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TheÂ optic lobe forms a prominent compartment of the Drosophila adult brain that processes visual input from the compound eye. Neurons of the optic lobe are produced during the larval period from two neuroepithelial layers called the outer and inner optic anlage (OOA, IOA). In the early larva, the optic anlagen grow as epithelia by symmetric cell division. Subsequently, cells convert into neuroblasts in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually moves towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing the lineages of neurons that populate the different parts of the optic lobe. In this paper we have reconstructed the complex morphogenesis of the optic lobe during the larval period, and established a role of the Notch and JAK/STAT signal pathway during the step in which epithelial cells of the OOA are converted into neuroblasts. We found that after an early phase of complete overlap in the OOA, signaling activities sort out such that JAK/STAT is active in the lateral OOA that gives rise to the lamina, and for upd is transiently expressed in the OOA, resulting in JAK/STAT activation and subsequently, Notch signaling. During early developing OOA, continuous Notch signaling is needed to maintain NE cells in the OOA. Both Notch and JAK/STAT are necessary for proper timing of NE differentiation. At the onset of NE-NB transition, JAK/STAT is activated in younger NE cells and Notch, in older NE cells that are ready to differentiate into asymmetric NB. Here we propose a model to describe the dual requirement of JAK/STAT and Notch signaling for NE cellular differentiationÂ in vivo.
Keywords: optic lobe, larval neurogenesis, neuroblasts, neuroepithelial, Notch, JAK/STAT, Unpaired, Delta, lineages
Neurons and most glial cells of the Drosophila brain are generated by a population of progenitor cells called neuroblasts. Neuroblasts divide asymmetrically in a stem cell mode, producing with each mitosis another neuroblast and a smaller daughter cell, the ganglion mother cell, which after one more round of division differentiates into neurons or glial cells. Neuroblasts of the central brain and ventral nerve cord (analog of the vertebrate spinal cord) are born in the early embryo; these neuroblasts are relatively few in number (less than 500 in all), and each one produces an invariant "lineage" of neurons/glial cells. By contrast, the optic lobe, in terms of cell number by far the largest part of the insect brain, is formed by neuroblasts that are born in the late larva from two neuroepithelial layers called the inner and outer optic anlagen (IOA, OOA). The optic anlagen arise in close proximity to the eye imaginal disc in the embryonic head ectoderm (Green et al., 1993). In the early larva, both IOA and OOA grow by symmetric cell division. With the beginning of the third instar (about half way through the larval period), epithelial cells convert into neuroblasts in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually moves towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing the lineages of neurons that populate the different parts of the optic lobe. Optic anlagen, neuroblasts and lineages derived from them together form the complex optic lobe primordium that accounts for fully half of each late larval brain hemisphere.
Neuroblasts of the central brain and ventral nerve cord are specified in a two-step process. During the first step, discrete clusters of neurectodermal cells ("proneural clusters") express a combination of regulatory genes, the proneural genes, which makes the cells "comptetent" to form neuroblasts. Proneural genes encode DNA binding proteins that belong to the large family of basic helix-loop-helix (bHLH) transcription factors, including the Achaete-Scute proteins and their vertebrate homologs (e.g., Mash-1 in mouse; Campuzano and Modolell, 1992; Kageyama et al., 1995; Guillemot, 1999; Lo et al., 2002). In the second step of neuroblast specification, called lateral inhibition, cells of each proneural cluster "compete" with each other to become a neuroblast. On the molecular level, this competition is mediated by the Notch signaling pathway, whose members are encoded by the so-called neurogenic genes (). Expression of the Notch ligand Delta is upregulated within proneural clusters by the proneural bHLH genes. Binding of Delta causes a conformational change followed by a cleavage of the Notch receptor. The released intracellular Notch fragment (NICD) moves into the nucleus and upregulates the expression of the Enhancer of split-complex (E(spl)-C; consisting of the loci mÎ´, mÎ³, mÎ², m3, m5, m7, m8 and groucho [Delidakis et al., 1991; Delidakis and Artavanis-Tsakonas, 1992; Knust et al., 1992; Schrons et al.,1992]). The expression of these genes, which also encode transcription factors of the basic helix loop helix family (bHLH), down-regulates the transcription of the AS-C-gene complex, which then causes the cell to abandon its neural fate and become epidermal instead. In neurogenic mutants ( Lehmann et al., 1983), in which lateral inhibition is perturbed, expression of the proneural genes does not become restricted to individual cells, but persists in all cells of the proneural cluster (Brand; Cabrera; Skeath and Ruiz; Martín-Bermudo et al., 1995). As a result all cells of the neurogenic region become neuroblasts, a phenotype called neural hyperplasia.
It stands to reason that the epithelial-to-neuroblast transition that occurs in the optic anlagen also requires the proneural gene-neurogenic gene cassette. A recent study () demonstrated that the proneural gene lethal of scute (l'sc) is indeed expressed in a narrow band of cells that fall within the epithelium-to-neuroblast transition zone of the OOA. Furthermore, l'sc expression was shown to be negatively regulated by the Janus Kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascade (Yasugi et al., 2008). Thus, loss of JAK/STAT activity in clones results in excess cells expressing l'sc, and, consecutively, becoming neuroblasts. This finding matches similar results with JAK/STAT signaling in the vertebrate retina, where it is also required to inhibit premature formation of neurons from neuroepithelial cells (Farkas and Huttner, 2008; Huttner and Kosodo, 2005; Huttner and Brand, 1997 which one?).
In this paper we have investigated the role of Notch signaling and its interaction with JAK/STAT in the Drosophila optic lobe. Here we report of an additional regulator of the proneural wave: Notch signaling that is regulated on two levels. At the most lateral edge, Notch is antagonized by JAK/STAT. NE cells free from JAK/STAT signaling express Delta (Dl), which in turn activates Notch in adjacent NE cells. A gradient of Notch signaling is then necessary for the synchronous NE-NB transition; cells with high Notch repress the proneural genes through E(spl)-C, similar to that of the SOP (Lai et al., 2004; Kopan and Ilagan, 2009). As with JAK/STAT signaling, Notch moves from medial to lateral where the most medial NE cells are free from Notch repression, resulting in L(1)sc expression and NE-NB transition. The dual spatial and temporal regulation of Notch and JAK/STAT are thus necessary to specify the diverse cells types that constitute the Drosophila optic lobe.
Flies were grown at 25oC otherwise noted. The following mutant and transgenic strains were used in this study: UAS 2X YFP (Bloomington, IN), temperature-sensitive alleles: Nts1 (Xu et al., 1992), Nts2 (Bloomington, IN), stat92EF (Baksa et al., 2002); enhancer trap lines: upd-GAL4 (Halder et al., 1995; Tsai and Sun, 2004) and dome-GAL4 (Ghiglione et al., 2002; Brown et al., 2001) is an enhancer trap line of upd and dome, respectively; reporter lines: Gbe+Su(H)m8-lacZ (Furriols and Bray, 2001), 10XStat92E-GFP (Bach et al., 2007). stat92E85C9 is a strong hypomorphic allele of stat92E. stat92E85C9/stat92EF and Nts1/Nts2 flies were raised at 29oC.
For all lineage expression analysis, we used the Gal4 Technique for Real-time and Clonal Expression (G-TRACE), a novel in vivo lineage tracing system to mark real-time and cells derived from a particular lineage using fluorescent proteins (Evans et al., 2009; Figure 3A).
Samples were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS, Fisher-Scientific, pH = 7.4). Tissues were permeabilized in PBT (phosphate buffer saline with 0.1% Tween-20, pH = 7.4) and immunohistochemistry were performed using standard procedures (Ashburner 1989). The following antibodies were provided by the Developmental Studies Hybridoma Bank (Iowa City, IA): Dachshund anti-mouse (mAbdac2-3, 1:600), Delta anti-mouse (1:10), Neurotactin (Nrt) anti-mouse (BP106, 1:10), DN-Cadherin anti-rat (DN-EX #8, 1:100; Iwai et al., 1997), Shotgun anti-rat (DCAD2, 1:100), Armadillo anti-mouse (N2 7A1, 1:40), FasciclinII anti-mouse (1D4, 1:500). Î²eta-Galactosidase anti-mouse (Î²-Gal, 1:100) and Î²-Gal anti-rabbit (1:100) were obtained from Cappel. Secondary antibodies, IgG (Jackson ImmunoResearch; Molecular Probes) were used at the following dilutions: Cy3-conjugated anti-mouse (1:200), Cy5-conjugated anti-mouse (1:200), Fluorescein (FITC)-conjugated anti-mouse (1:200), Cy3-conjugated anti-rabbit (1:200), Cy5-conjugated anti-rabbit (1:200), FITC-conjugated anti-rabbit (1:200), Alexa Fluor 488-conjugated anti-mouse (1:200), AlexaFluor 546-conjugated anti-mouse (1:200). Phalloidin 547 and Phalloidin 633 (Molecular Probes), used to visualize actin filaments were diluted in PBTA (pH = 7.4, phosphate buffer saline, 0.1% Triton X-100, 2% BSA), 1:100. TO-PRO-3 (Invitrogen, 1 ÂµM in PBTA) was used as a nuclear stain. Tissues were mounted in Vectashield mounting medium (Vector, Burlingame, CA, #H1000).
Structure and development of the larval optic lobe
The structure of the optic lobe primordium of the larva is highly dynamic and, towards later stages, very complex. As a result, we have currently only a rudimentary understanding of how the different neuropiles and cell types of the adult optic ganglia map onto the larval optic lobe primordium. Furthermore, the dynamic changes in shape that characterize the optic lobe at different larval stages make it very difficult to interpret mutant phenotypes of genes controlling optic lobe development. We will in the following provide first a description of the late larval optic lobe primordium and its topological relationship to the mature optic lobe, and then show the developmental changes occurring in the optic lobe primordium between the early and late larval stage. We will mainly focus on the outer optic anlage and its derivatives, the distal (outer) medulla, and the lamina.
For didactic purposes, the OOA can be pictured as a rectangular sheet of epithelial cells (Fig.1A1). Symmetric division increases the size of the OOA along both x-axis and y-axis. (As discussed further below, x corresponds to the medio-lateral axis of the OOA, y, when discounting the curvature of the OOA, represents the dorso-ventral axis.) During the second larval instar, a polarization of the OOA takes place that is accompanied by dramatic differences in morphogenesis and gene expression patterns along the x-axis (Fig.1A2, 1A3). The lateral fringe of the OOA (cells lateral of the boundary, called lamina furrow, indicated by green line in Fig.1A) bends outward; this lateral OOA (OOAl) gives rise to the lamina. The (much larger) medial OOA (OOAm) will form the distal medulla.
At around the time when the lamina furrow divides the OOA into a lateral and medial domain, epithelial cells along the edges of these domains convert into asymmetrically dividing neuroblasts (; Fig.1A3, 1A4). Neuroblasts "bud off" progeny in the direction perpendicular to the plane defining the OOA. Because of this directed proliferation, neurons born first come to lie at ever increasing distances from the neuroblast/OOA (Fig.1A4-8, shown only for OOAm). At the same time as the neuroblasts divide, new rows of neuroblasts appear as, one by one, rows of epithelial cells along the y-axis convert into neuroblasts (Fig.1A5-8). This gradual temporal progression of epithelial-to-neuroblast conversion can be seen most clearly, and has been described in previous works (), in the large medial OOA. It is less clear for the OOAl which, from the beginning, contains only few rows of epithelial cells. However, also in the OOAl, epithelial cells and budding neuroblasts coexist throughout the larval period ().
The description of OOA development above indicates that two spatio-temporal gradients are built up along the medial and lateral edge of the OOA. One gradient, directed along the x-axis of the OOA ("x-gradient"), describes the sequence in which rows of neuroblasts are formed; the second gradient, directed perpendicular to the plane of the OOA ("z-axis"), underlies the order in which each neuroblast produces neurons ("z-gradient"). It is not yet fully clear how these gradients are translated into the structure of the emerging optic lobe neuropiles. It has been reported in the literature that for the OOAl, the z-gradient corresponds to the antero-posterior axis of the lamina (). It is well possible that both z-gradient and x-gradient correlate with the ap-axis of the lamina, but due to the absence of conclusive data we will not further comment on this part of the OOA, For the medial OOA, giving rise to the distal medulla, the by far largest optic ganglion, it is clear that the it is mostly the x-gradient that correlates with the ap-axis of the medulla. Thus, axons that grow towards the first born OOAl neurons, derived from the most medial row of neuroblasts, are the R7/8 axons originating from posterior retina, as well as L- neurons from the posterior lamina (Fig.1A5). The next set of axons, arriving later, captures neurons of the next (more lateral) row of OOAm neuroblasts, etc. What this implies is that the progression of epithelial-to-neuroblast transition may match the progression of ingrowing axons, and that this matching is important for the formation of an ordered medulla neuropile. We show evidence for this hypothesis in the sections further below.
The OOA is attached to the lateral surface of the larval brain hemisphere in such a way that its x-axis is aligned with the medio-lateral brain axis, and the y-axis, in principle, with the dorso-ventral axis (Fig.1B1). What adds to the structural complexity of the larval optic lobe is the fact that the OOA bends along the y-axis (Fig.1B2). As a result, instead of forming straight rows along the y (dorso-ventral) axis, cells are aligned in C-shaped curves (Fig.1B, C). What this spatial transformation means when looking at optic lobes sectioned along the "standard" frontal plane (Fig.1D) is that the OOA is sectioned twice, once dorsally, and once ventrally.
Fig.2 shows in some detail the different components of the late larval optic lobe primordium. The epithelial part of the OOA (blue solid lines) is flanked medially and laterally by neuroblasts (blue circles) that produce the distal medulla (Md) and lamina (L), respectively. The medial neuroblasts generate lineages of the distal medulla that are directed centro-laterally (hatched lines with arrows). The oldest lineage (l1) is the one situated furthest medially; the youngest, most recently borne one (ln) is the one furthest laterally. Within each lineage, central neurons (nc) are older than peripheral ones (np). Neurons form bundles of axons that gather at the base of the lineages. Along with ingrowing axons from lamina neurons (L) and retinal axons (not shown) this mass of fibers gives rise to the neuropile of the distal medulla (Mdnp; shaded blue).
The inner optic anlage (IOA), which was not considered so far, also undergoes a transition from epithelium to neuroblasts, and a bending along the y-axis, similar to what has been described above for the OOA. The details of this transformation are described elsewhere (); briefly, in the late larva, the IOA consists of a C-shaped epithelial component (IOAep; red lines; note that, as for the OOA, the IOA sectioned along the frontal plane appears twice). Further laterally, neuroblasts derived from the IOA (IOAnb; red circles) form a mass of cells that is also bent, and therefore sectioned twice. The IOA neuroblasts produce two populations of neurons. Neurons pushed anteriorly (or outward, taking into account the curvature of the IOA) become the proximal medulla (Mp); those pushed interiorly, or centrally, the lobula (Lo). Fig.2 illustrates the arrangement of the optic lobe neuropiles in the late larva (), and the correspondence to their adult counterparts.
Activity of Notch and Jak/Stat in the larval optic lobe primordium
A pivotal step in the devepment of the optic lobe is the formation of neuroblasts from a neuroepithelium. As established for neuroblast formation in the embryo, the number of neuroblasts emerging at any given time point appears to be tightly regulated. It has been recently documented that JAK/STAT signalling plays a role in the transition from epithelial cells to neuroblasts (). We wanted to investigate the function of Notch signalling, a common player during the selection of neuronal progenitors, and its interaction with the JAK/STAT pathway.
In the early larva, expression of the Notch ligand Delta and Notch activity (monitored by..), as well as Stat activity, are found ubiquitously at moderate levels in the OOA and IOA (Fig.). Delta and m8-lacZ are also expressed in all neuroblasts of the central brain and their early progeny; Stat activity is high in glial cells (Fig.). At the time when the OOA has expanded and is beginning to differentiate into a lateral and medial domain, Stat becomes increasingly restricted to the lateral domain (OOAl). In the late larva, Stat is exclusively in the OOAl and the lamina progenitors/lamina neurons derived from it (Fig.). Both the JAK/STAT ligand upd3(?) and its receptor, domeless, after an initial phase of widespread expression in the OOA and IOA, also become restricted to the OOAl and lamina (Fig.). The restriction of expression can be confirmed experimentally by using the G-TRACE construct (Gal4 Technique for Real-time and Clonal Expression) (Evans et al., 2009), which combines the Gal4/UAS gene expression system in concert with the FLP/FRT recombination system (Theodosiou and Xu, 1998) to visualize real-time and lineage-traced gene expression pattern (Fig. ). While real time upd expression is restricted to the OOAl and lamina during late larval development (marked by RFP in Figure 3B'), a large proportionÂ of the optic lobe is labelled by GFP, demonstrating that these cells are derived from cells that an earlier stage expressed upd.
At the stage when the epithelium-to-neuroblast transition begins at the medial edge of the OOA, the activity of the Notch/Delta pathway becomes centered aroundthis transition line. Delta remains always restricted to the epithelial part of the OOAm. Laterally, it partially overlaps with the domain of Stat92E expression, i.e., it reaches into part of the OOAl, but does not extend all the way to its lateral rim (Fig.). Medially, Dl expression declines at the epithelium-neuroblast border line. It is still expressed in the most recently formed neuroblasts (right adjacent to the epithelium), but strongly declines in older, more medial neuroblasts. Notch activity, monitored by expression of m8-lacZ, is high in the OOAm, overlapping with the expression of Dl. It is highest in the newly formed neuroblasts, and (in contrast to Dl) stays on in older neuroblasts and their progeny (Fig.). No expression of m8-lacZ is observed in the OOAl and the neuroblasts/neurons of the lamina that develop from it.
Loss of Notch leads to a precocious release of neuroblasts from the OOA
The dynamic expression of Delta and M8-lacZ in the optic anlagen suggests that Notch signalling is involved in the ordered release ("delamination") of neuroblasts from the anlage. This hypothesis predicts that in larval brains where Notch activity is reduced, the epithelial optic anlagen are smaller, and neuroblasts (as well as their progeny) appear precociously. In order to investigate the function of Notch during larval neurogenesis a 29Â°C temperature shift of hatching Nts 1st to late 3rd instar larvae was performed. 29Â°C is the restrictive temperature where the Notch-TS allele begins a conformational change and is not functional anymore. This state is reversible by dropping the temperature to the permissive temperature of 18Â°C. Raising embryos at the permissive temperature resulted in wild-type early larvae, with a normal number of neuroblasts and normal neural differentiation. The Notch-loss-of function phenotype resulting from the temperature shift evolved during the larval period and was investigated in late (wandering) third instar larvae. These late 3rd instar larvae were dissected and stained with markers for neurons, neuropile, and optic lobe epithelia. For the latter, we used an antibody against the membrane protein Crumbs which highly specifically labels the apical surfaces of the optic anlagen (Tepass et al., 1992?).
In wild-type, Crumbs expression (Figure 10B) labels C-shaped belt that demarcates the outer optic anlage (Fig.). In Nts mutant brains the Crumbs-positive domain was largely decreased in size and did not show its typical C-shape (Figure 10/11). The inner optic anlage of the mutant was not visible at all. This reduced size of the optic anlagen in late larval brains is caused by the premature conversion of epithelial cells into neuroblasts. Optic lobe neuroblasts and their progeny (GMCs and neurons) are positive for BP106 (Fig.: wt control), whereas the optic anlagen are distinctively BP106-negative. The misshapen optic lobe of Nts1 mutant larvae are strongly positive for BP106. As in wild-type controls, DNcad comes on in deep layers of the medulla primordium, suggesting that differentiation of these cells sets in. At the same time, the evenly distributed columns of neurons (most easily seen in the medulla primordium) and axons that represent the nascent medulla columns in the wild-type brain are disrupted in the mutant. Here, medulla neurons form more irregularly sized clusters with thicker axon bundles emanating from them (Fig.).
Notch related defects in optic lobe connectivity
Photoreceptor axons R1-R6 start growing into the brain during the middle of the 3rd instar (Hofbauer and Campos-Ortega, 1990) and make connections with cells of the lamina. R7 and R8 axons continue past the lamina and terminate in the external medulla. Lamina neurons (L-neurons) also extend axons towards the external medulla. External medulla neurons connect to the lobula complex. The internal medulla forms axons towards the central brain (posterior optic tract); likewise, the lobula complex projects multiple tracts towards the optic foci of the central brain (refs: Ito, ). Several of these fiber systems can be labeled with an antibody against Fasciclin II (FasII): all retinal axons; the lamina-medulla pathway, the posterior optic tract, and the lobula-central brain connection.
Not surprisingly, connectivity defects were profound in Nts mutants, given that Notch is known to be required for the pattern of retinal cell types (). Previous studies had shown that almost all photoreceptor neurons express the fate of R8 cells, whereas the later born R1-6 and R7 are reduced or absent (Cagan; other refs). The R8 neurons of Nts1 mutant brains form axons that do not properly bundle into thin fascicles, but form thick and irregular bundles that completely bypass the lamina and terminate at a deep level within the medulla primordium. Note that the medulla primordium, aside from the fact that it receives more massive input from the retina, is also misshapen due to the fact that medulla neuroblasts/neurons are born in an abnormal temporal pattern. Thus, in wild-type, the epithelial OOA grows to a large size and forms the "dome" covering the entire lateral surface of the brain (see previous section). Subsequently neuroblasts are released in a well ordered succession from the margin of the OOA. As a result the medulla primordium is large in surface area and small in depth. This is not the case in the Nts1 mutant: here the OOA does not grow to a large size because cells are prematurely converting into neuroblasts. The medulla is larger in depth and smaller in surface area. This altered topolgy, in addition to altered retinal input, may account for the entirely different architecture of the medulla neuropile (mark on fig).
The effect of loss of Notch function on the lamina primordium is also complex. FasII labeling of nascent lamina neurons is visible in wild-type at the lateral margin of the OOA (i.e., the lateral proliferation center; Fig.). Axons of these neurons fasciculate with the afferent retinal axons (Fig.; note that it is not possible to distinguish between retinal and lamina-derived axons in anti-FasII-labeled brains). In Nts1 mutants, FasII-positive neurons appear to be absent. Thus, FasII-label cannot be detected in the immediate vicinity of the rudimentary OOA (Fig.). This interpretation is further confirmed by the absence of the lamina neuronal marker, Dachshund (Dac; see section below). In wild-type, Dac expression appears at a low level in the LPC and is strongly upregulated in postmitotic lamina neurons (Fig.). This lamina-specific expression of Dac is reduced or absent in Nts1 mutant brains (Fig.). Lamina neurons could be absent in Nts1 mutant brains because of the change in retinal axonal identity, and/or because of an autonomous effect of Notch on lamina neuronal identity. Thus, previous work had clearly demonstrated an inductive effect of retinal axons on their target, the lamina. Eyeless mutations, for example, lack lamina neurons (refs). In nts1 mutants, most/all retinal axons have an R8 identity and pass through the lamina, thereby unable to provide the inductive effect needed for lamina neurons to become specified (is there anything in the literature to this effect?). On the other hand, it cannot be excluded that Notch acts directly on the lamina neuroblasts directly, although it seems unlikely that such an effect by itself would explain the lack of lamina neuronal specification: in the medulla, after all, neurons seem to appear in large numbers in the Nts1 mutant.
Loss of Jak/Stat signaling mimicks many aspects of reduced Notch signaling in the larval optic lobe
The use of molecular markers that switch expression as epithelial cells undergo transformation into neuroblasts had shown that the Jak/Stat pathway, similar to Notch signaling, may act as an inhibitor of neuroblast formation (). To verify that the structural phenotype ensuing from loss of Jak/Stat signaling follows this prediction, we used markers for optic lobe epithelium, neuroblasts, and neurons in the background of a temperature sensitive mutation of Stat (Statâ€¦). Late larvae that had developed at the restrictive temperature from hatching onward showed a significant reduction of the OOA neuroepithelium (Fig.5..). At the same time, neuroblasts and their lineage occupied the entire surface area of the optic lobe primordium (Fig.). This phenotype was confirmed by applying short BrdU pulses () to Stat ts larvae. In wild type controls, BrdU incorporation is mostly confined to the medial OOA where cells have converted into neuroblasts, which cycle much more rapidly than neuroepithelial cells (Fig.). In Statts mutants, BrdU incorporation is seen over the entire surface of the OOA, supporting the conclusion that these cells have prematurely converted into rapidly cycling neuroblasts (Fig.).
To test whether JAK/STAT has the potential to inhibit Notch activation in lateral NE cells; we placed the Notch transcriptional reporter, Su(H)m8-LacZ, in the background of Stat92EF/Stat92E85C9 mutants. Loss of STAT signaling resulted in Notch activation in all cells along the neuroepithelial layer (Figure 8J-K'). These results suggested that high JAK/STAT signaling is needed to elicit a proper Notch inhibitory response along neuroepithelial layer. Both Notch and JAK/STAT are thus necessary for the proper timing of NE-NB conversion. Cells in which JAK/STAT signaling is compromised resulted in precocious NB differentiation. Basal JAK/STAT signaling activates Dl, resulting in Notch activation along the most medial NE cells, which are ready to differentiate into medullar NB.
Neural progenitors give rise to the diversity of cell types seen in the central nervous system (CNS). Intrinsic factors expressed in progenitors, as well as extrinsic cues from neighboring cells specify cell fate. In both vertebrates and invertebrates, the specification of cell types follows a highly invariant spatio-temporal pattern. Typically, one can distinguish an early phase where the pool of progenitors (e.g., the neuroepithelium of the neural tube in vertebrates) expands by symmetric cell division. Subsequently, progenitors start leaving the pool of expanding cells and either directly differentiate into specific cell types, or undergo asymmetric divisions where one daughter cell keeps the properties of a progenitor, whereas the other differentiates (). Neurogenesis in the Drosophila optic lobe follows a similar pattern. Segregating from the embryonic neuroectoderm as a small epithelial placode, the optic lobe undergoes a phase of growth by symmetric cell division in the early larva, followed by a highly ordered transition into asymmetrically dividing neuroblasts. The medio-lateral gradient characterizing this transition is correlated with the posterior-anterior gradient of eye development: photoreceptor axons of the earliest developing (posterior) row of ommatidia arrive first and capture the first born neurons, formed (in case of the medulla) from the medial edge of the OOA. Later born axons occupy medulla neurons forming later, at increasingly lateral levels. It is to be assumed that this temporal match between target neuronal development and afferent axonal development plays an important role for correctly wiring the optic lobe; this hypothesis, though, requires rigorous testing.
Notch activity controls the epithelium-neuroblast transition in the optic lobe
We show in this paper that the Notch signaling pathway is critically involved in the ordered epithelium-neuroblast transition. The most significant effect resulting from decreasing Notch function in the larval brain was the reduction of the OOA. Given the fact that medulla neurons (the progeny of the OOA) are still present in normal, if not increased, numbers, the most likely explanation for this phenotype is a precocious transition of neuroepithelial cells into neuroblasts The inner and outer anlagen start out as two separate epithelial layers in the late embryo. During the first half of larval life these epithelia grow by symmetric division of epithelial cells. From approx. 60h of larval life onward epithelial cells of the anlagen convert to neuroblasts; as shown here, by the late third instar, only about 30% of the OOA and IOA remain epithelial. In N mutants, epithelial cells are all but gone from the optic anlagen. It is therefore likely that the function of N in the optic anlagen is to maintain its epithelial state. This would match a similar function of N in the embryonic neurectoderm, where N activity is also required for cells to stay epithelial (). The only difference is the topology of the neuroblast (ie, cell that moves out of the epithelium): in the embryonic neurectoderm, neuroblasts are mostly scattered cells, surrounded on all sides by epithelial cells. In the optic anlagen, there is a continuous front where all epithelial cells convert to neuroblasts.
However, this difference aside, the way in which N signaling acts and is controlled could be quite similar. From the first appearance of the optic anlagen onward, both Notch activity and expression of the N ligand Dl is high. It has been reported previously () that in the late embryo, the optic anlagen, as well as a few other "placode-like" neuroepithelia, are the only tissues that maintain expression of the E(spl), which are direct downstream targets activated by N (). In the larva, Delta expression and N activity remains high in the epithelium, with the exception of its lateral margin, the OOAl, where both are reduced possibly as a result of Jak/Stat activity (see below). The continuously high N activity maintains the OOA epithelium. Starting during mid larval life, the proneural gene l'sc is expressed at the medial margin of the OOA (). This expression sets in motion a cascade of genes, including other proneural genes (ase), that promote first the conversion of OOA epithelium to neuroblasts, followed by rapid asymmetric division and neuronal differentiation. At the same time, once cells have converted to neuroblasts, l'sc, Dl and N are downregulated, even though N stays on in a dynamic manner in neuroblasts and neurons. L'sc remains constantly high in a narrow fringe at the medial OOA margin. How initiation and maintenance of l'sc is controlled is unknown. Wingless, a known activator of proneural genes in other tissues () is expressed in a fairly restricted pattern in the apices of the OOA (). It is possible that a long range effect of Wg could be responsible for l'sc activation along the OOA margin.
A continued, interdependent expression of l'sc and Dl in the OOA could be the mechanism that accounts for the slow, gradual release of neuroblasts from the OOA margin. Thus, l'sc is known to upregulate Dl (), and this could be the case also in the OOA. A relative peak in Dl levels at the OOA margin, coinciding with the position where l'sc and ase are expressed, would support this interaction. High Dl would then signal to its neighbors laterally, increasing N activity, and thereby preventing a premature advance of l'sc towards lateral, and conversion to neuroblasts.
Interdependency of Notch and Jak/Stat Activity in OOA development
As previously reported, we find that reduction in Stat activity has a similar phenotype as reduction in Notch, consisting in a premature loss of the epithelial state of the OOA. This is accompanied by accelerated proliferation and gross abnormalities in the architecture of the optic lobe neuropile, as also seen in Notch mutant brains. Our data indicate multiple phases of where Notch and JAK/STAT activity function in an interdependent manner. At an early stage, both pathways are globally expressed in the OOA, and in regulating cell fate decisions for NE-NB transition. The use of the G-TRACE lineage reporter construct made it possibly to experimentally demonstrate the early, widespread expression of the Jak?Stat signal upd. In late larva and later stages, upd and upd-Gal4 are limited to the narrow OOl, which gives rise to the lamina. At the same time, expression of the lacZ reporter (?), turned on stably upd-Gal4, attests to an early phase where upd is active in the entire OOA. The transient, moderate-level activation of JAK/STAT may be part of the mechanism that maintains Notch activity. That would explain why in mutants with reduced Stat activity, the OOA also converts prematurely into neuroblasts (Yasudi et al., 2008; this study). At the same time, the proliferative activity of the growing OOA epithelium might be stimulated by both pathways.
At the stage when the OOA becomes visibly subdivided into a lateral domain (progenitor to the lamina) and a larger, medial domain (progenitor of the distal medulla), Jak/Stat and Notch levels increase and at the same time sort out into these two domains. It is likely that during this later phase, JAK/STAT and Notch enter into a inhibitory relationship, which might act to stabilize the boundary between OOAm and OOAl. Given the fact that neurons are produced from both medial and lateral OOA, the absence of Notch activity and of proneural genes in the lateral OOA is interesting, yet enigmatic, because it would constitute the only neurogenic tissue known so far that does not require proneural/neurogenic gene activity. Even though a number of studies have documented neurogenesis in the lamina (), the structural features of this process have not been revealed. The early studies cited above failed to differentiate between the different cell types, i.e., neuroepithelium, neuroblasts, and neurons. It is possible that neuroblasts divide in a mode that is quite different from that one of the OOAm-derived neuroblasts, or the neuroblasts of the central brain. This may explain a lack of proneural/neurogenic gene function in this domain. Another significant difference between lamina and the rest of the nervous system lies in the fact that proliferation of lamina precursors relies heavily on extrinsic signals, such as Hh supplied by the ingrowing retinal axons. It is possible that in the presence of such extrinsic "pacemakers" of neuron production, the mechanism provided elsewhere by the proneural/neurogenic gene cassette is dispensable.
The role of Notch and Jak/Stat signaling in other systems
The fundamental role of N in the developing nervous system appears to be to maintain cells in an undifferentiated (neuroepithelial) state at any given moment. Cells released from N activity enter a differentiative pathway (typically accompanied by structurally visible changes, such as a switch from epithelial cell to neuroblast, and/or a switch in mitotic behavior (symmetric vs asymmetric). The temporally controlled release/birth of neurons from the neuroepithelium is often tied to different cell fates. This has been shown very convincingly in the retina of vertebrates and Drosophila. For example, in the vertebrate retina, the first wave of differentiation results in ganglion cells, the second wave of differentiation at a later point includes photoreceptors, followed by bipolar cells, and so on (). If N activity is reduced at an early time point, the number of ganglion cells produced increases massively, at the expense of later born cell types (). In Drosophila, the first retinal cell type to be born behind the morphogenetic furrow is the R8 photoreceptor. If at the time of R8 specification, N function is decreased, the number of R8 cells is increased, and other cell types born later are decreased. With later pulses of N depletion, one gets different phenotypes; what they all have in common is that the cell types born at the time of the pulse are increased I number, the ones born later decreased.
In the Drosophila OOA investigated in this paper, the temporal progression of neuroblast formation that is controlled by N activity is linked to the coordinated growth between eye (and lamina) and medulla, derived from the OOA. However, it is well possible that the temporal progression is also tied into the control of different cell fates. As outlined in the results and Fig.1, the way in which the multitude of different medulla cell types map onto the larval optic lobe is not clear. It is more likely that they are specified along the z-axis, which would imply that each part of the OOA (in the x-y dimension) would produce the same cell types. However, it is well possible that some cell types which are actually not found in all medulla columns, such as wide field tangential neurons (), are produced by different parts of the OOA. Such cell types then might be affected by premature or delayed conversion of the OOA into neuroblasts; identifying specific markers that label cell types at early stages, and using such markers in the background of Notch loss or overactivity, will help clarifying this question.
It should also be mentioned that a role of the N pathway has been described for later stages in neural development, that is, the specification of neurons from ganglion mother cells (GMCs). Asymmetric neuroblast proliferation in the ventral nerve cord and brain produces a series of GMCs which each divides one more time into two, often different, neurons/glial cells. It has been shown that this fate choice between sibling pairs depends on N activity (). Such may also be the case for the neuroblasts emerging from the OOA. The relatively high level of the N reporterâ€¦ maintained in the OOA-derived lineages would speak for a continued role of N in these cells; however, detailed investigations of the neurogenesis of these lineages (do they have GMCs dividing once only? Is the division pattern of neuroblasts and GMCs fixed or variable?) need to be carried out in order to address this potential later N function.
The role of N suggested by the findings where N activity controlled an ordered succession of different cell fates is permissive, rather than instructive. Thus, the specific neuronal or glial fate of the cells that either lose N activity, or express a high level of it, is most likely under the control of other factors. In mammalian telencephalon, one potential signal is the cytokine JAK/STAT pathway. Telencephalic neural progenitors produce the different types of neurons first, followed by glial cells (astrocytes). Astrocyte fate depends on high Notch levels, but differentiation along the astrocyte lineage can only be induced in late neural progenitors by Notch activation in cells that have the competence to activate the JAK/STAT pathway. In this context, Notch function upstream to activate JAK/STAT by demethylation of astrocyte-specific promoters. The demethylation event is crucial for astrocyte cell fate induction and is triggered by the nuclear factor IA, which prevents binding of the methyl transferase DNMT1 to astrocytic promoters (ref). The murine telencephalon thus is a prime example of how the coordinated activities of both pathways are necessary to regulate a neural progenitor versus astrocyte cell fate decision.
Interdependency between Notch and Jak/Stat has been reported for many other developmental scenarios. Unfortunately, the types of genetic interactions between these pathways are as diverse as the developmental events or cell fates which they control. In the Drosophila ovary, mutual inhibition of the two set up the boundary between the stalk (Jak/Stat dependent) and the main-body follicle cells (Notch dependent; ). In adult intestinal stem cells, the relationship between the two pathways is also antagonistic (Jak/Stat promoting proliferation of stem cells, Notch inhibiting it; ), but the epistatic relationship between these pathways is not clear (). The same applies to the Drosophila eye, where according to one study Notch activates Jak/Stat by acting on upd transcription (), whereas another study reported that Jak/Stat acts upstream of Notch ().