BIOLOGICAL OVERVIEW

Bifid, more familiarly known as Optomotor blind, and T-related gene (Brachyenteron) are two Brachyury homologs in the fly. Brachyury has a major role in vertebrates in the differentiation of the notochord and in the formation of the mesoderm. optomotor blind is involved in differentiation of the brain, the CNS, the wing and in patterning of adult abdominal segments. These are all epidermally derived tissues. In the nervous system, omb is found in both neurons and glia.

Optomotor blind plays a prominent role in control of cell fate and polarity in the adult segments of Drosophila. Each abdominal segment produces a large dorsal cuticular plate (the tergite) and a smaller ventral plate (the sternite). Each tergite can be divided into three regions: an acrotergite that contains undecorated sclerotized cuticle, a central region containing an array of microchaetes, and a posterior region that contains a dark pigment band as well as a row of large macrochaetes at the posterior edge of this posterior region. All of the tergite, except the acrotergite, is covered with trichomes. For convenience, the posterior boundary of the tergite is defined to be the posterior edge of the pigment band. The intertergal cuticle is unpigmented and composed of an anterior trichome-bearing region (the posterior hairy zone or PHZ) and a posterior region of naked cuticle (the intersegmental membrane or ISM). All trichomes and bristles in the abdomen are oriented from the anterior to the posterior. The tergite and anterior portion of the PHZ develop from the anterior dorsal histoblast nest; the rest of the PHZ and the ISM develop from the posterior dorsal nest (Kopp , 1997).

Hedgehog protein secreted by posterior compartment cells plays a key role in patterning the posterior portion of the anterior compartment in adult abdominal segments. This patterning function of Hh is mediated by optomotor-blind. omb- mutants mimic the effects of loss-of-function alleles of hh: structures from the posterior of the anterior compartment are lost; often this region develops as a mirror image of the anterior portion. Structures from the anterior part of the posterior compartment are also lost. In the pupa, omb expression in abdominal histoblasts is highest at or near the compartment boundary, and decreases in a shallow gradient toward the anterior. This gradient is due to activation of omb by Hh, secreted by posterior compartment cells. In contrast to imaginal discs, this Hh signaling is not mediated by dpp or wg. Several hh gain-of-function alleles have been described that cause ectopic expression of omb in the anterior of the segment. Most of these cause the anterior region to develop with posterior characteristics without affecting polarity. However, an allele that drives high level ubiquitous expression of omb (QadroondFab) causes the anterior tergite to develop as a mirror-image duplication of the posterior tergite, a pattern just the opposite of that seen in omb- mutants. The Qd Fab allele has a dramatic effect on both polarity and bristle patterning. In Qd Fab hemizygotes and heterozygotes, the anterior tergite and intersegmental membrane (ISM) are deleted and replaced with a mirror-image duplication of the posterior tergite and PHZ. Ectopic macrochaetes are often, but not always, present at the anterior edge of the duplicated tergite structures, and sometimes also in the central tergite. The lines of polarity reversal are not fixed precisely with respect to cuticular pattern. In the most extreme phenotype, polarity is reversed exactly in the middle of the tergite and in the middle of the PHZ. More frequently, the line of polarity reversal is shifted anteriorly in the tergite and posteriorly in the PHZ. The phenotype is stronger in hemizygous males than heterozygous females, and is stronger in more posterior segments. The intertergal region is often compressed, and the dorsal longitudinal muscles underlying the tergites show irregular spacing and attachment sites (Koop, 1997).

omb alleles cause defects that are reciprocal to those of the Qd alleles. Hemizygotes for omb loss-of-function alleles mostly die as late larvae or early pupae; only a small percentage survive to the late pharate adult stage. Among the latter, the loss of structures that lie within the posterior region of the anterior compartment and the anterior region of the posterior compartment have been observed. In many hemisegments, especially those more anterior in the animal, posterior tergite and PHZ are deleted and replaced with a mirror-image duplication of the anterior tergite. This phenotype is exactly reciprocal to the phenotype of Qd Fab (Koop, 1997).

Ubiquitous expression of hh causes double-posterior patterning similar to that of Qd gain of function alleles. omb- alleles suppress this effect of ectopic hh expression and posterior patterning becomes independent of hh in the QdFab mutant. These observations indicate that omb is the primary target of hh signaling in the adult abdomen. However, it is clear that other targets exist. One of these is likely to be Scruffy, a novel gene, which acts in parallel to omb. To explain the effects of omb alleles, it is proposed that both anterior and posterior compartments in the abdomen are polarized by underlying symmetric gradients of unknown origin. It is suggested that omb has two functions: (1) it specifies the development of appropriate structures both anterior and posterior to the compartment boundary and (2) it causes cells to reverse their interpretation of polarity specified by the underlying symmetric gradients (Koop, 1997).

The role of Dpp signaling in maintaining the Drosophila wing anteroposterior compartment boundary: Omb regulates the segregation behavior of cells at the A/P boundary

The subdivision of the developing Drosophila wing into anterior (A) and posterior (P) compartments is important for its development. The activities of the selector genes engrailed and invected in posterior cells and the transduction of the Hedgehog signal in anterior cells are required for maintaining the A/P boundary. Based on a previous study, it has been proposed that the signaling molecule Decapentaplegic (Dpp) is also important for this function by signaling from anterior to posterior cells. However, it has not been known whether and in which cells Dpp signal transduction is required for maintaining the A/P boundary. The role of the Dpp signal transduction pathway and the epistatic relationship of Dpp and Hedgehog signaling in maintaining the A/P boundary has been analyzed by clonal analysis. A transcriptional response to Dpp involving the T-box protein Optomotor-blind is required to maintain the A/P boundary. Further, Dpp signal transduction is required in anterior cells, but not in posterior cells, indicating that anterior to posterior signaling by Dpp is not important for maintaining the A/P boundary. Finally, evidence is provided that Dpp signaling acts downstream of or in parallel with Hedgehog signaling to maintain the A/P boundary. It is proposed that Dpp signaling is required for anterior cells to interpret the Hedgehog signal in order to specify segregation properties important for maintaining the A/P boundary (Shen, 2005).

For many years, it was thought that En and Inv regulated the segregation of A and P cells by specifying a P-type cell segregation in a cell-autonomous fashion. Recent work has challenged this view by showing that a unidirectional Hh-mediated signal from P to A cells is required to specify the A-type segregation behavior of A cells and that the role of En and Inv is mainly to control Hh signaling. Based on the findings that A cells signal back to P cells via Dpp and that wings from flies hypomorphic for dpp have a distorted A/P boundary, it has been proposed that A to P signaling by Dpp might also be important to maintain the A/P boundary. However, whether Dpp signal transduction is required for the maintenance of the A/P boundary and in which cells the Dpp signal is required remained unknown. By analyzing clones mutant for tkv, mad, and omb, several independent lines of evidence are provided that Dpp signal transduction is required to maintain the A/P boundary and that it is only required in A cells, but not in P cells. Thus, the results do not support the hypothesis that A to P signaling by Dpp is required to maintain the A/P boundary. Instead, the results suggest that Dpp signaling within Dpp-producing A cells is required to maintain the A/P boundary (Shen, 2005).

Through analysis of mutant clones located at the A/P boundary lacking the activity of the type I Dpp receptor Tkv, evidence is provided that the reception of the Dpp signal in A cells is required to maintain the A/P boundary. When generated in the P compartment, a few tkv⁻bsk⁻ clones displace the A/P boundary to a small extent: this is attributed to the unusual round shape of these clones. However, the majority of P tkv⁻bsk⁻ clones do not displace the A/P boundary, suggesting that the reception of the Dpp signal is not required in P cells to maintain the A/P boundary. In contrast, mutant clones generated in the A compartment at the A/P boundary displace the position of the A/P boundary toward P, indicating that the reception of the Dpp signal is required in A cells to maintain the A/P boundary (Shen, 2005).

How does the reception of the Dpp signal control cell segregation at the A/P boundary? Although the molecular basis is unknown, a cell's segregation behavior presumably depends on its cytoskeletal or surface properties (cell affinity). Members of the TGFβ superfamily have been observed in other systems to be able to activate regulators of the actin cytoskeleton independently of Mad/Smad transcription factors, raising the possibility that Dpp reception could control cell segregation by directly altering structural components of the responding cells. Alternatively, Dpp could control the segregation of cells by regulating the transcription of one or several target genes. To distinguish between these possibilities, the role of downstream components of the Dpp signal transduction pathway were analyzed. Three independent lines of evidence is provided that a transcriptional response to the Dpp signal is required to maintain the A/P boundary. (1) The segregation behaviors of mad⁻bsk⁻ and tkv⁻bsk⁻ clones are indistinguishable. Like tkv⁻bsk⁻ clones, A mad⁻bsk⁻ clones displace the A/P boundary toward P, indicating a role for the transcription factor Mad in A cells to maintain the A/P boundary. (2) mad⁻brk⁻ clones respect the A/P boundary, indicating that repression of brk transcription by Mad is important for normal A/P cell segregation. (3) A omb⁻ clones displace the A/P boundary toward P. The frequency and extent of the boundary displacement of A omb⁻, tkv⁻bsk⁻, and mad⁻bsk⁻ clones is comparable, suggesting that the Dpp target gene omb is the main mediator of this aspect of the Dpp signal. In contrast to omb⁻ clones, most A clones mutant for the Dpp target gene sal do not displace the A/P boundary, indicating that sal does not play an important role in maintaining the A/P boundary. Together, these data suggest that the transduction of the Dpp signal controlling the maintenance of the A/P boundary bifurcates at the level of the Dpp target genes (Shen, 2005).

Cells of tkv⁻bsk⁻, mad⁻bsk⁻, and omb⁻ clones displacing the A/P boundary do not appear to intermingle well with P cells. In fact, within the entire wing disc pouch, these mutant clones have a round shape and smooth borders, suggesting that these mutant cells in general do not intermingle freely with wild-type cells. Similar clone shapes have been reported upon mutation or misexpression of several genes, including mutants in the Dpp target gene sal and misexpression of a constitutively active form of Tkv. The round shapes and smooth borders of clones have been attributed to differences in the affinity of clone cells for their neighbors, suggesting that Tkv, Mad, and the Dpp target genes omb and sal may affect some aspects of wing pouch cell affinity. Therefore, the inability of A tkv⁻bsk⁻, mad⁻bsk⁻, and omb⁻ clones displacing the A/P boundary to intermingle well with P cells is attributed to this particular role (Shen, 2005).

Taken together, this analysis indicates two roles for Dpp signal transduction: (1) it provides some aspects of the cell affinity of both A and P wing pouch cells; (2) it is required in A cells to specify an A cell affinity important for maintaining the A/P boundary. These two roles of Dpp signal transduction could either be related or distinct. The finding that the Dpp target gene sal is required for the first role, but not the second, provides a first indication that these two roles are implemented by partially distinct molecular mechanisms (Shen, 2005).

How might Omb regulate the segregation behavior of cells at the A/P boundary? Recent work has shown that Omb has at least two roles during the patterning of the Drosophila wing. First, Omb is required for the expression of two Dpp target genes sal and vestigial (vg) (del Alamo Rodriguez, 2004). Since sal mutant clones do respect the A/P boundary, the role of Omb in maintaining the A/P boundary cannot depend on sal induction. Since Vg is required for wing cell proliferation, its role in maintaining the A/P boundary cannot be tested. Second, Omb is involved in shaping the expression pattern of tkv along the A/P axis of the wing disc (del Alamo Rodriguez, 2004). The expression of tkv is reduced in Dpp-producing A cells along the A/P boundary. This reduction of tkv expression is mediated by the transcription factor Master of thickveins (Mtv, also known as Brakeless and Scribbler, which is expressed in these cells in response to the Hh signal. Since both tkv and mtv are upregulated in omb mutant clones, it has been proposed that Omb is required for Mtv to repress tkv (del Alamo Rodriguez, 2004). However, reduction of tkv transcription in A cells does not seem to be important for the segregation of cells at the A/P boundary, because A clones either mutant for mtv, in which tkv levels are increased, or overexpressing tkv, respect the A/P boundary. Thus, neither the role of Omb in repressing tkv nor in activating sal transcription appears to be important for Omb's function in maintaining the A/P boundary. Therefore, other target genes of Omb must exist that mediate Omb's function in maintaining the A/P boundary (Shen, 2005).

Anterior cells at the A/P boundary have been shown to require Hh signal transduction to segregate from P cells. Evidence is provided that A cells in addition need to transduce the Dpp signal for normal segregation. What is the epistatic relationship between Hh and Dpp signaling? The activity of the Hh transduction pathway is not affected in either tkv⁻bsk⁻ or mad⁻bsk⁻ clones as monitored by the expression of the Hh target gene ptc, indicating that Hh signal transduction does not require Dpp signal transduction components for its activity. However, the Dpp target gene omb appears to be important for A cells to interpret the Hh signal because the ability of Ci to specify A-type segregation properties depends, in part, on the activity of Omb. Thus, Dpp signaling acts either downstream of or in parallel with Hh signaling in maintaining the A/P boundary (Shen, 2005).

Previously, three transcription factors, a transcriptional activator form of Ci (hereafter referred to as Ci[act]), En, and Inv, have been shown to be required for the segregation of A and P cells. Evidence exists for the involvement of a fourth transcription factor, the T-box protein Omb. Omb is further shown to act downstream of or in parallel with Ci. How could these four transcription factors regulate the segregation of A and P cells? In a simple model, Ci[act], En, Inv, and Omb could regulate the segregation of A and P cells by controlling the transcription of the same set of target genes that may encode cell affinity molecules or regulate the activity of cell affinity molecules. Omb is activated in both A and P cells in a broad domain centered around the A/P boundary by Dpp signaling where Omb may upregulate the expression of this putative target gene(s). The activity of Ci[act] is restricted to Hh-responding A cells along the A/P boundary. In these A cells, the target gene(s) would be further induced. En and Inv expressions are mainly confined to P cells in which they are known to act as repressors of transcription. Thus, En and Inv would repress the putative target gene(s) in P cells. The abrupt difference in the expression of putative target gene(s) would contribute to the segregation of A and P cells. Anterior clones (but not P clones) of cells lacking Omb would displace the A/P boundary because normally the putative target gene would be highly expressed in A cells, but not in P cells, where it would be repressed by En and Inv. Omb may therefore provide a basal affinity to cells in the center of the wing disc that is modified by Ci[act], En, and Inv to create a sharp difference of this affinity in cells on both sides of the A/P boundary. In an alternative model, Omb, Ci[act], En, and Inv would regulate distinct sets of genes. To distinguish among these models, it will be necessary to identify the Ci[act], En, Inv, and Omb target genes mediating cell segregation (Shen, 2005).

The precise position and shape of the Dpp organizer along the A side of the A/P boundary are important for normal growth and patterning of the wing. It has been proposed that the segregation of cells at the A/P boundary contributes to maintain this precise position and shape of the Dpp organizer in the growing wing disc epithelium. It is intriguing to notice that the Dpp-organizing activity itself plays a role in the segregation of A and P cells, suggesting that the Dpp-organizing activity contributes to maintain its own position. It will be interesting to investigate whether other organizers associated with compartment boundaries have similar functions (Shen, 2005).

Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila

Visual motion detection in sighted animals is essential to guide behavioral actions ensuring their survival. In Drosophila, motion direction is first detected by T4/T5 neurons. Their axons innervate one of the four lobula plate layers. How T4/T5 neurons with layer-specific representation of motion-direction preferences are specified during development is unknown. This study shows that diffusible Wingless (Wg) between adjacent neuroepithelia induces its own expression to form secondary signaling centers. These activate Decapentaplegic (Dpp) signaling in adjacent lateral tertiary neuroepithelial domains dedicated to producing layer 3/4-specific T4/T5 neurons. T4/T5 neurons derived from the core domain devoid of Dpp signaling adopt the default layer 1/2 fate. Dpp signaling induces the expression of the T-box transcription factor Optomotor-blind (Omb), serving as a relay to postmitotic neurons. Omb-mediated repression of Dachshund transforms layer 1/2- into layer 3/4-specific neurons. Hence, spatio-temporal relay mechanisms, bridging the distances between neuroepithelial domains and their postmitotic progeny, implement T4/T5 neuron-subtype identity (Apitz, 2018).

Visual signals received by the retina are generally not stationary because objects in the environment and/or the bodies of animals move. To detect motion, visual circuits perform complex spatio-temporal comparisons that convert luminance changes collected by photoreceptors into signals containing information about direction or speed. Despite the seemingly divergent anatomy of vertebrate and insect visual systems, they display remarkable parallels in the computations underlying motion vision and the neuronal elements performing them. In most sighted animals, this involves neurons that respond to motion signals in specific directions. Direction-selectivity emerges from differences in the connectivity of their dendrites. Motion-direction preferences by their axons are represented by layer-specific innervation. Thus, anatomical characteristics such as layer-specificity seem to be intricately linked with motion-directionality. However, how these are implemented during circuit development is poorly understood (Apitz, 2018).

The Drosophila visual system has emerged as a powerful model for elucidating the neural circuits and computations underlying motion detection. Photoreceptors (R-cells) in the retina extend axons into the optic lobe consisting of the lamina, medulla, lobula plate, and lobula. Neuronal projections in these ganglia are organized into retinotopically arranged columnar units. The medulla, lobula plate, and lobula are additionally subdivided into synaptic layers. They are innervated by more than a 100 neuronal subtypes that extract different visual features in parallel pathways. T4 and T5 lobula plate neurons are the first direction-selective circuit elements. Each optic lobe hemisphere contains ~5300 T4/T5 neurons. T4 dendrites arborize within medulla layer 10, and T5 dendrites in lobula layer Lo1. Their axons project to one of the four lobula plate layers, thereby defining four different neuron subtypes each. Axons segregate according to their motion-direction preferences. Thus, front-to-back, back-to-front, upward, and downward cardinal motion directions are represented in lobula plate layers. T4 neurons are part of the ON motion detection pathway reporting brightness increments, while T5 neurons are part of the OFF pathway reporting brightness decrements. Distinct neuron sets in the lamina and medulla relay ON and OFF information to T4 and T5 neurons. Direction-selectivity emerges within T4/T5 dendrites and involves the non-linear integration of input from these upstream neurons for enhancement in the preferred direction and suppression in the null-direction. Dendritic arbors of the four T4 neuron subtypes have characteristic orientations, that correlate with the direction preferences of lobula plate layers innervated by their axons. Thus, direction-selectivity involves the establishment of neuron subtypes, each with distinct spatial connectivities. This study addresses when and how T4 and T5 neuron subtypes with different layer identities are specified during development (Apitz, 2018).

Optic lobe neurons originate from two horseshoe-shaped neuroepithelia, called the outer and inner proliferation centers (OPC and IPC). These are derived from the embryonic optic lobe placode and expand by symmetric cell divisions during early larval development. At the late 2nd instar larval stage, neuroepithelial (NE) cells from the medial OPC edge begin to transform into medulla neural stem cells, called neuroblasts (Nbs). These undergo asymmetric divisions to self-renew and give rise to ganglion mother cells (GMCs), which divide to generate two neurons or glia. Apposing the OPC, two dorsal and ventral NE domains, called the glial precursor cell (GPC) areas, produce neuron subtypes associated with all ganglia. At the mid 3rd instar larval stage, the lateral OPC begins to generate lamina neurons (Apitz, 2018).

The IPC generates lobula and lobula plate neurons, including T4/T5 neurons from the early 3rd instar larval stage onward. Recent studies showed that NE cells in one domain, the proximal (p-)IPC, convert into progenitors in an epithelial-mesenchymal transition (EMT)-like process. Progenitors migrate to a second proliferative zone, the distal (d-)IPC, where they mature into Nbs. These transition through two competence windows to first produce C and T neurons, corresponding to C2 and C3 ascending neurons connecting the medulla and lamina, as well as T2/T2a and T3 neurons connecting the medulla and lobula, and then T4/T5 lobula plate neurons. Cross-regulatory interactions between Dichaete (D) and Tailless (Tll) control the switch in Nb competence defined by the sequential expression of the proneural bHLH transcription factors Asense (Ase) and Atonal (Ato). The latter is co-expressed with the retinal determination protein Dachshund (Dac). The molecular mechanisms that control layer-specific T4/T5 neuron subtype identities within this sequence of developmental events occurring at different locations have remained elusive (Apitz, 2018).

T4/T5 neuron diversity resulting in differential layer-specificity could be achieved by postmitotic combinatorial transcription factor codes upstream of distinct guidance molecules. Although not mutually exclusive, layer-specificity of T4/T5 neurons could also be determined by temporal differences in the expression of common postmitotic determinants, similar to the birth-order dependent R-cell growth cone segregation strategy described in the medulla. This study provides evidence for another mechanism, whereby layer-specific T4/T5 neuron subtype identity is determined early in the p-IPC neuroepithelium. Their specification depends on two relay mechanisms involving Wnt and Bone morphogenetic protein (Bmp) signaling and transcription factor interactions. These establish and translate the spatial patterning of NE cells into postmitotic neuronal subtype identities to bridge distances inherent to this particular neurogenesis mode (Apitz, 2018).

The spread of Wg is dispensable for patterning of many tissues. However, this study uncovered a distinct requirement for diffusible Wg in the nervous system, where it orchestrates the formation of T4/T5 neurons innervating lobula plate layers 3/4. Their generation depends on inductive mechanisms that are relayed in space and time. The spatial relay consists of a multistep-signaling cascade across several NE domains: Wg from the GPC areas induces wg expression in the s-IPC and Nb lineage adjacent to ventral and dorsal p-IPC subdomains; this secondary Wg source activates dpp expression. Dpp signaling mediates EMT of migratory progenitors from these subdomains. The p-IPC core produces Dac-positive layer 1/2 specific T4/T5 neurons. Dpp signaling in p-IPC NE subdomains triggers a temporal relay across intermediate cellular states by inducing omb. Omb in turn suppresses Dac, conferring layer 3/4 identity to postmitotic T4/T5 neurons (Apitz, 2018).

When Wg is membrane-tethered, the first step of this cascade is disrupted. This defect is not caused by decreased signaling activity of NRT-Wg protein in wg{KO;NRT-wg} flies. First, wild-type Wg signaling activity inside the GPC areas and the adjacent OPC was not affected. Second, in allele switching experiments, ectopic expression of a highly active UAS-NRT-wg transgene in the GPC areas was unable to rescue. By contrast, restoring wild-type wg function in the GPC areas was able to rescue, supporting the notion that Wg release and spread from the GPC areas are required to induce its own expression in the s-IPC and the Nb clone (Apitz, 2018).

Although Wg release is essential, the range of action is likely limited. Wg expression in the s-IPC commences in early 3rd instar larvae, when it is still in close proximity with the GPC. Half of the wg{KO;NRT-wg} flies showed residual dpp expression in one progenitor stream at the 3rd instar larval stage and a 25% reduction of T4/T5 neurons, correlating with three lobula plate layers in adults. The other half lacked dpp-lacZ expression and showed a 50% reduction of T4/T5 neurons correlating with two remaining layers. While this partial phenotypic penetrance is not fully understood, NRT-Wg likely partially substituted for Wg because of the initial close proximity of the GPC areas and the s-IPC and Nb clone. Occasional residual NRT-Wg expression in the s-IPC argues against an all-or-nothing inductive event and suggests a model, whereby cell-intrinsic signaling thresholds have to be reached. Theoretically, the dpp expression defect in the p-IPC of wg{KO;NRT-wg} flies could reflect the dependence on long-range Wg from the GPC areas. However, as this study has shown, IPC-specific wg knockdown leads to dpp loss in the p-IPC. Propagation of sequential Wnt signaling could explain long-range activities. Moreover, sequentially acting primary and secondary sources of Wg have been described in the developing Drosophila eye, suggesting that the regulatory mechanism observed in the optic lobe might be employed in several contexts. The different outcomes of early and late allele wg to NRT-wg allele switching indicate that Wg secretion is required for the induction but not long-term maintenance of wg expression in the s-IPC. The GPC areas become rapidly separated from the s-IPC and Nb clone by compact rows of newly generated neurons. As part of a relay system, diffusible Wg may therefore be required to bridge distances over a few cell diameters during the initial phase of neurogenesis. The s-IPC in wg{KO;NRT-wg} flies expressed Hth and generated two neuron clusters as in wild-type. Thus, the sole function of wg in the s-IPC is to relay the GPC-derived Wg signal to induce dpp expression in the p-IPC. Since Wg release is not required in the GPC areas to induce dpp in the adjacent OPC, this secondary wg function in the s-IPC is most likely juxtacrine (Apitz, 2018).

Compared to approximately 80 medulla neuron subtypes derived from the OPC, the specification of 13 distinct subtypes originating from the p-IPC appears simple. However, the distinct mechanisms employed are surprisingly complex. Previous work has shown that cross-regulatory interactions between D and tll regulate a Nb competence switch from generating early-born C2, C3, T2, T2a, and T3 neurons to eight distinct layer-specific T4/T5 subtypes. Ato and Dac are expressed in the second Nb competence window and depend on tll. Functional studies showed that dac mutant T4/T5 neurons adopted early-born T2/T3 neuron-like morphologies. Similarly, ato mutant T4/T5 neurons displayed neurite connectivity defects. Notably, simultaneous knockdown of dac and ato resulted in the absence of T4/T5 neurons, demonstrating that both are required together for the ability of d-IPC Nbs to produce new neuron subtypes in the second competence window (Apitz, 2018).

Dac is initially expressed in all T4/T5 neurons but only maintained in layer 1/2 innervating subtypes. This suggests that an essential step for the specification of layer 3/4 innervating neurons is the downregulation of Dac and the suppression of the T4/T5 default neuron fate, i.e., layer 1/2 identity. Although the mode of this inhibitory mechanism depends on the outcome of the Nb-specific switching mechanism in the d-IPC, it is already primed in p-IPC NE cells. Thus, layer-specificity and therefore motion-directionality are determined early in the NE precursors of T4/T5 neurons. Molecularly, it involves the Omb-mediated relay of Dpp-signaling-dependent NE cell patterning information across intermediate cell states to postmitotic T4/T5 neurons resulting in the repression of Dac. In contrast to the OPC, this study found no link between NE patterning in the p-IPC and Notch-dependent differential apoptosis of region-specific T4/T5 subtypes. Instead, Notch controls the choice between T4 and T5 identity, likely during the second competence window, indicating that the distinction between layer 1/2 and 3/4 fates precedes T4 and T5 neuron specification (Apitz, 2018).

The mechanisms controlling the maintenance of omb expression, and Omb-mediated downregulation of Dac are unclear. Hypotheses regarding the latter have to be reconciled with the fact that dac, together with ato, is required for the formation of all T4/T5 neurons and hence is expressed in all d-IPC Nbs during the second competence window. Omb and Dac are initially co-expressed in Nbs and young T4/T5 neurons, suggesting that Omb does not directly repress dac transcription. Yet, expression of the dacp7d23 enhancer trap Gal4 line showed that dac is only transcribed in layer 1/2 neurons in adults. A possible scenario is that Omb could break Dac autoregulation by triggering degradation of Dac. Since T-box genes can act as transcriptional activators and repressors and their effects are influenced by various co-factors, future studies will need to explore the molecular details underlying Omb-mediated repression of Dac. It will also be important to determine whether layer 3/4 specification is mediated solely by Dac downregulation, or whether omb has additional instructive roles (Apitz, 2018).

Consistent with the observation that C2 and C3 neurons have distinct developmental origins, this study found that Nbs derived from the Dpp-expression domain produce C2 and possibly T2a neurons during the first Nb competence window, while the core p-IPC generates C3, T2, and T3 neurons. dac mutant T4/T5 neurons adopt T2/T3-like morphologies suggesting that this is the default neuron fate in this neuron group. While Omb is maintained in C&T neurons derived from the Dpp-expression domain, Dac is not expressed, suggesting that Omb interacts with other molecular determinants in these neurons. While this study did not explore how layer 1 and 2 neurons or layer 3 and 4 neurons become distinct from each other because of the lack of specific markers, the data suggest a possible contribution of Ato/Dac and Notch signaling, as these are active within the d-IPC. Findings in a concurrent study of Pinto-Teixeira (2018) align with the current data concerning the role of Dpp and Notch signaling. Furthermore, a second study of Mora (2018) reported an additional role for Ato in controlling the transient amplification of d-IPC Nbs by symmetric cell division to ensure that the correct number of T4/T5 neurons is produced. It will be fascinating to identify the transcriptional targets of Notch, Ato/Dac, and Omb that mediate ganglion- and layer-specific targeting of T4/T5 dendrites and axons, respectively. Finally, future behavioral studies of layer 3/4-deficient flies will address to what extent direction selectivity is affected or compensatory mechanisms are in place (Apitz, 2018).

Signaling centers, also called organizers, pattern tissues in a non-autonomous fashion. The vertebrate roof plate and the cortical hem, for instance, both release Wnts and Bmps to pattern NE cells in the developing dorsal spinal cord and in the surrounding forebrain, respectively. In the Drosophila visual system, the GPC areas express wg and pattern the OPC by inducing dpp expression in adjacent dorsal and ventral OPC subdomains. Together with the current insights into the function of GPC-derived wg in IPC patterning and neurogenesis, this firmly establishes the GPC areas as local organizers of optic lobe development. At the onset of neurogenesis, wg is first expressed in the GPC areas followed by the s-IPC, explaining the well-established delay in neurogenesis between the IPC and OPC. Wg release from the GPC areas could coordinate the timely onset of neurogenesis in the OPC and IPC to safeguard the alignment of matching partner neurons across several retinotopically organized neuropils. The intercalation of new-born neurons between both neuroepithelia may have driven the need for a relay system using primary and secondary sources of Wg. Wg induces Dpp to subdivide the adjacent OPC and p-IPC NE into distinct regions as basis for generating neuronal diversity. The temporal relay mediated by Omb represents an efficient strategy to pass the memory of spatial NE patterning information by Dpp signaling on to postmitotic neurons generated at a distance. It is thus intricately tuned to the distinct neurogenesis mode of the p-IPC essential for spatially matching birth-order-dependent neurogenesis between the OPC and IPC. Interestingly, the progressive refinement of NE patterning by the induction of secondary signaling centers plays a central role in vertebrate brain development. Furthermore, similar signaling cascades have been recently identified in mammalian optic tissue cultures where sequential Wnt and Bmp signaling induces the expression of the Omb-related T-box transcription factor Tbx5 to specify dorsal retinal NE cells. Hence, such cascades could represent conserved regulatory modules that are employed repeatedly during invertebrate and vertebrate nervous system development (Apitz, 2018).

GENE STRUCTURE

optomotor blind DNA spans about 120 kb; the transcript extends 80 kb, with a 40 kb 3' regulatory region (Pflugfelder, 1990, 1992a and 1993).

PROTEIN STRUCTURE

Amino Acids - 974

Optomoter blind is a T-box DNA-binding protein. The T-box family is an ancient group that appears to play a critical role in development in all animal species. These genes were uncovered on the basis of similarity to the DNA binding domain of murine Brachyury (T) gene product, the defining feature of the family. Common features shared by T-box family members are DNA-binding and transcriptional regulatory activity, a role in development and conserved expression patterns, most of the known genes in all species being expressed in mesoderm or mesoderm precursors.

date revised: 3 DEC 97 

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