odd-skipped


DEVELOPMENTAL BIOLOGY

Embryonic

ODD transcripts accumulate in a dynamic pattern during early embryogenesis, with two temporally distinct modes of expression. The first mode results in a 'pair-rule' pattern of seven stripes at the blastoderm stage, representing the expected double segment periodicity. This pattern comes into being with a temporal gradient of anterior to posterior and ventral to dorsal. There is also early expression in the anterior part of the embryo. These transcripts alternate with Fushi tarazu. During gastrulation [Image], a second mode of expression is initiated when the seven primary stripes are supplemented by secondary stripes which appear in alternate segments, resulting in the equivalent labeling of every segment in the extended germ band. Similar double to single segment transitions have now been reported for four of the six pair-rule genes analyzed (Coulter, 1990).

See Chris Doe's Hyper-Neuroblast map site for information on the expression of odd-skipped in specific neuroblasts.

Though odd-skipped is initially expressed in a striped pattern that reflects its function within the segmentation hierarchy, it is also expressed in a variety of patterns during later stages of embryogenesis. To identify the cells and tissues that correspond to these latter patterns, the distribution of the Odd protein at all embryonic stages was examined. Odd is a specific and persistent marker for subsets of cells in developing mesoderm, ectoderm, and neural tissue. It is concluded that Odd is a useful tool for studying cell specification, cell migrations and morphogenetic movements during organogenesis of the heart, gut and central nervous system (Ward, 2000a).

Moving from anterior to posterior within the gut, Odd is expressed in the following regions: a ring around the salivary duct, within the proventriculus, the posterior region of the midgut, and the proximal Malpighian tubules. The salivary duct connects the salivary glands to the pharynx. Odd is expressed in a ring of cells midway along the length of this duct. Distinct expression within the salivary duct primordium can be traced back to stage 11, when six Odd-positive cells (three per hemisegment) appear within the ventral epidermis of parasegment 2. Following their association into a cluster, these migrate interiorly and dorsally to assume their final position within the duct. The proventriculus is a complex, triple-layered structure where the foregut and the midgut join together to form a gastric valve. Odd is expressed in the outermost layer of the proventriculus. Although the proventriculus is not formed until stage 16, Odd expression in these cells can be traced back to cellularization, when cells at the anterior terminus begin expressing Odd. These cells enter the body via the stomodeum and move together as a narrow band of cells as the foregut extends caudally. Odd continues to be expressed as the gastric valve is formed and moves into its final destination within the animal. The Malpighian tubules perform an excretory and osmoregulatory function in the animal. The two pairs of branched tubules each join in a common ureter that empties into the hindgut. Odd is expressed in each ureter and in the posterior midgut. From stage 11 onward Odd is expressed in cells that will become incorporated into the ureters and posterior midgut. Analysis of third instar larvae reveals that Odd expression is not maintained in the gut throughout larval development (Ward, 2000a).

Odd is expressed in the garland cells associated with the proventriculus, in the pericardial cells, and the lymph glands associated with the heart. Together, these three cell types comprise a subpopulation of cells called nephrocytes, which are phagocytic cells that along with the Malpighian tubules form the excretory system of insects. The origin of the garland cells has not been described, however, they can be observed at stage 11, where they are loosely attached to the foregut cells expressing Odd. The garland cells remain attached to these foregut cells throughout the remainder of development. The progenitors of the lymph glands arise during stage 11 in the dorsal mesoderm of thoracic segment 1 through abdominal segment 1 (T1-A1). During subsequent stages additional lymph gland progenitors arise and these cells migrate posteriorly to the A1/A2 boundary, where they form clusters of cells corresponding to the lymph glands. At the end of development the Odd pericardial cells form continuous rows of cells flanking either side of the contractile heart tube. The anterior of each row terminates at a lymph gland. Double labeling shows that Odd and Eve, another pericardial cell marker, are expressed in distinct pericardial cells, with the Odd-positive cells located ventral to the Eve-positive cells. Analysis of third instar larvae indicates that nephrocyte-specific expression persists during larval development (Ward, 2000a).

Odd is expressed in a subset of cells within the central nervous system. At the end of development Odd is expressed in three discrete clusters within the brain. From stage 11 onward Odd can be observed continuously in the supraoesophageal ganglion, a region which gives rise to the brain. Odd is also expressed in a few neuroblasts and neurons within each segment of the developing nerve cord. The majority of the Odd positive cells within the neuroectoderm have been well characterized. Odd accumulates transiently during stage 9 in a subset of midline glial cells. The exact identity of the Odd expressing glial cells is not known (Ward, 2000a).

Somatic muscles are attached to the exoskeleton via tendon-like junctions called apodemes. In the trunk of the animal, there is one Odd-expressing apodeme per hemisegment, located midway along the dorsal/ventral axis. The muscle(s) attached to this apodeme are unknown. Odd is also expressed in the vicinity of the pharynx. Based upon position this expression corresponds to either the pharyngeal muscles or to the apodemes attaching the pharyngeal muscles to the exoskeleton (Ward, 2000a).

In summary, Odd expression identifies distinct subsets of cells in the gut, mesoderm, CNS and ectoderm. Furthermore, because it marks these cells throughout their development, Odd is a useful tool for following the morphogenetic movements of these tissues. Interestingly, these cells are readily identified in a strong odd mutant which makes a truncated protein recognized by the antiserum. Because this mutant appears to be a functional null, this observation indicates that the Odd-expressing tissues may not require Odd function for their initial specification and development, but does not preclude an essential role for Odd in other aspects of the structure or physiology of these cells (Ward, 2000a).

The Drosophila heart is a simple organ composed of two major cell types: cardioblasts, which form the simple contractile tube of the heart, and pericardial cells, which flank the cardioblasts. A complete understanding of Drosophila heart development requires the identification of all cell types that comprise the heart and the elucidation of the cellular and genetic mechanisms that regulate the development of these cells. A new population of heart cells is reported here: the Odd skipped-positive pericardial cells (Odd-pericardial cells). Descriptive, lineage tracing and genetic assays were used to clarify the cellular and genetic mechanisms that control the development of Odd-pericardial cells. Odd skipped marks a population of four pericardial cells per hemisegment that are distinct from previously identified heart cells. Within a hemisegment, Odd-pericardial cells develop from three heart progenitors and these heart progenitors arise in multiple anteroposterior locations within the dorsal mesoderm. Two of these progenitors divide asymmetrically such that each produces a two-cell mixed-lineage clone of one Odd-pericardial cell and one cardioblast. The third progenitor divides symmetrically to produce two Odd-pericardial cells. All remaining cardioblasts in a hemisegment arise from two cardioblast progenitors, each of which produces two cardioblasts. Furthermore, numb and sanpodo mediate the asymmetric divisions of the two mixed-lineage heart progenitors noted above (Ward, 2000b).

Odd is expressed in two rows of mesodermal cells that flank the dorsal midline of late stage Drosophila embryos. The two rows of Odd-expressing cells begin at the boundary of abdominal segments 1 and 2 and extend to the boundary between abdominal segments 6 and 7. Analyses of embryos double-labeled for Odd and Mef2 expression demonstrate that the rows of Odd-expressing cells are in close physical contact to, and flank, the cardioblasts. Odd-positive cells also reside slightly ventral to the cardioblasts. Immediately anterior to these two rows Odd expression has been detected in two bilaterally symmetric clusters of ~20 cells, which correspond to the lymph glands. The spatial dynamics of the Odd expression pattern in the dorsal mesoderm parallels that of proteins known to identify subsets of pericardial cells. For example, Eve and Tin label subsets of pericardial cells and these cells are found in two rows that flank the cardioblasts in late stage embryos. Based on the similarity between the spatial pattern of Odd expression and that of known pericardial cell markers, the Odd-expressing cells are identified as pericardial cells and they are therefore called Odd-pericardial cells (Ward, 2000b).

Two markers, Zfh1 and mAb3, label all pericardial cells. To determine whether Odd-pericardial cells express proteins or epitopes consistent with a pericardial cell fate, the expression of Odd and either that of Zfh1 or the epitope recognized by mAb3 have been followed in late-stage wild-type embryos. Odd-pericardial cells have been found to co-express Zfh1 and the antigen recognized by mAb3. These results indicate that Odd-pericardial cells express markers common to pericardial cells and are consistent with the identification of these cells as pericardial cells (Ward, 2000b).

It was determined whether Odd identifies a new population of pericardial cells or constitutes an additional marker for a previously identified subset of pericardial cells. To investigate this question, late-stage wild-type embryos were double labeled to detect Odd expression and the expression of either Eve or Tin. No co-expression of Odd with either Tin or Eve was detected. Since between them, Eve and Tin label all previously identified pericardial cells, it is concluded that Odd identifies a new subpopulation of pericardial cells that also express Zfh1 and mAb3. Odd-pericardial cells reside ventral and medial to Eve-pericardial cells and dorsal and lateral to Tin-pericardial cells. These results demonstrate that Odd identifies a new population of pericardial cells. Based on their position in the heart, Odd-pericardial cells probably correspond to the 'classical' pericardial cells that are defined by morphological studies to reside immediately lateral to the cardioblasts (Ward, 2000b).

Odd is the first molecular marker identified for this population of pericardial cells. Thus, Odd expression was used as a tool to determine (1) the embryonic origin of Odd-pericardial cells and how they develop, as well as (2) the cellular and molecular mechanisms that control their development. Thus, a careful developmental analysis of Odd-pericardial cell development was carried out in wild-type embryos. To increase the precision of these studies, the development of Eve-mesodermal cells during the early time points in these studies was simultaneously followed. Eve-mesodermal cells produce the Eve-pericardial cells and the Eve-positive DA1 muscle. Odd-pericardial cells could not be detected in a reproducible manner until stage 12/2 even though Eve-mesodermal cells are detected by stage 10. By stage 12/2, zero to two Odd-pericardial cells were detected in each hemisegment. These cells are located just posterior to the ectodermal Odd stripe, roughly midway between the AP position of Eve-mesodermal cells. Eve-mesodermal cells develop underneath the wg expression domain and thus mark the AP location of wg-expressing cells. By stage 13, on average three Odd-pericardial cells are detected beneath and just posterior to the Odd-ectodermal stripe per hemisegment and by stage 14, an average of four Odd-pericardial cells are detected aligned end to end along the AP length of a hemisegment. Odd-expressing cells that arise from the dorsal mesoderm in the thoracic and first abdominal segments give rise to part of the lymph gland. The development of these cells differs from the development of Odd-pericardial cells in the abdominal segments (Ward, 2000b).

Having established a wild-type profile of Odd-pericardial cell development it was of interest to identify the genetic regulatory mechanisms that govern Odd-pericardial cell development. Genes that control asymmetric divisions regulate Eve-pericardial cell development. Thus, whether loss of sanpodo or numb function affect Odd-pericardial cell and cardioblast development was examined. Normally 4.2 Odd-pericardial cells and 6.0 cardioblasts develop within each abdominal hemisegment of late-stage embryos. In numb mutant embryos, 6.0 Odd-pericardial cells and 4.2 cardioblasts were detected per hemisegment. Conversely, 7.6 cardioblasts and 2.7 Odd-pericardial cells per hemisegment were detected in sanpodo mutant embryos. Thus, in numb mutant embryos roughly two extra Odd-pericardial cells and two fewer cardioblasts were detected per hemisegment. Conversely, in sanpodo mutant embryos roughly two fewer Odd-pericardial cells and two additional cardioblasts form per hemisegment (Ward, 2000b).

These results demonstrate that sanpodo promotes Odd-pericardial cell development and opposes cardioblast development. Conversely, numb opposes Odd-pericardial cell development and promotes cardioblast development. In addition, they suggest that two cardioblasts and two Odd-pericardial cells arise via the asymmetric divisions of numb/sanpodo dependent heart progenitors. These results are consistent with the known requirement for Notch in pericardial cell development. Loss of numb function disrupts the precise alignment of cardioblasts leading to 'broken rows' of cardioblasts in numb mutant embryos (Ward, 2000b).

Multiple models can explain the reciprocal effects of sanpodo and numb on cardioblast and Odd-pericardial cell development. For example, one model predicts that two mixed-lineage heart progenitors each divide to yield one cardioblast and one Odd-pericardial cell. A second model predicts the existence of four progenitors: two would divide with each producing one Odd-pericardial cell and one cell of unknown fate; the other two progenitors would divide each producing one cardioblast and one cell of unknown fate. In these and other models, loss of numb or sanpodo function would equalize all asymmetric divisions and could result in the observed Odd-pericardial cell and cardioblast phenotypes (Ward, 2000b).

Lineage-tracing assays were used to distinguish between these models and to determine whether any Odd-pericardial cells and cardioblasts share a common ancestry. It was reasoned that if individual heart progenitors divide to produce both cardioblasts and Odd-pericardial cells, then lineage clones that contain both cell types should be found. Conversely, if cardioblasts and Odd-pericardial cells do not arise from a common progenitor, then clones should contain one of these cell types but not both. To trace the lineage of cardioblasts and Odd-pericardial cells the FLP/FRT lineage tracing system was used to create random clones of tau-lacZ reporter gene expression (Ward, 2000b).

Clones were induced during stages 8-9 just as the general pan-mesodermal cell divisions are being completed. Thus, it was expected that clones would be induced in mesodermal cells prior to the emergence of cardioblast and pericardial progenitors. To identify the lineage of Odd-pericardial cells embryos were double labeled for ß-galactosidase (to mark clones), and Odd (to identify Odd-pericardial cells). To identify the lineage of cardioblasts, embryos were double labeled for ß-galactosidase, to mark clones, and Mef2, to identify cardioblasts. Mef2 labels both cardioblasts and somatic muscles. However, one can use Mef2 to identify cardioblasts unambiguously, owing to the juxtaposition of Mef2 cardioblasts and the dorsal midline. 52 clones were identified that contained at least one Odd-pericardial cell and 36 that contained at least one Mef2-labeled cardioblast. Odd-pericardial cell clones fell into two major classes: those that contained two Odd-pericardial cells (36%) and those that contained both one Odd-pericardial cell and one cell that did not express Odd (48%). The Odd-negative cell was located medial, slightly dorsal and posterior to the Odd-pericardial cell. Odd-negative cells were identified as cardioblasts because cardioblasts are the only heart cells located medial, slightly dorsal and physically adjacent to Odd-pericardial cells. Rare larger clones were observed that consisted of either two pericardial cells and two cardioblasts (4%), three pericardial cells and one cardioblast (6%), or two Odd-pericardial cells and one cardioblast (6%) (Ward, 2000b).

Four Odd-pericardial cells develop per hemisegment. The simplest model by which the two major classes of clones could produce four Odd-pericardial cells predicts that one Odd-pericardial cell progenitor and two mixed lineage progenitors develop within each hemisegment. The Odd-pericardial cell progenitor would divide to produce two Odd-pericardial cells and the two mixed-lineage progenitors would each divide to produce one Odd-pericardial cell and one cardioblast. This model predicts a 2:1 ratio of mixed lineage to Odd-pericardial cell progenitors (or clones) and the data most closely fit this model, even though they yield an approximate 1.5:1 ratio of these clone types. The clonal analysis of cardioblast clones and the descriptive analysis of the development of these three heart progenitors support the predicted 2:1 ratio of mixed lineage heart progenitors/clones to Odd-pericardial cell progenitors/clones (Ward, 2000b).

These data also argue against a strict lineal relationship between any of the heart progenitors that produce Odd-pericardial cells. Most notably, the rare four cell clones identified fall into two classes: those that contain two Odd-pericardial cells and two cardioblasts and those that contain three Odd-pericardial cells and one cardioblast. The presence of these two clone types is incompatible with a strict lineal relationship between any two of the three Odd-pericardial cell progenitors (Ward, 2000b).

Specification of cell fates in the dorsal mesoderm appears to occur during early stage 11. Two general pan-mesodermal cell divisions precede these events and occur during stages 7 and 9, while spatially more distinct mesodermal cell divisions occur during stage 11 and stage 12. Clones were induced towards the end of the second pan-mesodermal cell division and only two and four cell clones were detected. Four cell clones are interpreted as arising from clones induced prior to the second pan-mesodermal division and two cell clones as arising from clones induced after the second pan-mesodermal division. Taken together these data appear to favor a model whereby the initial pan-mesodermal divisions produce a pool of uncommitted mesodermal cells upon which patterning and cell fate specification mechanisms act during stage 11 to commit these cells to specific fates. In this model, any two of the three progenitors of Odd-heart cells could be siblings at some frequency even though they are not specified in a lineage dependent manner (Ward, 2000b).

Clones that contained Mef2-labeled cardioblasts also fell into two major classes: those that contained two adjacent cardioblasts (44.4%); and those that contained one cardioblast and another cell located immediately lateral and anterior to the cardioblast (50%). The non-cardioblast cell in these clones was identified as Odd-pericardial cells because Odd-pericardial cells are the only heart cells that reside immediately lateral to cardioblasts. As with Odd-pericardial cell clones, rare larger clones were observed: one contained four cardioblasts, and one contained two cardioblasts and one Odd-pericardial cell. As noted, six cardioblasts develop per hemisegment. The simplest model by which the two major classes of cardioblast clones could yield six cardioblasts per hemisegment predicts that two cardioblast and two mixed lineage progenitors arise in a hemisegment. The two cardioblast progenitors would each divide to produce two cardioblasts and the two mixed-lineage heart progenitors would each divide to produce one cardioblast and one Odd-pericardial cell. This model predicts a 1:1 ratio between cardioblast and mixed lineage progenitors (or clones) and the data, which yield an approximate 1:1 ratio between these clone types, fit this model well. Together with the lineage data from Odd-pericardial cells these results indicate that five heart progenitors produce the six cardioblasts and four Odd-pericardial cells that develop in each hemisegment (Ward, 2000b).

The cardioblast lineage data do not exclude the possibility that the two cardioblast progenitors are strictly linearly related. However, only a single four-cell cardioblast clone was seen among the 36 cardioblast clones identified, even though flp expression was induced at a stage that should activate flp in the parental (or even grand-parental) cells of these progenitors. Thus, the model that all heart progenitors are selected from uncommitted pools of cells by patterning and cell-fate specification mechanisms is preferred. The future identification of four cell clones that contain three cardioblasts and one Odd-pericardial cell would support the idea that the two cardioblast progenitors do not develop in a lineage-dependent manner (Ward, 2000b).

An enhancer trap in the seven-up gene identifies the two mixed-lineage heart progenitors. Towards the end of the lineage analyses it was discovered fortuitously that an enhancer trap in the gene seven-up labels four heart cells in each abdominal hemisegment. This enhancer trap is referred to as svp-lacZ). Two of these cells reside at the dorsal midline and are cardioblasts since they express Mef2. The other two cells reside just lateral and slightly ventral and anterior to the svp-lacZ cardioblasts. These two cells are Odd-pericardial cells because they express Odd. The relative positioning of the svp-lacZ cardioblasts and Odd-pericardial cells closely resembles that of the sibling cardioblasts and Odd-pericardial cells marked by the mixed lineage heart clones. This suggests that the svp-lacZ heart cells may identify the four progeny of the two mixed lineage heart progenitors that arise in each hemisegment. If the four svp-lacZ heart cells are the progeny of these two progenitors, then loss of sanpodo function should convert all svp-lacZ heart cells to cardioblasts and loss of numb function should convert all svp-lacZ heart cells to Odd-pericardial cells. In sanpodo mutant embryos, all four svp-lacZ cells acquire the cardioblast fate and in numb mutant embryos all four svp-lacZ cells acquire the Odd-pericardial cell fate. The results from these experiments demonstrate that svp-lacZ identifies the progeny of the two mixed lineage heart progenitors and that numb and sanpodo mediate the asymmetric divisions of these mixed-lineage heart progenitors (Ward, 2000b).

Present models of heart development suggest that all heart cells arise from dorsal mesodermal cells that reside beneath the transverse stripe of ectodermal cells that express Wg protein. However, Odd-pericardial cells are first detect emerging roughly midway between Eve-pericardial cells, which themselves arise beneath the Wg-expressing ectodermal cells. The initial appearance of Odd expression in pericardial cells at the end of stage 12 precludes its use as a definitive marker of the embryonic origin of these cells. However, since svp-lacZ labels all four progeny of the two mixed lineage heart progenitors an assay was carried out to see whether svp-lacZ is expressed in the mixed lineage heart progenitors. If so, svp-lacZ could be used as a marker to identify the AP origin of mixed lineage heart progenitors (Ward, 2000b).

svp-lacZ is first detected in the dorsal mesoderm during stage 11 in two individual cells: one is located beneath the ectodermal Odd stripe midway between Wg-stripes; the other is found immediately anterior to, or just at the anterior edge of, Wg-expressing cells. During early stage 12 these two cells divide and produce four cells, all of which express svp-lacZ and Mef2; at this stage none of these cells express Odd. During stage 12 the four svp-lacZ heart cells congregate together to form a tight four-cell cluster. Using confocal microscopy, it was found that by stage 13, the four svp-lacZ-positive cells could be broken into two groups based on Mef2 expression: two cells express Mef2 at high levels and two cells express Mef2 at low levels. Because cardioblasts retain and Odd-pericardial cells extinguish Mef2 expression, the svp-lacZ heart cells with high-level Mef2 expression were identified as cardioblasts and those with low Mef2 expression as Odd-pericardial cells. These results suggest that the two svp-lacZ heart progenitors arise from two different AP locations in the dorsal mesoderm, at least one of which does not arise from dorsal mesodermal cells located beneath the ectodermal wg stripe, the postulated source of all heart cells. The svp-lacZ molecular marker also allows for distinguishing between svp-lacZ/Odd-pericardial cells and the Odd-pericardial progenitor and its progeny. This is facilitated the identification of the location of the Odd-pericardial progenitor just prior to its division. Odd-expression was first detected in the Odd-pericardial progenitor at stage 12/0. The Odd-pericardial progenitor is located beneath the ectodermal Odd-stripe and divides shortly after stage 12/0 to produce the remaining two Odd-pericardial cells per hemisegment. The Odd-progenitor and its progeny reside adjacent and anterior to the Odd/svp-lacZ-pericardial cells. These results suggest that the Odd-pericardial progenitor is specified from dorsal mesodermal cells located beneath the Odd ectodermal stripe. However, extensive mesodermal rearrangements occur prior to stage 12/0 (Ward, 2000b).

Thus, it is possible that the Odd-pericardial progenitor is specified in another AP domain (e.g. the wingless domain) and that the cellular rearrangements in the mesoderm place this cell beneath the ectodermal Odd stripe prior to the stage (stage 12/0), during which this cell stimulates Odd expression. Markers that identify the Odd-pericardial progenitor earlier in development are required to determine definitively whether the progenitor is born (1) beneath the Odd stripe, or (2) in a different AP domain, migrating beneath the Odd stripe later in development (Ward, 2000b).

Odd-skipped and leg segmentation

Flexible joints separate the rigid sections of the insect leg, allowing them to move. In Drosophila, the initial patterning of these joints is apparent in the larval imaginal discs from which the adult legs will develop. The later patterning and morphogenesis of the joints, which occurs after pupariation (AP), is described. In the tibial/tarsal joint, the apodeme insertion site provides a fixed marker for the boundary between proximal and distal joint territories (the P/D boundary). Cells on either side of this boundary behave differently during morphogenesis. Morphogenesis begins with the apical constriction of distal joint cells, about 24 h AP. Distal cells then become columnar, causing distal tissue nearest the P/D boundary to fold into the leg. In the last stage of joint morphogenesis, the proximal joint cells closest to the P/D boundary align and elongate to form a 'palisade' (a row of columnar cells) over the distal joint cells. The proximal and distal joint territories are characterized by the differential organization of cytoskeletal and extracellular matrix proteins, and by the differential expression of enhancer trap lines and other gene markers. These markers also define a number of more localised territories within the pupal joint (Mirth, 2002).

To identify distinct cell populations in the joints, the expression patterns of 10 joint markers were examined with respect to a posterior marker (engrailed lacZ) and a ventral marker (wingless lacZ). The leg discs of wandering larvae, and pupal legs at 24-28 and 34-38 h AP, were examined. Four of the joint markers were previously reported to be expressed in L3 and prepupal joints (Notch, disconnected lacZ, Nubbin, and odd-skipped lacZ). The rest were isolated for this study by screening Gal4 enhancer trap lines for those that drive expression of GFP in pupal leg joints (ckm78, ckm90, ckm239, ckm175, ok388, and ok483). Most of the joint markers do not change their expression domains between 24-28 and 34-38 h AP. Therefore, data is presented from wandering L3 discs only, and from legs at 34-38 h AP (Mirth, 2002).

In the L3 leg disc, joint markers fall into one of two categories, marking either the proximal joint territories (e.g., Nubbin) or the distal territories (e.g., Notch and odd-skipped lacZ). Of all the markers examined, only Nubbin (Nub), disconnected lacZ (disco lacZ), and odd-skipped lacZ (odd lacZ) are expressed in more than two joints in the L3 stage. Others mark one or two joints at this stage but are expressed in all joints during the pupal stage. Studies examining the expression of Notch and other elements of the Notch patterning cascade have also found that the joint seems to be divided into proximal and distal territories at this stage. Thus, proximal and distal joint domains have already been established by the late L3 (Mirth, 2002).

By 34-38 h AP, patterns of marker expression define three additional territories. First, a proximal-dorsal patch is high-lighted by two joint markers, ckm90 and ckm175, that drive GFP expression only in a patch above and includes the most proximal cells of the dorsal apodeme. The expression of GFP driven by ckm175 includes a greater number of cells than that driven by ckm90. The second domain identified was a mid-distal domain. Odd lacZ expression becomes largely restricted to a mid-distal group of cells in all but the tarsal joints. This corresponds to the region that does not accumulate collagen IV and marks the cells that push underneath the proximal joint cells. Odd lacZ is also expressed in the apodemes. Lastly, ok388 expresses GFP in the lateral anterior and posterior parts of the distal tibial/tarsal (but not tarsal) joint, but is excluded from the dorsal and ventral domains. This expression domain corresponds with the region of elongating cells seen in longitudinal sections of the leg (Mirth, 2002).

Two of the joint markers are expressed in both the proximal and distal portions in the developing adult joint: ckm239 and disco lacZ. Disco lacZ is expressed throughout the entire joint, and ckm239 is excluded from the ventralmost region (wingless lacZ-expressing region) (Mirth, 2002).

It seems likely that the domains of gene expression observed in the L3 leg disc correspond with those of the same genes in the developing adult joint, though this has not been verified directly. If so, proximal and distal joint domains are established before pupariation. These two joint territories separate cells that will invaginate [the cells in the odd lacZ domain, expressing the Notch target E(SPL)Mß] from those that will form the proximal palisade (the cells expressing Delta, Serrate, and Nubbin). During pupal development, the proximal and distal domains of the joint become further subdivided. Most of the enhancer trap markers identified are expressed in specific groups of cells within either the proximal or distal domain in the tibial/tarsal joint at 34-38 h AP. At the same time, the expression of some earlier markers becomes restricted to more specific territories. odd lacZ, which is expressed in some joints in the L3, is expressed most strongly in the mid-distal joint cells at 34-38 h AP. Ok388 expresses in the distalmost but not mid-distal joint cells, and is restricted to the lateral anterior and posterior sides. In the proximal joint, markers such as ckm90 and ckm175 express in only a small group of cells on the dorsal side. Thus, it seems that the tibial/tarsal joint may divided into three proximodistal domains based both on cell behavior and gene expression: proximal, mid-distal, and distalmost regions. Later during pupal development, the distalmost region subdivides into lateral anterior/posterior and dorsal/ventral domains and the proximal joint also subdivides into smaller territories. That further patterning and subdivision of the joint occurs after the prepupal stages is hardly surprising: the adult joint is too complex a structure to be derived simply from the proximodistal interactions that occur before pupal development (Mirth, 2002).

The Drosophila lymph gland as a developmental model of hematopoiesis

Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).

In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).

The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).

Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).

Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn06946). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).

The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca2+-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).

The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).

The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).

In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).

The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).

To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).

By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).

In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).

The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).

To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).

Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).

Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).

This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).

Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).

Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S+O+) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S+O+ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S+O+H+Pv+) pre-prohemocytes initiate the expression of dome-gal4 (dg4), thereby maturing into prohemocytes. The prohemocyte population (S+O+H+Pv+dg4+) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S+O+H+Pv+dg4lowM+). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S+O+H+Pv+dg4-M+), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).

Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).

Effects of Mutation or Deletion

odd-skipped mutant embryos exhibit pattern defects in anterior regions of odd-numbered segments (Coulter, 1990).

Odd-skipped genes specify the signaling center that triggers retinogenesis in Drosophila

Although many of the factors responsible for conferring identity to the eye field in Drosophila have been identified, much less is known about how the expression of the retinal 'trigger', the signaling molecule Hedgehog, is controlled. This study shows that the co-expression of the conserved odd-skipped family genes at the posterior margin of the eye field is required to activate hedgehog expression and thereby the onset of retinogenesis. The fly Wnt1 homologue wingless represses the odd-skipped genes drm and odd along the anterior margin and, in this manner, spatially restricts the extent of retinal differentiation within the eye field (Bras-Pereira, 2006).

The eye disc is a flat epithelial sac. By early third larval stage (L3), columnar cells in the bottom (disc proper: Dp) layer are separated by a crease from the surrounding rim of cuboidal margin cells. Margin cells continue seamlessly into the upper (peripodial; Pe) layer of squamous cells. The Dp will differentiate into the eye, while the margin and Pe will form the head capsule. In addition, the posterior margin produces retinal-inducing signals (Bras-Pereira, 2006).

By examining gene reporters it was found that the zinc-finger gene odd is expressed restricted to the posterior margin and Pe of L3 eye discs. Since the odd family members drumstick (drm), brother of odd with entrails limited (bowl) and sister of odd and bowl (sob) are similarly expressed in leg discs, they were examined in eye discs. In L2, before retinogenesis has started, odd and drm are transcribed in the posterior Pe-margin, and this continues within the posterior margin after MF initiation. bowl is transcribed in all eye disc Pe-margin cells of L2 discs, but retracts anteriorly along the margins and Pe after the MF passes. In addition, bowl is expressed weakly in the Dp anterior to the furrow. sob expression in L2 and L3 is mostly seen along the lateral disc margins. Therefore drm, odd and bowl are co-expressed at the posterior margin prior to retinal differentiation initiation (Bras-Pereira, 2006).

Odd family genes regulate diverse embryonic processes, as well as imaginal leg segmentation. In embryos, the product of the gene lines binds to Bowl and represses its activity, while Drm relieves this repression in drm-expressing cells. Since drm/odd/bowl expression coincides along the posterior margin around the time retinal induction is triggered, it was asked whether they controlled this triggering. First, bowl function was removed in marked cell clones induced in L1. bowl- clones spanning the margin, but not those in the DP, cause either a delay in, or the inhibition of, retinal initiation and the autonomous loss of hh-Z expression. Correspondingly, there is a reduction in expression of the hh-target patched (ptc). These effects on hh and ptc are not due to the loss of margin cells, since drm is still expressed in the bowl- cells. The requirement of Bowl for hh expression is margin specific, since other hh-expressing domains within the disc are not affected by the loss of bowl (not shown). As expected from the bowl-repressing function of lines, the overexpression of lines along the margin phenocopies the loss of bowl. Nevertheless, the overexpression of bowl in other eye disc regions is not sufficient to induce hh. This suggests that, in regions other than the margin, either the levels of lines are too high to be overcome by bowl or bowl requires other factors to induce hh, or both (Bras-Pereira, 2006).

drm and odd are expressed together along the posterior disc margin-Pe, and drm (at least) is required for Bowl stabilization in leg discs. Nevertheless, the removal of neither drm nor odd function alone results in retinal defects. odd and drm may act redundantly during leg segmentation and this may also be the case in the eye margin. To test this, clones were induced of DfdrmP2, a deficiency that deletes drm, sob and odd, plus other genes. When DfdrmP2 clones affect the margin, the adjacent retina fails to differentiate, suggesting that drm and odd (and perhaps sob, for which no single mutation is available) act redundantly to promote bowl activity at the margin (although the possibility that other genes uncovered by this deficiency also contribute to the phenotype cannot be excluded). To test the function of each of these genes, drm, odd and sob were expressed in cell clones elsewhere in the eye disc. Only the overexpression of drm or odd induced ectopic retinogenesis, and this was restricted to the region immediately anterior to the MF, which is already eye committed. Interestingly, bowl is also expressed in this region of L3 discs. The retina-inducing ability of drm requires bowl, because retinogenesis is no longer induced in drm-expressing clones that simultaneously lack bowl function. Therefore, it seems that in the eye, drm (and very likely also odd) also promotes bowl function (Bras-Pereira, 2006).

The expression of hh or activation of its pathway anterior to the furrow is sufficient to generate ectopic retinal differentiation. Since (1) bowl is required for hh expression at the margin, (2) this hh expression is largely coincident with that of odd and drm, and (3) drm (and possibly odd) functionally interacts with bowl, whether drm- and odd-expressing clones induced the expression of hh was examined. In both types of clones hh expression is turned on autonomously, as detected with hh-Z, which would thus be responsible for the ectopic retinogenesis observed. That the normal drm/odd/bowl-expressing margin does not differentiate as eye could be explained if margin cells lack certain eye primordium-specific factors (Bras-Pereira, 2006).

These results indicate that the expression of odd and drm defines during L2 the region of the bowl-expressing margin that is competent to induce retinogenesis. How is their expression controlled? wingless (wg) is expressed in the anterior margin, where it prevents the start of retinal differentiation. drm/odd are complementary to wg (monitored by wgZ) during early L3, when retinal differentiation is about to start, and also during later stages. In addition, when wg expression is reduced during larval life in wgCX3 mutants, drm transcription is extended all the way anteriorly. This extension precedes and prefigures the ectopic retinal differentiation that, in these mutants, occurs along the dorsal margin. Therefore, wg could repress anterior retinal differentiation by blocking the expression of odd genes in the anterior disc margin, in addition to its known role in repressing dpp expression and signaling (Bras-Pereira, 2006).

Interestingly, the onset of retinogenesis in L3 is delayed relative to the initiation of the expression of drm/odd and hh in L1-2. This delay can be explained in three, not mutually exclusive, ways. (1) The relevant margin factors (i.e. drm/odd, hh) might be in place early, but the eye primordium might become competent to respond to them later. In fact, wg expression domain has to retract anteriorly as the eye disc grows, under Notch signaling influence, to allow the expression of eye-competence factors. (2) Building up a concentration of margin factors sufficient to trigger retinogenesis might require some time. In fact, the activity of the Notch pathway along the prospective dorsoventral border is required to reinforce hh transcription at the firing point. (3) Other limiting factors might exist whose activity becomes available only during L3. Such a factor might be the EGF receptor pathway, which is involved in the triggering and reincarnation of the furrow along the margins during L3 (Bras-Pereira, 2006).

Hedgehog, but not Odd skipped, induces segmental grooves in the Drosophila epidermis

The formation of segmental grooves during mid embryogenesis in the Drosophila epidermis depends on the specification of a single row of groove cells posteriorly adjacent to cells that express the Hedgehog signal. However, the mechanism of groove formation and the role of the parasegmental organizer, which consists of adjacent rows of hedgehog- and wingless-expressing cells, are not well understood. This study reports that although groove cells originate from a population of Odd skipped-expressing cells, this pair-rule transcription factor is not required for their specification. It was further found that Hedgehog is sufficient to specify groove fate in cells of different origin as late as stage 10, suggesting that Hedgehog induces groove cell fate rather than maintaining a pre-established state. Wingless activity is continuously required in the posterior part of parasegments to antagonize segmental groove formation. These data support an instructive role for the Wingless/Hedgehog organizer in cellular patterning (Mulinari, 2009).

It has been reported that segmental groove formation requires the activity of engrailed (en) and hh and that en has a function that is independent of its role in hh activation. More recently, it was been found that en is not expressed in groove cells, thus creating a non-cell-autonomous requirement for en. To address this issue, the role of hh and en in segmental groove formation was reinvestigated (Mulinari, 2009).

It was found that segmental grooves do not form in hh mutants. When hh was overexpressed, the four to five cell rows posterior to the Hh source constricted apically, elongated their apical-basal axis and took on a shape characteristic of segmental groove cells. Very similar cell behavior was observed in patched (ptc) mutants or when activated Ci, which mediates hh activity, was expressed. These observations suggest that Hh can organize segmental groove formation. No cell constrictions were observed in the ventral epidermis, indicating that a different mechanism might regulate cell shape there (Mulinari, 2009).

To address the proposed hh-independent function of en, en, invected (inv) double mutants were investigated in which hh expression was maintained using prd-Gal4. Segmental grooves were rescued in these mutants, suggesting that en is not required for segmental groove formation independent of its role in hh activation. By contrast, it was found that en represses groove cell behavior when ectopically expressed together with hh. A previous study that reported a requirement of en in groove formation was based on the analysis of en, inv, wg triple mutants, in which hh expression was maintained but did not rescue groove formation. This result was confirmed, but it is proposed that wg may be required in en mutants to allow the morphological differentiation of grooves (Mulinari, 2009).

Analysis of ptc mutants, or embryos overexpressing hh, reveals that a broad region of cells posterior to the en expression domain are specified as groove cells. However, groove-like invaginations form only at the edges of these regions. This is even more obvious in double mutants of ptc and the segment polarity gene sloppy paired 1 (slp1), which is required for maintained wg expression. In slp1, ptc mutants, wg expression fades prematurely and Hh signaling is constitutively active. This results in a substantial expansion of the number of groove cells. However, furrows differentiate only at the edges of groove cell populations. It is proposed that the morphological differentiation of segmental grooves can occur only at the interface between groove and non-groove cells (Mulinari, 2009).

To test this, wg, ptc double mutants were used in which Hh signaling is active throughout the epidermis and all cells take on a groove fate. Interestingly, these embryos did not differentiate grooves. A similar observation has been reported in en, inv, wg mutants, in which hh expression is sustained, leading to the suggestion that en might be required for groove specification (Mulinari, 2009).

Analysis of cell behavior in wg, ptc mutants showed, however, that cells throughout the tissue constrict their apices but fail to form invaginating furrows. The failure of wg, ptc mutants and en, inv, wg; UAS-hh embryos to differentiate grooves might be due to the absence of non-groove cells in the epidermis and the concomitant absence of an interface with groove cells (Mulinari, 2009).

The pair-rule gene odd is initially expressed in 4- to 5-cell wide stripes in even-numbered parasegments. At early gastrulation, odd expression expands to segmental periodicity and is subsequently refined to a single row of prospective groove cells located posterior to en. Continued expression of odd in these cells requires hh. In odd5 mutant embryos, grooves are unaffected in odd-numbered parasegments, but partially missing in even-numbered parasegments, and residual grooves coincide with regions in which odd expression is detectable (Vincent, 2008). These observations have been interpreted as indicating that groove fate might be specified prior to the requirement of Hh and differentiation of the groove. Thus, the later activity of Hh might not induce, but merely maintain, groove cell identity that has been pre-established in the odd-expressing cell population (Vincent, 2008). However, this hypothesis is based on the presumption that odd has a function in groove cell specification and this has not been demonstrated (Mulinari, 2009).

Residual grooves in odd5 mutants have been attributed to the hypomorphic nature of the odd5 allele; however, the molecular lesion in odd5 is unknown. Therefore the nucleotide sequence of odd5 was determined and a substitution was found that mutates codon 84 from CAG to a TAG stop codon. The resulting truncated peptide, which lacks all four putative zinc fingers encoded by wild-type odd, is no longer restricted to the nucleus but uniformly distributed in the cell. Thus, odd5 is likely to be a null allele (Mulinari, 2009).

To exclude the possibility that groove formation may be rescued by read-through of the stop codon in odd5 mutants, or that odd may be required redundantly, segmental grooves were investigated in Df(2L)drmP2 mutants, in which odd and its sister genes drumstick (drm) and sister of odd and bowl (sob) are entirely deleted. In these embryos, normal grooves formed in odd-numbered parasegments in the complete absence of odd function (Mulinari, 2009).

Next even-numbered parasegments were investigated in which grooves are partially missing. odd encodes a transcriptional repressor that regulates the expression of other segmentation genes in the early embryo. In odd mutants, derepression of the en activator fushi-tarazu in even-numbered parasegments results in the formation of an ectopic en stripe posterior to the normal stripe. Simultaneously, wg expands anteriorly and becomes expressed adjacent to the ectopic en-expressing cells. This results in the formation of an ectopic parasegment boundary with reversed polarity. Thus, the outward-facing edges of both en stripes are genetically anterior and lined by wg-expressing cells that do not form grooves. The inward-facing edges of the normal and ectopic en stripes fuse in some areas, and these corresponded to areas in which grooves were missing, as cells that were genetically posterior to en and could respond to the Hh signal had been replaced by en-expressing cells. The fusion of normal and ectopic en stripes was more severe in Df(2L)drmP2 mutants; however, islands of invaginating groove cells could still be observed, demonstrating that groove fate is specified in the absence of odd, drm and sob function in all parasegments. It is concluded that all cells that are genetically posterior to en are specified as groove cells in the absence of odd function and the partial absence of grooves in even-numbered parasegments in odd mutants is a secondary consequence of the pair-rule phenotype of these embryos. The slightly more severe pair-rule phenotype seen in Df(2L)drmP2 mutants might be due to a contribution from one of the odd sister genes, most likely sob, to pair-rule function, or could be caused by low-level read-through of the stop codon in the odd5 allele (Mulinari, 2009).

Finally, to investigate whether odd is sufficient to trigger cell shape changes, a UAS-odd transgene was expressed either alone or together with hh in the epidermis. No induction of groove cell behavior other than that triggered by hh was observed. Together, the data show that odd plays no essential role in groove cell specification and that odd paralogs are unlikely to act redundantly in this process (Mulinari, 2009).

The identification of odd as a groove cell marker led Vincent to suggest that groove fate might be specified prior to Hh requirement and that Hh may merely maintain groove fate instead of having an inducing role (Vincent, 2008). This study demonstrate that grooves are specified in the absence of odd function; however, this could be due to an odd-independent, early-acting mechanism present in the cells from which grooves arise (Mulinari, 2009).

In order to address whether groove fate is pre-established in the odd-expressing cell population, it was asked if groove fate could be induced in cells of a different origin at a later point in time. lines (lin) mutants were used in which late wg expression is altered resulting in the formation of an ectopic segment boundary at the anterior edge of the en domain in the dorsal epidermis. Importantly, the early expression of pair-rule or segment polarity genes is not affected (Mulinari, 2009).

In lin mutants, ectopic expression of the groove marker odd was initiated at stage 12 in a single row of groove-forming cells anterior to en that are derived from a previously non-odd-expressing cell population that does not contribute to grooves in the wild type. Ectopic grooves require hh as they were not induced in hh, lin double mutants, and ectopic odd expression was not induced in this background. An increase in hh levels in lin mutants resulted in the specification of groove fate in all cells except those expressing en. These results suggest that hh is sufficient, late in development, to specify groove cell fate in cell populations of different origins and that earlier-acting factors present in the population of odd-expressing cells posterior to en are not required. Very similar results have been reported by Piepenburg (2000), who showed that segment border cells form solely in response to the Hh signal that emanates from the en domain (Mulinari, 2009).

The findings are consistent with the role of Hh in the regulation of cell shape in other systems. Thus, during Drosophila eye development, Hh has been shown to control cell shape in the morphogenetic furrow, and Hh activation in other tissues is sufficient to induce apical constriction and groove formation. It is likely that Hh plays a similar role in tissue morphogenesis in other organisms. During neural tube closure in vertebrates, cells undergo similar shape changes involving apical-basal elongation and apical constriction, which is likely to be in response to Hh sources in the notochord and floor plate. Accordingly, knockout of sonic hedgehog is associated with defects in neural tube closure in mice. These observations suggest that Hh might be a principal inducer of cell shape across species (Mulinari, 2009).

It has previously been established that wg antagonizes the activity of hh in the specification of segment border cells (Piepenburg, 2000). However, it is not clear whether wg has a similar role in segmental groove formation, and a late requirement of wg to antagonize Hh-mediated groove specification has been questioned (Vincent, 2008). To investigate a direct role of wg in groove specification, a dominant-negative form of the transcription factor pan (panDN), which suppresses Wg signaling, was expressed. For this, pnr-Gal4, which initiates expression in the dorsal epidermis at stage 10-11 and thus does not affect early wg function, was used. Embryos that express panDN formed a single row of ectopic groove cells anterior to the en domain, confirming the results in lin mutants. Strikingly, inactivating Wg signaling and increasing Hh levels at the same time by co-expression of panDN and hh resulted in the expansion of groove fate to all cells except those expressing en. These results show that Wg signaling is required after stage 10 to repress groove specification anterior to en, thus making the activity of Hh asymmetric. These results also confirm observations that Hh is sufficient to induce groove fate in cells from different positions along the anterior-posterior axis and suggest that groove fate is not determined before stage 10 (Mulinari, 2009).

To confirm the ability of wg to repress groove fate, wg was expressed posterior to en in cells that normally take on groove fate. This resulted in the loss of Odd from many cells, suggesting that wg indeed antagonizes hh activity. Interestingly, these cells still formed grooves. However, these grooves appeared much earlier than segmental grooves, suggesting that they are ectopic parasegmental grooves caused by ectopic wg expression, as recently suggested (Larsen, 2008). Together, these data therefore support the contention that Wg signaling is required to repress Hh-mediated induction of groove fate after stage 10, thus permitting the formation of segmental grooves posterior, but not anterior, to en in the wild type (Mulinari, 2009).


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odd-skipped: Biological Overview | Evolutionary homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 April 2014

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