hedgehog

DEVELOPMENTAL BIOLOGY

Larval - Hedgehog signals travel from the eye to the brain

The arrival of retinal axons in the Drosophila brain triggers the assembly of glial and neuronal precursors into a neurocrystalline array of lamina synaptic cartridges. Retinal axons arriving from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina target field. In the eye disc, photoreceptor cells assemble into ommatidial clusters behind the morphogenetic furrow (mf) as it moves to the anterior. The ommatidial clusters project their axon fascicles into the crescent-shaped lamina. Neuronal precursor cells of the lamina (LPCs) are incorporated into the axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow. Glia precursor cells (GPCs) are generated in two domains that lie at the dorsal and ventral anterior margins of the prospective lamina. These glial precursors migrate into the lamina along an axis perpendicular to that of LPC entry. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody staining. Like the eye, lamina differentiation occurs in a temporal progression on the anterioposterior axis. Axon fascicles from new ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow) and associate with neuronal and glia precursors in a vertical lamina column assembly. At the anterior of the lamina, at the trough of the lamina furrow, LPCs await a retinal axon-mediated signal in G1-phase and enter their terminal S-phase at the posterior margin of the furrow. Postmitotic (Dac-positive) LPCs assemble into columns at the posterior margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers as they become specified as the lamina neurons L1-L5. Lamina neurons L1-L4 form a stack in a superficial layer, while L5 neurons reside in a medial layer near the R1-R6 axon termini. These neurons arise at cell-type specific positions along the column's vertical axis. Lamina glial cells take up cell-type positions in the precartridge assemblies. Epithelial (E-glia) and marginal (Ma-glia) glia are located above and below the R1-R6 termini, respectively. Satellite glia are interspersed among the neurons of the L1-L4 layer. The Ma-glia and E-glia layers, both located ventral to the neuronal precursor column, sandwich the R1-R6 axon termini. The medulla neuropil serves as the target for R7/8 axons and is separated from the lamina by the medulla glia, situated just below the Ma-glia (Huang, 1998 and references).

Hedgehog is transmitted along retinal axons to serve as the inductive signal in the brain for differentiation of lamina neurons. The target of HH is wingless, which in turn targets decapentaplegic and Distal-less. The lamina is a ganglion layer of the visual center of the brain that processes information received from R1-R6 photoreceptor neurons located in the ommatidia of compound eyes. The lamina develops in a precise order, directly coupled to the arrival of retinal axons from the eye. Ectopic hh expression in the brains of eyeless flies induces lamina differentiation, but is not sufficient to induce Elav, a late marker. This suggests that HH alone is not sufficient for the later events of lamina development that include the specification of lamina neurons (Huang, 1996). For more information on the role of HH in lamina development, see the Brain site and below.

Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations (Huang, 1998).

A number of markers distinguish glial and neuronal precursor cells from the corresponding mature cell types. The expression of optomotor-blind (omb) labels both glial precursors in the dorsal and ventral anlagen and mature glia that have migrated into the lamina target field. The glia cell marker Repo and the enhancer-trap lacZ insertion 3-109 are expressed by glia once they have entered the lamina target field. Cubitus interruptus (Ci), a transcriptional mediator of Hh signaling is expressed by LPCs anterior of the lamina furrow and by the postmitotic neuronal precursors within the lamina. The nuclear protein Dachshund is expressed only by neuronal precursors that have begun terminal differentiation and lie posterior to the lamina furrow. Thus, Omb and Ci label the glial and neuronal precursors, respectively, while the mature cells, following their interaction with retinal axons, additionally express Repo and Dac. In the lamina target field of eyeless mutants (mutants that project no neurons toward the optic disc), such as eyes absent (eya) or sine oculis (so), Dac expression is not detected and Repo expression is greatly diminshed (Huang, 1998). The migration and early differentiation of lamina glia are independent of Hh. Enhanced transcription of the putative Hh receptor, patched (ptc) is a universal characteristic of Hh signal reception. All classes of glia in the lamina region upregulate ptc expression in an hh-dependent fashion. These cells are thus Hh-responsive. All three classes of lamina glia, as well as medulla glia, that express a ptc-lacZ reporter construct are in close proximity to Hh-bearing retinal axons. Glia cell ptc reporter gene expression is not observed in hh- animals. This raises the question of whether Hh signal reception is responsible for the migration and/or subsequent maturation of glia cells. To determine whether the migration of glial precursors into the lamina target field is Hh-dependent, the distribution of Omb-positive cells was examined in hh- animals. In the wild type, a trail of Omb-positive cells delineates a path of glia migration from the dorsal and vental anlagen. Is glia precursor migration Hh-dependent? This was investigated by examining the distribution of Omb-positive cells in hh¹ mutant animals. hh¹ is a regulatory mutation that specifically affects hh expression in the visual system. In hh¹ animals, approximately 12 columns of ommatidia initiate differentiation in the eye imaginal disc before the anterior progression of the morphogenetic furrow ceases. hh¹ retinal axons lack Hh immunoreactivity by the time they reach the lamina target field and thus the Hh-dependent steps of LPC maturation fail to occur in hh¹ animals. Omb staining reveals a relatively normal number of glia precursors in the lamina target field of hh¹ animals, despite the absence of Dac induction. The Omb-positive cells are distributed uniformly along the dorsoventral axis among the retinal axon fascicles, but appear more closely spaced than in the wild type. A likely explanation for this spacing defect is the absence of the neuronal precursors that would constitute the majority of lamina cells at this point in development. To determine whether the glial precursors that enter the lamina target field in hh- animals express a retinal innervation-dependent marker, their expression of Repo was examined. In hh¹ animals, the Omb-positive cells within the lamina also express Repo. Moreover, the Repo-positive cells occupy proper layers above and below the R1-R6 axon termini expected for satellite, marginal and epithelial glia, though the lack of markers specific for these three glia types precludes an unambiguous determination of glial cell type. The presence of marginal and epithelial glia is consistent with the observation that R1-R6 growth cones terminate in their proper positions between these layers in hh- animals. The ectopic expression of Hh in the brains of `eyeless' animals is sufficient to induce the initial steps of LPC maturation in the absence of retinal axons. However, neither Hh nor the Hh-mediated events of LPC maturation are sufficient for glia cell migration and maturation (Huang, 1998).

The activities of a number of Hh signal transduction pathway components are now well characterized. Mutations at these loci have been shown to either mimic or block Hh signal reception in a cell-autonomous fashion. Examining the cellular requirements for these genes in mosaic animals should help illuminate the cellular circuitry that mediates the Hh-dependent events of lamina development. The seven-pass transmembrane protein encoded by smoothened (smo) acts as a positive effector of Hh signal reception, downstream of the Hh receptor Patched. If Hh exerts its effects directly on LPCs, it would be expected that loss of smo function should block the entry of G1-phase LPCs into S-phase and/or prevent the expression of Hh-dependent markers of lamina differentiation such as Dac. Inducing smo mutant clones reveals that with respect to lamina differentiation, smo acts cell autonomously. smo clones that extended to the posterior of the lamina are rare. It is possible that LPCs that cannot respond to Hh are not readily incorporated into the lamina and displaced by smo+ LPCs. LPCs that are unable to respond to Hh might be eliminated by cell death (Huang, 1998).

A hallmark of Hh signal reception in many Drosophila tissues is an increase in immunoreactivity to the C-terminal portion of the protein Ci, a transcriptional mediator of Hh signaling. This enhanced Ci immunoreactivity is due to inhibition of Ci proteolytic processing, a cellular response to Hh signal reception. LPCs posterior to the lamina furrow display the enhanced Ci immunoreactivity that would be predicted for Hh signal reception by LPCs. In animals in which hh- retinal axons innervate the lamina target field, cells posterior to the lamina furrow display a level of Ci immunoreactivity equivalent to the basal level detected anterior to the furrow, indicating that the increased Ci observed in the wild type is Hh-dependent. In smo mosaic animals, smo cells either anterior or posterior to the lamina furrow display a basal level of Ci immunoreactivity, while smo + cells immediately adjacent to the portion of smo clones within the lamina display the high Hh-dependent level. The initial response of LPCs to the arrival of Hh-bearing retinal axons would appear to be entry into S-phase at the lamina furrow. To determine whether cell cycle progression is directly dependent on Hh signal reception, the incorporation of bromodeoxyuridine (BrdU) into S-phase cells was examined in smo mosaic animals. In the wild type, LPCs that have entered their terminal S-phase form a discrete and continuous band at the posterior margin of the lamina furrow. In animals lacking photoreceptor innervation (due to defective hh expression in the eye disc) or animals in which photoreceptor axons lacking functional Hh enter the lamina target field, only a low background of scattered S-phase cells are detected. It is unclear whether the products of these scattered divisions are incorporated into the lamina (i.e., that these cells are indeed LPCs). In smo 3 mosaic animals, mutant clones that include the posterior margin of the lamina furrow lack S-phase LPCs. In contrast, the scattered S-phase cells anterior to the lamina furrow, and the distribution of S-phase cells in other proliferation centers, such as the OPC, are unaffected by the loss of smo function. At the lamina furrow, smo+ cells bordering smo clones are often found in S-phase. Thus, in sum, smo+ behaves as a cell-autonomous requirement for LPCs to initiate the Hh-dependent steps of lamina differentiation (Huang, 1998).

The Hh receptor Ptc, a multiple-pass membrane protein, and the cAMP-dependent protein kinase (PKA) normally maintain the Hh signal transduction pathway in a repressed state. Loss-of-function mutations in either of these genes mimic Hh signal reception and result in the cell autonomous activation of Hh target genes in many tissues. LPCs harboring mutations for either pka or ptc undergo differentiation cell-autonomously and independently of retinal innervation. Mutant cells anterior to the furrow do not differentiate precociously. This observation is consistent with the consequences of ectopic Hh expression in an the lamina in mutants lacking retinal innervation of the lamina. Hh expression in regions anterior to the lamina furrow does not induce precocious lamina differentiation, as though competence to respond to Hh is acquired by G1-phase LPCs at the anterior margin of the lamina furrow. Within the lamina target field, wild-type cells neighboring the pka or ptc mutant cells are never observed to express Dac. Thus activation of the Hh pathway by loss-of-function in either gene results in a strictly autonomous induction of LPC maturation. These results permit the conclusion that the terminal cell division and differentiation of LPCs both require the direct reception of the Hh signal (Huang, 1998).

In a number of instances, pattern formation mediated by Hh is accompanied by cell division. The well-defined pattern of Hh-induced cell division in the lamina provides an opportunity to determine the point at which the Hh signal reception engages the cell cycle machinery. LPC cell cycle progression and cell fate determination are jointly controlled by the transcriptional regulator Cubitus interruptus. Biochemical and epistasis experiments have placed the zinc finger molecule Ci downstream of all other hh signaling pathway components. Ci has been shown to bind directly to the regulatory sequences of Hh-responsive genes. Should all Hh-mediated events of LPC maturation be found to depend on Ci function, it could be concluded that, at least with regard to cell proliferation and the expression of differentiation markers, there is no branchpoint within the signaling pathway. To examine the requirement for Ci, two recombinant constructs were used that result in either dominant Ci gain-of-function or loss-of-function phenotypes. Overexpression of the wild-type Ci gene results in a gain-of-function phenotype that mimics activation of the Hh signaling pathway. Expression of an amino terminal fragment of Ci (hereafter referred to as DN-Ci) results in a dominant loss-of-function phenotype, as the normal in vivo function of this portion of the molecule appears to be transcriptional repression of Hh target genes. With either construct, genetically engineered ectopic expression in the lamina region results in the expected phenotype with respect to the lamina differentiation marker Dac. Dac expression in cells posterior of the lamina furrow is strongly reduced or undetectable in cells expressing DN-Ci. Conversely, the ectopic expression of wild-type Ci results in the induction of Dac-positive cells in the lamina target field of animals lacking innervation from the developing eye. The effects observed with either construct are strictly cell autonomous. Thus the results with ectopic Ci and DN-Ci expression are consistent with the expectation that Ci modulates Hh signaling activity directly in LPCs (Huang, 1998).

To determine whether Hh signaling acts via Ci to regulate the G1- to S-phase transition of LPCs at the lamina furrow, the incorporation of BrdU into S-phase cells was examined in animals harboring clones expressing either of the two constructs described above. Cells expressing DN-Ci at the posterior margin of the lamina furrow fail to enter S-phase. Where clones of DN-Ci-expressing cells traversed the lamina furrow, S-phase LPCs are absent, while S-phase LPCs are observed immediately outside of the clone. Moreover, the effect on cell division is limited to the LPCs at the lamina furrow. No defects are observed in other proliferation zones such as the OPC or IPC, the other major proliferation centers of the optic lobe, when they contain DN-Ci-expressing cells. Conversely, the induction of lamina differentiation by ectopic Ci expression in flies lacking retinal input into the lamina is accompanied by the entry of LPCs into S-phase at the lamina furrow. At the point when lamina differentiation is induced in the absence of retinal axons by ectopic Hh expression, ectopic Ci expression triggers a posterior-to-anterior pattern of differentiation such that S-phase LPCs are found at the anterior margin. In sum, these observations indicate that the induction of cell division by Hh occurs via the transcriptional regulation of Hh target genes (Huang, 1998).

A C-terminal motif targets Hedgehog to axons, coordinating assembly of the Drosophila eye and brain

The developmental signal Hedgehog is distributed to two receptive fields by the photoreceptor neurons of the developing Drosophila retina. Delivery to the retina propagates ommatidial development across a precursor field. Transport along photoreceptor axons induces the development of postsynaptic neurons in the brain. Hedgehog is composed of N-terminal and C-terminal domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal cleavage product. This study shows that the N-terminal domain is targeted to the retina when synthesized in the absence of the C-terminal domain. In contrast to studies that have focused on cholesterol as a determinant of subcellular localization, this study found that the C-terminal domain harbors a conserved motif that overrides retinal localization, sending most of the autocleavage products into vesicles bound for growth cones or synapses. Competition between targeting signals at the opposite ends of Hedgehog apparently controls the match between eye and brain development (Chu, 2006).

The photoreceptor neurons of the Drosophila retina provide Hedgehog to receptive cells in both the eye and brain, and thus coordinate the development of a light-receptive and processing circuit. Release into the retina occurs via an apical pathway, while access to the brain requires transport along axons, a basal pathway. One might suppose that this pattern of release would arise by a mechanism that distributes Hh generally in the cell. However, the data suggest that this distribution involves opposing determinants at opposite ends of the polypeptide. Hh is composed of N-terminal signaling and C-terminal protease domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal signaling fragment (Hh-Np). This study found that the N-terminal product localized to the retina when it was synthesized in the absence of the C-terminal domain. This localization was not conferred by the Hh secretory signal motif alone, and hence must reflect the activity of a targeting signal elsewhere in the N-terminal domain. Conversely, the C-terminal domain, synthesized with or without the N-terminal domain, was strongly localized to the growth cones. Thus, the cellular distribution of the N-terminal signaling domain would appear to reflect opposing determinants; a targeting motif at the C terminus directs most of the N-terminal cleavage product into an axonal pathway (Chu, 2006).

The apical localization of the N-terminal domain was observed with Hh-Nu, the product of a 3'-truncated cDNA. Three explanations were considered for this localization; that axonal targeting requires: (1) the absent 3' mRNA sequences, (2) a polypeptide signal in the C-terminal domain, or (3) the cholesterol moiety added by Hh's autocleavage. It was found that the 3' region of the hh mRNA contributed modestly to axonal localization and was relatively ineffective in mediating growth cone localization. This region of mRNA may direct the mRNA and its translation to sites that enhance membrane localization of the product, as has been observed with other morphogens. In contrast, indelibly appending the C-terminal domain to the N terminus by mutating the autocleavage site produced strong axonal and growth cone localization in puncta shared with Synaptotagmin, a fate like that of wild-type Hh. Though all other explanations for the localization properties of these Hh isoforms cannot be precluded, the most straightforward is that the C-terminal domain sorts Hh into a pathway for axonal transport (Chu, 2006).

The C-terminal axon-targeting signal was mapped to a 30 amino acid region where the Hh family shares a well-conserved five amino acid motif. When an invariant tyrosine in this motif (Y452) was mutated to either phenylalanine or glutamic acid, autocleavage appeared normal, but the two products remained, for the most part, in the retina. When another well-conserved tyrosine residue outside the motif at amino acid 457 was similarly mutated, there was no effect on axon transport. The N-terminal domain thus relies on a signal in the C-terminal domain for its axonal localization, though autocleavage separates the two polypeptides prior to transport. It is therefore evident that targeting the N-terminal domain to axon terminals does not require autocleavage or cholesterol modification (Chu, 2006).

A number of studies have examined the intracellular trafficking, release, and extracellular movement of Hh or its mammalian counterpart, Sonic Hedgehog. They have relied on comparison between the N-terminal product of Hh autocleavage, Hh-Np, and the cholesterol-negative isoform, Hh-Nu, to draw conclusions on the role of cholesterol in intracellular trafficking and extracellular signaling activity. The current observations raise doubt but do not directly address the role of cholesterol modification in other developing tissues and organisms. Moreover this study does not address the role of lipid modification in Hh movement outside of the cell, where, for example, cholesterol underlies the protein's assembly into extracellular lipoprotein transport particles (Chu, 2006).

In the brain, Hh acts over several cell diameters to expand the pool of lamina precursors in rough proportion to the number of photoreceptor axon fascicles that arrive. These signaling events establish a temporal coordination between the differentiation programs of the eye and brain and, eventually, a numerical match between sensory axons and postsynaptic neurons. Is the partitioning of Hedgehog by the activity of the C-terminal motif necessary to produce this match? In support of this view, it was found that the C-terminal motif Y452 mutant is deficient in the induction of lamina development, while it is similar to wild-type Hh in the induction of retinal development. The chromosomal mutant hh² was also proficient for eye development and deficient in axonal localization and lamina induction. The hh² allele, unlike other alleles that affect the eye and lamina equivalently, is predicted to yield a truncated product lacking the C-terminal targeting motif. The C-terminal motif is conserved in the mouse Sonic Hedgehog (SHH), which regulates visual system development via delivery from retinal ganglion cell bodies and axons. Adult neuronal expression and anterograde transport of SHH have also been reported. Therefore, the C-terminal motif may have a conserved role in localizing Hedgehog, thus regulating its signaling activity in the development and function of neural circuitry (Chu, 2006).

The highly ordered assembly of retinal axons and their synaptic partners is regulated by Hedgehog/Single-minded in the Drosophila visual system

During development of the Drosophila visual center, photoreceptor cells extend their axons (R axons) to the lamina ganglion layer, and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal Hedgehog (Hh). This inductive mechanism helps to establish an orderly arrangement of connections between the R axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding R axons. The bHLH-PAS transcription factor Single-minded (Sim) is induced by Hh in the lamina neurons and is required for the association of lamina neurons with R axons. In sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with R axons. As a result, lamina neurons are set aside from R axons. The data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks (Umetsu, 2006).

Most axons in the brain establish topographic maps in which the arrangement of synaptic connections maintains the relationships between neighboring cell bodies. A notable model of topographic map formation is the visual system, where the relay of visual information from the retina to the visual center must be arranged in a spatially ordered manner through the topographic connections of retinal axons with their midbrain target, which is the optic tectum (OT) in lower vertebrates and the superior colliculus (SC) in mammals. This topographic map is termed a retinotopic map. Many studies have shown that Ephrin protein family members, acting through their Eph receptors, play pivotal roles in the establishment of the retinotopic map. In the mouse and the chick, for example, the retinal ganglion cells (RGCs) extend their axons to the OT/SC, and the low-to-high anteroposterior gradient of ephrin A in the target limits the posterior extension of growth cones at various positions, dependent on the EphA level of each RGC (Umetsu, 2006).

The Drosophila visual system has also provided insight into topographic mapping. In Drosophila, the projections of photoreceptor neurons (R cells) themselves induce development of the corresponding postsynaptic neurons. The Drosophila visual system consists of the compound eyes and the three optic ganglia: the lamina, the medulla and the lobula complex. Each of the approximately 750 ommatidial units comprising the compound eye contain six outer photoreceptors (R1-R6) and two inner photoreceptors (R7, R8). R1-R6 cells send their axons to the first optic ganglion, the lamina, whereas R7 and R8 cells send axons through the lamina to the second ganglion, the medulla. R1-R6 cells in each ommatidium make stereotypic connections with particular lamina neurons. Synaptic units in the lamina are referred to as lamina cartridges. During the initial step of the assembly of a lamina cartridge, an arriving photoreceptor axon (R axon) fascicle forms a pre-cartridge ensemble, the 'lamina column', with a set of five lamina neurons. Formation of the ensemble results in a one-to-one correspondence of ommatidia to column units, and is fundamental to the subsequent establishment of intricate synaptic connections. Development of the lamina is tightly regulated by the projection of R axons. Failure in eye formation results in concurrent loss of the lamina, as in a normal brain, lamina neurogenesis is directly coupled to the arrival of R axons. Both R cell differentiation and ommatidial assembly progress in a posterior-to-anterior direction across the eye disc. Differentiated R cells begin to send their axons to the brain in the same sequential order, reflecting their position in the retina along the anteroposterior and the dorsoventral axes. Wnt signaling plays a role in regulating projections along the dorsoventral axis (Umetsu, 2006).

As the axons from each new row of ommatidial R cell clusters arrive in the lamina, a corresponding group of lamina precursor cells (LPCs) undergo a final division and initiate differentiation into lamina neurons. In the first step of their neurogenesis, direct contact with R axons triggers the transition of G1-phase LPCs into S phase. Both the G1-S transition and the initial specification into a lamina neuron are induced by Hedgehog (Hh), which is delivered by arriving R axons, and the next step in lamina differentiation is induced by the Spitz signaling molecule, which is also delivered by R axons. Hh expressed in R cells functions as a signal for photoreceptor development as well: secreted Hh induces anterior precursor cells to enter the pathway of R cell specification (Umetsu, 2006).

Thus, the retinotopic map along the anteroposterior axis of the lamina seems to be established autonomously and in a posterior-to-anterior order, as newly specified R cells send their axons to the lamina layer and make lamina columns. Each ommatidial unit sends a set of R axons as a single bundle to the lamina along the pre-existing fascicle that has been just projected. Then, the axon bundles are enveloped by the processes of newly induced lamina neurons. This step is key to forming the one-to-one associations between R axon bundles and their corresponding lamina neurons. This study shows that the activity of Single-minded (Sim) is required for developing lamina neurons to establish an association with the corresponding R axons and, hence, to form the lamina column. sim encodes a basic-helix-loop-helix-PAS (bHLH-PAS) transcription factor and is induced by Hh provided by the R axons. In sim mutant brains, the developing lamina neurons fail to associate with R axon bundles, resulting in a failure to establish connections between R axons and lamina neurons. It is inferred that sim programs developing lamina neurons to express a molecule(s) that is required for the association with R axons (Umetsu, 2006).

Retinotopic mapping in Drosophila provides unique insights into neuronal network formation not only because of its tight coupling to the control of development, but also because of the interactions between axons and neuronal cell bodies. The interactions observed stand in sharp contrast to what has been found for other models of axon guidance, where the growth cones of axons respond to a variety of attractive or repulsive guidance cues to navigate to their synaptic target cells. The cues include the netrins, Slits, semaphorins and ephrins, and the restricted expression pattern of these cues and the reactivity of growth cones play pivotal roles in the establishment of the proper synaptic connections. In this context, postsynaptic cells are seen as mere providers of guidance/adhesion molecules, passively awaiting the arrival of a growth cone. In other words, it is conceivable that presynaptic growth cones seek their targets dynamically, whereas postsynaptic cells remain static. Unlike the roles of presynaptic axons, the cellular behaviors of postsynaptic cells in the establishment of synaptic targeting are poorly understood. This study proposes another possible model for the establishment of topographic neuronal connections in which postsynaptic cells dynamically interact with presynaptic axons (Umetsu, 2006).

Thus, Sim, a target of Hh, is required for at least the first step of lamina column formation; namely, the incorporation of developing lamina neurons into the area where R axons project and lamina columns mature, an area referred to as the assembling domain. This model for Sim is based on four observations. First, sim²/sim^ry75 brains have a reduced number of lamina neurons in the assembling domain, leaving an abnormally large number of premature lamina neurons behind in the pre-assembling domain. Second, in clonal analysis, sim² clones fail to be recovered in the assembling domain (similar to smo¹ clones). Third, lamina neuron-specific inhibition of Sim function causes R axon bundles to be tightly packed and lamina neurons to be excluded from R axon bundles. And fourth, overexpression of sim in lamina neurons causes precocious incorporation of lamina neurons into the assembling domain (Umetsu, 2006).

In case of overexpression, neither expansion of the assembling domain nor increase in the number of lamina neurons relative to the number of R axon bundles was observed, even though lamina neurons prematurely incorporated into the assembling domain. This is probably because a reduced number of lamina neurons were generated. In fact, loss of E2F expression was observed at the lamina furrow in NP6099-GAL4 UAS-sim brains. The onset of incorporating lamina neurons into the assembling domain might be linked to an inhibition of cell proliferation. However, this is thought to be unlikely for two reasons: (1) lamina neurons did not show any extra E2F signal in the sim mutant brain in spite of an increase in unincorporated lamina neurons; and (2) lamina neurons ectopically expressing a cell cycle-braking factor, the Drosophila p21/p27 homolog dacapo (dap) cause the precocious incorporation of lamina neurons. Thus, a direct link between cell cycle regulation and the incorporation of lamina neurons is less plausible (Umetsu, 2006).

An alternative model, the 'time lag' model, is proposed. There appears to be a lag between the onset of sim expression and the onset of incorporation of lamina neurons. Differentiating lamina neurons are held temporarily in the pre-assembling domain and then the proper amount of lamina neurons are coordinately integrated into columns as more R axons are projected. Thus, it is speculated that a certain degree of accumulation of the Sim/dARNT heterodimer in nuclei is needed to exert cellular function. Consistent with this idea, graded accumulation of Sim is observed, with lower Sim levels in anterior (younger) lamina neuron nuclei and higher levels in posterior (older) lamina neuron nuclei. Overexpression of Sim in lamina neurons would thus cause higher levels of accumulation of the protein in young lamina neurons and facilitate their incorporation into the assembling domain. Interestingly, overexpression of the wild-type dARNT did not have any detectable effects, suggesting that Sim accumulation is a limiting factor for cell incorporation (Umetsu, 2006).

The mechanism of neuronal maturation and that of assembly of lamina neurons are independent, although both are under the control of Hh signaling. Disruption of sim did not affect the differentiation and proliferation of lamina neurons. Correspondingly, neither the incorporation of lamina neurons into the lamina column nor the expression of sim were affected by dac mutation. The cellular function required for assembling the column or the function of Sim at the cellular level is still not known. Electron microscopic observations by have revealed an intriguing behavior of lamina neurons at the early pupal stage; large processes extending from lamina neurons engulf R1 and R6 axons of newly incoming R axon bundles. This may be the key step in lamina column formation and interaction between the R axons and lamina neurons. Sim may regulate genes required for process formation, interaction with R axons and/or events that follow shortly after, since lamina neurons seem to fail to make interactions with R axons from the beginning in the sim mutant background. Sim is expressed in the midline cells of the CNS throughout neurogenesis in the Drosophila embryo and is required for the proper differentiation of the midline cells into mature neurons and glial cells. Midline precursor cells undergo synchronized cell division and then transform into the bottle-shaped cells, in which the nuclei migrate internally and leave a cytoplasmic projection joined to the surface of the embryo. The sim mutant midline cells fail to delaminate from the epidermal cell layer. Cells do not make the normal bottle-like shape and, instead, they appear rounded. In addition, overexpression of sim can induce other cell types to exhibit midline morphology. sim may thus regulate the transcription of a set of genes required for morphological changes, which in turn are required for interaction between cells, both in the lamina and during embryonic CNS development (Umetsu, 2006).

Although sim expression is regulated by Hh signaling, this does not answer the question of whether sim function is sufficient to confer on cells the ability to be incorporated into the assembling domain. Whether smo mutant clones can be recovered in the assembling domain was examined by forcing sim expression in smo clones using the MARCM technique. However, smo mutant clones expressing sim were not recovered in the assembling domain. This suggests that additional factors are involved in lamina neuron assembly. Hh may also contribute to specification of the difference in affinity between lamina neurons and R axons and/or between anterior and posterior lamina neurons. In Drosophila wing discs, the Hh signal differentiates the affinity of anterior compartment cells from that of the posterior compartment cells, thereby maintaining the compartment border (Umetsu, 2006).

An active role is proposed for postsynaptic cells in making a topographic map of the Drosophila visual system. Targeted expression of the dominant-negative form of the Sim partner in the lamina neurons clearly showed a role for postsynaptic cells in assembling lamina columns. This presumably affects an early step of assembly. It is not known if Sim function is also required for later steps in more mature lamina neurons. The forced expression of the dominant-negative Sim partner in the posterior lamina neurons had no effect, although it may simply be that the level of expression of the dominant-negative form of dARNT was not sufficient to have an observable effect on Sim function. In the lamina column, the R axon bundle associates with a precisely arranged row of five lamina neurons. No mechanisms for the development and formation of this stereotypic structure have been revealed. Another signal might be provided from the R axons with lamina neurons, and/or intrinsic structures of the R axons might play a role in this architecture. An intriguing property of postsynaptic muscle cells for axonal targeting has been observed: the muscle cells bear numerous postsynaptic filopodia ('myopodia') during motoneuron targeting. They showed that postsynaptic cells actively contribute to synaptic matchmaking by direct, long-distance communication. Together with what has been learned about myopodia in neuromuscular synapse formation, the curent findings reveal an active role for postsynaptic cells for the establishment of precise neural networking (Umetsu, 2006).

Sim belongs to the family of bHLH-PAS transcription factors, whose members function in many developmental and physiological processes, including neurogenesis, tissue development, toxin metabolism, circadian rhythms, response to hypoxia, and hormone receptor function. bHLH-PAS proteins usually function as dimeric DNA-binding protein complexes. The most common functional unit is a heterodimer. These heterodimers consist of one partner that is broadly expressed, and another whose expression is regulated spatially, temporally or by the presence of inducers. Sim and the bHLH-PAS protein dARNT heterodimerize to bind to their responsive element, the CME (CNS midline enhancer element), to activate target gene transcription. In this complex, dARNT is the general dimerization partner and Sim is the tissue-specific partner (Umetsu, 2006).

The Drosophila Sim and mammalian Sim1 and Sim2 proteins are highly conserved in their amino-terminal halves, which contain a bHLH and a PAS domain. Murine Sim1 and Sim2 are also expressed in both proliferative and postmitotic zones of the central nervous system at different stages of neural development. These zones of expression include the longitudinal basal plate of the diencephalon (Sim1 and Sim2), the mesencephalon (Sim1), the zona limitans intrathalamica (Sim1 and Sim2) and the portion of the spinal cord that flanks the floor plate (Sim1). Sim2 maps to the region responsible for Down Syndrome (DS) on Chromosome 21. Interestingly, Sim2 is also expressed in non-neuronal tissues, including branchial arches and the developing limb, which are primordia of tissues and organs where DS abnormalities are frequently observed (Umetsu, 2006).

Given the important roles of sim in Drosophila development and the expression of Sim2 in cell types that are affected in DS individuals, it was proposed that Sim2 may play a causative role in DS. However, because of a lack of direct evidence and the existence of other candidate genes, this remains speculative. Cells expressing sim during Drosophila development and Sim2-positive cells affected in DS seem to be able to migrate. The conserved role of Sim may enable cells to migrate and/or interact with surrounding cells in the various tissues, including the central nervous system. It will thus be intriguing to search for extra cellular targets of Sim regulation with the hope of elucidating mechanisms that underlie the behavior of Sim-expressing cells (Umetsu, 2006).

Patterning axon targeting of olfactory receptor neurons by coupled hedgehog signaling at two distinct steps

Evidence is presented for a coupled two-step action of Hedgehog signaling in patterning axon targeting of Drosophila olfactory receptor neurons (ORNs). In the first step, differential Hedgehog pathway activity in peripheral sensory organ precursors creates ORN populations with different levels of the Patched receptor. Different Patched levels in ORNs then determine axonal responsiveness to target-derived Hedgehog in the brain: only ORN axons that do not express high levels of Patched are responsive to and require a second step of Hedgehog signaling for target selection. Hedgehog signaling in the imaginal sensory organ precursors thus confers differential ORN responsiveness to Hedgehog-mediated axon targeting in the brain. This mechanism contributes to the spatial coordination of ORN cell bodies in the periphery and their glomerular targets in the brain. Such coupled two-step signaling may be more generally used to coordinate other spatially and temporally segregated developmental events (Chou, 2010).

The central finding of this study is the coupled two-step action of Hedgehog in patterning ORN axon targeting. In the first step, differential Hh pathway activity in peripheral sensory organ precursors in larva and early pupa creates ORN populations with different levels of the Patched receptor. These Patched levels in ORNs then determine axonal responsiveness to target-derived Hh in the brain in the second step: only ORN axons that do not express high levels of Ptc are responsive to and require a second-step of Hh signaling for proper target selection. Multiple lines of evidence support this model. First, genetic loss-of-function studies indicate that ORNs fall into two groups based on their autonomous requirement for Smo, a classic Hh pathway component, as well as Ihog, a recently discovered positive receptor component for Hh. Second, Smo/Ihog-dependence for axon targeting coincides with Ptc levels for all 21 classes examined (11 high-Ptc and 10 low-Ptc). Third, knockdown of Hh from brain neurons only affects the targeting of low-Ptc ORN classes, with similar mistargeting preferences as compared to loss of Smo or Ihog in ORNs. Fourth, overexpression of Ptc in ORNs preferentially affects targeting of low-Ptc classes, whereas loss of Ptc in ORNs only affects targeting of high-Ptc classes. Fifth and perhaps most telling, loss of Hh in the antenna and maxillary palp preferentially affects targeting of high-Ptc classes; these mistargeting defects can be suppressed by Ptc overexpression. This result supports two important predictions of the model: Hh from the periphery is not directly required for axon targeting, at least for low-Ptc classes, but is required for the initiation and maintenance of high levels of Ptc in high-Ptc classes. Removing Hh from the periphery results in loss of Ptc expression in high-Ptc ORNs, which lifts Smo inhibition and causes axon mistargeting similar to loss of Ptc. Brain-derived Hh, by contrast, is required for low-Ptc classes but should not be read by at least 6 high-Ptc classes (Chou, 2010).

Vertebrate Sonic hedgehog has been proposed to act locally as an axon guidance cue whose action is dependent on the classic Hh pathway component Smo and the Robo related protein Boc, an Ihog homolog. The finding that Drosophila Hh also plays a role in ORN axon targeting that is dependent on Smo and Ihog suggests an evolutionarily conserved function of Hh in regulating axon development. A recent in vitro study supports the idea that Shh acts directly as an axon guidance cue in a rapid, transcription-independent manner (Yam, 2009). In the fly olfactory system, low-Ptc ORN classes originate from the En- and Hh-producing compartment, which are exposed to their own Hh yet do not show a transcriptional response. This is likely because ci expression is repressed by En. Brain-derived Hh may thus also act locally in axon targeting, as reported in vitro for Shh (Chou, 2010).

The data do not distinguish whether Hh acts instructively as an axon guidance cue, or permissively to modulate activities of other axon guidance receptors. The primary argument against an instructive model is the lack of spatial patterns of Hh proteins in the antennal lobe to account for the spatial distribution of glomerular targets of low- and high-Ptc ORN classes. This does not rule out the instructive model, however, as Hh activity can be modulated post-translationally such that the spatial distribution of Hh activity may differ from Hh protein levels. Alternatively, a permissive model for Hh action on ORN axons is also possible. For instance, Hh may regulate the cAMP/PKA pathway, which can in turn modulate axon guidance signaling. Indeed, it has recently been shown that Shh can modulate axon responsiveness to Semaphorins at the midline of the vertebrate spinal cord (Chou, 2010).

Whatever the downstream effector, the coupled two-step mechanism uncovered in this study can be used to coordinate cell body positions of ORNs in the sensory organs and their glomerular targets in the brain. The data indicate that it is essential both for ORN classes that depend on brain-derived Hh for axon targeting to express low levels of Ptc, and at least a subset of ORN axons that do not respond to brain-derived Hh for axon targeting to express high levels of Ptc, in order to ensure their targeting fidelity. Ptc expression levels thus create a code to diversify ORN classes according to their cell body positions in the sensory organ. Indeed, mistargeting of low-Ptc ORNs in the absence of Smo shows a significant preference for glomeruli that are normally high-Ptc ORN targets. This switch of axon target is by no means complete, suggesting that Hh signaling works together with other mechanisms to ensure axon targeting fidelity. It has previously been shown that transcription factors Atonal and Amos divide the ORN classes largely according to sensillar groups, which might regulate coarse correspondence of ORN cell body positions in periphery and their target glomeruli in the antennal lobe. The Notch system also diversifies ORN classes within each sensillum. This analysis indicates that Hh/Ptc demarcation of ORN cell bodies and their glomerular targets does not coincide precisely with the sensillar groups or with the Notch system, suggesting that the Hh system acts to diversify ORN classes independently, and likely at a step in between large sensillar group specification by Atonal/Amos and finer level diversification within each sensillum by the Notch system (Chou, 2010).

Hh was previously shown to coordinate the development of sensory neurons and their targets in the Drosophila visual system: Hh made in photoreceptors is transported down their axons to trigger neurogenesis of target laminar neurons. The olfactory system is constructed differently: target PNs are born and create a spatial pattern with their dendrites before ORN axon arrival. Consistent with this idea, Hh signaling is not required for PN development. Despite this fundamental difference from the visual system, Hh signaling is also used, but in a novel manner, to coordinate the ORN cell body position in the sensory organ with the glomerular map in the brain. Hh signaling in the periphery creates populations of ORNs with different Ptc levels such that cells that respond to the Hh signal in the first round are incapable of responding in the second round. Such a coupled two-step mechanism may be generally used for a single signaling pathway to coordinate spatially and/or temporally separate developmental events. Signal-induced expression of a positive or negative pathway component during an early phase of signaling could serve as a time-delayed cellular memory to specify responses at a later stage by rendering cells sensitive or insensitive to a second round of signaling (Chou, 2010).

Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye

In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. This study shows that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function (Fan, 2008).

In developing wing discs in which apoptosis was induced by expression of the pro-apoptotic gene hid, loss of the caspase inhibitor DIAP1, or by X-ray treatment, the accumulation of two major mitogens, Dpp and Wg, has been observed in dying cells. Key for this finding is the simultaneous expression of the caspase inhibitor P35. Under these conditions, the dying cells were kept alive ('undead'), allowing accumulation of Dpp and Wg. This accumulation appears to be dependent on the initiator caspase Dronc, because it cannot be blocked by expression of P35 which inhibits effector caspases but not Dronc. In addition, the Drosophila homolog of the tumor suppressor p53, Dp53, has been implicated downstream of Dronc for compensatory proliferation. Notably, these studies on mechanisms of compensatory proliferation were carried out in developing larval wing imaginal discs in Drosophila. Cells in wing discs proliferate extensively during larval stages, and the majority of these cells does not differentiate before they reach pupal development. Hence, the mechanisms of compensatory proliferation have so far only been investigated in situations where most cells are proliferating. Interestingly, apoptosis-induced compensatory proliferation in differentiating eye tissue of third-instar larvae. However, it is unclear whether this form of compensatory proliferation is controlled by a similar mechanism as reported for larval proliferating wing discs (Fan, 2008).

This study revealed that there are at least two distinct mechanisms that promote compensatory proliferation in response to apoptotic activity. The general difference between these two mechanisms lies in the developmental context of the tissue in which compensatory proliferation occurs. In proliferating wing and eye tissues, compensatory proliferation induced by extensive apoptosis is dependent on Dronc and Dp53, which induce Dpp and Wg expression. In contrast, in differentiating eye tissue, apoptosis induces compensatory proliferation through a novel mechanism requiring the effector caspases DrICE and Dcp-1, which induce Hh signaling in a nonapoptotic function (Fan, 2008).

When cells stop proliferating and become committed to adopt cell fate, dramatic changes in gene expression are occurring. Given these changes in developmental plasticity, it is not surprising that distinct mechanisms of apoptosis-induced compensatory proliferation are employed in proliferating versus differentiating tissues. However, it should be noted that the proliferating capacity of differentiating tissues is rather restricted. In GMR-hid eye discs, although hid is expressed in all cells posterior to the MF, compensatory proliferation occurs only in cells that are still undifferentiated. Yet, even though they are undifferentiated they have withdrawn from the cell cycle and, under normal developmental conditions (i.e., without GMR-hid), they would soon be recruited to adopt cell fate. However, the apoptotic environment causing increased Hh signaling appears to be able to trigger reentry of these cells into the cell cycle (Fradkin, 2008).

Interestingly, the Hh signal is specifically increased in photoreceptor neurons requiring a nonapoptotic activity of effector caspases. Hh signaling can then nonautonomously induce proliferation of undifferentiated cells at the basal side of the eye disc. However, overexpression of Hh posterior to the MF in wild-type eye discs alone is not sufficient to induce a comparable wave of compensatory proliferation as in GMR-hid eye discs. This suggests that cell-cycle reentry requires activation of additional factors/pathways stimulated in apoptotic cells (Fradkin, 2008).

Although hid can stimulate increased Hh expression in photoreceptor neurons throughout the posterior half of the eye disc, compensatory proliferation is restricted to a certain distance (six to ten ommatidial columns) from the MF. This corresponds to approximately 6-15 hr of developmental time, and might be the time required for cell-cycle reentry. Similarly, when mammalian cells that have exited the cell cycle are stimulated to reenter the cell cycle, they need about 8 hr to do this. The reason for this delay is unknown. Studying compensatory proliferation in GMR-hid eye discs might provide a genetic model to address this interesting problem (Fradkin, 2008).

It is not clear whether this novel effector caspase-, Hh-dependent pathway of compensatory proliferation also applies to other, or even all, differentiating tissues. However, what this study shows is that there are at least two distinct mechanisms of apoptosis-induced compensatory proliferation. It is also possible that other mechanisms of compensatory proliferation in different developmental contexts are going to be uncovered in the future. Interestingly, in developing larval wing discs, P35-dependent compensatory proliferation has been implicated in cell competition. This suggests that, even in tissue with the same developmental potential, compensatory proliferation can occur with distinct mechanisms (Fradkin, 2008).

How cells sense different developmental contexts and operate distinct proliferating mechanisms in response to apoptotic stress is unknown. Specifically, where is the specificity and selectivity for distinct caspases coming from in tissues of different developmental potential? What are the mechanisms engaged by these caspases to trigger secretion of either Dpp and Wg or Hh? These are questions which need to be addressed in the future (Fan, 2008).

This study has several implications for tumorigenesis. First, many tumors develop when quiescent cells reenter the cell cycle. The mechanisms for cell-cycle reentry are largely unknown. Second, evasion from apoptosis is a hallmark of cancer. Many tumor cells are induced to undergo apoptosis. However, they do not die, because they downregulate essential components of the apoptotic pathway such as Apaf-1 and caspases. Thus, these undead tumor cells might secrete mitogens which might induce compensatory proliferation similar to the Drosophila case. In this way, undead cells might contribute to the growth of the tumor. A similar argument can be made for chemotherapy, which in many cases attempts to activate the apoptotic program in a tumor cell. If the death of the tumor cell is blocked, or slow, mitogens might be produced and the tumor growth could be even more severe. This is very obvious in the apoptotic wing or anterior eye discs in Drosophila when apoptosis is blocked by P35. Under these conditions, overgrown wing and eye tissues are observed. Thus, evasion of apoptosis might directly contribute to tumor growth. Finally, although increased Hh signaling can lead to various cancers, how Hh induces cellular proliferation and tissue overgrowth is not well understood. Mutations in Patched1, a negative regulator of sonic Hh, frequently give rise to human tumors. The exact cause is unknown. These data imply that Hh signaling might be involved in cell-cycle reentry allowing cells to resume proliferation (Fan, 2008).

Homeotic proboscipedia function modulates hedgehog-mediated organizer activity to pattern adult Drosophila mouthparts

Drosophila proboscipedia (HoxA2/B2 homolog) mutants develop distal legs in place of their adult labial mouthparts. How pb homeotic function distinguishes the developmental programs of labium and leg has been examined. The labial-to-leg transformation in pb mutants occurs progressively over a 2-day period in mid-development, as viewed with identity markers such as dachshund (dac). This transformation requires hedgehog activity, and involves a morphogenetic reorganization of the labial imaginal disc. These results implicate pb function in modulating global axial organization. Pb protein acts in at least two ways. (1) Pb cell autonomously regulates the expression of target genes such as dac; (2) Pb acts in opposition to the organizing action of hedgehog. This latter action is cell-autonomous, but has a nonautonomous effect on labial structure, via the negative regulation of wingless and decapentaplegic. This opposition of Pb to hedgehog target expression appears to occur at the level of the conserved transcription factor cubitus interruptus/Gli that mediates hedgehog signaling activity. These results extend selector function to primary steps of tissue patterning, and leads to the notion of a homeotic organizer (Joulia, 2005).

The labial palps, the drinking and taste apparatus of the adult fly head, are highly refined ventral appendages homologous to legs and antennae. As for most adult structures, these mouthparts are derived from larval imaginal discs, the labial discs. Wild-type pb selector function acts together with a second Hox locus, Scr, to direct the development of the labial discs giving rise to the adult proboscis. In the absence of pb activity, the adult labium is transformed to distal prothoracic (T1) legs, reflecting the ongoing expression and function of Scr in the same disc. Though the pb locus shows prominent segmental embryonic expression, as for the other Drosophila homeotic genes of the Bithorax and Antennapedia complexes, it is unique in that it has no detected embryonic function and null pb mutants eclose as adults that are unable to feed. Thus, normal pb selector function is required relatively late, in the labial imaginal discs that proliferate and differentiate during larval/pupal development to yield the adult labial palps. Though the genetic pathway guiding development of the ventral labial imaginal discs to adult mouthparts remains relatively unexplored both in flies and elsewhere, study of P-D patterning has identified several genes subject to pb regulation in the labial discs (notably Dll, dac, and hth) and a distinct organization of normal labial discs has been indicated compared to other imaginal discs (Joulia, 2005).

This study pursued an investigation of how pb homeotic function distinguishes between labial and leg developmental programs. The results implicate pb function at the level of global axial organization. Employing identity markers such as dachshund (dac), a 2-day period late in larval development has been identified when normal pb function is required for labial development. The labial-to-leg transformation occurs during the third larval instar stage, involves a progressive morphogenetic reorganization of the labial imaginal disc, and is hedgehog-dependent. This analysis of the transformation indicates that normal pb action is required at least at two distinct levels. One is in the cell-autonomous regulation of target genes such as dac likely to be implicated in cell identity. A second level involves an autonomous action with a nonautonomous effect on labial structure, through the negative regulation of wingless and decapentaplegic downstream of hh signaling. This opposition to hh targets is likely to occur at the level of the transcription factor cubitus interruptus/Gli, a crucial and conserved mediator of hh signaling activity. These results led to a proposal that homeotic function may exist in intimate functional contact with the hedgehog organizer signaling system: the 'homeotic organizer' (Joulia, 2005).

Segmental organization in the imaginal discs involves the reiterated deployment of segment polarity genes that organize the fundamental segmental form. This involves a cascade proceeding from posteriorly expressed Engrailed protein through a short-range Hh morphogen gradient in anterior cells favoring the activator form of Ci transcription factor, which in turn activates wg and dpp to establish two concurrent, instructive concentration gradients that structure gene expression along the proximo-distal axis. In contrast with this elaborate choreography of the segment polarity genes, the homeodomain proteins encoded by Hox genes are expressed in a segmental register, which obscures how they can direct the differentiation of distinct cell types within the segment. The present investigation of homeotic proboscipedia function during labial palp formation indicates a multipronged action for pb in the labial disc. Pb acts cell-autonomously in the negative regulation of target genes including dac, which is normally extinguished in Pb-expressing cells of labial or leg imaginal discs but is activated in labial discs in the absence of pb activity. This activation of dac in mutant labial cells is hh-dependent and is likely a response to wg and dpp morphogen signals as for leg discs. The data further indicate that pb acts cell autonomously to regulate the level of both wg and dpp expression in response to hh. Thus, pb appears to negatively regulate dac expression directly, but also by withholding positive instructions from Wg and Dpp morphogens. The interweaving of homeotic selector proteins with strategic target genes including morphogens (wg, dpp) and targets of signaling activity (dac, Dll) may influence segment patterning from global size and shape to specific local pattern and cell identity. This positioning offers a powerful yet economical mode of selector function that helps to better understand how a single selector gene can integrate global patterning with cellular identity (Joulia, 2005).

This view invoking multiple and overlapping modes of regulation by a homeotic selector protein supports and extends the vision from analyses seeking to explain how Ultrabithorax (Ubx) selector function differentiates between the serially homologous wing and haltere appendages. This analysis supports a role for Ubx in fruit flies transforming a dorsal default state (wing) to haltere, by repressing the accumulation of Wg in the posterior part of the haltere, and by regulating a subset of Dpp or Wg activated targets such as vestigial and spalt related. Additionally, clear evidence has been presented for a nonautonomous action of Ubx via its activity in cells of the D-V organizer where wg is expressed. Ubx thus acts to down-regulate wg in the haltere, but also intervenes to modulate the expression of targets of both dpp and wingless signaling pathways. An analysis of mutants for maxillopedia (mxp), the Tribolium pb homolog, revealed augmented transcription of flour beetle wg within the transformed labial segment. This observation, in full accord with the above results for Ubx, and the current results for Drosophila pb, supports a conserved role for homeotic regulation of nonautonomous signaling input in appendage development. At the same time, mxp mutants show a precocious maxilla-to-leg transformation in larvae, demonstrating a prior, embryonic requirement for mxp. This result is of particular interest since it highlights a temporal aspect of pb action in the fly labial disc: the absence of pb function early has no apparent effect on the labial discs in early L3 larvae, which appear normal. It is only subsequently that these diverge toward leg structure. Thus, the globally conserved activity of mxp/pb in equivalent beetle or fly organs is nonetheless employed in temporally different ways among species. Though it is not clear whether this reflects the existence of species-specific co-factors or rather of the effects of expression dosage and timing, such modifications might offer important possibilities for changing form. Variations on all these themes can probably contribute to the diversification of organism form, within and among species (Joulia, 2005)

The roles of diffusible Wg and Dpp morphogens induced by Hh at the A-P boundary, and the transcriptional programs they induce according to their concentrations within a gradient, are considered central to organizing the group of cells constituting a segment. The present work indicates that pb normally acts downstream of Hh within the organizer, where it maintains Wg and Dpp at low levels in labial imaginal tissue. Overexpressing Wg or Dpp in the labial discs results in malformed, overgrown or transformed 'labial' tissue. These observations support the viewpoint that limiting morphogen accumulation is essential to ensuring that the labial program is correctly applied. This study underlines the potential importance of the absolute levels of wg and dpp-encoded signaling molecules deployed for tissue organization. While a gradient may in principle be formed from any source, part of the spectrum of threshold levels necessary for stimulating specific gene responses is likely removed from the repertoire in the labial environment. The absolute level of activation or inhibition of diverse signaling pathways thus may be in itself a tissue-specific property, allowing gradients of related form but with different instructive capacities that can be a distinctive element in guiding tissue formation and specifying ultimate identity. This integration of diverse sorts of information -- the hh organizer linked to the Hox selector -- may confer order to tissue organization and identity (Joulia, 2005).

The fine-tuning of morphogen signals by Hox selectors coupled with the concomitant regulation of downstream targets thus appears to offer a strategic control point for achieving reliable developmental control coupled with evolutionary flexibility. The modulation of different cell signaling pathways by pb activity implies it can regulate both the tissue “context” generated by the signaling pathways activated in a tissue, and the cellular response to this context. This capacity to meld large-scale patterning with cellular identities merits emphasis (Joulia, 2005).

While the logic described above appears to be conserved, its application leads to widely different results according to the species and the tissue. Quite recently, an analysis of vertebrate Hox function has led to the identification of an intimate developmental link between Hox selector function and hedgehog signaling. This analysis reveals a direct physical interaction between the mouse Ci homolog Gli and Hox homeodomain transcription factors. It thus provides a compelling complement to the present work, since the molecular framework of a direct link between Gli and Hox proteins goes far to rationalise the dose-sensitive interplay between Ci and Pb that was observed in Drosophila. If Hox proteins indeed compete for available nuclear Gli/Ci, this molecular mechanism may also help to understand other phenomena including phenotypic suppression in flies or posterior prevalence in mice. Correspondingly, the current data place Pb in antagonism to Ci within the hedgehog organizer, where it modulates output from the wg and dpp genes and the instructive morphogens they encode. These complementary observations from insect and vertebrate models suggest the existence of an evolutionarily conserved machinery with enormous potential for generating morphological diversity. It will be exciting to know more about how the homeotic selector function is integrated in known cascades that make use of conserved molecules both to ensure the fidelity of normal form, as well as to generate new form (Joulia, 2005).

Pupal - Hedgehog and the formation of adult abdominal segments

The cuticle of the adult abdomen of Drosophila is produced by nests of imaginal histoblasts, which proliferate and migrate during metamorphosis to replace the polyploid larval epidermal cells. In this report, a detailed description is presented of the expression of four key patterning genes, engrailed (en), hedgehog (hh), patched (ptc), and optomotor-blind (omb), in abdominal histoblasts during the first 42 h after pupariation, a period in which the adult pattern is established. In addition, there is a description of the expression of the homeotic genes Ultrabithorax, abdominal-A, and Abdominal-B; these genes specify the fates of adult abdominal segments. The results indicate that abdominal segments develop in isolation from one another during early pupal stages, and that some patterning events are independent of hh, wg, and dpp signaling. Pattern and polarity in a large anterior portion of the segment are specified without input from Hh, and evidence is presented that abdominal tergites possess an underlying symmetric pattern upon which patterning by Hh is superimposed. The signals responsible for this underlying symmetry remain to be identified (Kopp, 2002).

The dorsal cuticle of a typical abdominal segment contains a stereotyped sequence of pattern elements. At the anterior edge of each segment is the acrotergite, a narrow strip of naked sclerotized cuticle (a1). The remainder of the tergite is covered by trichomes, and can be subdivided into four regions. From anterior to posterior these regions are: a lightly pigmented region with no bristles (a2 fate); a lightly pigmented region that contains two to three rows of microchaetes (a3); a darkly pigmented region with one to two rows of microchaetes (a4); and a darkly pigmented region with a single row of macrochaetes (a5). The tergite is followed by the unpigmented posterior hairy zone (PHZ), which is composed of both anterior (a6) and posterior (p3) compartment cells. All trichomes and bristles in the segment are oriented uniformly from anterior to posterior. Finally, at the posterior edge of the segment is a zone of thin, naked intersegmental membrane (ISM), which can be subdivided into anterior smooth (p2) and posterior crinkled (p1) regions (Kopp, 2002).

The adult abdominal pattern is established in the first 2 days of pupal development, concurrent with the proliferation and migration of histoblasts and the destruction of the larval epidermal cells (LECs.) The spatial and temporal evolution of en, hh, ptc, and omb expression is followed during this critical period. The cuticle of each abdominal hemisegment is formed by three major histoblast nests. The anterior dorsal nest (aDHN) is composed of anterior compartment histoblasts and produces the tergite and part of the PHZ (a1-a6), whereas the posterior dorsal nest (pDHN) is composed of posterior compartment cells and produces the intersegmental membrane and the remainder of the PHZ (p1-p3). The ventral histoblast nest, which produces the sternite and pleura, contains both anterior and posterior compartment cells. en, hh, ptc, and omb are expressed in similar patterns in dorsal and ventral histoblasts, and the description is limited to the dorsal abdomen (Kopp, 2002).

en-lacZ and hh-lacZ are expressed throughout the pDHN, but are not expressed in the aDHN. hh-lacZ is expressed in a gradient within the pDHN, with expression highest at the anterior edge. A similar gradient can be detected in understained preparations of en-lacZ. ptc-lacZ expression is present in only a few cells at the posterior edge of the aDHN. omb-GAL4 expression is seen in the posterior of the aDHN and the anterior of the pDHN. omb-GAL4 expression is highest near the compartment boundary and decreases symmetrically in both anterior and posterior directions. By 20-24 h APF, the aDHN and pDHN fuse to form a combined dorsal histoblast nest (DHN). The gradients of en-lacZ and hh-lacZ expression within the posterior compartment become more pronounced at this stage. ptc-lacZ is expressed in a narrow stripe in the middle of the DHN, which is presumably located just anterior to the compartment boundary. The posterior border of this stripe is sharply defined, whereas a short gradient forms in the anterior direction; no ptc-lacZ expression can be detected at the anterior edge of the DHN at this time. omb-GAL4 is expressed in a wide, double-sided gradient in the middle of the DHN. Double labeling for ß-galactosidase and En protein in omb-GAL4²/UAS-lacZ pupae shows that omb-GAL4 is expressed in both compartments (Kopp, 2002).

At ~30 h APF, the DHN of consecutive segments begin to merge. Contact occurs as the border cells, a specialized row of LECs located at the posterior edge of each segment, are lost. At this stage, expression of en-lacZ and hh-lacZ is still highest at the compartment boundary, and lowest at the posterior edge of the segment. At high magnification, a clear gradient of En protein can be seen at this stage on a cell-by-cell basis. The ptc-lacZ stripe in the middle of the segment widens somewhat, but retains a sharp posterior limit. As the border cells are eliminated and histoblasts of consecutive segments come into contact, cells at the anterior edge of each segment activate ptc-lacZ. Activation occurs only where border cells have been lost; no expression of ptc-lacZ is detected posterior to persisting border cells. This pattern strongly suggests that the border cells insulate anterior histoblasts from the Hh protein secreted by the posterior compartment cells of the preceding segment. Consistent with such a role, the border cells do not express hh transcript, although they do express En. omb-GAL4 continues to be expressed in a symmetric, double-sided gradient at this stage (Kopp, 2002).

By 40-42 h APF, the border cells, which are the last LECs to be replaced by histoblasts, have been eliminated and segmental fusion has been completed. en-lacZ and hh-lacZ are upregulated at the posterior edge of the segment at this time, and soon the expression of both genes becomes uniform within the posterior compartment. For a short time, En levels are highest in cells at both edges of the posterior compartment, and lower in the middle cells, suggesting that en expression is upregulated by contact of anterior and posterior compartment histoblasts. In addition to the main ptc-lacZ stripe, a weak second stripe develops at the anterior edge of the segment. omb-GAL4 expression becomes asymmetric, with a well-defined posterior and graded anterior boundaries; based on the positions of muscle insertion points, most or all of omb-GAL4 expression at this stage is in the anterior compartment (Kopp, 2002).

To test whether Hh signaling is required for ptc and omb expression, homozygous hh^ts2 individuals were grown at 29°C for 48 h prior to dissection. Under these conditions, ptc-lacZ expression was completely eliminated at all stages. However, the effect on omb-GAL4 expression was different, depending on the stage of development. In early pupae, the symmetric expression of omb-GAL4 about the compartment boundary was only slightly reduced, while expression in the LECs appeared normal. In contrast, the later asymmetric expression of omb-GAL4 in the anterior compartment was virtually eliminated. No change was seen in the expression of en-lacZ or En protein in hh^ts2 pupae raised at 29°C, suggesting that the gradients of en expression in the posterior compartment are established independently of Hh function (Kopp, 2002).

After replacement of the LECs by the histoblasts, the pupal abdomen consists of a chain of alternating anterior and posterior compartments. Therefore, at this stage each anterior compartment can be exposed to Hh protein diffusing across both its anterior and posterior edges. It is well documented that Hh diffusing from the posterior (across the compartment boundary) plays a key role in patterning the posterior tergite (a4-a6 fates). Hh diffusing from the anterior (across the segment border) appears to be less important, playing a direct role in specifying the acrotergite (a1), but not other anterior tergite (a2 and a3) fates (Kopp, 2002).

However, it has been suggested that Hh diffusing across the segment border may act indirectly through a secondary signal to specify polarity throughout the anterior tergite. To test this model, smo mutant clones located at the segment boundary were analyzed. Such clones should be unable to receive the Hh signal, and according to the model would be predicted to alter cell polarity in the anterior tergite. smo² clones in the a1 region are transformed to a2 identity and secrete trichomes, making it possible to determine the polarity of each cell. Two types of clones were examined. The first type consists of large clones that abut the segment boundary and span the a1 and a2, and sometimes also the a3, regions. 33 clones of this type were examined, of which 16 could clearly be seen to contact the segment border along their entire width. All such clones had completely normal polarity both within the clone and in the surrounding wild-type cells, suggesting that no anterior Hh-responsive cells are required to polarize the a2 and a3 regions. Rather, these observations argue strongly that these regions are polarized independently of Hh (Koop, 2002).

The second type of clone consisted of small clones contained entirely within the a1 region, and separated from the a2 region by a strip of untransformed a1 cuticle. Of 13 such clones examined, 11 had completely normal polarity throughout, and 2 showed altered polarity in 1 or 2 cells along the posterior edge of the clone. It is suggested that these polarity reversals, which are the exception rather than the rule and extend for only one cell diameter, are a strictly local effect of a2 cells coming into contact with a1 cells improperly located to their posterior (Koop, 2002).

Several genotypes have been described in which abdominal tergites show mirror-symmetric patterning. A series of experiments was conduced to test whether this mirror symmetry is the result of Hh signaling. The results are uniformly negative, suggesting that abdominal tergites possess an underlying mirror-symmetric pattern that is specified independently of hh (Koop, 2002).

Ubiquitous expression of omb causes double-posterior patterning of the tergite (a6-a5-a4-a4-a5-a6), whereas loss of omb function can cause reciprocal, double-anterior patterning (a2-a3-a3-a2). Ubiquitous expression of omb driven by the gain-of-function allele Qd^Fab has no effect on expression of en-lacZ, hh-lacZ, hh transcript, or the omb-GAL4² enhancer trap. Moreover, pupae hemizygous for the null allele omb²⁸² show normal expression of hh-lacZ, en-lacZ, and En protein (Koop, 2002).

These observations indicate that omb does not regulate the expression of hh , en, or omb. ptc-lacZ expression is also unaffected in omb²⁸² pupae, indicating that omb is not required for Hh signaling. However, Omb may potentiate Hh signaling: in Qd^Fab, the level of ptc-lacZ expression is increased relative to that of wild-type at both edges of the anterior compartment, although the timing of ptc-lacZ activation is not affected (Koop, 2002).

In an earlier report, it was found that the phenotype of Qd^Fab is not suppressed in Qd^Fab;hh^ts2 double mutants raised at the restrictive temperature, suggesting that the mirror-symmetric phenotype of Qd^Fab is independent of Hh function. However, the new observation that ptc-lacZ expression is upregulated in Qd^Fab prompted a reexamination of these double mutants. A large number of Qd^Fab/FM6; hh^ts2/hh^ts2 animals shifted to 31^o, C at pupariation were compared to their identically treated Qd^Fab/FM6; hh^ts2/In(3LR)Cx, Sb siblings. In agreement with earlier results, no suppression is seen of the Qd^Fab phenotype by hh^ts2. In a reciprocal experiment, it was asked whether cell fates or polarity in Qd^Fab could be altered by ectopic hh expression. Flip-out hh-expressing clones were generated. These clones were not associated with any changes in cell fate or polarity. Taken together, these results argue strongly that mirror-symmetric patterning in omb mutants is established independently of hh (Koop, 2002).

Ectopic expression of en causes transformation of anterior compartment structures to posterior compartment identity, and produces a mirror-symmetric double-posterior pattern (p1-p2-p3-p3-p2-p1). This phenotype is seen in the en gain-of-function en mutant, which causes near-ubiquitous expression of en in the pupal abdomen and in T155-GAL4/UAS-en heterozygotes. Examination of En-expressing clones in otherwise wild-type flies reveals that the line of symmetry lies within the anterior compartment. En-expressing cells located posterior to this line orient to the posterior, whereas En-expressing cells located anterior to it orient to the anterior. This effect of En on cell fate and polarity is strictly cell autonomous. Whether Hh signaling plays a role in the symmetric polarization of en-expressing cells has been tested. No activation of en-lacZ is seen in the anterior compartment of gain of function en heterozygotes, although sporadic activation of hh-lacZ and hh transcript is observed. However, it is difficult to see how such variable activation of hh could be responsible for the highly regular mirror-symmetric cuticular pattern produced. ptc-lacZ expression is reduced at both edges of the anterior compartment in gain of function en, consistent with repression of ptc by En. omb-GAL4 expression appears unchanged relative to wild type (Koop, 2002).

To ask directly whether en-expressing cells in gain of function en flies are patterned by Hh, smo³ clones were generated in gain of function en heterozygotes. These clones had no effect on cell fate or polarity: smo mutant cells located posterior to the line of symmetry retained posterior orientation, whereas cells located anterior to this line retained anterior orientation. The affinity of smo mutant cells in a gain of function en background also appeared unchanged, since all clones interdigitated freely with surrounding cells (Koop, 2002).

In a reciprocal experiment, it was asked whether the patterning of en-expressing cells is affected by ectopic Hh expression. Flip-out Hh-expressing clones were generated in en gain of function heterozygotes. Hh-expressing clones had no effect on cell fate or polarity. Thus, Hh signaling does not appear to play a role in the mirror-symmetric polarization of en-expressing tergites (Koop, 2002).

Mirror-symmetric patterning is also caused by ectopic expression of Hh itself. Ubiquitous Hh expression driven by hs-hh or UAS-hh;T155-GAL4 results in a mirror-symmetric double-posterior pattern (p2-p3-a6-a5-a5-a6-p3-p2). Interpretation of this phenotype has been complicated by the observation that ectopic Hh induces localized expression of en-lacZ in the anterior compartment. This induction leaves open the possibility that the mirror-symmetric patterning may be mediated by changes in endogenous hh expression (Koop, 2002).

To test this possibility, the expression of en-lacZ, hh-lacZ, and ptc-lacZ was examined in the abdomens of UAS-hh;T155-GAL4 pupae that were shifted from 17°C to 29°C at pupariation to enhance GAL4-induced ectopic expression. During the early pupal stages, ptc-lacZ expression was strongly and evenly expanded to the anterior, while the expression of hh-lacZ, en-lacZ, and En protein was unchanged. However, by 40-42 h APF some pupae showed weak ectopic expression of en-lacZ and hh-lacZ in a narrow stripe in the middle of the anterior compartment. ptc-lacZ expression was upregulated to each side of this stripe as well as at both edges of the anterior compartment (Koop, 2002).

The mirror-symmetric posterior tergite in UAS-hh;T155-GAL4 animals (a6-a5-a5-a6) develops between the ectopic en stripe and the normal posterior compartment. This region is flanked by hh-expressing cells and has peaks of ptc-lacZ expression at both its anterior and its posterior edges. Therefore, the symmetric patterning of the tergite could be caused by symmetric expression of the endogenous hh gene, rather than by ubiquitous expression of UAS-hh. To test this possibility, the hh^ts2 mutation was used to block endogenous Hh activity. UAS-hh;T155-GAL4 hh^ts2/hh^ts2 animals were shifted to 31°C at pupariation. Endogenous Hh signaling, as detected by ptc-lacZ expression, is eliminated under these conditions. In the pharate adults that developed, the mirror-image patterning of posterior tergite and PHZ structures was unaffected relative to that of identically treated UAS-hh;T155-GAL4 hh^ts2/TM6 siblings, although the transformation of anterior tergite to intersegmental membrane was partly suppressed (Koop, 2002).

To confirm the inactivation of endogenous Hh, en-lacZ and ptc-lacZ expression was examined in UAS-hh;T155-GAL4 hh^ts2/hh^ts2 pupae raised at 29^o C. In this genotype, en-lacZ was activated in a stripe in the middle of the anterior compartment, as it was in UAS-hh;T155-GAL4 pupae. However, no separate peaks of ptc-lacZ expression were detected. Instead, ptc-lacZ was activated uniformly in the posterior half of the anterior compartment. Curiously, little or no expression of ptc-lacZ was seen in the anterior half (Koop, 2002).

Taken together, these observations suggest that localized activation of the endogenous hh gene is not responsible for the mirror-symmetric pattern caused by ubiquitous expression of exogenous Hh. However, in this case the results are not conclusive, as the hh^ts2 allele may allow residual Hh function at the restrictive temperature (Koop, 2002).

In conclusion, abdominal tergites display mirror-symmetric patterning in several different genotypes. These genotypes include loss-of-function mutants of omb or hh, and genotypes in which omb, en, or hh are expressed ubiquitously. It is thought that these cases reveal an underlying symmetric patterning of the tergite. However, after the loss of the border cells, anterior compartments are exposed to Hh from both anterior and posterior edges, raising the possibility that these mirror-symmetric phenotypes result from symmetric Hh signaling. Indeed, it has been suggested that a U-shaped gradient of Hh produced by diffusion across the compartment and segment boundaries specifies polarity throughout the tergite. This report, tested the role of Hh in three separate cases of mirror-symmetric patterning. The results are uniformly negative, and provide compelling evidence that abdominal tergites possess an underlying mirror-symmetric patterning that is specified independently of Hh (Koop, 2002).

There are two main conclusions which may be drawn from the work to define Hh requirements in abdominal patterning: (1) Hh signaling is not required to specify pattern or polarity in the a2 and a3 regions, which comprise most of the anterior tergite; (2) abdominal tergites possess an underlying mirror-symmetric patterning that is specified independently of Hh. The phenotypes of hh^ts2 and omb² mutants, in which the a2 and a3 regions are often duplicated in mirror image, imply that a single patterning system is responsible for specifying both the a2 and a3 regions and the underlying mirror symmetry of the tergite. The identity of this system remains to be determined (Koop, 2002).

The abdomen of adult Drosophila consists of a chain of alternating anterior (A) and posterior (P) compartments, which are themselves subdivided into stripes of different types of cuticle. Most of the cuticle is decorated with hairs and bristles that point posteriorly, indicating the planar polarity of the cells. This study has focused on a link between pattern and polarity. Previous studies have shown that the pattern of the A compartment depends on the local concentration (the scalar) of a Hedgehog morphogen produced by cells in the P compartment. Evidence is presented in this study that the P compartment is patterned by another morphogen, Wingless, which is induced by Hedgehog in A compartment cells and then spreads back into the P compartment. Both Hedgehog and Wingless appear to specify pattern by activating the optomotor blind gene, which encodes a transcription factor. A working model that planar polarity is determined by the cells reading the gradient in concentration (the vector) of a morphogen 'X' which is produced on receipt of Hedgehog, is re-examined. Evidence is presented that Hedgehog induces X production by driving optomotor blind expression. X has not yet been identified and data is presented that X is not likely to operate through the conventional Notch, Decapentaplegic, EGF or FGF transduction pathways, or to encode a Wnt. However, it is argued that Wingless may act to enhance the production or organize the distribution of X. A simple model that accommodates these results is that X forms a monotonic gradient extending from the back of the A compartment to the front of the P compartment in the next segment, a unit constituting a parasegment (Lawrence, 2002).

It has been concluded that Hh acts indirectly via another system (a gradient of 'X') to effect polarity. The evidence was based on clones that lacked such downstream genes as patched (ptc) or cAMP-dependent protein kinase 1 (Pka). In the A compartments, Ptc and Pka proteins act within cells to prevent the Hh pathway from being activated inappropriately; if either protein is removed the Hh pathway becomes constitutively activated within the mutant cells themselves. With respect to the type of cuticle (the scalar output of Hh) the results fit the model; the mutant cells make the cuticle normally made by cells responding strongly to Hedgehog and all the cells outside the clone make the normal type of cuticle (a cell-autonomous effect). However, with respect to polarity (the vectorial output of Hh), the results are different; polarity is altered in the wild-type cells up to several cell diameters away from the clone (a cell non-autonomous effect). Although it has been argued that these effects were not due to Hh itself, the possibility was not eliminated that low levels of ectopic Hh might be produced by the clone and diffuse out, being sufficient to repolarize the cells without changing the scalar. This study now disproves this possibility by making clones that lack both effective Ptc protein and the hh gene. These clones still cause repolarization in the back half of the clone and behind it arguing strongly that the Hh protein is a component of 'X' and raising again the question, what is X? X should be engendered downstream of Hh receipt, which is where the search is started (Lawrence, 2002).

omb encodes a transcription factor that is activated on receipt of high amounts of Decapentaplegic (Dpp) in both the A and the P compartments of the wing and elsewhere. It is expressed in each segment, both dorsally and ventrally, as a single stripe spanning the AP border and including the rear of the A compartment and the front region of the P. Accordingly, omb^- clones in other parts of the segment are normal (Lawrence, 2002).

Within the posterior half of the A compartment, Omb is required for the normal scalar response to Hh. At the extreme back, in the a6 region, where the Hh concentration is highest, the omb^- cells develop only a little abnormally: the unpigmented cuticle of that region (a6) is expanded a little anteriorly in the clone, but sometimes contains small 'a3' bristles. Note that specification of a6 cuticle normally requires engrailed activity, which is induced in A cells by peak levels of Hh. However, in omb^- clones that are situated more anteriorly, in the pigmented region at the back of the A compartment (a4, a5), there is a big effect: it appears that Hh acts through omb, because omb^- cells never make a4 cuticle or a5 bristles (pattern elements that signal a response to Hh), and in their stead make a3 cuticle (the type of cuticle made where there is little or no response to Hh). Also, Hh directly upregulates expression of ptc, which encodes a component of the Hh receptor and this also occurs in omb^- clones. This finding indicates that Omb is not required for Hh signal transduction per se, but for the appropriate response of cells (Lawrence, 2002).

With regard to polarity, the clones confined to the anterior and middle part of the A compartment are normal. However, clones just behind the middle of the A compartment usually show reversal at the front, with normal polarization at the back. More strikingly, clones confined to the very back of the A compartment, in the a6, a5 and a4 domains can be largely or entirely reversed and this reversal usually extends anterior to the clone (Lawrence, 2002).

To explain these polarity changes, it is suggested that Hh induces X production through the agency of Omb. It follows that little or no X can be produced within omb^- clones and therefore that the polarities of cells in or near such clones depend on X produced outside. Clones in the middle of the A compartment behave normally because most X is produced behind them and the gradients of X concentration are little changed. Clones located a little further back will have peaks of X both behind and in front, and this can cause localized reversal at the front of the clone. For a clone extending back to the AP boundary, the only source of X will be anterior to the clone, presumably because omb⁺ cells there will 'see' Hh protein that has passed through the clone. These cells should make X that spreads backwards into the clone, setting up a gradient of reversed polarity. There is corroborating evidence: in some clones there is dark pigmentation and large bristles develop anterior to the clone, confirming that Hh has indeed been received there. However, many omb^- clones are associated with anterior repolarizations that occur even where there is no dark pigmentation anterior to the clone, suggesting that the level of Hh required to stimulate some X production anterior to the clone is less than that needed to make a4 pigment. It follows that, in normal flies, some X is produced by cells anterior to the a4 pigmented zone. Finally, it is found that some clones, which extend nearly to the back of A, show reversed territory behind the clone, perhaps due to the domination of the X source that is anterior to the clone over any production of X behind it (Lawrence, 2002).

It is noted that the reversed polarity associated with omb^- clones located at the back of the A compartment usually extends only to the AP boundary, with polarity in the P compartment being normal. This result suggests that the AP boundary coincides with a barrier to the movement or action of X. The existence of such a barrier would provide an explanation for why X normally produced in cells at the back of the A compartment does not spread posteriorly into the P compartment, reversing the polarity in P. However, in rare cases, some reversed hairs were seen in what appeared to be adjacent P compartment cells, as marked independently by ptc.lacZ staining. It is not known whether these rare cases are artifactual, due to a slight posterior shift -- during mounting -- of the cuticle relative to the underlying epidermis, or are frank reversals of cells within the P compartment. If the reversed cells are indeed P cells, this raises a problem for the notion that the AP boundary constitutes a barrier to X movement (Lawrence, 2002).

If the production of X depends at least in part on omb, then ptc^- clones, in which the Hh pathway has been constitutively activated, should produce little or no X if they also lack omb. Clones were made that were both ptc^- and omb^-; these clones form a6 cuticle as do ptc^- clones. However, in the middle of the A compartment and unlike ptc^- clones in that position, they fail to repolarize behind, but reverse their polarity in front -- as do omb^- cells. Similarly, omb^- ptc^- clones situated at the back of the A compartment behave like omb^- clones, the whole being reversed in polarity (and not like ptc^- clones in the same location, that have normal polarity). Thus in terms of the type of the cuticle (the scalar), omb^- ptc^- behave as ptc^- clones, but in terms of the vector they behave as omb^- clones. These results confirm that Hh induces X production through the action of omb (Lawrence, 2002).

The model for X suggests that, if omb were ectopically activated in cells at the front of the A compartment, those cells could become a source of X. Indeed omb-expressing clones can repolarize the cells behind them -- as if there were a local peak in the X distribution (Lawrence, 2002).

smoothened (smo), is an essential component of Hh transduction; without it the cells cannot see Hh protein. As regards polarity, one would expect neither omb^- nor smo^- clones to produce X and for their phenotype to be the same. Although this is generally the case, the effects of smo^- and omb^- differ for clones located at the back of the A compartment. Polarity within these omb^- clones is completely reversed, consistent with the model, whereas the corresponding smo^- clones are reversed only within the anterior portion of the clone, polarity returning to normal at the very back of the A compartment. The preferred explanation for this discrepancy is that Smo protein perdures in smo^- clones, allowing partial rescue of the smo mutant phenotype, particularly at the back of the A compartment, where Hh is most abundant. This rescue could allow production of X, enough to restore normal polarity at the back of the clone, but not enough to specify a4 cuticle or to upregulate ptc.lacZ. For both smo^- and omb^- clones, some Hh would be expected to move forward across the clone and induce an ectopic peak of X production in more anterior, wild-type cells, accounting for the polarity reversals that are observed in both cases (Lawrence, 2002).

To test this explanation Hh receipt was blocked by a different method that is not so subject to perdurance: a marked clone was made that contained no wild-type Ptc, but provided instead a mutant form of Ptc that is ineffective at transducing the Hh signal. Such clones behave like smo^- clones in most respects, including making a3 cuticle instead of a4, a5 or a6 cuticle in the back half of the A compartment, and causing polarity reversals both within and anterior to the clone. However, unlike smo^- clones, the polarity at the back of these clones does not return to normal. Instead, in the majority of cases, polarity remains reversed all the way to the back edge of the clone, and sometimes beyond, as observed for omb^- clones in the same position. These results support the perdurance explanation for the smo^- clones and are consistent with the working model, which is based mainly on the results with omb (Lawrence, 2002).

In clones mutant for arm or arrow, the expectation was that the Wg pathway in these two types of clones would be blocked. Two effects were noted. (1)The clones in the dorsal epidermis differentiated cuticle characteristic of the ventral epidermis: they made pleural hairs, and patches of sternite. Clones in all portions of the tergite, in both the A and P compartments, were transformed in this manner, indicating a general requirement for Wnt signaling to specify dorsal as opposed to ventral structures. Thus, in the wild type, all dorsal cells are probably exposed to at least low levels of Wg or some other Wnt protein. (2) Such clones affect polarity: in the tergites, the mutant clones were normal at the rear of the clone but reversed in the front, with reversal extending outside the clone. One explanation for these polarity changes could be that, in the tergites, Wg normally acts to enhance the production of X. Thus cells deficient in the Wnt pathway would produce less X than normal, giving a dip in the concentration landscape for X, causing reversed polarity at the front of the clone. In the eye, both arm^- and arrow^- clones cause equivalent polarity reversals and a similar resolution has been offered: it is suggested that Wg might regulate the production of a secondary polarizing factor also dubbed X (Lawrence, 2002).

Thus, it is proposed that Wg helps to produce X, but that Wg itself is not X. If Wg were X, both arm^- and arrow^- clones should not be able to transduce it, and hence, should have random polarity within the clone. Moreover, the effects on polarity should be cell autonomous. Yet, as has been seen, these clones behave as if they have caused an altered distribution of X, rather than any failure to transduce X. Similar arguments apply to sgg^- clones. In this case, the Wg pathway should be constitutively activated in all cells within the clone, preventing them from detecting a gradient of Wg protein. However such clones are not randomly polarized, indicating that they can still respond to graded X activity (Lawrence, 2002).

It is useful to compare the roles of Omb and Wg on X production. Omb is apparently essential for X production: omb^- clones at the back of A show reversed polarity that extends all the way to the posterior edge of the compartment. By contrast, in arm^- and arrow^- clones, reversal occurs only in the anterior portions of such clones. Thus, it is inferred that arm^- and arrow^- cells located at the back of A can produce some X, even though they cannot activate the canonical Wnt pathway. Thus, it could be that Hh drives X production mainly through Omb, but also adds to the level of X produced through the induction and action of Wg. The combination of both Omb and Wg activity might extend the reach of the X gradient to encompass the whole A compartment, and possibly also further forward into the neighboring P compartment (Lawrence, 2002).

None of the previous studies has helped gain an understanding of how the P compartment is patterned or how its cells are polarized. smo^- clones have no phenotype in the P compartment, confirming that Hh has no function there. In the embryo and imaginal discs, Hh crossing over from the P compartment induces the expression of Wg and Dpp in line sources along the back of A. Both proteins then spread back into the P compartment where they act as gradient morphogens to control P growth and pattern. Wg and Dpp are also produced at the back of the A compartment in each abdominal segment (albeit in distinct dorsal and ventral domains). Hence, by analogy with the embryo and imaginal discs, these morphogens seem to be the most likely candidates to pattern the P compartment here as well. If so, it would be supposed that in the tergites, Hh induces Wg and this Wg moves posteriorly across the AP compartment boundary into the P compartment where it activates expression of omb, thus specifying the zone of hairy cuticle (p3) and distinguishing it from p2 cuticle, which is bald. This hypothesis was tested in the following experiments (Lawrence, 2002).

Loss-of-function omb mutants tend to lose the hairy, unpigmented cuticle characteristic of both posterior A (a6) and anterior P (p3) regions, whereas gain-of-function mutations tend to acquire it. Since it was observed that omb^- clones in the A compartment are able to make a6 cuticle, it seems likely that Omb is required specifically for the hairy, unpigmented cuticle (p3) that normally forms at the front of the P compartment. If so, one might expect omb^- clones at the front of the P compartment to transform the anterior type of cuticle (p3) into that found more posteriorly (p2). Although most omb^- clones were normal in this region, about one third of p3 clones lost some, but not all, of the hairs within the clone. Thus it appears that omb may be required in the p3 territory, as it is in the a5 and a4 territories, to specify the type of cuticle secreted (Lawrence, 2002).

If Wg activates omb in anterior regions of the P compartment, blocking the Wnt pathway in cells in the P compartment should block expression of omb. Expression of omb was therefore monitored in arrow^- clones. omb is sometimes, but not always, turned off autonomously in the clone. Conversely, ectopic activation of the Wnt pathway should transform bald cuticle (p2) at the back of P into hairy cuticle (p3) normally found at the front of P. Indeed, some clones lacking the sgg gene become hairy if situated in the bald areas of P, apparently causing a transformation from p2 to p3 cuticle. But, clones expressing either tethered Wg or activated Arm, which should behave similarly, have no clear effects. Even so the positive results with arrow and sgg give support to the hypothesis that Wg stratifies the P compartment by working through Omb (Lawrence, 2002).

The heart of the model requires that a cell's polarity be determined by reading the local slope, the vector of a morphogen, X. Within the A compartment, it is proposed that X is produced in a gradient with its peak at the back of the A compartment and its minimum at the front. Hh is the primary morphogen that patterns the A compartment, and, at the rear of this compartment, it acts through omb to produce X. X spreads further to the anterior, forming a monotonic gradient that extends from the back of the A compartment and could go as far as the front of the next P compartment, thus encompassing a parasegment. In this model there might need to be a barrier to the movement of X across the AP (parasegment) border in order to isolate the X gradients in neighboring parasegments from one another. This model is speculative; for example there is no evidence for X spreading forward into the P compartment. In an alternative scenario, X might be made near the AP border, spreading forward into A and backward into P to form a reflected gradient. In that case, cells in the A and P compartments would have to make hairs that point in opposite directions relative to the vector of X, since all hairs point toward the posterior (Lawrence, 2002).

Although it is proposed that X is a long range morphogen, the results do not exclude models in which polarity depends on short range interactions between cells. Recent models for planar polarity concentrate mostly on this aspect of how cells become polarized, particularly on how proteins within cells become asymmetrically localized, and how such molecular polarity might propagate from cell to cell by localized recruitment of other proteins at the abutting cell membranes. These models can provide explanations for the local, non-autonomous perturbations of polarity that occur along the borders of mutant clones, but they do not readily explain the longer range effects of such clones nor how polarity is determined globally in the wild-type fly (Lawrence, 2002).

The model for X can be further elaborated, for example, polarity could depend on two cooperating morphogens, each operating in different directions. While X could emanate forward from the back of the A compartment, another polarizing gradient, 'Y' could be sourced from the front, or from the P compartment, and move backwards. Hairs would be subject to two separate and mutually supportive influences, pointing up the gradient of X and down the gradient of Y. More complex hypotheses of this sort have two main appeals: they might help explain how the polarity is determined across the AP border and they also might help in understanding of why removal of genes needed for polarity, such as fz or four-jointed still gives near-normal flies with much of their polarity unscathed (Lawrence, 2002).

Adult - Hedgehog and oogenesis

The coordinated division of distinctive types of stem cells within an organ is crucial for organogenesis and homeostasis. Genetic interactions among fs(1)Yb (Yb), piwi, and hedgehog (hh) regulate the division of both germline stem cells (GSCs) and somatic stem cells (SSCs), the two constituent stem cell populations of the Drosophila ovary. Yb, coding for an ATP/GTP-binding site motif A (P-loop) domain protein, is required for both GSC and SSC divisions; loss of Yb function eliminates GSCs and reduces SSC division, while Yb overexpression increases GSC number and causes SSC overproliferation. Yb acts via the piwi- and hh-mediated signaling pathways that emanate from the same signaling cells to control GSC and SSC division, respectively. hh signaling also has a minor effect in GSC division (King, 2001).

Yb is expressed in terminal filament and cap cells to control GSC self-renewing divisions. The loss-of-function and overexpression phenotype of Yb reported suggests that Yb is also involved in regulating SSC divisions. It is possible that Yb achieves this dual function indirectly by regulating GSC division, which in turn affects SSC division via an unknown coordination mechanism, or vice versa. These possibilities seem unlikely, since all other mutations are known to only affect either GSCs or SSCs, but not both, as judged from their reported phenotype. For example, piwi and dpp mutations cause failure of GSC maintenance, while bam and bgcn mutations as well as piwi and dpp overexpression cause an accumulation of germline cells without a corresponding increase in somatic cells. Similarly, hh activity regulates SSC division without significant effect on GSC divisions. It is therefore unlikely that all these mutations, except for Yb, have a dual effect on GSC or SSC division and on the coordination mechanism between GSCs and SSCs. Thus, Yb appears to be the only known gene that plays a major role in regulating both GSC and SSC divisions. This dual role of Yb is further supported by the regulatory relationship between Yb, piwi, and hh (King, 2001).

The somatic function of Yb is very similar to that of hh. Like hh, Yb is specifically expressed in cap and terminal filament cells to regulate follicle cell division. Loss of either hh or Yb function leads to reduced follicle cell proliferation, while overexpression of either gene by heat shock leads to overproliferation of follicle cells that exceeds the need for egg chamber formation. The relationship between Yb and hh is further defined by observations that Yb is required for the expression of hh in cap cells and, to a lesser extent, terminal filament cells, and that Yb overexpression significantly elevates hh expression in cap cells and, also to a lesser extent, terminal filament cells. Yb overexpression causes less follicle cell overproliferation than hh overexpression. This can be explained by the fact that Yb overexpression only elevates HH expression in cap cells and terminal filament cells, while heat shock causes HH to be overexpressed all over the germarium. Since hh signaling is the main, if not the only, signaling pathway that controls SSC division, the similar mutant and overexpression phenotype between hh and Yb suggests that Yb is a positive regulator of hh expression in cap and terminal filament cells. In addition, these data provide strong evidence that cap cells play a central role in controlling SSC and GSC divisions, a hypothesis that has been proposed based on the expression pattern and function of Yb, hh, dpp, and piwi, as well as on the mitotic behavior of GSCs (King, 2001).

A parallel situation exists between Yb and piwi in controlling GSC division: (1) both Yb and piwi are expressed in cap and terminal filament cells, and this expression is essential for GSC maintenance; (2) Yb and piwi mutants share a very similar, if not identical, GSC phenotype; (3) overexpressing either Yb or piwi in somatic cells causes a similar increase in the number of GSC-like cells; (4) Yb is required for piwi expression in cap and terminal filament cells. These observations suggest that Yb is also a positive regulator of piwi expression in these somatic cells that controls GSC division. In addition, it suggests that cap cells may play a central role in GSC division, because these cells express higher levels of Yb and piwi and directly contact GSCs. The Yb-piwi mechanism apparently does not control the production of the DPP signal required for GSC maintenance, since overexpression of dpp does not produce similar effects as does Yb or piwi and does not rescue the piwi phenotype (King, 2001).

The hypothesis that Yb controls GSC and SSC divisions by regulating piwi and hh expression, respectively, in cap cells and terminal filament cells is favored. The Piwi protein, as a nuclear factor, in turn controls GSC division by promoting the production of a somatic signal 'S,' which is received by its receptor 'R' in GSCs. In parallel, the Hh signaling molecule suppresses the Ptc receptor activity in SSCs to promote SSC division. Meanwhile, Hh also participates in promoting GSC divisions through the Ptc receptor on the GSC surface, since either overexpressing Hh in Yb mutants or removing PTC activity from GSCs in Yb mutants has a similar effect in rescuing GSC division and maintenance. The expression of hh may also be controlled by engrailed (en), a known transcription regulator of hh that is also specifically expressed in cap cells and terminal filament cells. dpp appears to act independent of the Yb-mediated pathway in regulating GSC division. This bifurcating model with Yb as a common upstream regulator of both GSC and SSC divisions represents a working hypothesis to address how the coordinated division of two distinct types of stem cells is possibly controlled (King, 2001).

An interesting aspect of the above model is that the HH signaling pathway, in addition to its essential role in SSC division, is involved in regulating GSC division. This GSC function of hh, however, appears to be somewhat redundant, since the loss of hh function only affects the maintenance of ~20% of GSCs, while overexpression of hh only stimulates a slight increase in GSC-like cells. Despite this, hh overexpression is sufficient to restore GSC divisions in both Yb and piwi mutants. These observations suggest that the hh signaling pathway is a dispensable mechanism that safeguards the GSC maintenance. It remains to be determined whether other known regulators of hh, such as engrailed, are involved in regulating hh, piwi, or Yb expression in cap cells and the terminal filament. What is the somatic signal and what is its receptor also remains to be determined in the piwi branch of the bifurcating pathway. Finally, it awaits to be established whether or how the Yb-mediated extrinsic signaling mechanism regulates the asymmetric expression and activity of intracellular stem cell genes, such as pumilio, bam, and nanos, during GSC division. The study of these questions should significantly advance understanding of the stem cell mechanism in general (King, 2001).

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