grainy head

DEVELOPMENTAL BIOLOGY

Embryonic

Zygotic GRH protein is detected in nuclei of ectodermal derivatives. Expression of grh is first seen in stage 11 in both the epidermis and CNS (Bray, 1989).

During Drosophila embryonic CNS development, the sequential neuroblast (NB) expression of four proteins, Hunchback (Hb), Pou-homeodomain proteins 1 and 2 (referred to collectively as Pdm), and Castor (Cas), identifies a transcription factor network regulating the temporal development of all ganglia. The Zn-finger proteins Hb and Cas, acting as repressors, confine Pdm expression to a narrow intermediate temporal window; this results in the generation of three panneural domains whose cellular constituents are marked by expression of Hb, Pdm, or Cas. Seeking to identify the cellular mechanisms that generate these expression compartments, the lineage development of isolated NBs in culture were studied. The Hb, Pdm, and Cas expression domains are generated by transitions in NB gene expression that are followed by gene product perdurance within sequentially produced sublineages. These results also indicate that following Cas expression, many CNS NBs continue their asymmetric divisions and generate additional progeny, which can be identified by the expression of the bHLH transcription factor Grainyhead (Gh). Gh appears to be a terminal embryonic CNS lineage marker. Taken together, these studies indicate that once NBs initiate lineage development, no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of Hb followed by Pdm and then Cas, and subsequently Gh expression during NB outgrowth (Brody, 2000).

Underpinning the formation of NB lineages are spatially and temporally regulated transcription factor networks that play pivotal roles in establishing the unique cellular identities of NBs and their progeny. Prior to NB delamination, during the initial specification of NBs, two spatially regulated transcription factor networks subdivide the ventral neuroectoderm along its anterior/posterior (A/P) x axis and dorsal/ventral (D/V) y axis. Later, during NB lineage development, at least one additional network, acting over several hours, gives rise to sequentially formed multilayered basal (inner or dorsal) to apical (outer or ventral) neuronal subpopulations. Along the basal/apical z axis, neuronal subpopulations in all ganglia can be identified by their expression of the transcription factors Hb, Pdm and Cas. Hb marks a deeper, basally distributed population of neurons that are born early, Cas marks a superficial, apically distributed population of neurons that are born late, and Pdm marks an intermediate population arrayed between the Hb- and the Cas-expressing cells. Both genetic and molecular analysis indicates that two Zn-finger proteins, Hb and Cas, act as repressors to silence pdm expression. By restricting pdm expression primarily to intermediate-born neuronal precursors these structurally different Zn-finger proteins help establish three pan-CNS neural subpopulations whose cellular constituents are marked by the expression of Hb, Pdm, or Cas (Brody, 2000).

Triple-immunolabeling studies have revealed that many of the overnight NB clones contain a subset of cells that do not contain detectable levels of Hb, Pdm-1, or Cas. In many of these in vitro lineages the putative NB is also unstained. The bHLH transcription factor Gh is known to be expressed in CNS NBs but only after stage 14. In view of the late onset of Gh expression in NBs and the triple-staining results identifying cells in o/n clones that do not express Hb, Pdm-1, or Cas, it was hypothesized that these negative cells may represent an additional late NB expression window marked by Gh expression. To test this hypothesis, the spatial/temporal expression dynamics of Gh were compared to other members of the z axis network. Similar to its late activation during in vivo development, Gh expression was observed only in overnight cultures; when more than one Gh-positive cell was detected in a clone they were consistently found clustered together. Two-thirds of the Cas+ clones had at least one Gh+ cell and the average number of Gh+ cells in all clones was 2.3. Approximately 2/3 of the Gh+ clones also contained Hb-immunopositive cells. While no Hb-Gh coexpressing cells were observed, approximately 20% of the Gh+ cells also expressed Cas. Given the late onset of Gh expression in both the embryo and the cultured NB clones and the overlapping Cas and Gh expression, it is likely that Gh marks a fourth temporal window for NB transcription factor expression. In addition, because there was an average of more than one cell in an o/n clone that was immunopositive for Gh, it is likely that Gh is also expressed/maintained in a sublineage(s) born after the one marked by Cas expression (Brody, 2000).

The following model for the origin of the layer sublineages marked by these transcription factors has been suggested. As each NB divides, generating a succession of GMCs, it undergoes multiple transitions in transcription factor expression. In succession, the NBs express Hb, Pdm, Cas, and Gh. The first progeny generated by the early S1 and S2 NBs express Hb, and the presence of Hb protein persists in their neural progeny. These early S1 and S2 NBs go on to activate the expression of the Pdms that, like Hb, persist in neural sublineages generated during this temporal window. Subsequently Cas is activated in NBs, represses Pdm transcription, and likewise persists in neural sublineages. After Cas expression, a fourth neural subpopulation, generated by dividing NBs, expresses Gh. This Gh subpopulation most likely represents the terminal sublineage of the embryonic NB. The data also reveal that not all NBs generate cells that occupy all four layers, a result that reflects the unique set of lineages, generated by each NB. Most likely, each NB has a preprogrammed time of delamination, but the timing of transitions is synchronized in a global fashion. The model further suggests that late delaminating NBs can be distinguished from early NBs by their inability to activate Hb. Although Hb is activated shortly after the S1s and S2s have delaminated, Hb is never seen in the proliferative zone during late delaminations (Brody, 2000).

Effects of mutation or deletion

The major developmental defect of grh mutants is alteration to head skeleton and cuticular structures. The [Image] becomes "grainy," hence the gene's sobriquet (Bray, 1991).

The embryonic cuticle of Drosophila is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. Mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle have been characterized. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion being reversed within the eggshell (the 'retroactive' phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the 'blimp' phenotype). One of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. Two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and a new mutation, called zeppelin (zep), is described that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do (Ostrowski, 2002).

The cuticle defects, particularly the disruption of the head skeleton, are most severe in kkv and grh mutants. All alleles of kkv, both those isolated previously and those identified in this screen, produce similar phenotypes. When removed from the vitelline membrane, kkv and grh mutant embryos are very flaccid and are not motile although they are able to contract their body wall muscles. All three alleles of knk and the one available allele of rtv produce milder defects in the head skeleton and denticle belts. When removed from the vitelline membrane they are more robust than the kkv and grh mutants, and they are motile but die within hours after removal from the eggshell. The head skeleton and denticle belts of zep mutants are almost wild type and these embryos are sometimes able to hatch on their own, although they die at roughly the same stage as the knk and rtv mutants. The degree of cuticle expansion can vary among cuticle preparations due to uncontrollable differences in the mechanical devitellinization process. However, the severity of head defects, flaccidity, and motility are consistent within the alleles of each complementation group. Thus the phenotypic effects of the blimp class mutations can be ranked from most to least severe: kkv, grh > rtv, knk > zep (Ostrowski, 2002).

The identification of kkv as a chitin synthase and the ability of a chitin synthesis inhibitor to phenocopy kkv shows that disrupting synthesis or deposition of chitin alone can account for the blimp phenotype. However, it is believed that two of the blimp class genes, knk and zep, may function in the epidermis prior to cuticle deposition because both interact genetically with mutations in the Drosophila E-cadherin, encoded by shotgun (Ostrowski, 2002).

grainy head affects head skeleton and embryonic cuticle. grh encodes a GATA family transcription factor and activates the transcription of a number of genes during development, one of which is Dopa-decarboxylase (Ddc). This enzyme is synthesized in the cuticle-secreting layer of cells at the end of embryogenesis; the dopamine produced undergoes further metabolism and oxidation to produce quinones that crosslink cuticular proteins. Thus, loss of grh function would result in weakening of the cuticle indirectly through its failure to activate Ddc expression (Ostrowski, 2002).

Grainy head controls apical membrane growth and tube elongation during tracheal development

Epithelial organogenesis involves concerted movements and growth of distinct subcellular compartments. Apical membrane enlargement is critical for lumenal elongation of the Drosophila airways, and is independently controlled by the transcription factor Grainy head. Apical membrane overgrowth in grainy head mutants generates branches that are too long and tortuous without affecting epithelial integrity, whereas Grainy head overexpression limits lumenal growth. The chemoattractant Branchless/FGF induces tube outgrowth -- it upregulates Grainy head activity post-translationally, thereby controlling apical membrane expansion to attain its key role in branching. A two-step model for FGF in branching is favored: first, induction of cell movement and apical membrane growth, and second, activation of Grainy head to limit lumen elongation, ensuring that branches reach and attain their characteristic lengths (Hemphälä, 2003).

A characteristic feature of transporting and secretory tubular organs, such as lung, kidney and many glands, is the structural and functional compartmentalisation of their epithelium. Tubulogenesis and branching rely on extensive cell rearrangements and an immense increase of apical lumenal surface, yet in many cases the epithelium remains intact and functional during development. Thus, the driving forces for cell movement, shape changes and growth must act in the context of prefixed distinct subcellular compartments, and they must be highly co-ordinated with cell adhesion. Although the molecular determinants of epithelial cell architecture are becoming increasingly clear, the regulation of the different subcellular compartments during epithelial tissue morphogenesis remains largely unknown. Epithelial cell movement and morphogenesis are commonly induced and guided by secreted factors from the surrounding tissues. How then are these morphogenetic cues integrated to regulate the dynamic cell behaviours that underlie epithelial tube formation and organ growth (Hemphälä, 2003)?

The development of the Drosophila trachea, a complex network of epithelial airways that supplies oxygen to the entire animal, provides a well-defined system for the analysis of regulatory mechanisms that control cell migration and branching. The tracheal system arises from 20 independent sacks of approximately 80 cells each that undergo a distinct sequential program of branch sprouting, directed branch outgrowth and branch fusion. Initially, the actions of at least three independent signals, TGFß-like (Decapentaplegic; Dpp), Wingless (Wg) and EGF, subdivide the cells in each tracheal placode into branch-specific groups. Subsequent branch sprouting and outgrowth occurs without cell division as cells migrate towards localized sources of Branchless (Bnl), an attractant signal of the FGF family. Primary branch growth entails the initial extension of cytoplasmic processes towards the Bnl source, followed by movement of the cell body and a concomitant increase in apical cell surface to promote lumenal extension. The characteristic lengths and diameters of the newly formed branches of the larval trachea are stereotyped and become specified during distinct developmental intervals (Hemphälä, 2003).

Bnl is the key morphogen co-ordinating branching that acts via the receptor tyrosine kinase Breathless (Btl) and the adaptor protein Dof/Stumps. This pathway leads to phosphorylation and activation of MAPK, which in turn may alter the activity of regulatory proteins to control cell behavior. During primary branching, actin-rich basal extensions are sent by the tracheal cells towards the sources of Bnl, a process that is likely to involve cytoskeletal modulation by the Rho family GTPases. Bnl signalling is also required for the expression of cell-fate determining genes in specific subsets of tracheal cells in each primary branch. Analysis of these genes has identified key components of the patterning and guidance of the unicellular secondary and terminal branches. However, the role of Bnl in the movement of the cell bodies and the growth of the branch lumen remains unknown (Hemphälä, 2003).

The mechanisms that control the elongation of tracheal tubes have been investigated. Mutations have been characterized in three genes that affect branch growth, resulting in abnormally long tubes. Mutations in fasII and Atpalpha alter cell adhesion and the basolateral cell domains, causing aberrations in cell shapes, excessive tubular elongation and sporadic lumenal dilations and breaks. In contrast, the transcription factor Grainy head (Grh) is required to specifically control tube elongation. Both loss of function and overexpression of grh indicate that it is required to limit lumenal growth and control tubular length. Grh selectively affects the growth of the apical cell membrane, arguing that different genetic programs regulate distinct sub-cellular domains during branching morphogenesis. Grh is uniformly expressed in the trachea, but its activity is modulated by Bnl/Btl signalling and Grh counteracts the activity of Bnl induced branch growth. Thus, through its regulation of Grh, Bnl regulates epithelial apical membrane growth to accommodate its role in branching morphogenesis (Hemphälä, 2003).

Grh is expressed in a number of epithelial structures, including the embryonic epidermis where it has been suggested to be involved in the formation of the cuticular layer that covers the apical surface of epidermal tissues. Early descriptions of grh mutants also have revealed a tracheal defect, which led to an investigation of the expression and phenotype of grh in the trachea (Hemphälä, 2003).

Nuclear Grh is detected in all tracheal cells, appearing first at stage 11, just after they have invaginated from the epidermis, and persisting throughout embryogenesis. To investigate its function, an antibody that specifically stains the tracheal lumen (mAb2A12) and several cell fate markers were used to analyse the tracheal phenotype of three strong loss-of-function grh alleles (one EMS allele, grh^B37; two P-element insertions, grh^s2140 and grh⁰⁶⁸⁵). None of the grh mutations affect the patterning, outgrowth and connection of branches or the expression of terminal cell markers (DSRF) and fusion cell markers (fusion-3). It is only when primary and secondary branching is completed (during stage 16), that grh mutant embryos begin to display tubular irregularities. The first signs of a defect are that the dorsal trunk (main airway) appears convoluted and elongated compared to the wild type. This phenotype subsequently becomes exaggerated, and is also seen in additional branches, including the lateral trunk, transverse connectives and ganglionic branches. These convoluted branches represent an overgrowth in tracheal tube length, as indicated by an increase of 40% in the tube length of grh mutants. Despite this substantial increase in tubular lengths, the tubular continuity is not affected in grh mutant embryos. Grh is therefore required for the restriction or maintenance of tubular length (Hemphälä, 2003).

The tracheal phenotypes produced by alterations in Grh levels imply that Grh activity must be carefully controlled during branching morphogenesis to ensure branch extension at the right stage and to the right extent. Consequently, tracheal Grh activity is likely to be modulated during branching morphogenesis. To assay the in vivo activity of Grh, carrying a transgene with four high-affinity Grh response elements (GBE-lacZ) were used. GBE-lacZ expression is detected in all tissues where Grh is expressed, is absent in grh mutants, and becomes activated upon ectopic Grh expression. It is thus representative of Grh transcriptional activity in vivo. During tracheal development GBE-lacZ is expressed in all tracheal cells after invagination, and requires Grh for its expression. However, GBE-lacZ expression is not uniform: it becomes temporarily enhanced in the fusion and terminal cells during branching. Since Grh itself appears to be uniform in all tracheal cells, the enhanced expression of GBE-lacZ indicates that the activity of Grh is regulated post-translationally during branching (Hemphälä, 2003).

One possible mechanism for regulation of Grh activity is through Bnl signalling, which is instrumental in the formation and extension of all tracheal branches. Initially, it was established that apical cell surface growth is an intrinsic component of Bnl-induced tube extension, by combining alleles of grh and bnl. This revealed that a subset of the branch outgrowth defects seen in embryos that carry only one copy of the bnl gene are partially rescued by a reduction in grh function (grh^s2140/grh^s2140; bnl^P1/+). Thus, in embryos heterozygous for bnl, 40% of the ganglionic branches fail to reach the CNS, whereas the simultaneous removal of grh restores this phenotype so that 78% of the branches now enter the CNS. These data therefore show that Grh-mediated modulation of the apical cell surface has an active inhibitory role on Bnl-induced branch extension (Hemphälä, 2003).

In order to analyse whether tracheal Grh activity could be targeted by Bnl/Btl signal transduction, GBE-lacZ expression was analyzed in embryos with altered levels of Bnl and Btl activity. When Bnl is ectopically expressed in all tracheal cells, GBE-lacZ expression becomes significantly upregulated, although the levels of Grh protein are not altered. This suggests that Bnl controls Grh activity post-translationally, and surprisingly, upregulates the expression of this artificial Grh target. Nevertheless, the effects of Btl appear specific since with more limited Bnl expression using the Term-Gal4 driver, GBE-lacZ expression becomes enhanced specifically in the cells that respond to Bnl by ectopically expressing the terminal marker DSRF. Similar enhancement of GBE-lacZ expression is evident upon tracheal expression of an activated form of the Btl receptor itself (UASBtl-Tor). In all instances the augmented GBE-lacZ expression is dependent on Grh, since embryos that express ectopic Bnl or the activated form of Btl, but lack Grh activity, do not express GBE-lacZ. Furthermore, ectopic activation of Dpp, another signalling pathway that promotes the growth of dorsal and ganglionic branches during tracheal development, has no effect on GBE-lacZ, indicating that the effects on GBE-lacZ are specific for Bnl/Btl (Hemphälä, 2003).

Whether Bnl signalling is a prerequisite for the transcriptional activity of Grh was tested by analysing the levels of GBE-lacZ expression in mutants for bnl, btl or pointed (pnt). Tracheal GBE-lacZ expression is both reduced and uniform in bnl and btl mutant embryos, but is unchanged in pnt embryos that lack the activity of a downstream transcriptional effector of the ETS family. Since Grh is a substrate for activated MAPK (ERK2) in vitro, its activity could be modulated directly during branching by Bnl-induced phosphorylation. This would account for the fact that GBE-lacZ expression is affected by mutations in bnl and btl, but not by mutations in the nuclear effector pnt (Hemphälä, 2003).

The apparent upregulation of Grh activity by Bnl signalling and the fact that Grh and Bnl exert opposing effects on branch extension suggests that there are two possible models of Grh activity. The first assumes a two-step process, where upregulation of Grh activity represents a second function of Bnl to prevent excessive tube extension. Alternatively, the Bnl signalling augments some aspects of Grh function (e.g. activation of GBE-lacZ) but inhibits others (e.g. the restriction of apical membrane growth) allowing for branch extension. These two models are discussed below (Hemphälä, 2003).

It is concluded that Bnl signalling converts Grh to a more potent activator of its GBE-lacZ target. Since Grh becomes phosphorylated by MAPK in vitro, and MAPK is a downstream effector of Btl signal transduction, the alteration in Grh activity may be brought about by MAPK-mediated phosphorylation of the Grh protein (Hemphälä, 2003).

Currently, two ways of explaining the biological consequence of the regulation of Grh have been suggested. In the first model, the regulation of Grh by Bnl increases its activity, and thereby delimits lumen growth. This invokes a hierarchical two step function for Bnl in which it first promotes branching and tube elongation and it then activates Grh to halt excess apical surface growth and establish a functional lumen. In this model active restriction of morphogenetic processes is required to achieve stereotyped tube dimensions and is an intrinsic part of the program that induces branching morphogenesis. In the second model, regulation by Bnl has differential consequences on Grh, activating some functions (like the one necessary for GBE-lacZ expression) and inactivating others, necessary for inhibiting apical membrane growth. In this model, high levels of Btl signalling would temporarily inactivate Grh, in order to allow for apical membrane expansion during the process of branch extension. Both models are consistent with the genetic interactions, which indicate an antagonistic relationship between grh and bnl, and add the control of apical membrane growth to the repertoire of cellular activities regulated by FGF signalling during morphogenesis (Hemphälä, 2003).

Of the two models, the former, where Btl coordinates branching through a sequence of activities, is currently favored since this model is consistent with the activation of the GBE-lacZ reporter. It can also be well integrated with the apical overgrowth phenotype of grh mutants, which becomes apparent first in the branches that have reached their final length and only after the completion of branch elongation at stage 16. If Grh were acting to restrict membrane growth continuously, the grh mutant phenotype would be expected to appear at earlier stages. A two step model could also explain the inhibiting effect on tube elongation that is seen upon expression of activated forms of Btl receptors in all tracheal cells of wild-type embryos (Hemphälä, 2003).

Since restriction of apical membrane growth depends on Grh-mediated alterations in transcriptional activity, the induction of apical membrane expansion upon branch elongation may also rely on changes in gene expression. The nuclear factor Ribbon (Rib) is required for branch elongation, and may act as an activator of apical membrane growth. In rib mutants, the extension of basal cytoplasmic processes towards the Bnl source appears normal, but the movement of the cell body fails and the apical membrane does not expand, causing a tracheal phenotype that is reminiscent of that seen with ectopic Grh expression. It is thus conceivable that a balance between Rib and Grh activity determines the extent of apical membrane growth and is coordinated by Bnl through direct modulation of Grh, and perhaps also of the Rib protein. Such a regulation of apical cell surface size by signals deriving from the target tissue could coordinate branch elongation, and would provide an elegant allometric control of organ size depending on the signal strength, size and respiratory demand of the target tissue (Hemphälä, 2003).

Apart from its tracheal expression, Grh is found in the embryonic epidermis and all primary epithelial tissues. The epidermal expression of grh is also essential because grh mutant embryos show a 'blimp' phenotype, where the embryonic cuticle stretches to a much greater extent than the wild-type cuticle upon removal of the vitelline membrane. It is found that the epidermal cells in grh embryos also show an abnormal apical membrane expansion. This is associated with the production of an enlarged cuticle that lines the apical cell surface. Grh may therefore have a common biological function in the epithelial tissues where it is expressed, being required to regulate apical cell membrane growth. Grh protein is continuously expressed in epithelial tissues during larval life, a period of extensive organ growth to accommodate the dramatic increase in animal size. Thus, Grh is likely to be required not only for organogenesis, but also for the continuous modulations in organ size and shape that occurs throughout the animals life. However, the temporal and spatial control of Grh activity must be accomplished through distinct mechanisms in different tissues, since Bnl signalling does not operate in the epidermis (Hemphälä, 2003).

Grh belongs to a small family of transcription factors that is found only in higher eukaryotes. The specific, but basic function of Grh in the regulation of epithelial apical cell membrane growth raises intriguing questions as to its functional conservation in higher organisms. Two mammalian Grh homologs (MGR and BOM) have been recently identified (Wilanowski, 2002). Like Grh, MGR and BOM form dimers and MGR interacts specifically with Grh DNA binding sites in vitro. Intriguingly, these mammalian homologs display similar expression patterns to that of Grh. During mouse development MGR is expressed predominantly in the epidermis, and BOM is expressed in the epidermis as well as in several internal tubular organs including the kidney and lung. Thus the biological function of Grh may be conserved in its murine homologs. Given the functional conservation of FGF signalling in tracheal and lung morphogenesis, it will be of great interest to test whether the mammalian homologs of Grh participate in the growth of the lung and to investigate their functional relationship with FGF signalling (Hemphälä, 2003).

grainy head is essential for the function of the frizzled pathway in the Drosophila wing

The Drosophila wing is covered by an array of distally pointing hairs. This tissue planar polarity is regulated by the frizzled pathway. The function of the grainy head transcription factor is essential for the function of the frizzled pathway. grainy head mutant cells fail to localize planar polarity proteins at either the proximal or distal sides of wing cells and produce multiple hairs of abnormal polarity. Levels of the Starry night protein are strongly reduced in grainy head mutants in both larval wing discs and pupal wings, which is sufficient to account for much of the polarity phenotype. In addition, grh has frizzled pathway independent functions during the development of the adult cuticle (Lee, 2004).

grh function is required for several different processes during the differentiation of the adult Drosophila epidermis. These include the function of the fz dependent tissue polarity pathway, pigmentation, the timing of differentiation, epidermal hair morphogenesis and wing vein/blade specification. The Grh protein was originally isolated by virtue of its ability to bind to DNA in a sequence specific manner and to regulate the expression of target genes. These and later experiments led to the conclusion that grh functions as a transcription factor for development specific gene regulation. Experiments on vertebrate homologs of grh also suggest a similar cellular function. It is likely that it serves a similar function in the development of the adult epidermis (Lee, 2004).

The analysis of grh function in regulating gene expression appears complex. The first studies on grh argued that it acted as a positive regulator of Ddc and Ubx expression. Curiously, although Grh was isolated by virtue of its ability to bind to a sequence essential for the neuronal activation of Ddc, grh mutations alter the epidermal and not neuronal expression of Ddc. More recently it was found that grh positively regulates tll expression and negatively regulates ventral dpp expression (Lee, 2004).

The function of the grh transcription factor is shown in this study to be required for the function of the fz pathway in the wing. In the absence of grh function the Fz, Dsh and Vang proteins fail to accumulate apically and the levels of the Stan protein are dramatically decreased. Furthermore, Stan levels are increased in cells with two versus one copy of grh. Thus, stan expression is directly related to grh dose suggesting that stan might be a direct target of Grh. The direct relationship between stan expression and grh dose is seen in both pupal wing cells where Stan is localized assymetrically and in third instar wing disc cells where it is evenly distributed. Thus, it is concluded that the decreased levels of Stan protein in grh cells is not due to a failure of assymetric localization. Grh does not affect Stan stability; stan expression from the endogenous stan gene is altered, consistent with Grh having an important role in promoting stan transcription. It is suspected that this could be due to a direct interaction of Grh protein with stan genomic DNA. stan does not appear to be highly enriched in putative Grh binding sites but this may be a reflection of the variability in identified Grh binding sites not providing an ideal consensus site. It is also concluded that the decreased level of Stan protein is neither the cause or effect of the the delay in hair morphogenesis in grh cells. Thus far, all of the proteins that localize assymetrically are co-required for the asymmetric localization of the others, however only Stan is required for the apical accumulation of all of the other proteins. The alterations in tissue polarity protein localization seen in grh mutant cells could be explained entirely by the effect of grh on Stan expression. It remains possible however, that grh could be important for the expression of several or all members of the tissue polarity group. These experiments did not allow the assessment of possible changes in Fz or Vang levels due to altered expression of these genes, since the localization was examined of proteins produced from transgenes that did not utilize the normal promoters. Decreased levels of Dsh were not seen by antibody staining, however the staining background was relatively high in these experiments which could have hidden a modest effect on Dsh levels. The finding that Arm cortical localization is not altered in grh clone cells indicates that apical-basal polarity is not altered and suggests that gross cellular physiology is not altered in grh clones (Lee, 2004).

While it is possible that the grh mutant planar polarity phenotype could be due solely to a lack of stan expression in grh mutant cells, this may not be the case since there are a number of differences between the phenotypes of grh and stan clones. For example, the multiple hair phenotype of grh is much stronger than stan. There is also a difference in the non-autonomy of grh and stan clones. For mutations in both of these genes the domineering nonautonomy of clones is much weaker than that of fz or Vang. However, the weak domineering nonautonomy is seen much more frequently with grh than stan clones, suggesting that grh mutations alter the expression of additional tissue polarity genes or other cellular genes that interact with the planar polarity system. Genetic screens for enhancers or suppressors of the dominant negative grh^FK2131 allele could be useful in identifying such genes (Lee, 2004).

grh has both fz pathway dependent and independent functions during wing development. Epistasis experiments showed that the ectopic wing vein, cuticle pigmentation, disturbed marginal bristle row and extreme multiple hair cell phenotypes of grh mutations are not altered in a null fz, in or mwh mutants. Thus, it is quite likely that some of the target genes whose transcription is altered by grh mutations are not part of the fz pathway (Lee, 2004).

grh cells are often dramatically delayed in hair morphogenesis. This is not seen in cells mutant for fz or stan and hence is unlikely to be an indirect consequence of a failure of stan expression or in the inactivation of the frizzled pathway. The time course of pupal development is controlled by ecdysone and it is possible that grh functions as part of the ecdysone cascade. The delay in hair morphogenesis could be due to a failure to induce the expression of genes such as kojak, where a loss of function results in a similar delay (Lee, 2004).

The grh multiple hair cell phenotype differs from that of downstream members of the fz pathway such as inturned, in not showing the typical fz/in abnormal polarity pattern and in the hairs being much more erect. The identity of the targets responsible for this phenotype are unkown. The grh hair phenotype is somewhat reminiscent of that seen with mutations in genes such as Rho kinase or crinkled (myosin VII) suggesting these or related genes as possible targets (Lee, 2004 and references therein).

The transcription of the Ddc gene has previously been shown to be regulated by grh and Ddc activity is required for melanization. Is ddc likely to be the target gene whose altered expression leads to the lowered pigmentation of grh clone cells? This is certainly possible but it seems unlikely to be the entire story. Ddcts2 flies raised at the restrictive condition have more profound pigmentation defects than grh clones. However, clones of ddc null alleles typically have a less severe pigmentation phenotype than grh clones due to partial rescue of the pigmentation phenotype by neighboring cells (i.e. ddc displays submissive cell non-autonomy). Based on these observations it is argued that grh must have other targets that contribute to the decreased pigmentation (Lee, 2004).

The data reported in this paper argue that grh has multiple functions during the development of the adult epidermis. In this context it is not clear to what extent grh functions in a permissive fashion to promote the expression of developmentally important genes and/or to promote changes in gene expression that are associated with the differentiation of the adult cuticle. The data are consistent with grh functioning in both ways. The requirement for grh for the expression of stan was seen at multiple stages consistent with grh having a permissive role. The effects on the timing of hair morphogenesis are consistent with, but do not demand an instructive role (Lee, 2004).

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date revised: 25 June 2007   

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