Wg repression by mid/H15? Mid and H15 are members of the T box family of transcription factors and therefore presumably modulate target gene expression directly. The target genes of Mid/H15 are currently unknown. Although it is conceivable that wg is a direct target gene, other scenarios are possible: Mid/H15 may positively or negatively regulate the expression of unidentified genes and thereby modulate Wg or Hh pathway activities, and hyperactivity of either pathway could produce an ectopic stripe of Wg expression. It is noteworthy that a different group of T box genes, the dorsocross genes, has been identified as a negative regulator of Wg expression in the dorsolateral epidermis. However, the Dorsocross target genes are also unknown. Further studies are required to elucidate the mechanisms by which T box proteins negatively regulate Wg expression (Buescher, 2004).
In the Drosophila CNS, combinatorial, interdependent, sequential genetic programs in neuroectodermal (NE) cells, prior to the formation of neuroblasts (NBs), determine the initial identity of NBs. Temporal factors are then sequentially expressed to change the temporal identity. It is unclear at what levels this positional and temporal information integrates to determine progeny cell identity. One idea is that this is a top-down linear process: the identity of a NB determines the identity of its daughter, the ganglion mother cell (GMC), the asymmetric division of the GMC and the fate specification of daughter cells of the GMC. Results with midline (mid), which encodes a T-box protein, in a typical lineage, NB4-2->GMC-1->RP2/sib, suggest that at least part of the process operates in GMCs. That is, a GMC or a neuronal identity need not be determined at the NB or NE level. This is demonstrated by showing that Mid is expressed in a row 5 GMC (M-GMC), but not in its parent NB or NE cell. In mid mutants, M-GMC changes into GMC-1 and generates an RP2 and a sib without affecting the expression of key genes at the NE/NB levels. Expression of Mid in the M-GMC in mid mutants rescues the fate change, indicating that Mid specifies neurons at the GMC level. Moreover, a significant plasticity is found in the temporal window in which a neuronal lineage can develop. Although the extra GMC-1 in mid mutants is born ~2 hours later than the bona fide GMC-1, it follows the same developmental pattern as the bona fide GMC-1. Thus, a GMC identity can be independent of parental identity and GMC formation and elaboration need not be strictly time-bound (Gaziova, 2009).
That two cells converge to the same fate from different lineages has been well documented in C. elegans: except in the gut and germline, identical cells in all other tissues originate from multiple lineages. Body wall muscle cells that are almost identical morphologically and physiologically come from four different founder cells. Similarly, identical neurons can be specified by different lineages. For instance, for bilateral neurons among the six sensory neurons involved in mechanosensation, although derived from the same founder cell, their lineages diverge four cell divisions prior to the terminal division. However, Drosophila is not driven by lineages, except for NBs, but they produce distinct progeny lineages specific to a given NB. Therefore, the above conclusion as drawn from studies in C. elegans was not an obvious, or expected, one in Drosophila and this makes the current findings with mid significant. The fate of a cell is not specified simply by a single transcription factor, but instead by a complex combination of cell-autonomous and cell-non-autonomous genetic circuitry. The results indicate that Mid plays a central role in this process in M-GMC, preventing it from becoming GMC-1 of the RP2/sib lineage. Absence of Mid activity initiates a cascade of events in M-GMC that ultimately transforms M-GMC into GMC-1 (Gaziova, 2009).
A NB undergoes multiple self-renewing asymmetric divisions, each time producing a GMC of specific identity, which then generates two neurons of distinct identities. The identity of the first GMC from a NB is dependent upon the gene expression program in the NE cells from which the parent NB is delaminated, and this identity is thought to be invariant. Following division to generate a GMC, the gene expression program in the NB changes so that it produces a second GMC of different and distinct identity from the first GMC. Based on these and several other similar studies, it is currently believed that the identity of a GMC and its neuronal pairs is already determined in the NE and NB levels, i.e. it is ancestry-dependent. However, the current results with mid show that this ancestry-dependent fate specification is not as stringent as once thought, and that the identity of a GMC can be altered without altering the gene expression program in the NB or NE level. Thus, a specific set of neurons (in this case RP2/sib) can be derived or specified from a GMC other than the bona fide GMC by altering the activity of a single gene, in this case mid. One should keep in mind that the ultimate specification of the identity of a GMC (in this case M-GMC/eGMC-1) certainly depends on a complex interplay of many gene products. This study, however, identifies Mid as a key player in preventing M-GMC from becoming GMC-1 of the RP2 lineage. One should also point out that some GMCs, although being generated by different NBs, may have similar potentials and that there might be only one gene responsible for their differences; it is believed that mid as one such gene (Gaziova, 2009).
The results also show that duplication of the RP2/sib lineage, an extensively studied neuronal lineage, can occur by a mechanism or route that is different from those previously described. There are several ways the RP2 lineage can be duplicated. The most common way is through a second NB changing its identity into NB4-2, the parent of the RP2/sib lineage. RP2 lineage duplication can also occur when a GMC-1 divides symmetrically to produce two GMC-1s, each producing an RP2. A GMC-1 can also divide asymmetrically to self-renew and generate an RP2, and the self-renewed GMC-1 divides again to generate another (or more) RP2 or sib. A GMC-1 can also divide symmetrically to generate two RP2s. All these scenarios are different from the one described in mid mutants, in which an unrelated GMC (M-GMC) in a relatively distant location changes its identity to GMC-1 and generates a second set of RP2/sib cells at this distant site. This occurs without changing the expression of any of the genes known to be crucial for fate determination in the precursor NB or NE cells. This has not been observed before and as such adds to the novelty of the results (Gaziova, 2009).
A third set of results that are novel comes from the fact that a second GMC-1-->RP2/sib lineage can be formed 2-2.5 hours after the formation of the bona fide GMC-1-->RP2/sib lineage. This type of plasticity in the timing of formation of a lineage has never been shown before for this or any other lineage in the CNS. There is a certain degree of plasticity in the timing of formation and elaboration of a lineage in the CNS between hemisegments. For example, formation of NB4-2 and its division can be delayed by ~15 minutes between hemisegments. In the case of gsb or en/invected mutants, for example, NB5-3 (which is located close to NB4-2) transforms into NB4-2, thus duplicating the RP2 lineage. NB5-3 (whether transformed into NB4-2 as in these mutants, or not) is formed ~30 minutes prior to the formation of NB4-2. Thus, the sequential production of the duplication can be delayed by as much as 45 minutes in an embryo. A similar interval in the sequential production of the RP2 lineage is also observed in embryos mutant for lottchen (Drop), in which a second NB (possibly NB3-2, located adjacent to NB4-2) changes into NB4-2. The results with mid indicate that an additional GMC-1-->RP2/sib lineage can be formed as much as ~2 hours later than normal for this lineage, and at a site relatively distant from the original location of this lineage. This indicates considerable plasticity in terms of the developmental timing of a neuronal lineage, and that the nerve cord is capable of generating an early forming neuronal lineage also at a later point in time. Moreover, in all previous cases in which a second RP2/sib lineage was formed, it was always formed close to the bona fide RP2/sib lineage. The duplication of the RP2/sib lineage in mid mutants is the first case in which the second lineage is formed at an ectopic site (Gaziova, 2009).
These results are also interesting from another angle. A NB loses it ability, later in development, to produce earlier neurons. In other words, there is a temporally guided progressive restriction on the ability of a NB to generate earlier-born neurons. Indeed, a previous study showed that NBs indeed gradually lose competence to generate earlier-born cells. Although it is not clear whether this is true for all lineages, the current results show that at an organismal level, an earlier lineage can be generated at a later point in development. Thus, whereas the same NB, later in its life, may lose its ability to generate an earlier-born neuron, an earlier-born neuron can still be generated in the CNS at a later point in development, albeit in a different NB or GMC lineage (Gaziova, 2009).
The results show that Mid plays a unique role in preventing M-GMC from becoming GMC-1, ~2 hours after the formation of the bona fide RP2 lineage. It is possible that in the wild type, during evolution a combination of gene expression patterns converged at this ~2-hour time point with the potential to push the M-GMC into GMC-1, but because a nerve cord does not need two RP2s, evolution found a way to prevent this from occurring via expression of Mid in this M-GMC. It is suspected that a similar mechanism might exist in many more lineages than just the M-lineage (Gaziova, 2009).
It has been suggested that the extra cell is not an RP2 neuron. However, this conclusion was based on the observation that this cell does not have an axon projection similar to that of RP2. Since the location of this eRP2 is at the periphery of the nerve cord, one would not expect to observe an ipsilateral projection from this neuron. It was found that the growth cone from this neuron projects anterior and towards the midline, where a choice point for an RP2 projection might exist. This growth cone often fasciculates with the ISN along with the projection from the bona fide RP2. A number of experiments were employed involving different markers, mutant combinations and a very detailed and thorough analysis of this extra lineage. This analysis reveals that it is indeed an RP2: the GMC divides into a larger and a smaller cell akin to the division of the GMC-1 into an RP2 and a sib. One of the two cells, similar to a sib, loses the expression of Eve and does not express RP2-specific markers such as Zfh1. Furthermore, in a mid, insc double-mutant embryo, the esib adopts an RP2 fate, with both cells being the same size and expressing the same RP2 markers as the bona fide RP2 lineage in insc mutants. Similarly, in mid, numb double mutants, both cells become sibs. The two POU genes, pdm1 and pdm2, are required for the specification of GMC-1 of the RP2/sib lineage. In mid, pdm1, pdm2 triple mutants, the eGMC-1 fails to adopt a GMC-1 identity just as the bona fide GMC-1 also fails to adopt a GMC-1 identity (Gaziova, 2009).
However, there are temporal differences in the gene expression pattern between the bona fide RP2/sib lineage and the eRP2/sib lineage. For example, Eve expression begins later in the eRP2 lineage in at least 50% of the hemisegments, as late as subsequent to the eGMC-1 division. Thus, hemisegments are often found with no Eve-positive esib. Since loss-of-function for Eve has no drastic effect on the RP2/sib lineage, this late expression of Eve is likely to be non-consequential to the development of the lineage (Gaziova, 2009).
The bona fide GMC-1-->RP2/sib lineage originates from NB4-2, an S2 NB formed at ~4.5 hours of development (at 22°C). The GMC-1 is formed at 6-6.5 hours of development, although it becomes Eve-positive at ~7 hours of development; it then divides at ~7.45 hours into an RP2 and a sib. The cells undergo a complex migration and then settle within the anterior commissure. An RP2 begins to project its axon growth cone at ~10 hours of development. The eGMC-1 appears to be formed at ~8 hours, becoming Eve-positive at ~9 hours of development. It then divides at ~9.5 hours and begins to project its axon at ~12 hours of development. This indicates that there is significant plasticity in terms of developmental timing as far as the ability of the embryo to generate an RP2 lineage is concerned. All the requisite genetic pathways must still be operational even after 2 hours of development of the bona fide RP2/sib lineage (Gaziova, 2009).
The results indicate that the M-GMC from a row 5 NB (most likely NB5-4) is transformed into GMC-1, as opposed to a NB being transformed into NB4-2. It has previously been shown that in order to specify a NB as NB4-2, that cell should be Gsb-negative. First, 'suppression' results indicate that the eRP2 is generated by a row 5 NB and not a row 4 or 6 NB. However, none of the NBs in row 5 is Gsb-negative in mid embryos; row 5 NBs also had normal expression of three other markers: Wg, Slp and Hkb. This indicates that the identity of these NBs is unlikely to be affected in mid mutants. Second, whereas none of the NBs in row 5 expresses Mid, a row 5 GMC that generates the neuron that transforms into an RP2 in the mutant expresses Mid. It is possible that Mid is expressed in the parent NB of M-GMC but at an undetectable level. However, the conclusion was based on three sets of results: (1) by RNA in situ hybridization using a mid probe, no mid-positive NBs were detected at this location; (2) mid-promoter-lacZ transgenic lines were generatedand the expression of lacZ was basically the same as expression observed with the Mid antibody; and (3) it was possible to rescue/suppress the mid phenotype (i.e. the formation of an extra RP2 lineage) by expressing Mid in M-GMC in mid mutants. Finally, the timing of NB versus GMC specification is also consistent with the conclusion that the transformation occurs at the NB level. It is concluded that a row 5 GMC becomes GMC-1 of the RP2/sib lineage in the absence of wild-type Mid function (Gaziova, 2009).
One issue that was not possible to resolve conclusively is the identity of the parent NB for the M-lineage. The current results indicate that it is NB5-4; the first GMC of this NB gives rise to the M-lineage. Alternatively, it might be NB5-5, in which case the NB has to generate the M-GMC within 1 hour, or it could be a later-born GMC of NB5-3, although based on the position of the M-GMC this latter possibility is unlikely. It was not possible to address the ultimate fate of the M-neuron or its sibling, as to whether they are motoneurons, interneurons or some other cell type (it is unlikely to be glial as they do not express Repo, a glial cell marker), or the function of these cells (Gaziova, 2009).
These results indicate that row 5 NBs are affected in wg mutants, not just rows 4 and 6 as was previously thought. In previous work, a temperature-sensitive (ts) mutant was used and an allele of wg, wgCX4. Whereas the ts mutation is likely to be a hypomorph and retains some Wg activity, wgCX4 is considered a null. However, it was noticed that this allele carries a background mutation(s) that suppresses the wg loss-of-function effect; a partial recombination did eliminate the background suppressor mutation(s) and this 'cleaned up' wgCX4 mutation in trans to another allele of wg, wgIG22, did have the missing row 5 NB defect. It is believed that because of the effect of wg mutation on row 5 NBs, the wg phenotype is mostly epistatic to the mid phenotype in wg, mid double mutants in terms of the extra RP2 lineage defect (Gaziova, 2009).
The T-box-binding element (TBE) was first defined as a 20-bp degenerate palindromic sequence with the highest affinity for the Brachyury protein. However, analysis of the actual target genes reveals that the TBE is highly variable in sequence, number and distribution within their promoters. In the current experiments, with the consensus TBE only the Org-1 protein showed strong activation of the reporter gene, whereas Mid or H15 showed only an ~2-fold increase in transcriptional activation over the GFP control. However, with the gsb-n promoter, which contains a degenerate palindromic TBE sequence, activation by Org-1 was only slightly greater than that by the control protein. By contrast, there was a significant level of activation (~4-fold that of the control) by H15 from the same promoter element (H15 shares 62% identity with Mid), and the level of activation by Mid was ~1.5-fold that of the control, which is slightly more than the stimulation by Org-1. That Org-1 behaves differently to Mid and H15 is consistent with the fact that Mid and H15 belong to the Tbx20 subfamily, whereas Org-1 belongs to the Tbx1 subfamily. This result also shows that although these proteins are all in the Tbx family, they diverge significantly in their sequence preference with regard to the activation of transcription. The Tbx family of proteins is also known to repress transcription. Whereas the Tbx domain binds to DNA, albeit with different specificities according to variations in DNA sequence in the binding site, the rest of the protein is likely to be responsible for either activation or repression (Gaziova, 2009).
Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. This study shows that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. It is proposed that Midline controls axon pathfinding through coordinating the two guidance systems (Liu, 2009).
To address how Mid activates expression of the three axon guidance genes, the binding sequence of Mid was determined using an in vitro binding site selection method. Mid-binding sequence was selected from a pool of random oligonucleotides using Mid protein affinity-purified from an embryonic extract. The consensus sequence deduced from the selected oligonucleotides was (G/A/T)NA(A/T)N(T/G)(A/G)GGTCAAG. This sequence was found in the upstream regions or an intron of slit, frazzled, and robo, and all of these sites were conserved among several Drosophila species. To determine whether Mid binds to these regions in vivo, chromatin immunoprecipitation (ChIP) was performed using anti-Mid antibody. In all three genes, Mid was present around the Mid-binding sites, but not on regions without the binding site. In contrast, a potential Mid-binding site 32-kb upstream of the commisureless gene, whose expression is not affected in mid mutants, was not occupied by Mid (Liu, 2009).
The importance of the Mid-binding sites in frazzled and slit was assessed by transgenic reporter assays. To test the role of the Mid site in frazzled, reporter genes were constructed that contain the transcription start site of frazzled and an upstream region including a wild-type Mid-binding site (fraPlacZ) or a mutated site (fraMPlacZ). Compared with the wild-type reporter gene, the reporter with a mutated binding site showed reduced expression levels (33% reduction). Thus Mid-binding site is indeed required for the proper expression of frazzled. Mutating the Mid-binding site in slit also caused a severe effect on slit expression. The lacZ expression in sliPlacZ is driven by the slit regulatory element and the endogenous promoter. While sliPlacZ with the wild-type binding site recapitulated the slit expression in the midline glia and lateral cells, base substitutions in the Mid-binding site in sliMPlacZ abolished the lacZ expression. It is possible that the Mid-binding site resides in an essential promoter element of slit, and hence, the base substitutions abolished slit transcription in all cells. However, the same results were obtained using sli4.5HHlacZ and sliM4.5HhlacZ in which the slit regulatory element is fused to a heterologous hsp70 promoter. Since mid was expressed in the lateral cells but not in midline glia, these results suggest that Mid-binding sites in slit control slit transcription via binding to multiple factors: Mid in lateral cells and unknown factor(s) in midline glia. Taken together, these results demonstrate a direct role for Mid in the regulation of frazzled and slit, and suggest that Mid governs the expression of multiple axon guidance genes through directly binding of the Mid sites in their regulatory regions (Liu, 2009).
This study has shown that Mid directly controls transcription of key components of the two major axon guidance systems: the Netrin/Frazzled system and the Slit/ROBO system. Because these two systems are considered to have opposing outputs, it is interesting that the expression of both systems are induced by the same transcription factor, Mid. Dynamic expression of Frazzled and ROBO is required for growth cones to simultaneously respond to both attractants and repellents, integrate these signals, and then respond to the relative balance of forces. These molecules also provide nonautonomous functions required for cell motility, such as mediating cell adhesion and promoting axon elongation. The coordination of axon guidance systems by Mid may thus ensure cooperative actions of multiple guidance molecules in growth cone dynamics, axonal adhesion, and elongation. The role of Mid in the transcriptional regulation of axon guidance might be a conserved function, because its orthologs of human, mouse, and zebrafish Tbx20 are also expressed in motor neurons (Liu, 2009).
Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the JAK/Stat pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, this study shows that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. The E(spl)-C gene HLHm5 is shown to be expressed in a pattern complementary to tin during phase 3, and this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain (Johnson, 2011).
tin expression can be divided into four distinct spatial-temporal phases. Phase 1 tin expression initiates after gastrulation during which Twist (Twi) activates pan-mesodermal tin expression via the enhancer tinB. Phase 2 begins after FGF-mediated mesoderm spreading in which Dpp signals produced by the dorsal ectoderm maintain tin expression throughout the dorsal mesoderm via a second enhancer, tinD. It is during phase 2 that Tin specifies the major dorsal mesoderm derivatives. Phase 3 initiates after dorsal mesoderm progenitor specification and is characterized by a pronounced restriction of tin to the cardiac and visceral muscle progenitors. Upd and Upd2 are expressed in the ventral ectoderm during the transition from phase 2 to phase 3 expression. Phase 4 initiates after precursor specification and is characterized by further restriction of tin to the cardiac precursors that give rise to the contractile cardiomyocytes and the noncontractile pericardial nephrocytes. Phase 4 expression further directs heart cell diversification and maturation and is dependent on a third enhancer element, tinC (Johnson, 2011 and references therein).
To test the hypothesis that the JAK/Stat pathway functions in the cardiac-specific gene regulatory network, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. The JAK/Stat pathway regulates final cardiac morphology as well as heart precursor diversification. Stat92E loss-of-function analysis identified a discrete function for the JAK/Stat pathway in restricting tin during the transition from phase 2 to phase 3 expression. In addition, Stat92E embryos have an expanded pericardial cell domain arguing that the JAK/Stat pathway regulates tin to ensure proper heart precursor diversification. Mechanistically, it was found that the E(spl)-C gene HLHm5 is expressed in a complementary pattern to tin during phase 3 expression and that the JAK/Stat pathway activates HLHm5 expression in the dorsal mesoderm. The E(spl)-C genes in turn repress twi expression to preserve cardiac morphology and restrict tin and H15 expression to direct heart precursor diversification. These findings provide the first evidence of a role for the JAK/Stat pathway in cardiogenesis and identify a novel tin autoinhibitory circuit involving Stat92E and E(spl)-C (Johnson, 2011).
Stat92E is a direct Tin target gene during phase 2 expression; however, Stat92E is expressed in segmented stripes at this stage whereas tin is expressed throughout the dorsal mesoderm. In addition, embryos lacking only the maternal contribution of Stat92E have mesoderm patterning defects. Tin-regulated Stat92E zygotic transcription is therefore insufficient to coordinate mesoderm development. These data suggest that maternally contributed Stat92E is activated in response to segmented Upd and Upd2 activity, binds the Stat92E locus and co-activates Stat92E zygotic transcription with Tin. In addition, ChIP-chip experiments identified Stat92E binding activity and a conserved Stat92E consensus binding sites (SCBS) within the Tin-responsive Stat92E mesoderm enhancer. It is concluded that Stat92E and tin co-regulate a brief, spatially restricted JAK/Stat signaling event that patterns the mesoderm (Johnson, 2011).
Phase 3 tin expression promotes cell-type diversification and differentiation within the dorsal mesoderm and is indirectly activated by Wg via the T-box transcription factors in the Dorsocross complex and the GATA factor Pannier. A key finding of this study is that the JAK/Stat pathway activates the transcriptional repressor HLHm5 in the dorsal mesoderm to establish phase 3 tin expression. Because the HLHm5 cis-regulatory region lacks a conserved SCBS, it is predicted that Stat92E regulates HLHm5 expression through a non-consensus binding site. Alternatively, Stat92E acts at long distances to regulate gene expression. The SCBSs in E(spl)-C could be a platform from which Stat92E regulates multiple E(spl)-C genes that, in turn, regulate HLHm5 expression. In either event, Stat92E-mediated activation of E(spl)-C genes restricts tin in the dorsal mesoderm to establish phase 3 expression. Tin, therefore, establishes an autoinhibitory circuit by activating its own repressors in the JAK/Stat pathway and in E(spl)-C (Johnson, 2011).
Both Stat92E and Df(3R)Esplδmδ-m6 embryos show an increased number of Tin+ pericardial cells and an expanded H15 expression domain. Misexpressing mid or H15 in the mesoderm expands the number of Tin+ cells in the dorsal vessel and embryos misexpressing mid show a phenotype strikingly similar to Stat92E and E(spl) embryos. As mid, and presumably H15, are positively regulated by Tin during St11/12, unrestricted tin expression in Stat92E or Df(3R)Esplδmδ-m6 embryos expands the H15 expression domain. Ectopic H15 then specifies supernumerary Tin+ pericardial cells. Because mid expression is not affected in Stat92E embryos, the expression of mid and H15 must be controlled by distinct mechanisms and might have non-redundant functions (Johnson, 2011).
Although the Twi target genes directing mesoderm development and subdivision have been studied in detail, the molecular mechanism that restricts twi expression after gastrulation remains unclear. One regulator of twi is the Notch signaling pathway, which acts through E(spl)-C genes to restrict twi expression. However, Notch signaling appears to be active throughout the mesoderm after gastrulation. This study suggests that segmented JAK/Stat signaling activity differentially upregulates E(spl)-C gene expression in concert with Notch to produce periodic twi expression in the mesoderm. In addition, pan-mesodermal twi expression causes cardiac defects independently of cell fate specification, suggesting that the cardiac morphology defects in Stat92E embryos are due to dysregulated twi expression. These results highlight a previously unrecognized role for the JAK/Stat pathway and Twi in regulating cardiogenesis (Johnson, 2011).
Pericardial cell hyperplasia without a concomitant loss of cardioblasts has been reported for dpp hypomorphic embryos and lame duck (lmd) embryos. A late Dpp signal, which occurs after the Dpp signal that regulates phase 2 tin expression, instructs the Gli-like transcription factor Lmd to repress Tin expression in fusion competent myoblasts (FCMs). Tin expression appears to expand into the FCM domain in Stat92E embryos; however, the closest Stat92E chromatin binding domain is over 120 kb distal to the lmd transcriptional start site. This study highlights the possibility that sequential JAK/Stat and then Dpp signals regulate Lmd function to direct heart precursor diversification (Johnson, 2011).
In vertebrates, skeletal myogenesis initiates with the periodic specification of somites in the presomitic mesoderm. Cyclical expression of hairy1 in the chick, the hairy- and E(spl)-related family (Her) in zebrafish, and the orthologous Hes family in mice are under the control of Notch-Delta signaling. Loss of her1 and her7 alters the periodicity with which somite boundaries are specified in fish, and artificially stabilizing Hes7 causes somites to fuse in the mouse. Thus, mesoderm segmentation is governed by Notch-Delta regulation of the E(spl)-C genes in both insects and vertebrates indicating that the two processes share molecular homology. A cell culture model of somitogenesis shows that oscillating Hes1 expression is dependent on Stat activity. This study supports the exciting possibility that JAK/Stat signaling and E(spl)-C form a conserved developmental cassette directing mesoderm segmentation throughout Metazoa (Johnson, 2011).
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