retained

DEVELOPMENTAL BIOLOGY

Embryonic

Maternal DRI mRNA is distributed throughout the embryo during the syncytial cleavage divisions, while at cellularization, mRNA is found in broad bands at the termini and in a central band. At germ band extension, mRNA is found predominantly in the mesoderm. Dri protein is found to be localized to the nucleus whenever present. It is found evenly distributed among syncytial nuclei. The only instance in which mRNA and protein distribution differ is in late blastoderm embryos. At this stage, the stripped appearance of mRNA distribution contrasts with ubiquitous distribution of protein, presumably reflecting the persistence of maternal protein after the degradation of maternal mRNA. At germ band extension, protein distribution again reflects mRNA localization, both appearing primarily in the mesoderm. Germ band-retracted embryos exhibit organ-specific expression, including expression in the pharyngeal muscles, discrete rows of cells in the hindgut epithelium, the amnioserosa, the ring gland, a ring of cells at the midgut-hindgut junction, and several distinct cells in the posterior region of each brain lobe. Expression is also observed in cells of the salivary gland duct but not in cells of the salivary gland itself; in a ring of cells at the foregut-midgut junction, and in a segmentally repeated pattern in the central nervous system (Gregory, 1996).

The Drosophila salivary gland is a simple tubular organ derived from a contiguous epithelial primordium, which is established by the activities of the homeodomain-containing proteins Sex combs reduced (Scr), Extradenticle (Exd), and Homothorax (Hth). EGF signaling along the ventral midline specifies the salivary duct fate for cells in the center of the primordium, while cells farther away from the source of EGF signal adopt a secretory cell fate. EGF signaling works, at least in part, by repressing expression of secretory cell genes in the duct primordium, including fork head (fkh), which encodes a winged-helix transcription factor. Fkh, in turn, represses trachealess (trh), a duct-specific gene initially expressed throughout the salivary gland primordium. trh encodes a basic helix-loop-helix PAS-domain containing transcription factor that has been proposed to specify the salivary duct fate. In conflict with this is the idea that trh specifies salivary duct fate: three genes, dead ringer (dri), Serrate (Ser), and trh itself, are expressed in the duct independently of trh. Expression of all three duct genes is repressed in the secretory cells by Fkh. Ser in the duct cells signals to the adjacent secretory cells to specify a third cell type, the imaginal ring cells. Thus, localized EGF- and Notch-signaling transform a uniform epithelial sheet into three distinct cell types. In addition, Ser directs formation of actin rings in the salivary duct (Haberman, 2003).

dead ringer (dri; also known as retained) and Serrate (Ser), are expressed to high levels in the salivary duct. dri encodes an ARID-box transcription factor whose role in the salivary duct has not yet been determined. Ser encodes a ligand for the Notch receptor, whose role in this tissue is also unknown. Expression levels of both dri and Ser are unaffected in trh mutants. Dri protein is present in the uninvaginated salivary duct cells that remain on the surface of trh mutants. Similarly, both Ser RNA and ß-galactosidase expressed under the control of a Ser enhancer (Ser-lacZ) are expressed in salivary duct cells in trh mutants. Thus, trh is neither required for its own expression nor for the expression of at least two other salivary duct genes (Haberman, 2003).

Since dri and Ser are expressed independently of trh, it was asked whether there is any regulatory relationship among the three genes. trh expression is not altered in embryos mutant for dri or Ser. Similarly, Ser expression is not altered in dri mutants, and Dri expression is not altered in Ser mutants. Thus, all three genes are expressed in the salivary duct independently of the other two (Haberman, 2003).

trh is initially expressed throughout the salivary gland, in both duct and secretory cell primordia, but becomes restricted to the duct cells by fkh. It has been suggested that Fkh acts through repression of trh to limit expression of all duct genes to only the ventral preduct portion of the salivary gland primordium. Since it has been shown that expression of at least three genes is trh-independent, it is unclear how their expression is limited to the duct. Whether or not expression of the trh-independent duct genes is affected by Fkh was tested. Since salivary gland cells undergo apoptosis in fkh mutants, the experiments were performed in the background of the H99 deficiency, which blocks apoptosis by removing the apoptosis-activating genes hid, grim, and reaper. As in fkh mutants alone, all salivary gland cells remain on the surface of the embryo in fkh H99 embryos. In these embryos, secretory cells express the secretory marker Pasilla (PS) and Trh is expressed in all salivary gland cells. Similarly, expression of both Dri and Ser expanded into the secretory cells of fkh H99 embryos, suggesting that fkh is required to prevent secretory cell expression of multiple duct genes independently. Expression of all three genes is also observed throughout the salivary gland primordium of fkh mutants without the H99 deficiency, demonstrating that the observed expression profiles are not affected by the H99 deficiency. Also, expression of all of these genes is unchanged in H99 homozygous embryos, further indicating that the changes in gene expression are due to fkh (Haberman, 2003).

Given the role of trh in salivary duct morphogenesis, what is the role of the two Trh-independent salivary duct genes? Staining of dri mutants with the duct markers Trh, Ser, or Crb did not reveal any overt morphological changes from wild-type embryos. Staining of Ser mutants with Dri revealed only a subtle, partially penetrant defect, where the distal ends of the individual ducts are slightly enlarged. Differences between Ser and wild-type embryos in the distal ends of the salivary ducts are more apparent with staining for cytoplasmic Ser-lacZ, which reveals that the ends of the individual ducts are splayed in the region where they contacted the secretory cells (Haberman, 2003).

To test for any potential cell fate changes at the ends of the individual ducts in Ser mutants, expression was analyzed of several salivary gland markers. By coimmunofluorescence with Ser-lacZ, it was found that the cells at the duct ends still express Dri and do not express the secretory cell markers dCrebA and PS. Thus, the change in duct morphology is likely not due to a change in duct cell fate. No change in staining for the phosphorylated form of histone H3 was detected, indicating that loss of Ser does not cause a change in cell proliferation (Haberman, 2003).

Effects of Mutation or Deletion

Homozygotes for P-element insertion alleles dri⁷ and dri⁸ are embryonic lethal, but have only mild phenotypes. The pattern of cuticle structures in these embryos is normal both in zygotic and germline clone mutant embryos, but the pattern of dri-expressing cells in the hindgut, marked by the expression of a lacZ reporter gene, is highly disrupted. In situ hybridization with DIG-labeled dri cDNA and immunohistochemical staining with specific anti-Dri antibody (Gregory, 1996) shows that both of these P-insertion alleles retain mRNA and protein expression in most tissues, indicating that they are likely to be hypomorphic alleles. In an attempt to generate amorphic alleles, ethylmethane sulfonate (EMS) mutagenesis has been used to create alleles that fail to complement dri⁷ and dri⁸. Immunochemical staining with an anti-Dre antibody reveals that two of the resulting six alleles, dri¹ and dri², show no zygotic dri product. A Western blot of protein derived from dri germline and zygotic mutant embryos also shows the absence of the Dri protein. It has been concluded that dri¹ and dri² are amorphic alleles. This conclusion is supported by the observation that the phenotypes observed with the two alleles are indistinguishable. Embryos homozygous or trans-heterozygous for these alleles, or trans-heterozygous for either allele and Df(2)tid, are embryonic lethal, but appear to have a normal cuticle pattern. Disruption of the pattern of dri-expressing hindgut cells in these lines, marked (in this case) by expression of lacZ from the enhancer trap line 18-13, closely resembles disruptions in dri⁷ and dri⁸ (Shandala, 1999).

Maternally derived Dri is uniformly distributed throughout the syncytial cleavage divisions and during early gastrulation (Gregory, 1996). It was thought likely that the presence of maternal dri product would decrease the severity of the zygotic phenotypes. To abolish the maternal dri contribution dri¹ and dri² germline clones were generated. Embryos lacking both maternal and zygotic products were produced using this approach, but the efficiency of egg production was much lower than expected of a gene that plays no role in oogenesis. In addition, many eggs that were produced were unfertilized or exhibited early syncytial proliferation defects. Consistent with this, dri has been shown to be expressed during oogenesis in the germinal vesicle and in the nuclei of nurse cells and follicle cells. dri mutant germline clone embryos could be rescued by a paternal dri⁺ allele, as judged by the appearance of normal embryos carrying a lacZ marker on the dri⁺ paternal chromosome and by the appearance of viable and fertile heterozygous germline clone progeny. Rescue is only partial, however, as only 16% of germline embryos with a wild-type paternal allele survived to the first instar larval stage. As predicted, embryos lacking both the maternal and the zygotic dri product exhibit much stronger phenotypes than those that lack the zygotic component alone. Analysis of embryos lacking maternal and zygotic dri function, but with a normal nuclear distribution, reveals varying levels of disruption to segment formation, particularly in the posterior regions of the embryos. A majority of embryos also exhibit abnormal germ-band retraction phenotypes that are not always rescued by a wild-type paternal allele (Shandala, 1999).

Dri is uniformly expressed throughout the mesoderm during germ band extension (Gregory, 1996). Anti-muscle myosin staining reveals variable levels of disruption to somatic muscle development in dri mutant embryos. Many fibers are missing; unfused myoblasts are frequently observed and some myotubes have formed aberrant attachments with epidermal cells. In addition, all dorsally closing dri mutant embryos lack pericardial cells, Variable expressivity of the muscle phenotype makes it impossible to define a specific group of muscles affected (Shandala, 1999).

The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum. Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell rows arise at the juxtaposition of a domain of Engrailed- plus Invected-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe (Iwaki, 2002).

At its anterior, the hindgut joins the posterior midgut; at its posterior, it forms the anus. Along this AP axis, the hindgut of the mature embryo consists of three morphologically distinct domains: the wide, looping small intestine, the long and narrow large intestine, and the tapered rectum. Beginning at stage 13, these domains are demarcated at their junctions by rings of unusually high accumulation of the apical surface protein Crumbs (Crb). The ring at the small intestine/large intestine junction is designated the anterior boundary cell ring, and the ring at the large intestine/rectum junction is designated the posterior boundary cell ring (Iwaki, 2002).

Patterning of the hindgut in the DV axis is detected at stage 10 (germ band extension) when the hindgut develops an interiorly directed (dorsal) convexity. The side of the hindgut closest to the interior of the embryo is dorsal and expresses both En and Inv; that closest to the exterior is ventral and expresses dpp. By the completion of germ band retraction, the convexity at the anterior of the hindgut has shifted toward the left side of the embryo. Thus at the anterior of the hindgut, the initially dorsal, En- and Inv-expressing side comes to lie on the outer (left-facing) curve, while the initially ventral, Dpp-expressing side of the hindgut comes to lie on the inner (right-facing) curve; the DV relationship is retained at the posterior connection to the rectum. These initially DV patterned domains of the large intestine persist to the end of embryogenesis and into the larval stages; they are referred to as large intestine dorsal (li-d) and large intestine ventral (li-v). At each of the two boundaries between li-d and li-v, there is a single row of cells with high levels of Crb expression running the length of the large intestine, from the anterior boundary cell ring to the posterior boundary cell ring. These are designated the 'boundary cell rows'. In addition to their high level of Crb expression, the boundary cell rows and rings express the nuclear protein Dead ringer (Dri). Double antibody staining reveals that boundary cell rows at the border of the En/Inv-expressing li-d domain and the Dpp-expressing li-v domain express Dri in their nuclei and have strong Crb expression at their apical surfaces (Iwaki, 2002).

In addition to expressing Dpp, the li-v domain expresses the Notch ligand Delta (Dl); Dl is also expressed in the anterior of both the rectum and the small intestine. Fringe (Fng), a modulator of Notch signaling, is expressed opposite Dl in the Drosophila wing and eye; in the hindgut, Fng is expressed in li-d and the boundary cell rows, opposite the domain of Dl expression in li-d (Iwaki, 2002).

Interestingly, the Dri- and Crb-expressing boundary cells delimit both AP and DV boundaries in the hindgut. The rings form borders at the anterior and posterior ends of the large intestine, while the rows form borders between the dorsal (li-d) and ventral (li-v) regions of the large intestine. This study focusses primarily on the establishment and characteristics of the boundary cell rows (Iwaki, 2002).

Staining with both anti-Crb and anti-ß_HEAVY Spectrin shows that the boundary cell rows are significantly more elongated along the AP axis than other hindgut epithelial cells. Staining of byn^apro/+ embryos (containing a P-element insert in byn) with anti-ß-Gal antibody reveals that the nuclei of the cells of the boundary rows (identified by strong staining with anti-Crb) are also elongated in the AP axis (Iwaki, 2002).

The dramatically higher level of Crb expression in the boundary cells (both rings and rows) suggests that their apical surface may differ from that of other hindgut epithelial cells, and/or that, in the boundary cells, Crb may be present in cellular compartments in addition to the apical surface. Both of these expectations are borne out by a higher magnification examination of the boundary cells. In cross-sections of the large intestine viewed by electron microscopy, short microvilli on the apical surfaces of two cells on opposite sides of the hindgut lumen were observed; these cells most likely correspond to the boundary cell rows. The microvilli of the presumed boundary cell rows appear more organized and parallel than the irregular protrusions on the surfaces of the other cells of the hindgut epithelium. Because of their apical microvilli, the presumed boundary cell rows have a larger apical membrane surface and are expected to be labeled more strongly with anti-Crb. Consistent with this, cross-sections of anti-Crb-stained embryos viewed by light microscopy reveal two cells on opposite sides of the large intestine lumen with a higher level of Crb on their apical surfaces. In addition to their stronger apical labeling with anti-Crb, these presumed boundary cell rows also display an accumulation of Crb in their cytoplasm; this is strongest apical to the nucleus. The cytoplasmic accumulation of Crb suggests that Crb is produced at a higher level, or is more stable, in the boundary cells (Iwaki, 2002).

In conclusion, differences in gene expression demonstrate that the boundary cells are a separately patterned (fated) group of cells in the large intestine. The unique fate of the boundary cells is manifested both molecularly, in their expression of Dri and high cytoplasmic accumulation of Crb, and morphologically, in their marked AP elongation and development of apical microvilli (Iwaki, 2002).

The boundary cell rows form at the junction of the li-d and li-v domains, which express different genes. To investigate whether the spatially restricted gene expression observed in these domains is essential for establishment of boundary cell rows, embryos homozygous for loss-of-function alleles of en, inv, dpp, dri, Dl, Ser, Notch, or fng were examined. The presence or absence of boundary cells was assessed by anti-Crb staining, since this delineates their characteristic morphology, and also detects one of their unique differentiated features (i.e. the cytoplasmic accumulation of Crb) (Iwaki, 2002).

In embryos homozygous for a strongly hypomorphic dri allele (dri null mutants lack a discernable hindgut), the hindgut is of roughly normal diameter but only about one-third its normal length. Even in these severely reduced dri hindguts, however, boundary cells can still be observed; this phenotype is similar to that described for embryos lacking both maternal and zygotic dri function. Since reduced hindgut size is observed in embryos that lack zygotic, but retain maternal dri function, it is concluded that zygotic expression of dri (most likely the uniform expression at the blastoderm stage) is required to establish or to maintain the normal-size hindgut primordium. Neither blastoderm expression of dri, nor its later expression in the boundary cells, however, appears to be required to establish the boundary cells (Iwaki, 2002).

The data presented here support the following model. En/Inv is expressed in li-d and represses Dl in that domain; Dl expression is thereby restricted to the li-v domain. At the li-v/li-d transition, the Dl-expressing cells induce, by Notch signaling, a row of Dl-non-expressing cells to become a boundary cell row. Since En/Inv is not detected in differentiated boundary cells, Notch activation likely represses En/Inv expression. Notch activation also leads to Dri expression and an upregulation of Crb expression. While all of these transcriptional changes could be mediated by Su(H), they could also be further downstream (Iwaki, 2002).

Dead ringer is required for positioning of the longitudinal glia in the embryonic CNS

The Drosophila gene dead ringer (dri) [also known as retained (retn)] encodes a nuclear protein with a conserved DNA-binding domain termed the ARID domain (AT-rich interaction domain). dri is expressed in a subset of longitudinal glia in the Drosophila embryonic central nervous system and dri forms part of the transcriptional regulatory cascade required for normal development of these cells. Analysis of mutant embryos reveals a role for dri in formation of the normal embryonic CNS. Longitudinal glia arise normally in dri mutant embryos, but they fail to migrate to their final destinations. Disruption of the spatial organization of the dri-expressing longitudinal glia accounts for the mild defects in axon fasciculation observed in the mutant embryos. The axon phenotype includes incorrectly bundled and routed connectives, and axons that sometimes join the wrong bundle or cross from one tract to another. Consistent with the late phenotypes observed, expression of the glial cells missing (gcm) and reversed polarity (repo) genes was found to be normal in dri mutant embryos. However, from stage 15 of embryogenesis, expression of locomotion defects (loco) and prospero (pros) was found to be missing in a subset of LG. This suggests that loco and pros are targets of Dri transcriptional activation in some LG. It is concluded that dri is an important regulator of the late development of longitudinal glia (Shandala, 2003).

After the initial migration and pioneer axon navigation, however, the behavior of dri-expressing glial cells becomes aberrant. The normal final positions of these cells are never adopted and the cells exhibit cell shape defects. The mild misplacement of LG in dri mutants is probably caused by defects in glia-glia and axon-glia contacts, resulting at least in part from downregulation of the glial cell surface marker Neuroglian. These defects may interfere with correct migration of glia along the axon bundles which, in turn, causes the axon tract defects (Shandala, 2003).

The similarity between the dri phenotypes and those of repo, loco and pnt suggests that gene regulatory relationships might exist between these genes. A considerable amount of information already exists about the nature of the transcriptional cascade required to establish longitudinal glial cells. The glial fate is induced by expression of gcm, while later expression of transcription factors encoded by repo and pnt direct glial differentiation. dri expression was examined in embryos mutant for genes required for glial formation and differentiation. As expected, dri expression in all of the dorsal glia (but not in the dri-expressing lateral neural cells) depends on gcm. Moreover, with the probable exception of the subperineural glia (A/B SPG), normal levels of dri glial expression requires repo, since repo mutant embryos show a significant reduction in the levels of dri expression and in the numbers of LG that contain Dri. However, dri expression in all dorsal glia does not depend on pnt. Analysis of the dri promoter region did not reveal any consensus binding sites for GCM (A/GCCCGCAT) or REPO (NNATTA), suggesting that dri might be not a direct transcriptional target of these genes. The finding that dri expression in all glia is not affected in embryos mutant for faint little ball (flb), a null allele of the Drosophila Egfr gene, is in line with previous observations that only the midline glia require EGFR signalling (Shandala, 2003).

In a complementary set of experiments, the expression of glial differentiation markers was examined in a dri mutant background. In dri loss-of-function mutants, repo, pnt and cut continue to be expressed in the appropriate glia. However, a reduction in the number of pros- and loco-positive glial cells is apparent. One pros-positive glial cell was found to be consistently missing in dri mutant embryos, while there was frequent, if irregular, reduction or loss of pros expression in three or four other LG. Similarly, loco expression was reduced or lost in some LG, although this phenotype also exhibited variability in different segments. The number of loco-positive cells was scored in two neuromeres of abdominal segments from a total of ten stage 15 embryos. The average number of dorsal glia per hemineuromere in dri¹/CyOwglacZ heterozygotes was 9.8, not significantly different from wild-type numbers. By contrast, there was an average of 4.8 loco-positive cells per hemineuromere in dri¹ homozygotes, confirming the significance of the apparent loss of loco-expressing cells (Shandala, 2003).

What is the molecular basis of the mutant phenotype found in dri mutants? Dri is a transcription factor, so the link between loss of dri function and the failure to differentiate properly is likely to be indirect, mediated through misregulation of dri targets required for normal longitudinal glial development. The most informative data came from an analysis of the position of dri in the glial transcriptional regulatory cascade. In general terms, dri activity was found to be downstream of gcm and repo, and independent of pnt and cut. It was also found to be upstream of two genes, loco and pros, which are essential for normal development of some glial cells. In this developmental context dri acts as an activator of downstream targets (Shandala, 2003).

The requirement for Dri in the activation of loco is unexpected. loco has been found to be a transcriptional target of Pnt but not of Repo, while dri expression depends on Repo and not on Pnt. It is possible that expression of loco is co-dependent on Pnt and Dri in some cells and that the reduced level of dri expression observed in repo mutants is enough to permit loco expression (Shandala, 2003).

The genetic analysis presented here strengthens the hypothesis that there are different genetic controls for different subsets of dorsal glia. For example, dri expression in all glial cells requires GCM activation, but only some of them requires Repo. The Repo-independent dri-positive cells, two per hemineuromere, appear to correspond to the A and B subperineural glia (A/B SPG). These derive from neuroglioblast NB1.1, suggesting that Repo is required for the expression of dri only in cells derived from the lateral glioblasts. Unlike dri, pnt and its downstream target loco are not expressed in the medialmost cell body glia, which do not have a lateral glioblast origin. This suggests that there are different pathways for pnt and dri induction downstream of gcm (Shandala, 2003).

At least some of these hierarchical transcriptional interactions may explain the phenotypes observed. The axon and mild positional defects of glia in dri mutants resemble phenotypes of other known late gliogenesis factors, such as those observed in pnt, repo, loco or pros embryos. It is known that early distribution of the glycoprotein Neuroglian is perturbed in pros mutant embryos. loco encodes a regulator of G-protein signalling (RGS) that has been shown to bind to a Galphai-subunit and could regulate a G-protein signalling pathway involved in LG migratory behavior. In addition, expression of the Drosophila FGF receptor Heartless in LG, and similarities between the loco and heartless mutant phenotypes, leaves open the possibility that FGF could trigger final migration of glia along the longitudinal connectives. This hypothesis is strengthened by the recent finding that subcellular redistribution of Neuroglian from the plasma membrane to cytoplasm, which normally happens during final glial migration to enwrap axon bundles, is disrupted in heartless mutants. Alternatively, it remains possible that additional targets of dri mediate the role of this gene in longitudinal glial differentiation (Shandala, 2003).

These studies add dri to the list of genes, including pnt, repo, loco and pros, that exhibit phenotypes that are much milder than those of the gcm, glide2 and Drop/Ltt genes at the head of the dorsal glia hierarchy. It appears that diversification of these downstream regulators produces different types of glial cells. Nonetheless, each plays an essential role in driving the required behavior of glial cells during CNS development. In the case of the Dri transcription factor, this role includes fine tuning the cell shape and migration characteristics of longitudinal glia that enable them to establish a normal axon scaffold (Shandala, 2003).

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