Gene name - glass

Synonyms -

Cytological map position - 91A1-2

Function - transcription factor

Keywords - eye morphogenesis

Symbol - gl

FlyBase ID:FBgn0004618

Genetic map position - 3-63.1

Classification - zinc finger

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Bernardo-Garcia, F.J., Fritsch, C. and Sprecher, S.G. (2016). The transcription factor glass links eye field specification with photoreceptor differentiation in Drosophila. Development [Epub ahead of print]. PubMed ID: 26952983
Eye development requires an evolutionarily conserved group of transcription factors, termed "retinal determination network" (RDN). However, little is known about the molecular mechanism by which the RDN instructs cells to differentiate into photoreceptors. This study shows that photoreceptor cell identity in Drosophila is critically regulated by the transcription factor Glass, which is primarily expressed in photoreceptors and whose role in this process was previously unknown. Glass is both required and sufficient for the expression of phototransduction proteins. Data demonstrate that the RDN member Sine oculis directly activates glass expression, and that Glass activates the expression of the transcription factors Hazy and Otd. Hazy was identified as a direct target of Glass. Induced expression of Hazy in the retina partially rescues the glass mutant phenotype. Together, these results provide a transcriptional link between eye field specification and photoreceptor differentiation in Drosophila, placing Glass at a central position in this developmental process.

Liang, X., Mahato, S., Hemmerich, C. and Zelhof, A. C. (2016). Two temporal functions of Glass: Ommatidium patterning and photoreceptor differentiation. Dev Biol [Epub ahead of print]. PubMed ID: 27105580
The downstream mechanisms that maintain identity and regulate differentiation of retinal cells remain poorly understood. This study report that the transcription factor Glass has a dual role in establishing a functional Drosophila eye. Persistent defects in ommatidium patterning combined with cell death correlate with the overall disruption of eye morphology in glass mutants. In addition, Glass exhibits a separable role in regulating photoreceptor differentiation. In particular, the apparent loss of glass mutant photoreceptors is not only due to cell death but also a failure of the surviving photoreceptors to complete differentiation. Moreover, the late reintroduction of Glass in these developmentally stalled photoreceptors is capable of restoring differentiation in the absence of correct ommatidium patterning. Mechanistically, Glass is necessary for the expression of many genes implicated in differentiation, i.e. rhabdomere morphogenesis, phototransduction, and synaptogenesis. Specifically, Glass was shown to directly regulate the expression of Pph13, which encodes a transcription factor necessary for opsin expression and rhabdomere morphogenesis. Finally, Glass choreographing of photoreceptor differentiation is conserved between Drosophila and Tribolium, two holometabolous insects. Altogether, this work identifies a fundamental regulatory mechanism to generate the full complement of cells required for a functional rhabdomeric visual system and provides a critical framework to investigate the basis of differentiation and maintenance of photoreceptor identity.
Bernardo-Garcia, F. J., Humberg, T. H., Fritsch, C. and Sprecher, S. G. (2016). Successive requirement of Glass and Hazy for photoreceptor specification and maintenance in Drosophila. Fly (Austin): [Epub ahead of print]. PubMed ID: 27723419
Development of the insect compound eye requires a highly controlled interplay between transcription factors. However, the genetic mechanisms that link early eye field specification to photoreceptor terminal differentiation and fate maintenance remain largely unknown. This study deciphered the function of two transcription factors, Glass and Hazy, which play a central role during photoreceptor development. The regulatory interactions between Glass and Hazy suggest that they function together in a coherent feed-forward loop in all types of Drosophila photoreceptors. While the glass mutant eye lacks the expression of virtually all photoreceptor genes, young hazy mutants correctly express most phototransduction genes. Interestingly, the expression of these genes is drastically reduced in old hazy mutants. This age-dependent loss of the phototransduction cascade correlates with a loss of phototaxis in old hazy mutant flies. It is concluded that Glass can either directly or indirectly initiate the expression of most phototransduction proteins in a Hazy-independent manner, and that Hazy is mainly required for the maintenance of functional photoreceptors in adult flies.

The glass gene is required for normal development of photoreceptor cells in three different organs: a larval photoreceptor known as Bolwig's organ, the adult compound eye and adult simple eyes, known as ocelli. Although the glass gene is expressed in all cell types in the developing eye, its function is impaired in certain cells. Glass function is activated in photoreceptors but not cone cells, indicting that there is specific repression of the target genes of Glass in non-neural cells (Moses, 1991 and Ellis, 1993).

The glass mutation has been used to analyze the nature of guidance clues used by axons growing into the brain from the developing eye. In glass mutants, retinal axons project aberrantly and the larval optic nerve is absent. During embryogenesis, the larval optic nerve contacts several different cell types, including optic lobe pioneer (OLP) neurons and a number of glial cells. OLP neurons are present and project normally in glass mutant embryos in which the larval optic nerve fails to develop, suggesting that they do not depend on interactions with the larval optic nerve for differentiation and proper axonal projection (Kunes, 1993 and Campos, 1995).

In glass mosaics, (animals in which patches of cells with the wild type glass gene lie adjacent to patches with the mutant glass gene), retinal axons project normally, despite the misrouted projections of neighboring mutant glass axons. These results indicate that growing axons can make pathfinding decisions independently of their mutant neighbors. It appears that incoming axons are responding to position specific information eminating from the target rather than from positional information relative to other incoming axons . In other words axons don't care what neighboring axons are doing but take their cues from target cells (Kunes, 1993).

Evidence is provided for the existence of an extraocular light perception in Drosophila that does not use the same phototransduction cascade as the adult photoreceptors. The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results were obtained when these responses were measured in strains where the larval photoreceptor neurons were ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. The existence of an extraocular light perception is suggested that does not use the same phototransduction cascade as the adult photoreceptors. These results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene (Busto, 1999).

The larval visual system was first described in the house fly Musca domestica by Bolwig and henceforth was named the Bolwig's organ. Similarly, in D. melanogaster, the larval visual system is composed of two bilateral groups of 12 photoreceptor cells located in the more anterior part of the head, juxtaposed to the mouth hooks. The axons of the photoreceptor cells form the larval optic nerve that innervates the optic lobe primordium area of the brain lobes. The early development and the establishment of connectivity in this system has been described previously. In the Drosophila larva a light stimulus modulates the direction of movement as well as quantitative aspects of locomotion such as path length and frequency of turning. Mutations that disrupt phototransduction in the adult eye disrupt aspects of the larval response to light measured in this assay. These results suggest that the larval and adult visual systems are similar from the functional point of view. These mutations, however, fail to abolish all perception of light, suggesting the existence of a light detection mechanism that does not require these gene products. The analysis of developmental mutants and of strains where the cell death genes are ectopically expressed suggests that this novel light detection mechanism is not located in the Bolwig's organ (Busto, 1999).

In the wild-type strains tested, change of direction when the light is turned off is greater than in the absence of light transitions, suggesting that turning off the light is a transition perceived by the animal. This observation supports the notion that a simple mechanism for the perception of light exists in the Drosophila larva that distinguishes changes in light conditions from absence of light transitions but is unable to distinguish whether the light is being turned on or off. This light response is mediated by the blue-absorbing rhodopsin (Rh1) because it is abolished in part by mutations in the ninaE gene. Interestingly, it does not rely on the same phototransduction pathway as that of the adult visual system as seen by the wild-type response of norpA and NinaC mutant larvae. The results indicate that these hypothetical photoreceptors are not housed within the Bolwig's organ, defined as the larval photoreceptors that depend on the gl gene function for differentiation. However, the observation that the function of this visual system is impaired in larvae where the cell death gene rpr is expressed under the control of the gl promoter demonstrates that these are cells in which the gl transcription factor is functional. Thus it is possible that this novel function is performed by a small number of cells that express Rh1 and the gl gene product but whose differentiation and Rh1 expression are not under the control of the gl gene (Busto, 1999).

Two different groups of cells are likely to be involved in this novel light perception. The observation that the ability of these cells to register this type of light perception is dependent on the so gene function but not gl suggests that these cells are included in the optic lobe primordium. The ablation of this proposed function by expression of the cell death gene rpr under the gl promoter suggests that the central brain neurons that express the gl gene are also involved in this behavior. A precedent for a light detector that does not rely on known elements of the phototransduction machinery in adults is the photic input pathway required for the entrainment of the circadian rhythm. The novel visual system function proposed in this paper presents other parallels with cells involved in the control and generation of circadian rhythms. Mutations in the gl gene do not abolish circadian rhythms. However, the expression of the period (per) gene under the control of the gl promoter is sufficient to restore circadian rhythmicity in per mutant flies. These results strongly suggest that the gl-expressing cells that are not the photoreceptors house the circadian pacemaker. It is possible that this novel visual function, which distinguishes changes in light condition from absence of light transitions but is unable to distinguish whether light is being turned on or off, is also involved in the control of pacemaker oscillation (Busto, 1999).


Bases in 5' UTR - 959

Exons - five

Bases in 3' UTR - 894

Amino Acids - 604

Structural Domains and Evolutionary Homologs

Glass has five consecutive Cys2 - His2 zinc fingers (Moses 1989).

The Glass protein is a transcriptional activator. A comparison of the sequences of the glass genes from D. melanogaster and D. virilis and a detailed functional domain analysis of the D. melanogaster glass gene reveal that both the DNA-binding domain and a bipartite transcriptional-activation domain are highly conserved between the two species. The putative minimal activation domain consists of residues 131-214, with two regions of conservation. Within the more N terminal conserved region are seven hydrophobic residues in a pattern that resembles a motif also found in the activation domains of several other transcriptional activators, including p32, VP16 and SP1. The seven hydrophobic regions do not seem to be as important in the functioning of the Glass activation domain as they are in Sp1, VP16 and p53. Analysis of the DNA-binding domain of Glass indicates that on their own, the three carboxyl-terminal zinc fingers are necessary and sufficient for DNA binding. A deletion mutant of glass containing only the DNA-binding domain can behave in a dominant-negative manner both in vivo and in a cell culture assay that measures transcriptional activation (O'Neill, 1996).

Chemotaxis to water-soluble chemicals such as NaCl is an important behavior of C. elegans when seeking food. ASE chemosensory neurons have a major role in this behavior. che-1, defined by chemotaxis defects, encodes a zinc-finger protein similar to the Glass transcription factor required for photoreceptor cell differentiation in Drosophila, and che-1 is essential for specification and function of ASE neurons. Expression of a che-1::gfp fusion construct is predominant in ASE. In che-1 mutants, expression of genes characterizing ASE such as seven-transmembrane receptors, guanylate cyclases and a cyclic-nucleotide gated channel is lost. Ectopic expression of che-1 cDNA induces expression of ASE-specific marker genes, a dye-filling defect in neurons other than ASE and dauer formation (Uchida, 2002).

Glass has five zinc-finger domains and the last three C-terminal zinc finger domains alone are necessary and sufficient for DNA binding. The first to the fourth zinc-finger domains of CHE-1 correspond to the second, third, fourth and fifth zinc-finger domains of Glass, respectively. These combinations show higher similarities (71%, 71%, 68% and 57%) than any other combinations. These high similarities and the same order of corresponding zinc-finger domains along the primary structure indicate that CHE-1 may be a C. elegans counterpart of Glass. The other regions of CHE-1 apart from zinc-finger domains did not show a significant homology to any proteins or motifs (Uchida, 2002).

The molecular signature and cis-regulatory architecture of a C. elegans gustatory neuron

Taste receptor cells constitute a highly specialized cell type that perceives and conveys specific sensory information to the brain. The detailed molecular composition of these cells and the mechanisms that program their fate are, in general, poorly understood. Serial analysis of gene expression (SAGE) libraries have been generated from two distinct populations of single, isolated sensory neuron classes, the gustatory neuron class ASE and the thermosensory neuron class AFD, from the nematode C. elegans. By comparing these two libraries, >1000 genes were identified that define the ASE gustatory neuron class on a molecular level. This set of genes contains determinants of the differentiated state of the ASE neuron, such as a surprisingly complex repertoire of transcription factors (TFs), ion channels, neurotransmitters, and receptors, as well as seven-transmembrane receptor (7TMR)-type putative gustatory receptor genes. Through the in vivo dissection of the cis-regulatory regions of several ASE-expressed genes, a small cis-regulatory motif, the 'ASE motif,' was identified that is required for the expression of many ASE-expressed genes. The ASE motif is a binding site for the C2H2 zinc finger TF CHE-1 (homolog of Drosophila Glass), which is essential for the correct differentiation of the ASE gustatory neuron. Taken together, these results provide a unique view of the molecular landscape of a single neuron type and reveal an important aspect of the regulatory logic for gustatory neuron specification in C. elegans (Etchberger, 2007).

Cis-regulatory mechanisms of left/right asymmetric neuron-subtype specification in C. elegans

Anatomically and functionally defined neuron types are sometimes further classified into individual subtypes based on unique functional or molecular properties. To better understand how developmental programs controlling neuron type specification are mechanistically linked to programs controlling neuronal subtype specification, a neuronal subtype specification program was characterized that occurs across the left/right axis in the nervous system of the nematode C. elegans. A terminal selector zinc finger transcription factor, CHE-1, a homolog of Drosophila Glass, is required for the specification of the ASE neuron class, and a gene regulatory feedback loop of transcription factors and miRNAs is required to diversify the two ASE neurons into an asymmetric left and right subtype (ASEL and ASER). However, the link between the CHE-1-dependent ASE neuron class specification and the ensuing left-right subtype specification program is poorly understood. This study shows that CHE-1 has genetically separable functions in controlling bilaterally symmetric ASE neuron class specification and the ensuing left-right subtype specification program. Both neuron class specification and asymmetric subclass specification depend on CHE-1-binding sites ('ASE motifs') in symmetrically and asymmetrically expressed target genes, but in the case of asymmetrically expressed target genes, the activity of the ASE motif is modulated through a diverse set of additional cis-regulatory elements. Depending on the target gene, these cis-regulatory elements either promote or inhibit the activity of CHE-1. The activity of these L/R asymmetric cis-regulatory elements is indirectly controlled by che-1 itself, revealing a feed-forward loop configuration in which che-1 restricts its own activity. Relative binding affinity of CHE-1 to ASE motifs (Etchberger, 2007) also depends on whether a gene is expressed bilaterally or in a left/right asymmetric manner. This analysis provides insights into the molecular mechanisms of neuronal subtype specification, demonstrating that the activity of a neuron type-specific selector gene is modulated by a variety of distinct means to diversify individual neuron classes into specific subclasses. It also suggests that feed-forward loop motifs may be a prominent feature of neuronal diversification events (Etchberger, 2009).

The following conclusions can be drawn from these studies: (1) CHE-1 is not only required to induce ASE fate embryonically, but is also continuously required throughout the life of the neuron to maintain L/R asymmetric ASE cell fate; (2) in contrast to bilaterally expressed target genes of CHE-1, the regulation of asymmetrically expressed target genes involves the modulation of the activity of bilaterally expressed CHE-1 at the cis-regulatory level by additional regulatory motifs which restrict CHE-1 activity in a target gene-dependent manner to either ASEL or ASER; (3) the binding affinity of CHE-1 to its target site plays an important role in restricting the CHE-1 activity to ASEL or ASER; (4) the cis-regulatory architecture that restricts CHE-1 activity in a L/R asymmetric manner is remarkably diverse. Rather than using a simple cis-regulatory motif (as is the case for ASE-motif/che-1-dependent ASEL/R bilateral specification), different L/R asymmetrically expressed genes appear to use distinct cis-regulatory strategies (Etchberger, 2009).

The amenability of C. elegans to RNAi and the possibility to time its delivery allowed the fundamental question to be asked: whether gene regulatory factors that turn on a specific terminal neuronal fate also are continuously required to maintain this fate throughout the life of the neuron. This is indeed the case for the che-1 transcription factor. Using a temperature-sensitive allele, such sustained function could also be demonstrated for the unc-4 homeobox gene, which acts to determine specific synaptic inputs, but this issue has not been addressed for other regulatory factors that control neuronal differentiation in any system. Consistent with a maintenance function, che-1 is expressed in the ASE neurons throughout the life of the animal. Sustained che-1 activity is ensured by che-1 autoregulating its own expression via an ASE motif (Etchberger, 2009).

The continuous expression and requirement for che-1 suggests that CHE-1 does not only have a role in initiating the terminal fate of ASE neurons, which is, immediately after the birth of the ASE neurons, initially bilaterally symmetric. Rather, che-1 also appears to be required for the progression of the hybrid precursor state to the L/R asymmetric terminal state. This is inferred not only from the continuous expression and requirement of che-1, but also from the presence of functionally required ASE motifs in the cis-regulatory regions of L/R asymmetrically expressed genes. The possibility can be excluded that CHE-1 acts first to initiate L/R asymmetric genes via the ASE motif, and then to only indirectly restrict expression of L/R asymmetric genes in either ASEL or ASER, via the activation of intermediary transcription factors. If this were the case, the promoter analysis would have identified cis-regulatory motifs that can instruct ASE expression independently of the ASE motif. Even though motifs were found that are required for activation of L/R asymmetric genes, these motifs only act in conjunction with the ASE motif. Moreover, the elimination of repressor motifs 'bilateralizes' the expression of normally L/R asymmetric genes, again arguing that bilateral CHE-1 is continually able to drive gene expression in both ASEL and ASER, but is prevented from doing so in a target gene-dependent manner (Etchberger, 2009).

CHE-1 acts as a terminal selector gene that determines overall ASE fate by directly activating the expression of a large battery of bilaterally expressed genes (Etchberger, 2007). In addition to this neuron class specification function of CHE-1, several cis-regulatory mechanisms have been uncovered that restrict CHE-1 activity on several target gene promoters to either the left or right ASE neuron, thereby driving a subtype specification program that diversifies these two cellular subtypes from one another. The cis-regulatory mechanisms that restrict CHE-1 activity are promoter dependent and remarkably diverse. In three promoters (lim-6, gcy-7, lsy-6), evidence was found for the existence of three distinct unrelated activator elements with which CHE-1 cooperates to promote L/R asymmetric expression. Notably, these activator elements are only required in the context of the complete regulatory module, as the ASE motif alone can drive bilateral ASE expression when in complete isolation, but is apparently not able to do so if in the context of the whole regulatory element. In such context, a cooperating activator motif is required for ASE expression (K50 binding site in gcy-7 and bHLH binding site in lsy-6). It is possible that bilaterally expressed ASE motifs also require such positively cooperating factors (Etchberger, 2009).

Regulation of the gcy-7 and lsy-6 promoters, even though relying on distinct motifs, appears to share a similar logic. CHE-1 appears to be engaged in a tug-of-war with repressor elements in the promoter, which it can only overcome by the presence of an additional activator motif that cooperates with the ASE motif. The activator motif is only required if the repressor elements are present. If both are removed, CHE-1 exerts bilateral control over the promoter in both ASEL and ASER (Etchberger, 2009).

By contrast, no discrete repressor motifs were found in lim-6 and no discrete co-activator motifs were found in gcy-5. It appears striking that none of the four promoters analyzed in this study uses similar strategies, as neither of the identified activator or repressor motifs show any similarity to one another. Moreover, even though some repressor motifs found to be required in one promoter (gcy-7) are present in other promoters (gcy-14, gcy-20), these motifs are not required in these other cases. These findings may indicate an independent evolutionary recruitment of the many L/R asymmetrically expressed gcy genes into the regulatory network that controls L/R asymmetry of these neurons. This diversity is consistent with differences in the expression of gcy genes in different nematode species. This plasticity in the composition of the L/R asymmetric terminal features may relate to the sensory function of ASE neurons that may need to adapt to distinct environmental cues in a species-specific manner (Etchberger, 2009).

Previous genetic analysis has revealed several candidates for activator and repressor factors that may act through the cis-regulatory elements that were described here to restrict CHE-1 activity. These include the zinc-finger transcription factor DIE-1 [genetically required to activate ASEL-expressed genes and repress ASER-expressed genes, the LIM homeodomain protein LIM-6 [required to repress gcy-5 in ASEL, the zinc-finger protein FOZI-1 [required to repress gcy-7 in ASER, and two other, as yet uncloned lsy genes with similar phenotypes to lim-6 [lsy-20 and lsy-26. DNA-binding sites are not known for DIE-1, LIM-6 or FOZI-1, and in vitro gelshift assays have not detected binding of these factors to ASEL or ASER-specific promoters. However, the putative K50-homeodomain binding site identified in the gcy-7 promoter is a likely binding site for the CEH-36/Otx homeodomain protein. CEH-36 binds to the motif in vitro and gcy-7 fails to be activated in ceh-36 mutants. The asymmetric, ASEL-fate inducing activity of CEH-36 is also evident at the cis-regulatory level as the CEH-36 binding site will convert a bilaterally expressed ASE motif into a regulatory motif that is more strongly expressed in ASEL than in ASER. Therefore, CEH-36 activity must be somehow lateralized, even though CEH-36 is expressed in both ASEL and ASER (Etchberger, 2009).

Another factor that contributes to the restriction of CHE-1 activity is the affinity of the CHE-1/ASE motif interaction, which is important for some, but not all, promoters. The affinity argument stems largely from three observations. (1) Swapping a high-affinity ASE motif from a bilateral promoter into that of two different asymmetric promoters results in the partial bilateralization of promoter activity. This is interpreted to mean that, at least in some cases, CHE-1 binding to the ASE motif is weak so as to make it susceptible to the effect of repressor motifs. Increases in the affinity of the CHE-1-binding site counteract the repressive effect. (2) Increases in CHE-1 expression disrupt L/R asymmetric promoter activity. (3) An unusual class of alleles of che-1 have been identified, that separate the activity of che-1 on bilaterally expressed versus L/R asymmetrically expressed promoters. These alleles cause a general decrease in ASE motif affinity and lead to a disruption of L/R asymmetric gene expression, while leaving bilateral expression intact. It is noted, however, that the importance of affinity may not be a general theme as not all motif swaps yielded the same results and the effect of the unusual che-1 alleles does not extend to every single L/R asymmetrically expressed gene (Etchberger, 2009).

Although CHE-1 activity must somehow be integrated with other transcription factors such as CEH-36, which are genetically required for L/R asymmetry, another, as yet completely unexplored, layer of regulatory control is noted that may play a role in the system. All cis-regulatory elements described in this study (including the ASE motifs) are located in remarkable vicinity to predicted translational start sites and hence may be components of the RNA-Pol II-binding core promoter. Unfortunately, owing to the phenomenon of trans-splicing, transcriptional start sites and hence core promoter sites are difficult to map in C. elegans. As previous work in other systems has shown shifts in core promoter selectivity during development, it will be intriguing to investigate how the regulatory elements defined in this study relate to Pol II function (Etchberger, 2009).

The findings described in this study provide insights into how cellular fates become progressively restricted during development. Transcriptional regulators often define broad domains of gene expression that become further restricted, refined and diversified through added layers of regulatory control. In the context of terminal neuronal differentiation, an important class of regulatory proteins are what have been termed 'terminal selector genes'. These encode transcription factors that determine the terminal identity of individual neuron types by directly controlling the expression of terminal gene batteries. CHE-1 is such a terminal selector gene, which directly controls the expression of a large battery of cell fate markers that are shared by ASEL and ASER. Other examples for terminal selector genes can not only be found in C. elegans but also in vertebrates, particularly in the brain. Although terminal selector genes define the properties of individual neuron classes, ensuing subtype specification events further diversify neuron classes, as is the case in the diversification of the ASEL and ASER subtypes (Etchberger, 2009).

Together with previous analysis of gene regulatory factors in ASE, the data presented in this study demonstrates that terminal selector genes participate directly in the subtype diversification that follows neuron type specification. The way that CHE-1 achieves this feat may reveal a common theme in gene regulatory networks that serve to diversify gene expression programs. CHE-1 interacts with other regulatory modules in a feed-forward loop (FFL) motif configuration. A conventional FFL consists of a transcription factor A, controlling factor B, and factor A and B controlling together a target C. Such simple FFL motifs can have specific properties such as persistence detection or response acceleration. CHE-1 acts in a more complicated version of the FFL. Besides activating a single transcription factor (CEH-36) with which it collaborates to regulate the expression of a target gene (gcy-7), CHE-1 also activates multiple components of the bistable regulatory loop; the regulatory loop provides a net activity output that then cooperates with CHE-1 in a promoter-specific manner on a given target gene. For some genes, the loop provides a positive output with which CHE-1 needs to interact to be able to turn on a target gene (e.g. lim-6 in ASEL). For other target genes, the loop provides a negative output (e.g. in the form of the LIM-6) that restricts the ability of CHE-1 to turn on a target gene (e.g. prevents the activation of gcy-5 in ASEL) (Etchberger, 2009).

Apart from whatever precise kinetic properties such network motif configuration may convey, one may view such network architecture as being reflective of the evolution of gene regulatory networks. The two ASE neurons may have initially been identical, with CHE-1 controlling the exact same set of genes in ASEL and ASER. This ancestral state may still be reflected ontogenetically in the hybrid precursor state through which CHE-1 passes after the ASE neurons are born. As a segregation of certain features (such as chemoreceptors) into distinct cells (i.e. ASEL and ASER) can convey beneficiary selective advantages to an animal (increases in discriminatory properties); additional regulatory mechanisms may have been implemented downstream of CHE-1 to restrict CHE-1 activity to a subset of target genes (Etchberger, 2009).

Even though not dissected to the same extent as the ASE system, FFL-loop dependent subtype specification mechanisms also occur in other systems, such as the vertebrate retina (Hsiau, 2007 or in the fly ventral nerve cord (Baumgardt, 2007), and may provide a commonly used regulatory logic for subtype specification (Etchberger, 2009).

Germ-Granule Components Prevent Somatic Development in the C. elegans Germline

Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans. In Drosophila, germ granules have been implicated in germline determination. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination, but their components are still needed for fertility. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 (Drosophila homolog: Held out wings) or upon neuronal induction by CHE-1 (Drosophila homolog: Glass). However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, P granules were severly compromised by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization [GLH-1 (see Drosophila Vasa) and GLH-4] and an investigation was carried out to see whether this causes germ cells to lose totipotency and initiate somatic reprogramming. It was found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate (Updike, 2014).

Coordinated control of terminal differentiation and restriction of cellular plasticity

The acquisition of a specific cellular identity is usually paralleled by a restriction of cellular plasticity. Whether and how these two processes are coordinated is poorly understood. Transcription factors called terminal selectors activate identity-specific effector genes during neuronal differentiation to define the structural and functional properties of a neuron. To study restriction of plasticity, this study ectopically expressed C. elegans CHE-1 (see Drosophila glass), a terminal selector of ASE sensory neuron (see Drosophila sensory neurons) identity. In undifferentiated cells, ectopic expression of CHE-1 results in activation of ASE neuron type-specific effector genes. Once cells differentiate, their plasticity is restricted and ectopic expression of CHE-1 no longer results in activation of ASE effector genes. In striking contrast, removal of the respective terminal selectors of other sensory, inter-, or motor neuron types now enables ectopically expressed CHE-1 to activate its ASE-specific effector genes, indicating that terminal selectors not only activate effector gene batteries but also control the restriction of cellular plasticity. Terminal selectors mediate this restriction at least partially by organizing chromatin. The chromatin structure of a CHE-1 target locus is less compact in neurons that lack their resident terminal selector and genetic epistasis studies with H3K9 methyltransferases suggest that this chromatin modification acts downstream of a terminal selector to restrict plasticity. Taken together, terminal selectors activate identity-specific genes and make non-identity-defining genes less accessible, thereby serving as a checkpoint to coordinate identity specification with restriction of cellular plasticity (Patel, 2017).


Promoter Structure

glass contains a TATA-box deficient (TATA-less) promoter. Such promoters have a conserved sequence motif, A/GGA/TCGTG, termed the downstream promoter element (DPE). This is located about 30 nucleotides downstream of the RNA start site of many TATA-less promoters, including glass. DNase I footprinting of the binding of epitope-tagged TFIID to TATA-less promoters reveals that the factor protects a region that extends from the initiation site sequence (about +1) to about 35 nucleotides downstream of the RNA start site. There is no such downstream DNase I protection induced by TFIID in promoters with TATA motifs. This suggests that the DPE acts to mediate transcription of TATA-less promoters in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box (Burke, 1996).

In a comparison of the Drosophila melanogaster promoter region of glass with the D. virilis promoter, seven perfectly conserved sequence elements were found of 15 base pairs or longer. Three element were found to promote eye specific expression, but these differ in time and location of expression. One element was found to be inhibitory (Liu, 1996)

Transcriptional Regulation

Glass positively regulates itself by binding to identified sites in its own promoter (Moses, 1991).

Targets of Activity

glass positively regulates the rhodopsin gene by binding to the rhodopsin promoter at both a proximal site and an upstream enhancer site (Moses 1991). Non-sensory cells are unable to express a rhodopsin reporter construct in response to glass expression, because another unidentified DNA binding factor inhibits glass activity in non-sensory cells (Ellis 1993).

BarH1 and BarH2, homeobox genes required in two photoreceptor cells, are regulated by rough and glass (Higashijima 1992).

Directly or indirectly glass controls the expression of approximately 25 % of all enhance trap lines expressed in the eye disc. The phenotype of eye discs doubly mutant for glass and rough suggest that glass is required for subtype specification and for recruitmant of cells to the ommatidial cluster. rough and glass appear to act on common target genes . However the is no simple relationship between ro and gl. While RO represses both seven up and boss in R2 and R5 precursors, and GL activates both in cells which normally express them, in the absence of both ro and gl it is found that svp is expressed but boss is not. One possibility is that GL is critical for the establishment of R8-specific genes and RO for the establishment of R2/5-specific genes and for the repression of inappropriate genes in these cells (Treisman, 1996).

rough encodes a homeobox transcription factor required for proper specification of photoreceptor cells R2 and R5 in Drosophila eye development. To identify the transcriptional targets through which ro acts to specify the R2/R5 neuronal sub-type, enhancer trap lines expressed in developing photoreceptors were screened for those whose expression patterns are altered when ro function is inactivated. In this way two potential ro targets were identified; these are also targets of the zinc finger transcription factor Glass. An enhancer trap line was identified that exhibits altered morphogenetic furrow expression in a ro mutant background. Finally, an enhancer trap line, AE33, was molecularly characterized that was identified in earlier screens as a target of both ro and gl. The transcript interrupted by AE33 shares similarity with the mammalian vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases that is associated with actin filaments, focal adhesions, and dynamic membrane regions. There is also similarity with Enabled, a substrate of the Drosophila Abl tyrosine kinase and with two human Expressed Sequence Tags (ESTs) (DeMille, 1996).

In the course of a screen designed to identify genes regulated by the photoreceptor transcription factor Glass, a set of lethal non-complementing P-element insertions mapping to 62E6-7 were isolated. Expression of lacZ from these insertions is completely Glass-dependent in photoreceptors. This gene has been named misshapen (Treisman, 1997).

Glassis a zinc-finger transcription factor that is required for the differentiation of all photoreceptor cells. It is expressed in all cells behind the morphogenetic furrow; however, glass-dependent gene transcription is restricted to only the R cells. Since onecut probably participates in late differentiation events and is expressed exclusively in all R cells, it was of interest to investigate if its expression in the developing eye is dependent on glass. Immunostaining of third instar larval eye discs from a glass mutant, with Onecut antibodies reveals that onecut expression is not affected. It is interesting to note that the expression of several other photoreceptor-specific genes that are required for proper differentiation of R cells, for example, Orthodenticle (Otd), a homeodomain protein, and Calphotin, a calcium-channel protein, are also independent of Glass. This observation suggests that glass may regulate only some aspects of neuronal differentiation in the eye. Indeed, in null glass mutants, the expression of some neural-specific antigens such as those recognized by monoclonal antibody 22C10 and anti-HRP antibody is not affected. Thus, onecut is not downstream of glass, but may act in a parallel regulatory pathway in the control of photoreceptor cell differentiation (Nguyen, 2000).

Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference

Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, this study performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, direct TF-DNA interactions were derived using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, network motifs, cis-regulatory codes, and regulators of eye development were found. The predicted target regions of Grainyhead were validated by ChIP-seq and this factor was identified as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions (Potier, 2014).

The development of the Drosophila eye is a classical model system to study neuronal differentiation and patterning. The TFs that represent the core of the retinal determination network are Eyeless (Ey), Twin of Eyeless (Toy), Dachsund (Dac), Sine Oculis (So), and Eyes Absent (Eya). Although many regulatory interactions are known between these TFs, as they intensively cross-regulate each other, knowledge about interactions with downstream target genes and of other TFs involved in the eye-antennal gene regulatory network (GRN) is sparse. This study aimed at combining classical reverse genetics-starting from a mutant allele and analyze its (molecular) phenotype-with genomics. Doing so, attempts were made to unveil genetic regulatory interactions in an unbiased way, and many regulators of the eye and antennal developmental programs were identified; most of these did not require or use any mutation or direct perturbation (Potier, 2014).

The mapping approach began by systematically perturbing the developmental system. Attempts were made to include multiple perturbations into one data matrix to obtain a broad spectrum of expression profile changes. These perturbations included TF mutants, TF overexpression, TF knockdown, and cell sorting (Potier, 2014).

Eye-antennal discs were dissected at the stage where in the WT discs about half of the eye disc contains pluripotent cells that are dividing asynchronously, while the other half contains differentiating PR neurons, in consecutive stages of differentiation. Simultaneously, the antennal disc contains neuronal precursors that are undergoing specification. The expression changes induced by the perturbations often result from a shift in proportion of cell types. This is trivial for the cell-sorting experiments; for example, the GMR>GFP-positive cells show, as expected, a very strong enrichment of genes related to PR differentiation. TF mutants and TF perturbations can also result in cell type shifts; for example, overexpression of Atonal yields more R8 PRs, and the glass mutant results in fewer differentiated PRs. Other TF perturbations cause changes in gene expression downstream of the TF without changing the cell type composition, such as Retained, which disturbs axonal projection. The key technique that was applied, however, was not to compare each TF perturbation with WT discs to identify differentially expressed genes. Rather, linear and nonlinear correlations of gene expression profiles were used across the entire vector of 72 gene expression measurements. This TF-gene coexpression network contains both direct and indirect edges, and although this network is informative, a second layer of predicted TF-DNA interactions was added, thus making this a direct GRN. To increase the sensitivity, a very large collection was used of TF motifs, also including position weight matrices derived for yeast and vertebrate TFs and including computationally derived motifs (e.g., highly conserved words). Using motif-motif similarity measures and TF-TF orthology relationships, each motif was linked to a candidate binding factor. This yielded a large network with 335 TFs and their predicted direct targets. The only functional network of comparable size and comparable directedness to this in vivo network is the TH17 GRN that was derived in vitro in a recent study (Yosef, 2013). That study used a microarray time course of naive CD4+T cells differentiating into TH17. From these gene expression data, they derived TF-gene interactions by clustering and filtered those with TF-DNA interactions obtained by ChIP-seq data, TF perturbations, and cis-regulatory sequence analysis (Potier, 2014).

The predicted direct and functional eye-antennal GRN includes many previously reported interactions, such as known target genes for Eyeless and Sine Oculis. Target genes in the network were also found for late factors (e.g., Glass, Onecut) and very late factors (e.g., Pph13). The fact that information was captured at different time points during development is because several cell populations were sorted that are loosely correlated with the temporal axis of development, consisting of undifferentiated pluripotent cells anterior to the furrow, all PR cells undergoing differentiation posterior to the MF, R8 PR cells, and late populations of chp-positive cells. However, the temporal information encoded in the network is limited to these broad domains, and a more detailed reconstruction of the time axis would require higher resolution cell sorting or microdissection experiments. Although the perturbed TFs were mainly chosen for their development of the retina, master regulators of antennal development, such as aristaless were also identified (Potier, 2014).

Interestingly, general factors like Grainyhead were found that were ubiquitously expressed. Grh was found as one of the TFs with the largest number of target enhancers and its binding correlates with open chromatin. Previous studies have shown that Grainyhead may interact with Polycomb and Trithorax proteins to regulate (both activate and repress) target gene expression. It is speculated that this observed correlation can be explained by the fact that Grh is present ubiquitously in the eye disc, thus yielding many sequence fragments from bound and nucleosome-free enhancers by FAIRE-seq (Potier, 2014).

It is well known that network motifs such as FFLs play an important role in biological networks. One such network motif was examined in more detail, namely the TF pair Glass-Lozenge, and their common targets. These TFs constitute a double-feedback loop (Glass regulates Lozenge, Lozenge regulates Glass, and they together regulate 36 targets). For this network motif, it was found that Glass and Lozenge motifs co-occur at the same enhancer, where they furthermore overlap; this may indicate competition for binding between Glass and Lozenge. Given that Lozenge, an important regulator of cone cell differentiation, could be a repressor and Glass, an important regulator of PR differentiation, could be an activator, such a competition at the CRM level could indeed be a plausible mechanism for their regulatory action (Potier, 2014).

Another interesting feature that can be derived from a GRN is the proportion of autoregulatory TFs (108 autoregulatory TFs in the eye network) and the proportion of activating versus repressive TFs. A recent large-scale study in yeast found a small majority of yeast TFs to have a repressive role. In that study, each individual TF was perturbed, thereby providing information on positive versus negative edges from the TF to its direct predicted targets, whereby TF-DNA information was used from ChIP-chip data. Since the eye GRN was started from a coexpression TF-gene network, the correlations between TFs and their candidate targets were revisited, and 151 TFs were found that have their motif enriched in the positively correlated target genes, but not in the negatively correlated targets, and 127 TFs showing the opposite; 62 TFs show enrichment in both. This finding agrees, to some extent, with the results in yeast concerning the high amounts of gene-specific repressors. On the other hand, the eye network suggests relative more TFs with a dual activator/repressor function, while the yeast study found only a few such cases (Potier, 2014).

In conclusion, starting from an expression matrix derived from large-scale perturbations and combining TF-gene coexpression with TF-DNA interactions based on motif inference enabled drawing an extensive eye-antennal GRN. All predicted regulatory interactions, target genes, and candidate regulatory regions are stored in a Neo4J database and can be queried from a laboratory website. The database can also be accessed directly from Cytoscape using the CyNeo4j plugin or can be queried programmatically using the Neo4j query language Cypher. Although many known regulators and cis-regulatory elements were uncovered and several other ones were revealed, a large part of the predicted network, including how the dynamics of the developmental program are encoded in the cis-regulatory regions and in the topology of the network, remains to be explored (Potier, 2014).


Embryonic development of the Drosophila corpus cardiacum, a neuroendocrine gland with similarity to the vertebrate pituitary, is controlled by sine oculis and glass

The development of the Drosophila neuroendocrine gland, the corpus cardiacum (CC) was investigated, along with the role of regulatory genes and signaling pathways in CC morphogenesis. CC progenitors segregate from the blastoderm as part of the anterior lip of the ventral furrow. Among the early genetic determinants expressed and required in this domain are the genes giant (gt) and sine oculis (so). During the extended germ band stage, CC progenitor cells form a paired cluster of 6–8 cells sandwiched in between the inner surface of the protocerebrum and the foregut. While flanking the protocerebrum, CC progenitors are in direct contact with the neural precursors that give rise to the pars intercerebralis, the part of the brain whose neurons later innervate the CC. At this stage, the CC progenitors turn on the homeobox gene glass (gl), which is essential for the differentiation of the CC. During germ band retraction, CC progenitors increase in number and migrate posteriorly, passing underneath the brain commissure and attaching themselves to the primordia of the corpora allata (CA). During dorsal closure, the CC and CA move around the anterior aorta to become the ring gland (see Image). Signaling pathways that shape the determination and morphogenesis of the CC are decapentaplegic (dpp) and its antagonist short gastrulation (sog), as well as hedgehog (hh) and heartless (htl; a Drosophila FGFR homolog). Sog is expressed in the midventral domain from where CC progenitors originate, and these cells are completely absent in sog mutants. Dpp and hh are expressed in the anterior visceral head mesoderm and the foregut, respectively; both of these tissues flank the CC. Loss of hh and dpp results in defects in CC proliferation and migration. Htl appears in the somatic mesoderm of the head and trunk. Although mutations of htl do not cause direct effects on the early development of the CC, the later formation of the ring gland is highly abnormal due to the absence of the aorta in these mutants. Defects in the CC are also caused by mutations that severely reduce the protocerebrum, including tailless (tll), suggesting that additional signaling events exist between brain and CC progenitors. The parallels between neuroendocrine development in Drosophila and vertebrates are discussed (De Velasco, 2004).

In the larva, the ring gland forms a large and conspicuous structure located anterior to the brain and connected to the brain by a pair of tracheal branches and the paired nerve of the corpus cardiacum (NCC). Three different glands, the corpus allatum (CA; dorsally), prothoracic gland (laterally), and corpus cardiacum (CC; ventrally) form part of the ring gland. By far, most of its volume is taken up by the prothoracic gland whose cells, the source of ecdysone, grow in size and number as larval development progresses, whereas the cells of the CC remain small and do not appear to proliferate. Both the CC and CA, as well as axons innervating the ring gland, are FasII positive from the late embryonic stage onward. Labeling of the CC is stronger and starts earlier (stage 11) than that of the CA (stage 15), which makes it easy to distinguish between the two structures in the embryo. Another convenient marker of the CC is adipokinetic hormone (AKH), which is expressed exclusively in the CC from late embryonic stages onward (De Velasco, 2004).

The ring gland of the mature embryo is situated posterior to the brain hemispheres. The CC and CA occupy their positions ventral and dorsal to the aorta, respectively. The prothoracic gland cannot yet be recognized as a separate entity, possibly due to the fact that its precursors are small and few in number. Cells of the CC number around eight on each side and are arranged in a U-shape around the floor of the aorta. All cells are spindle shaped and send short processes ventromedially where they meet and form a bundle attached to the ventral wall of the aorta (subaortic processes) (De Velasco, 2004).

The homeobox gene glass (gl) is expressed in the CC from stage 10 onward. Glass-positive CC precursors first appear as two pairs of cells located between the roof of the stomodeum and the inner surface of the brain primordium. Several populations of head mesoderm cells internalized during gastrulation as part of the anterior ventral furrow form a sheet of cells covering the inner surface of the brain primordium; the CC precursors form part of this cell group. Between stages 11 and 15, CC progenitors migrate posteriorly, gradually increasing in number (3–4 cells by stage 11; 6-8 cells by stage 13). The movement of the CC precursors parallels the invagination and elongation of the esophagus. During stages 11 and 12, the primordium of the stomatogastric nervous system (SNS) appears as three invaginating pouches in the roof of the esophagus. The CC precursors maintain a position laterally adjacent to the first SNS invagination on their posterior course. By stage 15, they have passed underneath the brain commissure and join up with CA precursor cells derived from the gnathal mesoderm to form the ring gland. On their migration, posteriorly the CC precursors are always in contact with the medial surface of the developing brain. One group of neurons transiently in contact with the CC precursors is the FasII-positive P3m cluster, which becomes part of the pars intercerebralis (PI) and is likely the source of some of the NCC axons innervating the CC. This close contact between PI and CC provides the opportunity for inductive interactions between the two structures (De Velasco, 2004).

A previously undescribed population of head mesoderm cells expressing the tinman (tin) gene represents another group of cells that surround the CC precursors during their migration. The Tin-positive cells, for which the term 'cephalic vascular rudiment' (CVR; an evolutionary vestige of the cephalic aorta which forms a prominent component of the vascular system in other insects) is suggested, form a loose cluster that extends backward dorsal of the esophagus and eventually establishes contact with the Tin-positive trunk aorta. The CC precursors are initially close to the anterior (trailing) end of the CVR, but they appear to 'catch up' and lead the CVR during later stages (De Velasco, 2004).

Based on reports from other insects, it had been anticipated that the CC is derived from the foregut as part of the invaginating stomatogastric primordium. However, this is not likely to be the case in Drosophila because the CC is present in embryos mutant for forkhead (fkh) in which both esophagus and SNS are eliminated. The expression and phenotype of numerous head gap genes were subsequently investigated to determine the origin of the CC. The results of this analysis indicate strongly that the CC originates from the anterior lip of the ventral furrow (AVF). The CC is deleted in mutations in the genes sine oculis (so), giant (gt), and twist/snail (twi/sna). Each of these genes is expressed in several domains at the blastoderm stage and during gastrulation, but the AVF is the only place of overlap between the three. Furthermore, giant expression, which is particularly strong in the AVF and persists slightly longer than expression of so or twi, visualizes the AVF cells as they spread out and form the anterior part of the head mesodermal layer that lines the inner surface of the brain primordium and includes the glass-positive CC precursors (De Velasco, 2004).

Besides sine oculis, giant, and twist/snail, one more head gap gene, tailless (tll) affects CC development. Tll is expressed in the anlage of the protocerebrum and only appears faintly, if at all, in the AVF. In tll mutant embryos, the CC is absent, whereas the SNS appears normal in size. It is speculated that the effect of tll on the CC is indirect, caused by the elimination of the protocerebrum (including the PI) in tll mutants. Another head gap gene, orthodenticle (otd), is expressed similarly to tll but leaves the CC intact. Otd mutant embryos also show a reduction in size of the protocerebrum but still possess the PI contacted by the CC precursors. Taken together, these findings (which need further follow-up analysis) hint at the possibility of inductive interactions between protocerebrum and CC (De Velasco, 2004).

In the embryo, glass is expressed in the primordium of the larval eye (Bolwig's organ), a small group of protocerebral neurons, and the CC precursors. Loss of gl in the allele gl2 results in the absence of both larval eye and the CC, as shown in labelings with anti-FasII and AKH probe. Interestingly, this phenotype is a dominant effect since 75% of embryos derived from crossing balanced gl parents have no CC or larval eye. In stages 11 and 12 gl mutant embryos, the gl probe still gives a signal in CC and larval eye precursors. The signal becomes patchy (first in CC precursors, slightly later in larval eye) during stage 12 and has disappeared by stage 13. This finding suggests that gl is required for CC migration and/or differentiation, and that the absence of the CC in gl mutants as assayed for by the differentiation marker AKH is caused by transformation and/or apoptosis of initially correctly specified CC precursors (De Velasco, 2004).

Two other regulatory genes that were found to play an important role in vertebrate pituitary development, are Lhx3 and goosecoid (gsc). The Drosophila homologs of both of these genes are expressed in the SNS and possibly the ring gland. To investigate the role of Lim3 and gsc during CC development, their expression and phenotype were analyzed. Lim3 appears in the precursors of the SNS at a relatively late stage (stage 11), following the complete separation of these cells from the esophagus. In addition, lim3 is expressed in several small clusters in the brain primordium. Comparison with the expression of gl makes it clear that the Lim3 expressing cells are distinct from the CC progenitors. No CC defects were found in lim3 mutant embryos. Goosecoid is expressed in the SNS and, in late stage embryos, the CC. However, no CC defects were detected in the gsc0534lacZ allele, which does cause structural abnormalities in the SNS. It is possible that gsc plays a role in later CC differentiation (De Velasco, 2004).

Several signaling pathways, notably Shh, BMP, and BMP antagonists, Wnt and FGF, specify the fate map of the head in vertebrates and also control later morphogenetic events shaping head structures. The same signaling pathways are active at multiple stages in Drosophila head development, and the pattern of activity and requirement of these pathways in regard to CC development was therefore investigated (De Velasco, 2004). .

The first signal acting zygotically in the Drosophila head is the BMP homolog Dpp, which forms a dorsoventral gradient across the blastoderm. The homolog of the BMP antagonist Chordin, short gastrulation (Sog), is expressed in the ventral blastoderm, overlapping with the ventral furrow. Loss of sog results in the absence of the CC, while the SNS is still present, which reflects ventral origin of the CC. Sog seems to be the only signal, of those tested, required for CC determination, since mutation of all other pathways does not eliminate the CC but merely effects its size, shape, or location (De Velasco, 2004).

Following its early widespread dorsal expression, Dpp becomes more confined during gastrulation to a narrow mid-dorsal stripe and an anterior cap that corresponds to parts of the anlagen of the esophagus and epipharynx. From this domain segregates the most anterior population of head mesoderm cells that give rise to the visceral muscle of the esophagus and which maintain Dpp expression. The visceral mesoderm of the esophagus flanks both CC and SNS. Loss of Dpp causes absence of the SNS; the CC is still present and expresses AKH but does not migrate posteriorly (De Velasco, 2004).

Both Hh and Wg are expressed from gastrulation onward in a similar pattern in the developing foregut. The pattern resolves into two domains, a posterior one covering the posterior esophagus, and an anterior one overlapping with the epipharynx. The esophageal domain, which shows a higher level of expression than the anterior domain, is located posterior to the precursors of CC and SNS. No significant abnormality in CC and SNS was obvious in Hh mutants. Wingless mutants show defects in the SNS but the CC is present, if misshapen and mislocalized, in the strongly distorted head of late wg mutant embryos (De Velasco, 2004).

Activity of the MAPK signaling pathway is widespread in the Drosophila head from gastrulation onward. Beside a wide anterior and posterior domain traversing the lateral and dorsal domain of the head ectoderm, the primordia of the foregut, including the SNS, and head mesoderm show a dynamic MAPK activity. At least two RTKs, EGFR and FGFR/heartless, drive the MAPK pathway in the embryonic head. EGFR is responsible for activation in the ectoderm and foregut. Loss of EGFR causes widespread cell death in the head and the absence of the SNS. The CC is still present, although reduced in size. Activation of MAPK by Heartless (Htl) occurs in a narrow anterior domain of head mesoderm that gives rise to the dorsal pharyngeal muscles. The foregut, SNS, and CC develop rather normally in htl mutants. However, the CC shows variable defects in shape and location, which are most likely due to the absence of the aorta and CA, both of which are derivatives of the dorsal mesoderm, which is defective in htl loss of function and to which the CC is normally attached (De Velasco, 2004).

This study has identified several early acting genes functioning in the development of the corpus cardiacum; among them sine oculis, giant, and glass are essential for its development. The apparent origin of the CC from the anterior ventral furrow, rather than the SNS placode as surmised in other studies, came as a surprise. In Manduca, CC precursors seem to delaminate from the posterior part of the neurogenic foregut ectoderm that gives rise to the SNS. In Drosophila, CC precursors are also close to the SNS placode as soon as they express the marker glass. Since this marker is not expressed during the segregation of CC precursors, it could not be directly observed from which ectodermal domain of the head they derive. It is therefore still possible that they originate from the SNS placode located in the roof of the foregut primordium. However, genetic data argue strongly against this possibility. Thus, the CC is present in a mutation of fkh, which is expressed and required in the foregut primordium and which is essential for the SNS. Similarly, the CC forms normally in mutations of EGFR, which entirely eliminate the SNS. By contrast, the CC is deleted in twist;snail and giant mutations, both of which are not expressed in the SNS placode and do not affect SNS development. The apparent discrepancy between Drosophila and Manduca indicates that the CC may originate from slightly different domains in different insect groups (the distance between presumptive SNS placode and anterior ventral furrow in the blastoderm is minimal); alternatively, the Manduca CC might also delaminate from the ventral furrow and only secondarily come to lie next to the SNS precursors (De Velasco, 2004).

The proposed origin of the CC from the anterior ventral furrow, which also gives rise to most of the anterior endoderm, underlines the close relationship between endodermal and neuroendocrine lineages. Such relationship also seems to exist in vertebrates. Numerous peptide signaling factors in vertebrates are expressed in cells of the digestive tract, in particular the pancreas, and the pituitary and/or hypothalamus. Among these are cholecystokinin (CKK), as well as the glucagon-like peptide (GLP) 1. GLPs and glucagon itself are the closest vertebrate counterparts to the CC-derived insect hormone AKH. Both AKH and glucagon, besides numerous other hormones released from the neuroendocrine system, coordinately control energy metabolism and behaviors associated with food uptake and processing. It is reasonable to assume that in the simple Bilaterian ancestor, cells that carried out the food uptake and digestive activities, that is, principal cells of the digestive tract, were identical with or spatially close to those cells that regulated these activities, among them endocrine and nerve cells (De Velasco, 2004 and references therein).

The pars intercerebralis/corpora cardiaca complex of insects has been repeatedly compared to the hypothalamus-pituitary axis in vertebrates. This comparison is usually based on clear similarities between the two on a gross anatomical and functional level. Thus, in both insects and vertebrates, neurosecretory neurons located in the anteromedial brain produce peptide hormones that are transported along axons to a peripheral gland. The axons either terminate on gland cells and modulate the release of glandular hormones, or they terminate in a separate secretory part of the gland where they release their products directly into the blood. Functional similarities include a role of both insect and vertebrate neuroendocrine factors in energy metabolism, growth, water retention, and reproduction. However, to what extent do these functional similarities represent true homologies, which would imply the presence of the homologous genes in the homologous cells in the Bilaterian ancestor (De Velasco, 2004)?

The main hormone produced by the CC is adipokinetic hormone (AKH), a peptide that acts on the fat body and mobilizes lipids and carbohydrates. AKH also stimulates the nervous system and activates locomotor activity. A peripheral feedback loop controls AKH release, in that sugars (e.g., trehalose in the hemolymph) inhibit AKH release; centrally, several PI-derived neuropeptides controlling AKH secretion have been identified, among them FMRFamide, tachykinin, and crustacean cardioactive peptide. FMRFamide inhibits AKH release, whereas cardioactive peptide and tachykinins (both of which also influence contractility of the heart and visceral muscles) stimulate AKH release (De Velasco, 2004).

AKH shares common functions with the vertebrate glucagon and has some sequence similarity with the N-terminus of glucagons. However, comparison of the genes encoding AKH and glucagons, respectively, provides no clear evidence for homology of these peptides on the molecular level. Glucagon, along with two other growth factors, GLP1 and GLP2, is encoded by the proglucagon gene for which true homologs have so far only been identified among vertebrates. The arthropod AKH gene may have been traced further back to the protostome root with the recent finding of significant sequence similarity with the mollusk gene encoding the APGWamide peptides. However, no significant sequence similarity exists between proglucagon and the AKH/APWHamide genes. The expression pattern of the proglucagon and AKH/APGWamide genes is too widespread to add meaningfully to the question of common ancestry. Glucagon is produced in the endocrine pancreas, as well as the intestinal epithelium, but the GLP growth factors (and therefore the proglucagon gene) are expressed in many cells, including neurons, of the developing and mature vertebrate. AKH is expressed mainly in the corpora cardiaca but is also found in the brain of various insect species. Thus, the molecular sequence of the specific secreted products of the CC and pituitary can currently provide no support for or against the notion that both structures are homologous (De Velasco, 2004 and references therein).

The vertebrate pituitary and Drosophila CC show significant similarities during development. Precursors of both are derived from an anterior anlage; following segregation from this anlage, CC precursors contact the part of the anteromedial forebrain primordium from which they will receive innervation. Shared regulatory genes and signaling pathways add to the overall similarity. In this regard, the role of sine oculis is particularly striking. The expression pattern of so in Drosophila is fairly restricted, including the eye field, stomatogastric anlage, and anterior lip of the ventral furrow that give rise to the CC. Another gene of the sine oculis/six family, optix, is expressed in an anterior unpaired domain close to the SNS, but not the CC. In the early vertebrate embryo, six3/6 (the ortholog of optix) is specifically expressed in the eye field and the anlage of the pituitary; six1/2, orthologs of Drosophila sine oculis, are expressed in sensory placodes of the vertebrate head, but no pituitary expression has been reported yet. In both systems, a sine oculis/six gene plays an early and essential role in the specification of the CC and pituitary, respectively. In Drosophila, both CC and SNS are absent in so mutants; in vertebrate, loss of six3/6 causes severe reduction and posteriorization of the forebrain region though not mention of the pituitary effect has been described (De Velasco, 2004).

Two other regulatory genes, goosecoid and Lhx3/lim3, are relevant in the comparison of the vertebrate and Drosophila neuroendocrine systems. Gsc appears in the ventral neural tube and foregut of postgastrula mouse embryos and is required for ventral neural tube patterning.Drosophila gsc is also expressed at an early stage, but it appears exclusively in the anlage of the SNS and comes on in the CC primordium only late. Loss of gsc results in mild structural defects in the SNS and no morphologically apparent phenotype in the CC. Lhx3 is a transcription factor of the Lim family that is triggered by Shh and FGF8 in the vertebrate pituitary primordium and required for its invagination. Drosophila lim3 is expressed at a late stage in part of the SNS primordium, but not the CC primordium. As stated for gsc, no structural phenotype associated with the SNS or CC has been noted in lim3 mutants, but more careful analysis, using additional late differentiation markers for these structures, will be required to establish the role of these two genes in Drosophila neuroendocrine development (De Velasco, 2004).

glass represents a homeobox gene expressed in the eye, nervous system, and as shown in this study, the corpus cardiacum. glass, which is absolutely required for the Drosophila CC since loss of one copy of the gene causes complete absence of the CC at late embryonic stages, has vertebrate cognates but so far no eye function of these genes has been reported (De Velasco, 2004).

Several signaling pathways are expressed in similar patterns in and/or around the developing neuroendocrine system of vertebrates and Drosophila. In both, Hh/Shh is expressed posteriorly adjacent to the CC/Rathke's pouch in the primordium of the foregut/oral epithelium. Vertebrate BMP2/4 comes on in the mesenchyme surrounding the base of Rathke's pouch. Similarly, Drosophila Dpp appears in the mesoderm flanking foregut primordium, CC, and SNS. FGF8 is derived from the hypothalamus floor; the FGF receptor homolog Htl is expressed in the myogenic head mesoderm that is anteriorly adjacent to the CC/SNS complex. In vertebrates, the ventral-to-dorsal BMP gradient and dorsal-to-ventral FGF8 gradients control the differentiation of pituitary cell types; Shh is also required in the proliferation and differentiation of the pituitary primordium. The role of these signaling pathways in Drosophila is less apparent. The CC is still present in dpp, hh, or htl mutant embryos, although it exhibits abnormalities in shape and location. This may constitute an indirect effect of these genes, given their widespread role in head morphogenesis (in case of Dpp and Hh) or mesodermal migration (for Htl). It is anticipated that with the advent of additional markers for subsets of CC cell types, the role of the Hh and Dpp signaling pathways will become clearer (De Velasco, 2004).

A mutation in the Dpp antagonist Sog was the only signaling mutant analyzed in this work that was able to completely remove the CC. This is surprising, given the relatively mild phenotype of sog mutants in the primordium of the ventral nerve cord. Here, only removal of both sog and brinker (brk) together are able to suppress the appearance of most neuroblasts. However, certain domains in the ventral head (that include the precursors of the CC) may be more sensitive to a shift in balance of the Sog-Dpp antagonism. It is speculated that the loss of the CC precursors in sog mutants results from an expanded Dpp gradient, although more experiments would be required to rule out the possibility that sog (the Drosophila homolog of chordin) directly affects CC precursor fate (De Velasco, 2004).

In conclusion, this study presents evidence for a number of conserved properties in the way the progenitors of the neuroendocrine system in vertebrate and Drosophila embryos are spatially laid out and employ cassettes of signaling pathways and fate determinants. This suggests that fundamental elements of a primordial “neuroendocrine system” were already present in the Bilaterian ancestor. Current ideas on pituitary evolution are compatible with this notion. Sensory structures proposed to represent the homologs of the vertebrate pituitary are present in cephalochordates, urochordates, and hemichordates. In amphioxus, for example, these cells form the so-called Hatschek's pit, located in the roof of the pharynx in close contact with the anterior neural tube. Molecules characteristic of the vertebrate pituitary, such as GnRH and Pit-1, are found in Hatschek's pit and in the proposed homolog in urochordates. It is thought that the pituitary originated as a chemosensory structure that senses environmental cues and produced hormones controlling gametogenesis and reproductive behavior, as well as fundamental metabolic functions. Subsequently, the pituitary lost its sensory function and was taken under the control of the CNS, which was able to assimilate sensory information more efficiently. It is likely that stage one, that is, a sensory-endocrine pituitary forerunner, was present in the Bilaterian ancestor. This forerunner probably formed part of the pharynx, which would explain the conserved developmental origin in Drosophila and vertebrates. The sensory-neuroendocrine state of the pituitary homolog is still preserved in present-day protochordates. Loss of sensory function and the taking-over of pituitary control by the CNS occurred during vertebrate evolution. In arthropods or other protostomes, evidence for a sensory forerunner of the neuroendocrine gland has not yet been described; guided by situation in protochordates, one would expect to find such a structure among the sensory organs of the head (De Velasco, 2004).


glass transcripts are present in the third instar eye-imaginal disc, starting in the morphogenetic furrow and extending to the posterior margin of the disc (Moses 1991). In addition to the retina photoreceptor cells glass is found in two other organs in Drosophila: the larval photoreceptor (Bolwig's organ) and the ocelli (adult simple eyes). There are two groups of Glass-positive nuclei in each brain hemisphere (Ellis 1993).

Effects of Mutation or Deletion

In glass mutants, retinal axons project aberrantly and the larval optic nerve is absent. In mosaic animals, wild-type retinal axons project to proper dorsoventral position despite the misrouted projections of neighboring glass axons (Kunes, 1993).

The Drosophila rotund (rn) gene is required in the wings, antenna, haltere, proboscis and legs. Previously identified in the rotund region was a member of the Rac family of GTPases, denoted the RacGAP84C or rotund racGAP gene. However, rotund racGAP is not responsible for the rotund phenotypes. The rotund gene has now been isolated. It is a member of the Krüppel family of zinc finger genes. The adjacent roughened eye locus specifically affects the eye and is genetically separable from rotund. However, roughened eye and rotund are tightly linked, and thanks to this connection, the roughened eye transcript was isolated. Intriguingly, roughened eye is part of the rotund gene but is represented by a different transcript. The rotund and roughened eye transcripts result from the utilization of two different promoters that direct expression in non-overlapping domains in the larval imaginal discs (St Pierre, 2002 and references therein).

Little is known about the genetic cascades within which roe and rn are acting. The results from eye-antennal imaginal discs indicate that roe acts at the morphogenetic furrow, as evident both from its expression and from the effects on Delta and Scabrous expression in roe mutants. Both Dl and sca play roles in spacing the array of ommatidial preclusters in the morphogenetic furrow, and it is interesting to note that the expression of roe at the furrow is not evenly distributed and appears stronger in clusters of cells. Genetic screens for modifiers of the Nspl mutation have identified roe as an enhancer, and sca and Dl as suppressors of the Nspl eye phenotype. Given the dynamics of N signaling, these results support models where Roe acts to either positively or negatively regulate Dl and Sca. A genetic interaction screen for enhancers of glass also identified roe, an interesting finding given that ectopic expression of roe using GMR-GAL4 leads to a glass-like phenotype with a loss of bristles and pigment cells (St Pierre, 2002 and references therein).

Circadian rhythms can be entrained by light to follow the daily solar cycle. In adult flies a pair of extraretinal eyelets expressing immunoreactivity to Rhodopsin 6 each contains four photoreceptors located beneath the posterior margin of the compound eye. Their axons project to the region of the pacemaker center in the brain with a trajectory resembling that of Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Kruppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. It is thus demonstrated that the adult eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LNv), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues to be expressed in the adult eyelet. Action spectra of entrainment were compared in different fly strains: in flies lacking compound eyes but retaining the adult eyelet (so1), lacking both compound eyes and the adult eyelet (so1;gl60j), and retaining the adult eyelet but lacking compound eyes as well as Cryptochrome (so1;cryb). Responses to phase shifts suggest that, in the absence of compound eyes, the eyelet together with Cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in the eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring (Helfrich-Forster, 2002).


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date revised: 25 March 2015
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