Glia and axonogenesis

Separate sections of The Interactive Fly group genes according to their involvement in glia morphogenesis and axonogenesis.

Determination of neuroblast identity in the neurectoderm

The Drosophila central nervous system (CNS) develops from a bilateral neuroectoderm that lies on either side of a narrow strip of ventral midline cells. Single neuroectodermal cells delaminate from the surface epithelium, in a fixed pattern, and move into the interior of the embryo to form neural precursor cells called neuroblasts. The early neuroblasts form an orthogonal grid of four rows (1, 3, 5, and 7) along the anterior-posterior (AP) axis and three columns (ventral, intermediate, and dorsal) along the dorsoventral (DV) axis. Subsequently, each neuroblast expresses a characteristic combination of genes and contributes a stereotyped family of neurons and glia to the CNS. Thus the earliest steps in patterning the CNS are the formation and specification of neuroblasts.

Neuroblast formation is regulated by two phenotypically opposite classes of genes: Proneural genes promote neuroblast formation, whereas the neurogenic genes inhibit neuroblast formation. Proneural genes encode a family of basic helix-loop-helix transcription factors that are expressed in 4-6 cell clusters at specific positions within the neuroectoderm. Embryos lacking the proneural genes achaete/scute or lethal of scute have a reduced number of neuroblasts (for review, see Skeath, 1994). Conversely, neurogenic genes are expressed uniformly in the neuroectoderm, and embryos that lack any one neurogenic gene function, such as Notch or Delta, develop an excess number of neuroblasts (for review, see Campos-Ortega, 1995).

Neuroblast identity is determined in the neuroectoderm. Neuroblasts delaminate in five waves spanning approximately three hours. The generation of neuronal diversity begins with the specification of unique neuroblast identities along both the anterior-posterior (AP) and dorsal-ventral (DV) axes. The pair-rule genes wingless, hedgehog, gooseberry, and engrailed are expressed in stripes of neuroectoderm that subdivide the AP axis. These genes are required for establishing AP row identity within the neuroectoderm and neuroblasts (Chu-LaGraff, 1993; Zhang, 1994; Skeath, 1995; Bhat, 1996; Matsuzaki, 1996; Bhat, 1997 and McDonald, 1997). For example, gooseberry is expressed in row 5 neuroectoderm. Embryos lacking gooseberry function have a transformation of row 5 into row 3 neuroectoderm and neuroblast identity, whereas misexpression of gooseberry results in the converse row 3 to row 5 transformation (Zhang, 1994 and Skeath, 1995). Similarly, wingless encodes a protein secreted from row 5 and required for specifying the fate of the adjacent rows 4 and 6 neuroectoderm and neuroblasts (Chu-LaGraff, 1993). For information on the expression of segmentation genes and neuroblast identity genes in specific neuroblasts, see Chris Doe's Hyper-Neuroblast map.

Three genes are expressed in restricted domains along the DV axis within the neuroectoderm: ventral nervous system defective (vnd) is an NK2 class homeobox gene expressed in the ventral column neuroectoderm (Jimenez, 1995 and Mellerick, 1995) and muscle segment homeobox (msh) is a homeobox gene expressed in the dorsal column neuroectoderm and neuroblasts (D'Alessio, 1996 and Isshiki, 1997). Mutations in vnd cause defects in neuroblast formation and lead to severe defects later in neurogenesis (White, 1983 and Skeath, 1994). Mutations in msh result in a partial transformation of dorsal neuroblasts into a more ventral or intermediate column identity, without affecting neuroblast formation (Isshiki, 1997). Signaling via the EGF receptor is required to establish ventral and/or intermediate column fates in the neuroectoderm (Rutledge, 1992; Raz, 1993; Schweitzer, 1995; Skeath, 1998; Udolph, 1998 and Yagi, 1998). A newly cloned homeobox gene, intermediate neuroblasts defective (ind) is the first gene known to be expressed specifically in the intermediate column of neuroectoderm and neuroblasts. ind function is required for the establishment of intermediate column identity in the neuroectoderm, and for the formation of intermediate column neuroblasts. There is a hierarchical cascade of transcriptional repression. Vnd represses ind expression to establish the ventral boundary of ind transcription, and ind represses msh to establish the ventral boundary of msh transcription. The homeobox genes expressed in columns within the Drosophila neuroectoderm--vnd, ind, and msh--each have gene homologs expressed in corresponding domains along the DV axis of the vertebrate neural ectoderm. On this basis it appears that fundamental molecular mechanisms of DV patterning may be similar in Drosophila and vertebrates (Weiss, 1998 and McDonald, 1998).

Control the temporal sequence of neuroblast specification

Stage-specific inductive signals in the Drosophila neuroectoderm control the temporal sequence of neuroblast specification. To test when identity specification of the various neuroblasts takes place and whether extrinsic signals are involved, heterochronic transplantation experiments were performed. Single neuroectodermal cells from stage 10 donor embryos (after S2) were transplanted into the neuroectoderm of host embryos at stage 7 (before S1) and vice versa. The fate of these cells was examined by determining the makeup of their lineages at stage 16/17. Transplanted cells adjust their fate to the new temporal situation. Late neuroectodermal cells are able to take over the fate of early (S1/S2) neuroblasts. The early neuroectodermal cells preferentially generated late (S4/S5) neuroblasts, despite their reduced time of exposure to the neuroectoderm. Furthermore, neuroblast fates are independent from divisions of neuroectodermal progenitor cells. It is concluded from these experiments that neuroblast specification occurs sequentially under the control of non-cell-autonomous and stage-specific inductive signals that act in the neuroectoderm (Berger, 2001).

The segmented CNS (ventral nerve cord) of the Drosophila embryo is relatively simple, consisting of approximately 400 cells per hemineuromere. These originate after gastrulation from the ventral neurogenic region of the ectoderm. About 25% of the neuroectodermal cells delaminate into the embryo as CNS progenitor cells, called neuroblasts (NBs). The singling out of the NBs from among neuroectodermal cells is achieved by the activity of proneural and neurogenic genes. In each hemisegment approximately 30 NBs delaminate from the neuroectoderm according to a stereotyped spatiotemporal pattern. Each NB delaminates from a specific region of the neuroectoderm to occupy a particular place within the subectodermal NB layer. The process of delamination has been divided into five successive waves (S1-S5) with particular subpopulations of identified NBs delaminating during each wave. Thus, each NB is characterized by a typical position and time of delamination. Furthermore, each NB expresses a specific set of molecular markers. Finally, the unique identity of each NB is revealed by the production of a characteristic cell lineage (Berger, 2001 and references therein).

Crucial steps in the specification of the various NB identities appear to take place before delamination by the interpretation of positional information in the neuroectoderm encoded by segmentation genes and dorsoventral patterning genes. Heterotopic transplantation experiments have shown that neuroectodermal cells become committed by these spatial cues to different degrees. For example, whereas dorsal neuroectodermal cells are able to adjust their fate when transplanted to more ventral positions, ventral neuroectodermal cells exhibit firm commitment and produce lineages consistent with their origin. These experiments refer to a given developmental stage (early gastrula, stage 7). However, the time of delamination differs between NBs, and the identity of a given NB correlates with a certain time of delamination. This implies that NB specification requires temporal cues in addition to positional information (Berger, 2001 and references therein).

The mechanisms behind the temporal sequence of NB specification are unknown. Different modes of regulation could be envisaged. For example, all NB identities, including the respective times of delamination, might become firmly determined at an early stage and are cell-autonomously expressed during further development. Alternatively, progenitor cells might acquire NB-identities sequentially under the influence of extrinsic signals. To test whether the developmental potencies of neuroectodermal progenitor cells change over time and whether inductive signals are involved, the temporal axis was manipulated independently from spatial cues by performing heterochronic transplantations of neuroectodermal cells. Neuroectodermal cells were transplanted from stage 7 donors (early gastrula, before S1) into stage 10 hosts (after S2), and vice versa. The identities assumed by these cells were determined by analyzing their lineages in the host embryos at stage 16/17. In both experimental situations, neuroectodermal cells are able to adjust their fate to the new environment. Late neuroectodermal cells can generate early (S1, S2) NBs. Early neuroectodermal cells preferentially produced late (S3-S5) NB lineages, despite having been exposed to the neuroectoderm for a significantly reduced period of time. Late NB fates are independent of previous divisions of neuroectodermal progenitor cells. These data suggest that extrinsic inductive signals exist in the neuroectoderm that change over time to control the specification of temporal subsets of neuroblasts (Berger, 2001).

In one set of experiments, neuroectodermal cells from stage 10 embryos were heterochronically transplanted into the neuroectoderm of 2 hours younger, early gastrula (stage 7) hosts. The transplanted cells gave rise to CNS clones, or to epidermal clones, or to mixed CNS/epidermal clones. This shows that despite their more advanced age, the implanted cells participate in the cell interaction process that leads to the decision of neurectodermal cells between an epidermogenic and a neurogenic fate. Remarkably, however, among the cells that follow the neural pathway, about 50% produced lineages typical for early NBs (S1, S2), as for example, NB1-1, MP2, NB2-2 or NB4-2. This indicates that neuroectodermal cells at stage 10, which normally only give rise to late NB lineages, have not lost the potency to assume identities of early NBs. Taken together these data indicate that late ectodermal cells (stage 10) are not irreversibly specified, and that signals exist in the early neuroectoderm (stage7) that are sufficient to induce early NB fates. Thus, instead of being merely based on cell-autonomous properties, the temporal regulation of early NB determination appears to be mediated by extrinsic inductive signals that are active in the early neuroectoderm (Berger, 2001).

Reduced time of exposure to the neuroectoderm does not prevent formation of late NBs. Having shown that the determination of early NB fates depends on stage specific inductive signals, whether inductive signals are also involved in the generation of late NB fates was tested. Cells from the early neuroectoderm (stage 7) were heterochronically transplanted into the neuroectoderm of stage 10 host embryos. Among 132 identifiable clones obtained from these cells, 24 (19%) were CNS clones and 108 (81%) epidermal clones. Closer analysis of the 24 CNS clones revealed that about 80% (n=19) of them corresponded to lineages typical for late NBs, like 2-1, 5-4, 6-4 or 7-3, and only 20% (n=5) to early NB lineages. Therefore, the transplanted cells tend to adopt to the new temporal environment regarding the identities of NBs to be formed. Although having skipped two hours of exposure to the neuroectoderm, a significant proportion of them can compensate for this lack of time. Thus, the cells are not bound to an intrinsic timer to become specified as late NBs, but are able to react to inductive signals in the late neuroectoderm. The 20% of cells that developed an early NB fate might point to differences in the degrees of commitment of neuroectodermal cells at a given stage or to an insufficient exposure to signaling in the late neuroectoderm under the experimental conditions (Berger, 2001).

Determination of late NBs does not depend on previous division in the neuroectoderm. As opposed to early NBs the lineages of S4 and S5 NBs, and some of the S3 NBs have an epidermal sister clone. This is due to the postblastodermal division pattern of neuroectodermal progenitors. Progenitors developing as S1 and S2 NBs do not divide before delamination from the neuroectoderm: some of those giving rise to S3 NBs divide, and those giving rise to S4 and S5 NBs (NBs 1-3, 2-1, 2-4, 3-3, 4-3, 4-4, 5-1, 5-4, 5-5 and 7-3) always divide in the neuroectoderm. Only one of the daughter cells that results from this division subsequently delaminates as a late NB, the other remains in the periphery to develop as an epidermoblast. Is this neuroectodermal division required for late NBs to form and become properly specified? When neuroectodermal cells are heterochronously transplanted from stage 7 donors into stage 10 hosts, they are deprived from the phase in which the first wave of divisions normally runs through the neuroectoderm. Most of the CNS clones obtained from these cells corresponded to lineages of late NBs. However, whereas S4 and S5 NBs normally have an obligatory epidermal sister clone, the situation is variable under the experimental conditions. Some of these clones have a sister clone consisting of epidermal cells, whereas the other clones lack an epidermal sister clone (Berger, 2001).

These data show that: (1) proliferation of individual neuroectodermal progenitors can be influenced by surrounding tissue; (2) late NBs can segregate from the neuroectoderm without having previously divided; and (3) late NBs do not depend on a previous division to acquire an individual identity and to produce their specific and complete CNS lineage. These observations lend further support to the idea that the temporal pattern of NB determination depends on inductive signals in the neuroectoderm instead of following a stereotype cell autonomous clock (Berger, 2001).

There is ample evidence that the specification of NBs crucially depends on positional information in the neuroectoderm provided by the products of segmentation genes and dorsoventral patterning genes. Part of this information becomes integrated into the cell-autonomous program of the cells before neurogenesis. Another part, however, is subsequently provided by extrinsic signals. For example, the segment polarity gene wingless (wg) is segmentally expressed in a single row of neuroectodermal cells and the secreted Wg protein is non-autonomously required in adjacent anterior and posterior neuroectodermal cells for the formation and specification of NBs. Along the dorsoventral axis, the secreted Spitz and Vein proteins are involved in conferring NB identities. These heterochronic transplantation experiments show that extrinsic signals are also involved in NB specification along the temporal axis. Although neuroectodermal cells of stage 10 embryos normally never produce NBs belonging to the group of S1 and S2 NBs, they do so after being transplanted into stage 7 neuroectoderm. The possibility that the cells follow this fate autonomously after being released from signals that normally inhibit these fates in the late neuroectoderm is incompatible with the following evidence. Cells from the non-neurogenic dorsal ectoderm of stage 10 donors are able to adopt a CNS fate upon heterotopic transplantation, and to become specified as early NBs. However, they are unable to autonomously develop as a NB in cell culture. Thus, the transplanted late cells do react to signals in the early neuroectoderm and adjust their development accordingly. This also seems to be possible in the other direction. Upon transplantation of stage 7 neuroectodermal cells into the neuroectoderm of hosts at stage 10, most of the CNS lineages obtained are typical for NBs that normally delaminate late. Similar to the situation in Drosophila, heterochronic transplantations using the developing ferret brain have revealed an interaction scenario of extrinsic cues and intrinsically changing properties for the sequential birth of neuronal cell types from ventricular zone progenitor cells. Progenitor cells from very young embryos can adjust their fate to older host tissues. By contrast, cells from older tissue transplanted into younger host brains adopt only fates typical of their origin. The latter experiment reveals an irreversible intrinsic change of the developmental properties of older cells. Intrinsic changes over time are likely to occur also in the Drosophila neuroectodermal cells; however, they are reversible under the influence of external signals. It remains to be tested as to how far this is also the case for NBs once they have delaminated from the neuroectoderm (Berger, 2001 and references therein).

These experiments suggest that the entire temporal sequence of delamination of specific subsets of NBs is not readily determined in the early neuroectoderm but is controlled by the dynamic expression of stage specific signals. Segment-polarity genes play an important role in the formation and identity specification of NBs. They are segmentally expressed in particular rows of neuroectodermal cells. As the expression domains of some of these genes evolve dynamically and, hence, differ at different stages, they are also good candidates for being involved in the temporal control of NB formation/specification. The differential commitment of the late neuroblasts NB 6-4 (S3) and NB 7-3 (S5) has been shown to be mainly controlled by the interplay of the segment polarity genes naked (nkd) and gooseberry (gsb). Mutation of either nkd or gsb leads to the transformation of one NB fate to the other. Interestingly, however, the temporal sequence of their delamination is maintained, i.e. independent from these genes. This suggests that formation and specification of these two NBs is under independent control. Further work will have to test whether this is also the case for other NBs and to uncover the signals that regulate the temporal pattern of NB fate determination (Berger, 2001 and references therein).

Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells

Stem cell asymmetric division requires tight control of spindle orientation. To study this key process, Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles, were examined. Early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function (Rebollo, 2007).

Immediately after cytokinesis, the single dot revealed by both PCM and centriole reporters splits in two, strongly suggesting that centrosome duplication has taken place. The YFP-Asl marker, like other centriolar markers in Drosophila, does not allow for resolution of individual centrioles within a centrosome in larval NBs. Therefore, timing of centriole duplication in these cells remains uncertain. The two resulting centrosomes migrate together within the single major aster of the cell to the apical cortex. Later on, one of the centrosomes loses PCM and starts to migrate. At this early time point in the cell cycle, unequal centrosome fate is already established: one, apical, will remain in the stem cell; the other will go into the differentiating daughter. Migration of the downregulated centrosome (revealed by the centriolar marker), initially within the apical side of the cell and more basally later on, occupies most of the cell cycle and is the most variable stage, its duration being dependent on cell-cycle length. The apical centrosome organizes the only aster found in the NB for most of the cell cycle. As mitosis onset approaches, the moving downregulated centrosome becomes stabilized at the basal side and starts to accumulate PCM and organize the second aster. As a direct consequence, the spindle is assembled already in alignment with the polarity axis of the cell. In Drosophila male germline stem cells (Yamashita, 2007), one of the centrosomes is also consistently located adjacent to the hub from early interphase onward. Only this centrosome maintains a robust aster through the cell cycle. The other, associated with only a few microtubules, moves away from the hub and is inherited by the gonialblast. In these cells, the oldest centriole is always in the centrosome that is proximal to the hub and is therefore retained by the stem cell (Yamashita, 2007). It has not yet been possible to determine which of the two centrosomes contains the oldest centriole in larval neural stem cells. In pins NBs, unequal centrosome fate and function are established, but, eventually, the stable, aster-forming apical centrosome is downregulated and starts to behave like the other, migrating across the cell. Like the other too, it organizes an aster only shortly before NEB. The place of assembly of the two asters in pins mutant NBs is not fixed and consequently spindle orientation is randomized, and so is the size difference between the two daughter cells (Rebollo, 2007).

It is still unclear how NB polarity is maintained from one cycle to the next; a distinct Baz apical crescent is only assembled at prophase. The permanent positioning of the NB centrosome in the apical side of the cell, through the cell cycle, suggests that it could be contributing to specifying the apical cortex after mitosis (Rebollo, 2007).

In summary, four main conclusions can be derived from these observations: (1) the two centrosomes of asymmetrically dividing Drosophila larval NBs become unequal early in the cell cycle in terms of mobility, MTOC activity, and fate; (2) such elaborated unequal centrosome regulation provides a means to position the asters, thus ensuring spindle alignment along the polarity axis of the cell; (3) Pins contributes to spindle orientation in NBs by preventing the downregulation of the MTOC capability of the apical centrosome, thus maintaining the apical aster in place, and (4) spindle orientation is predetermined and can be accurately predicted as soon as the aster reaches the apical cortex during the initial stages of the cell cycle. Altogether, these observations reveal that asymmetry in Drosophila neural stem cells goes beyond the polarized localization of a number of protein complexes during mitosis and may affect entire organelles such as the centrosome, which exerts a major effect on cell architecture and function throughout the cell cycle (Rebollo, 2007).

Spindle alignment is achieved without rotation after the first cell cycle in Drosophila embryonic neuroblasts

Spindle alignment along the apicobasal polarity axis is mandatory for proper self-renewing asymmetric division in Drosophila neuroblasts (NBs). In embryonic NBs, spindles have been reported to assemble orthogonally to the polarity axis and later rotate to align with it. In larval NBs, spindles assemble directly aligned with the axis owing to the differential spatiotemporal control of the microtubule organising activity of their centrosomes. This study has recorded embryonic NBs that express centrosome and microtubule reporters, from delamination up to the fourth cell cycle, by two-photon confocal microscopy, and it was found that the switch between these two spindle orientation modes occurs in the second cell cycle of the NB, the first that follows delamination. Therefore, predetermined spindle orientation is not restricted to larval NBs. On the contrary, it actually applies to all but the first cell cycle of embryonic NBs (Rebollo, 2009).

In terms of centrosome behaviour and microtubule-organising center (MTOC) activity, NBs in their first cell cycle are somewhat between the neuroectodermal cells from which they derive and older NBs. In epidermoblasts and delaminating NBs, centrosomes duplicate long before mitosis and both centrosomes have feeble MTOC activity. MTOC activity has been shown to be very weak during interphase in many Drosophila cell types (Rogers, 2008). At mitosis onset, the centrosomes of epidermoblasts and delaminating NBs start to gain MTOC activity and to migrate to opposite sides of the nucleus defining a line that is nearly orthogonal to the apicobasal axis along which the spindle assembles. However, while spindle orientation remains unchanged through mitosis in epidermoblast, it changes in the newly differentiated NB at metaphase to an apicobasal orientation. In most cases, rotation occurs in the direction that positions the slightly larger aster on the apical side. Completion of the first cytokinesis results in the basal delivery of the first GMC and leaves the NB centrosome on the apical side of the cell. This is the landmark of the switch to the predetermined alignment mode in which differential centrosome behaviour leads to spindle assembly directly along the apicobasal axis, and in which no major spindle rotation occurs. The apical centrosome contains a considerable amount of PCM, organises a large microtubule aster, and stays at the apical cortex of the cell. The other centrosome, almost totally devoid of PCM and microtubules, moves away from the apical cortex and remains motile, mostly around the basal half of the cell, until mitosis onset, when it starts to accumulate PCM and to nucleate microtubules near to the basal cortex. As a result, the spindle assembles directly along the apicobasal axis, and once more, but this time without significant spindle rotation, asymmetric division delivers a basal GMC. This process of asymmetric centrosome behaviour and aligned spindle assembly is repeated in the following cycles in the embryo and indeed in larval NBs. This mode has been observed in all the post-delamination NB cell cycles that could be unequivocally identified in the embryo by two-photon microscopy. It is therefore likely that such a mode operates in all but the first cell cycle in Drosophila NBs (Rebollo, 2009).

When a NB delaminates from the epithelium, the apical stalk carries the Par complex from the apical cortex of the corresponding epidermoblast, which triggers the recruitment cascade that establishes apicobasal polarity during the first round of NB asymmetric cell division. However, because the Par complex and other known polarity markers fade away from the cortex after mitosis, it is unclear how cortical polarity orientation is passed on in the following cell cycles. The current results strongly suggest that the aster that stays anchored to the cortex during interphase might convey such information (Rebollo, 2009).

Temporal control of the development of neural sublineages

What mechanisms control the sequential generation of neurons -- that is, how are unique fates acquired by the successive daughters of a neuroblast? After a neuroblast has been specified positionally, by the actions of segment polarity genes and a network of homeodomain genes, it delaminates from the ectoderm and begins to divide unequally into one large and one small daughter cell. The large cell (still called a neuroblast) continues to go on this way for a variable number of rounds. The small cell, called a ganglion mother cell (GMC), typically divides equally one more time to generate a pair of postmitotic neurons. Often these neurons form a stack on top of the neuroblast from which they originated. As postmitotic neurons in the insect CNS do not generally migrate, the position of a neuron in the CNS depends on whether it was generated early or late. In this way, a histogenetic order is built into the cellular cortex of the insect CNS, with early neurons deep and close to the neuropil and late neurons next to the surface of the brain (Kambadur, 1998; Harris, 2001).

This arrangement of cells according to relative birth date is also observed in laminated structures in the vertebrate CNS, the best example being the cerebral cortex. In the mammalian cortex, cells acquire their fates at the ventricular surface at the time they are born, and these postmitotic neurons cells then migrate to their specified laminar destinations (McConnell, 1995). In both the cortex and the retina, it is thought that progenitors are pluripotent and realize their particular fates by being exposed to an extracellular environment that changes with time (Harris, 1997). Whether intrinsically or extrinsically controlled, particular combinations of transcription factors are expressed in the neuroblasts of both the vertebrate retina and fly CNS over the course of development, and these factors appear to restrict the competence of neuroblasts to the fates that are appropriate (Harris, 2001 and references therein).

The first insights into this problem in the fly CNS were made in Odenwald's laboratory at the NIH. They showed that expression of the transcription factor genes hunchback (hb), pdm, and castor (cas) occur sequentially in the embryonic CNS of Drosophila (Kambadur, 1998). Furthermore, the CNS neuroblasts themselves sequentially express these three genes in a conserved order (Brody, 2000), and whichever of the three genes is expressed in the neuroblast when it divides continues to be expressed in the progeny. The transcription factor Grainyhead (Gh) appears to mark the NB after it has generated lineages marked by Hb, Pdm and Cas, and the Gh positive NB also generates a fourth lineage (Brody, 2000). Thus, the earliest generated neurons in the fly CNS tend to express hb, while later generated neurons express pdm, and still later generated neurons express cas followed by gh. By analogy to the spatial coordinate genes, these can be called 'temporal coordinate genes (Harris, 2001).

Isshiki (2001), working in Doe's laboratory, followed individual neuroblasts and their progeny. It appears that each neuroblast examined express four temporal coordinate genes -- hb, Kruppel (Kr), pdm, and cas, in that invariant order. By following the GMCs and their daughter neurons, Isshiki confirmed on a cellular level that each GMC maintains the expression profile of the temporal coordinate genes that its parent neuroblast displayed at the time the GMC was generated. The relevance of these temporal coordinates to neuronal fate was addressed with misexpression constructs and loss of function mutants in hb and Kr. These experiments led to respecification of GMCs and their progeny to earlier or later fates, as expected if these genes really are important to fate. Thus, these temporal coordinate genes play a similar role in fate specification along a histogenetic axis as the spatial coordinate genes play in the positional axes (Harris, 2001).

What, one might wonder, are the mechanisms that could generate these temporal transitions in the parent neuroblast? The first possibility is that global temporal cues, such as circulating hormones, or intracellular signaling such as Notch, Egf, Wingless, or Hedgehog, trigger these transitions. Since transitions in transcription factor expression take place in isolation, in cultured clones, it is concluded that once NBs initiate lineage development no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of transcription factor expression during NB outgrowth (Brody, 2000; Harris, 2001).

Second, the cascade mechanism itself, in which each of these genes is responsible for turning on the next in the series, functions to regulate the transcription factor transitions. Overexpression of Hb activates Kr and represses Pdm and Cas; overexpression of Kr activates Pdm, represses Cas, but has no effect on Hb expression; Pdm positively regulates Cas expression; and Cas repressed Pdm expression (Kambdadur, 1998 and Isshiki, 2001). Although there are appropriate sequential regulatory interactions of this kind, mutations in any of the earlier genes only subtly affect the temporal expression of subsequently expressed genes. Thus, although these interactions refine sequential expression, there must be additional elements to temporal regulation. Isshiki (2001) has shown that individual NBs go through the same sequence of expression in their own sweet time, independent of the developmental stages at which they delaminate. A third possibility is therefore suggested, a more mysterious clock mechanism may also be responsible for generating the order. The clock in this case appears to be directly related to the cell cycle, since arresting cell division with the Cdc25 mutant, string, freezes the pattern in time (Cui, 1995; Weigmann, 1995; Harris, 2001 and references therein).

The addition of a time axis adds to understanding of how different neuronal types arise in the Drosophila CNS, and it also raises the intriguing problem of how multiple inputs regulate the expression of the temporal coordinate genes (Harris, 2001).

Patterns of embryonic neurogenesis in a primitive wingless insect, the silverfish; comparision with those in flying insects

Neurogenesis was examined in the central nervous system of embryos of the primitively wingless insect, the silverfish, Ctenolepisma longicaudata, using staining with toluidine blue and the incorporation of bromodeoxyuridine. The silverfish has the same number and positioning of neuroblasts as seen in more advanced insects and the relative order in which the different neuroblasts segregate from the neuroectoderm is highly conserved between Ctenolepisma and the grasshopper, Schistocerca. Of the 31 different neuroblasts found in a thoracic segment, one (NB 6-3) has a much longer proliferative period in silverfish. Of the remainder, 14 have similar proliferative phases, while 16 neuroblasts have extended their proliferative period by 10% of the duration of embryogenesis (10%E) or greater in the grasshopper, as compared with the silverfish. Both insects have similar periods of abdominal neurogenesis except that in the silverfish terminal ganglion, a prominent set of neuroblasts continues dividing until close to hatching, possibly reflecting the importance of cercal sensory input in this insect. This comparison between silverfish and grasshopper shows that the shift from wingless to flying insects was not accompanied by the addition of any new neuronal lineages in the thorax. Instead, selected lineages undergo a proliferative expansion to supply the additional neurons presumably needed for flight. The expansion of specific thoracic lineages was accompanied by the reduction of the terminal abdominal lineages, specifically NB6-3, as flying insects began to de-emphasize their cercal sensory system. This neuroblast is notable in that it is the only neuroblast from the grasshopper set that is missing in Drosophila. A reasonable speculation is that NB 6-3 makes interneurons that deal with ascending information from the cercus (Truman, 1998).

Although abdominal neurogenesis in Ctenolepisma is roughly equivalent to that in the grasshopper, except in the terminal ganglion where it is higher, in the thorax it falls well below that seen in the grasshopper. In the silverfish, the last thoracic NBs stop dividing by about 70% of the way through embryonic development (70%E), whereas in grasshopper embryos, selected thoracic NBs continue dividing until slightly after 90%E. Comparison of the neurogenic periods between individual grasshopper and silverfish neuroblasts shows that only some of the neuroblasts have participated in this expansion. The greatest differences are seen for NBs 1-1, 5-1, and 6-4; these proliferate for 25%E to more than 30%E longer in the grasshopper as compared to the silverfish. An additional 14 neuroblasts have their proliferative period extended by at least 10%E in grasshopper embryos. The one neuroblast that goes counter to the overall trend is NB6-3, which has a proliferative period that extends for over 20%E longer in the silverfish. Hence, this lineage has become smaller in more advanced insects rather than becoming larger or staying the same. It is argued, however, that an increase in lineage size alone is not sufficient to conclude that that particular lineage produces neurons associated with flight (Truman, 1998).

Using Fas2 to chart the structure of the neuropile

Insect neurons are individually identifiable and have been used successfully to study principles of the formation and function of neuronal circuits. In Drosophila, studies on identifiable neurons can be combined with efficient genetic approaches. However, to capitalize on this potential for studies of circuit formation in the CNS of Drosophila embryos or larvae, it is necessary to identify pre- and postsynaptic elements of such circuits and describe the neuropilar territories they occupy. A strategy for neurite mapping is presented, using a set of evenly distributed landmarks labelled by commercially available anti-Fasciclin2 antibodies that remain comparatively constant between specimens and over developmental time. By applying this procedure to neurites labelled by three Gal4 lines, neuritic territories are shown to be established in the embryo and maintained throughout larval life, although the complexity of neuritic arborizations increases during this period. Using additional immunostainings or dye fills, Gal4-targeted neurites can be targetted to individual neurons and they can be characterized further as a reference for future experiments on circuit formation. Using the Fasciclin2-based mapping procedure as a standard (e.g., in a common database) would facilitate studies on the functional architecture of the neuropile and the identification of candiate circuit elements (Landgraf, 2003).

Working with defined pre- and post-synaptic neurons is a prerequisite for the study of mechanisms that underlie circuit formation. The fact that such neurons establish synaptic contacts with one another requires that some of their neurites project to a common region. Thus, proximity of neurites is a criterion that can be used towards the identification of putative pre- and postsynaptic neurons. In Drosophila (like in other insects), synaptic contacts are restricted to the neuropile, a cell body-free area, which also contains the ascending, descending, and commissural fibers. Unlike the gray matter in the vertebrate spinal cord (where cell bodies and synapses are intermingled), neuronal cell bodies of the Drosophila CNS are restricted to the synapse-free 'cortex' from where they send monopolar projections into the neuropile. These neuropilar accumulations of neurites of CNS neurons (i.e., efferent and interneurons) are joined by projections from peripheral sensory neurons. The functionality of thus established neuronal circuits demands that the spatial arrangements of synapse-bearing neurites in the neuropile are fairly reproducible between different individuals, as has been learned from analyses in larger insects. In order to map these reproducible neurites in the Drosophila neuropile, predominantly anatomical landmarks of the neuropile have been used to date as reference points for the relative positions of neuronal projections. Such landmarks are segmentally repeated nerve roots and commissures, or easily identifiable fiber tracts (so far applied only in the imaginal CNS (Landgraf, 2003).

This study capitalizes on a set of axon tracts that are labelled by the commercially available antibodies against the intracellular domain of Fasciclin2. These provide a set of standard landmarks that are evenly distributed throughout the neuropile. As shown by double-labellings with presynaptic markers, all Fasciclin2-positive fiber tracts are fully contained within the synaptic neuropile. They can be used in a very easy and efficient way for the charting of neurites in the neuropile. So far, Fasciclin2 fibre tracts have served as one-dimensional (mediolateral) landmarks in younger embryos. This approach has been extended by using the set of Fasciclin2 tracts in three dimensions and at different developmental stages. These analyses were exclusively centered on abdominal neuromeres for two reasons: predominantly, the abdominal motorsystem contributes to larval movement, and abdominal neuromeres face only minor reorganization during larval life (Landgraf, 2003).

Each Fasciclin2 fascicle has been named according to its relative dorsoventral (D, dorsal; C, central; V, ventral) and mediolateral (M, medial; I, intermediate; L, lateral) position. Such a nomenclature is neutral and can therefore be applied to any set of axon fascicles. The pattern of Fasciclin2 tracts remains relatively constant throughout larval development and thereby permits comparisons and extrapolations across different developmental stages. The main change to the embryonic pattern of Fasciclin2 in the neuropile is the addition of further elements, particularly five transverse projections (TP1-5) per neuromere in larval stages, which provide added reference points for the anteroposterior axis. From their association with different motor axons in the larva (TP1 with RP2 and VUM; TP2 with aCC), it is concluded that TP1 represents the pISN and TP2 the aISN nerve root (Landgraf, 2003).

In order to facilitate comparisons with published work, attempts were made to relate the Fasciclin2 pattern of the late embryo and larval stages to existing descriptions. For example, the Fasciclin2 pattern has frequently been used for work on the ventral nerve cord of earlier embryos, usually at 13 h of development. At this stage, three tracts can be resolved in the horizontal plane, of which the intermediate Fasciclin2 tract is formed or at least joined by axons of the MP1-interneurons (targeted by C544-Gal4), the medial tract by MP2-interneurons (targeted by 15J2-Gal4;. A split of the three tracts into vertically distinguishable bundles occurs during the next ca. 3 h. During this interval, it is still possible to trace the MP2/pCC- and MP1-axons via the C544- and 15J2-Gal4 lines when visualizing the Gal4-expressing neurons with the Uas-CD8-GFP reporter gene; later their Gal4-expression patterns change dramatically. Thus, despite the highly dynamic changes in the neuropile during this period (i.e., nerve cord condensation, closer apposition of neuropile at the midline and the fact that the intracellular Fasciclin2 domain vanishes from many cell bodies and axons), it is possible to map the MP2/pCC-axons to the DM (dorsointermediate) axons, and the MP1 interneuron axons to the dorsal CI-fascicles (Landgraf, 2003).

Classical neurobiological work on neuronal circuitry in other insects has been based on mapping strategies that used morphologically distinguishable axonal tracts in the neuropile and relates these to projection patterns of neurons. Similar strategies have been used for the thoracic adult CNS of Drosophila. The DM- and VMd-fascicles serve as reliable landmarks for distinguishing dorsal, intermediate, and ventral commissural tracts. The distinct patterns of sensory projections of different modalities, linked to the classical neurobiological literature, reveal a partitioning of the larval Drosophila neuropile. In an effort to relate the pattern of Fasciclin2 tracts to neuropilar regions, use has been made of three different Gal4 lines that target different subpopulations of sensory neurons (C161-, MJ94-, MzCh-Gal4). Sensory projections are confined to ventral regions, while neurites of motorneurons occupy the very dorsal neuropile. Thus, there is little, if any, physical overlap and contact between afferent sensory projections and central motorneuron neurites during larval stages. However, some overlap might occur lateral to the DM-fascicle, most likely with projections of the dbd and vbd-neurons. Thus, the data suggest that there are few, if any, monosynaptic connections between sensory and motorneurons in the embryonic and abdominal larval ventral nerve cord of Drosophila. However, this is a fairly rough estimation that will have to be tested by more detailed studies in the future (Landgraf, 2003).

Having described some spatial features of the neuropile with the help of the Fasciclin2 pattern, this charting strategy was next applied to three selected Gal4 driver lines. This effort is intended to identify and characterize neurons that are genetically amenable and that could be used for the investigation of neural circuit formation in the embryonic and larval Drosophila CNS. Three neural Gal4 lines were analyzed with precision. Before presenting detailed characteristics of abdominal Gal4-labelled neurons, an overview of the three Gal4 lines is provided: Per abdominal half-neuromere eve-Gal4^RRK expresses Gal4 in two motor-(aCC and RP2) and one interneuron (pCC). DDC-Gal4 displays 9-11 Gal4-neurons, and MzVum-Gal4 12-14 cells plus 3 efferent VUM (Ventral Unpaired Median) neurons located at the ventral midline. In all three lines, Gal4 expression occurs in a defined sequence, and for most cells it is yet unclear to what extent a late onset of expression reflects a late birth and/or differentiation of those cells. Only in the aCC, pCC and VUM neurons is Gal4 expression initiated at the time of their respective births, thus making them amenable to genetic manipulations of axonal pathfinding and differentiation. Next, the relative strengths of Gal4 expression were compared and overall MzVum-Gal4 expresses strongest, followed by DDC-Gal4 and eve-Gal4^RRK. However, Gal4 levels of different neuronal subsets in each Gal4 strain can differ significantly (e.g., in MzVum-Gal4, GABAergic interneurons express low levels while VUM and leucokinin-1-positive neurons express high levels). Because of differences in timing and strength of expression, Gal4-based manipulations would not be expected to affect all cells alike (Landgraf, 2003).

By virtue of the Fasciclin2-positive landmarks, it was possible to work out detailed descriptions of the neuropilar positions of neurites labelled by the three Gal4 lines eve-Gal4^RRK, MzVum-Gal4, and DDC-Gal4. These studies clearly show that the Fasciclin2 framework allows spatial relationships between neurites to be pinpointed even across specimens: for example, neurites of the aCC and RP2 neurons (eve-Gal4^RRK) are concentrated to form an oval in each hemineuromere that is located at the level of the DL-fascicle, medial to the ascending section of transverse projection 2 and anterior to transverse projection 1. At the same level (of the DL-fascicle), MzVum-Gal4-labelled neurites form whirlwind-like arrangements that have oval holes in their centers. These holes map to the region where aCC and RP2 neurites are concentrated, as indicated by the transverse projection 2. Thus, by using Fasciclin2-positive tracts as landmarks, spatial relationships of neurites are reproducibly revealed in three dimensions (Landgraf, 2003).

Since the pattern of Fasciclin2-positive axon tracts remains relatively constant from the late embryo to larva, it can also be used to investigate how neuronal projections change during this developmental period. The larval patterns of neurites described above are prefigured in the late embryo. For example, at late embryonic stages, the central arborisations of aCC and RP2 at the level of the DL-fascicle are also concentrated anterior to pISN (equals TP1 in the late larva), which corresponds to the region that lacks neurites in MzVum-Gal4 embryos. Thus, the principle spatial relationships between these sets of neurites (of aCC and RP2 versus those of MzVum-Gal4) appear to be laid down in the late embryo and maintained to larval stages, though the complexity and the spread of neurites increases over developmental time. This is an important observation because it suggests that: (1) By late embryonic stages neuritic arbors define those territories in the neuropile from which they will elaborate and spread during subsequent larval stages. Thus, principle spatial relationships between neurites are laid down during embryogenesis. (2) Data on the distribution of neurites obtained at one stage of development can be extrapolated and used to interpret other stages (Landgraf, 2003).

As demonstrated so far, using Fasciclin2 stainings significantly improves descriptions of the characteristic patterns of central neurites targeted by different Gal4-lines. However, these patterns of neurites are composites of different neurons. With this in mind, attempts were made to define methods with which to resolve such complex neuritic patterns into their constituent parts (Landgraf, 2003).

The first approach to tackle this problem is to employ antisera, which would reveal the morphologies of particular subsets of neurons. By using antibodies against the neurotransmitter/neuromodulator Serotonin and the neuropeptides Corazonin and Leucokinin-1 on nerve cords displaying Gal4-driven CD8-GFP expression, it is possible to define these neurites among the composite of Gal4-targeted projections that correspond to Serotonin, Corazonin, and Leucokinin-1 immunoreactive neurons and the regions of the neuropile that these occupy. Anti-Serotonin stains two and anti-Corazonin one neuron per hemineuromere. These three cells are targeted by the DDC-Gal4 line and appear to give rise to most of the DDC-Gal4-labelled neurites in the abdomen. Anti-Leucokinin-1 labels Gal4-targeted efferent projections forming type-3 terminals on the VL1-muscle (type-3v, DDC-Gal4;) and on the segment border muscle (type-3u, MzVum-Gal4). Of these, only the type-3u neuron is revealed by Leucokinin-1-like immunoreactivity and can thereby be traced back to a ventrolateral cell body in the CNS extending side branches toward the VL-fascicle. There are additional Leucokinin-1-positive projections associated with the DM-fascicle that are not targeted by MzVum-Gal4 but seem to originate from 2-4 (Gal4-negative) neurons at the anterior tip of the nerve cord. This has been confirmed by targeting the cytotoxin Ricin to MzVum::CD8-GFP neurons. This selectively abolishes all MzVum-Gal4-specific CD8-staining and the VL- but not the DM-associated Leucokinin-1-like immunoreactivity (Landgraf, 2003).

In summary, it has been shown that a small range of antisera can readily be used to reveal the projections of particular subsets of neurons. Such specific stainings are well suited to serve as spatial reference points in their own right. Moreover, in this instance, the Serotonin, Corazonin, and Leucokinin-1 immunoreactive neurons were instrumental in revealing some of the constituent parts of the complex projection patterns of the DDC-Gal- and MzVum-Gal4-lines (Landgraf, 2003).

Next, efferent neurons targeted by the three Gal4 lines were characterized and their axonal projections (nerve root and branch), target muscles, and terminal types were described. Based on morphological, molecular, and ultrastructural characteristics of motor terminals, several types of efferent neurons can be distinguished in Drosophila. It should be emphasised that distinctions between terminal types are not only of importance to studies of the Drosophila motor system but also correlate with differences between the central dendritic arbors of particular efferent neuron types. To classify the Gal4-labelled efferent neurons with respect to terminal type, a range of immunohistochemical stainings was employed: Synaptotagmin, Cysteine string protein, and Synapsin all represent presynaptic proteins involved in regulation of synaptic vesicles; Discs large is a predominantly postsynaptic protein, which labels the subsynaptic reticulum; and anti-Leucokinin-1 antisera detect an insect neuropeptide (Landgraf, 2003).

While the visualization of Gal4-labelled neurites via immunostaining is efficient, it is at the same time limited to particular subsets of cells, leaving many neurons unidentified. This limitation can be overcome by using standard neuronal tracers. To reveal the morphologies of those Gal4::CD8-GFP neurons, the neural tracer dye Cascade Blue was iontophoretically applied to individual cells. Thus, it was possible to define the positions of somata and central projections of all efferent neurons and a number of interneurons (Landgraf, 2003).

For the efferent neurons, it was found that their central projections are restricted to the dorsal neuropile (dorsal to the CI-fascicles). The only exception to this was the efferent SBM-neuron (MzVum-Gal4; whose short central arbors reside in the ventral neuropile where they associate with the VL-fascicle (consistent with Leucokinin-1 staining). In addition, it was found that differences in terminal type are reflected by distinctions in the central arbors of efferent neurons: while type-1 motoneurons elaborate extensive dendritic arbors (aCC and RP2; VA), efferent neurons with type-2 and type-3 terminals form comparatively sparse and stunted central arbors (VUM and SBM; VL1). Finally, these analyses suggest that the central projections of the same motoneuron in consecutive neuromeres do not overlap, i.e., they seem to behave in accordance with the tiling principle (Landgraf, 2003).

The interneurons of two of the Gal4-lines have been identified previously: pCC (eve-Gal4^RRK) lies adjacent to the aCC motorneuron; three interneurons of DDC-Gal4 are serotonergic or corazonergic. In addition, two MzVum-Gal4 interneurons were identified via Cascade blue fill. These two intersegmental interneurons seem to contribute to most or all MzVum-Gal4-targeted neurites in the ventral neuropile, ventral to the CI-fascicle (except for intersegmental ascending and descending projections and the leucokinin-1-positive neurites associated with the VL-fascicle). It is possible that additional ventral neurites might be derived from the mVg- and GABAergic neurons of MzVum-Gal4 (Landgraf, 2003).

In summary, it was found that neurites targeted by MzVum-Gal4 segregate into a dorsal fraction, consisting primarily of motoneuronal side branches, and a ventral fraction derived almost exclusively from interneurons. This pattern simplifies interpretations of experimental results obtained with this Gal4-line (for example, if mutant backgrounds reveal selective impairment of only dorsal or ventral neurites). Having applied a combination of a standardized set of Fasicilin2-positive landmarks, specific antisera, and single cell tracings, it has been possible to (1) assign most neurites of the Gal4-lines to identified neurons, and (2) define the regions of the neuropile that they occupy. Future applications of a standardised mapping strategy to other Gal4 lines will considerably advance the understanding of the functional architecture of the Drosophila neuropile, and it will form a basis with which candidate pre- and postsynaptic circuit elements can be identified (Landgraf, 2003).

An important aspect of this study is that despite its limited scope it reveals an apparent partitioning of the neuropile into (possibly functionally) distinct regions. Facets of a functional architecture of the neuropile have already been documented such as the modality-specific sensory projections that partition the ventral neuropile. Due to the Fasciclin2-based mapping, these areas can now be named and the regions can be related to projection patterns of other neurons. In accordance with work published for other insects, the dorsal neuropile is predominantly occupied by the central arbors of efferent neurons (with the single exception of the ventral type-3u neuron arbors). There is little overlap with sensory areas so that direct connections between sensory and motor neurons will be the exception. In addition, different efferent neurons elaborate their central arbors in distinct anteroposterior regions of the dorsal neuropile. These territories seem to be defined in the embryo and they are maintained through larval stages, although areas of overlap between formally distinct territories increase as central arbors become more elaborate over time. This relative constancy of the topography of the neuropile over time also exists for Serotonin-, Corazonin-, and Leucokinin-1-positive neurons. An important consequence of such constancy for future research work is that neurites can be compared or descriptions extrapolated across different developmental stages (Landgraf, 2003).

Interestingly, neuropeptidergic projections seem to cluster in particular areas. Corazonin and Leucokinin-1 (and also Serotonin) are closely associated with the DM-fascicle. Published data suggest that antibodies against FMRF, molluscan neuropeptide SCP_B, and Substance-P reveal neural structures that might also be localized in this median area. A second neuropeptide 'hot spot' is the VL-fascicles, where staining with anti-Leucokinin-1 antibodies is found. Also antisera against Allatostatin and Insulin appear to stain in this region. The fascicles are innervated by the posterior ascending cells of MzVum-Gal4 and DDC-Gal4 and curiously are detected with antisera against muscle myosin heavy chain. Interestingly, both of these neuropeptidergic 'hot spot' areas bear very prominent Fasciclin2-labelled neurites (Landgraf, 2003).

Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila

In Drosophila most thoracic neuroblasts have two neurogenic periods: an initial brief period during embryogenesis and a second prolonged phase during larval growth. Adult-specific neurons that are born primarily during the second phase of neurogenesis. The fasciculated neurites arising from each cluster of adult-specific neurons express the cell-adhesion protein Neurotactin and they make a complex scaffold of neurite bundles within the thoracic neuropils. Using MARCM clones, the 24 lineages that make up the scaffold of a thoracic hemineuromere were identified. Unlike the early-born neurons that are strikingly diverse in both form and function, the adult specific cells in a given lineage are remarkably similar and typically project to only one or two initial targets, which appear to be the bundled neurites from other lineages. Correlated changes in the contacts between the lineages in different segments suggest that these initial contacts have functional significance in terms of future synaptic partners. This paper provides an overall view of the initial connections that eventually lead to the complex connectivity of the bulk of the thoracic neurons (Truman, 2004).

In insect embryos, the vast majority of neurons in each segmental ganglion arise from 30 paired and one unpaired neuroblasts. In basal insect groups, these segmental NBs show a single neurogenic period, each producing all of its progeny during embryogenesis. In insects with complete metamorphosis, however, most of the segmental NBs in the thorax have two neurogenic periods, involving a relatively brief phase of neurogenesis during embryonic development followed by a much more prolonged phase during larval life. Mapping of postembryonic NBs in the thoracic neuromeres of Drosophila larvae indicated that 23 out of the 31 segmental NBs showed this second, larval phase of neurogenesis. The count from the present study is that there are 24 such clusters per hemisegment (Truman, 2004).

The MARCM clones analyzed in this study were induced early in embryogenesis, and should include both the embryonic and postembryonic progeny from a given neuroblast. This, indeed, was seen when Actin-GAL4 or tub-GAL4 was used as a driver to make the MARCM clones. The diversity of morphologies and strength of GFP expression in the larval neurons, however, sometimes obscured some of the neurites arising from the associated adult-specific cluster. When similar clones were generated using the purported pan-neuronal driver line, elav [C155], the fully differentiated larval neurons in the clones typically failed to show GFP expression but expression was strong in the arrested, adult-specific cells. Although the reason that mature larval neurons fail to express under these conditions is not known, elav-based clones are invaluable for determining the exact projection patterns of the clusters of adult-specific neurons and how each contributed to the overall Neurotactin scaffold. Having established the morphology of the adult-specific region of the lineage, it was then possible to return to MARCM clones generated using tub-GAL4 and Actin-GAL4 drivers to associate the neurons of adult-specific clusters with their larval siblings. Since the larval progeny of all of the embryonic neuroblasts have been described, the larval neurons aided in identification of the embryonic neuroblast responsible for many of the adult-specific clusters (Truman, 2004).

The early neurons generated by a given NB typically show a great diversity in terms of their type and their axonal projections. Indeed, the projection patterns of the daughter cells can change dramatically from one GMC to the next. Later born cells, though, appear to be much more similar in their morphologies, transmitters and functions. The present study shows that the similarity in late-born progeny is a general rule for all lineages. Although each NB may show a high degree of diversity in the first few neurons that it produces, the vast majority of their progeny are similar in their pathfinding decisions, with typically only one or two initial targets for the neurites that leave a cluster. Indeed, only 33 major projection patterns were found for the thousands of neurons that are born within a thoracic hemineuromere (Truman, 2004).

The diversity of phenotypes in the early born cells of a lineage is accomplished through the sequential expression of a series of transcription factors (hunchback, kruppel, pdm and castor) that are passed from the NB to successive GMCs. This molecular specification of unique identities imposed by the neuroblast on the first few neurons in a lineage appears to be lacking in the later born neurons, all of which express grainyhead. It is suspected that the transition from uniquely specified GMCs to ones that express the same transcription factor marks the transition from generating unique individual neurons to generating neuronal classes. For the latter cells, interaction with other neurons, rather than factors supplied by their NB, may then be essential for establishing identity within their neuronal class. It should be noted that the transition between uniquely identified neurons to neuronal classes does not necessarily lie at the dividing line between the embryonic and postembryonic phases of proliferation. By feeding larvae on diet containing bromodeoxyuridine (BUdR) from the time of hatching, all of the neurons that are born during larval growth can be labelled. Analysis of Elav-based MARCM clones in these larvae showed some lineages in which some of the developmentally arrested neurons were unlabeled and, hence, were born prior to hatching. These were always the neurons in the clone that were nearest the neuropil (i.e., the oldest cells). Hence, the NBs do not necessarily stop dividing after they make the neurons that will be used in the larva, and they may depend on an extrinsic signal to terminate their embryonic phase of neurogenesis. These embryonically born cells may serve as pioneers to guide the growth of postembryonic members of their lineage (Truman, 2004).

An interesting feature of the adult-specific neurons is that each extends an initial neurite to a lineage-specific location but then their development stalls until pupariation. As illustrated in the developing hippocampus, a developing neuron often sends out a single, unbranched process with a growth cone to navigate to an initial target. This is followed by interstitial sprouting, which then enables interactions with secondary targets. Contact with the initial target may persist or it may be lost through stereotyped pruning but connections with final targets are often then refined through local cell-cell interactions. In the adult-specific neurons in Drosophila, the period of developmental arrest separates axon pathfinding and contact with the initial target from the phase of interstitial sprouting to secondary targets. This arrest is terminated at the start of metamorphosis, when the neurons show a profuse sprouting, accompanied by the appearance of the broad-Z3 transcription factor, and the onset of nitric oxide (NO) sensitivity. The latter observation is especially interesting because studies on other insect neurons show that the onset of NO sensitivity occurs as a neuron shifts from pathfinding to interacting with its synaptic targets. The appearance of NO sensitivity at the termination of arrest suggests that the neurons have switched into a new developmental mode in which interactions with future synaptic partners become of prime importance (Truman, 2004).

Hence, the larval CNS just prior to metamorphosis gives an unprecedented snap-shot of neuronal development. Thousands of neurons are arrested at their initial targets awaiting the hormonal signals that will initiate secondary sprouting. This probably represents a watershed in the development of the CNS. Up to this point in development, the identity of the neurons and their growth decisions may have been relatively 'hard-wired' by genetic information supplied by the NB and the ganglion mother cell. After this point, interactions with their primary and secondary targets probably dominate in shaping the final phenotypes of the cells (Truman, 2004).

The map of initial contacts depicted in this study is undoubtedly not a complete description of all of these contacts. In addition, at this time the polarity of the contacts is not known, i.e. who will be presynaptic and who will be postsynaptic. Nevertheless, this map probably provides a broad overview of the first step in establishing the connectivity for the bulk of the thoracic neurons. These initial contacts acquire some functional importance when the segmental variation in their pattern is considered. The patterns in neuromeres T1, T3 and A1 are compared with the situation in T2, since this is the only segment that possesses the full complement of 24 postembryonic lineages. Importantly, many of the segmental changes involve coordinated changes in the lineages that project to the same region of the neuropil. The most obvious example involves the lineages associated with the ventrolateral neuropil. These include the motor lineage (lineage 15) that makes motoneurons exclusively and projects to a leg imaginal disc. Lineage 15 is confined to the thoracic neuromeres as are nine other lineages that send their neurite bundles exclusively to the ventrolateral neuropil. With one exception, these lineages show no obvious variation in their projection patterns between the three thoracic neuromeres. The only lineage that shows a variable projection pattern is lineage 1, which also has initial targets in two adjacent neuromeres. Accordingly this lineage retains its homosegmental projection (bundle 1c) in T1 but it lacks the 1i bundle (i.e., no bundle projects to the SEG). All of the lineages that project to the ventrolateral neuropil are absent from A1, with again the exception of lineage 1. The lineage 1 neurons arising in A1, though, all project to the T3 neuropil (via bundle 1i) and the homosegmental 1c bundle is missing. Identification of the lineages in the subesophageal neuromeres is not complete but it appears that most, if not all, of these lineages are also lacking from the subesophageal ganglion. Apart from lineages that project exclusively to the ventrolateral neuropil, there are a few lineages, like lineages 3 and 19 that have one bundle projecting to this neuropil and another projecting into more dorsal regions. This is especially interesting in the case of lineage 19 because its 19i bundle makes contact with the expanded area of the lineage 15 bundle and therefore may represent premotor interneurons. These ventrolateral projections, though, are missing in the A1 version of lineages 3 and 19. The uniformity of projection patterns within the thorax and their absence outside of this region of the body suggests that all of the lineages that project to ventrolateral neuropil make neurons involved with the sensory or motor requirements of the legs. This functional interpretation is supported by the fact that lineage 14 is one of the above lineages and its proposed homologues in grasshoppers (from NB 4-1) process input from leg mechanosensory hairs and integrate locomotor reflexes of the leg (Truman, 2004).

Although the ventrolateral projections are relatively stable within the thoracic neuromeres, projections to intermediate and dorsal neuropils show striking segmental variation. For example, lineage 11 is absent from T3 and the two lineages that send neurite bundles that terminate next to those of lineage 11 in more anterior segments, have these bundles reduced (bundle 3id) or missing altogether (the 12im and 12id bundles of lineage 12) in this segment. T1 also has its unique set of changes. In T2 and posterior, the 0 bundle from the median NB projects to the aI commissure and appears to terminate between bundle 10c (ventral to it) and bundle 18c (dorsal). The 18c bundle is missing in T1 and we see that bundle 0 is redirected to the pI commissure. T1 also shows a marked reduction in the number of bundles that project to anterior neuromeres; bundle 18c is missing and bundle 19c is greatly reduced to only a few fibers. Thus, the neurons in the 18c and 19c bundles may be involved in coordination within the thorax rather than taking information to higher centers in the head. No obvious glial structures were found at the sites where the neurite bundles terminate. The correlated loss of converging bundles (such as seen for 12id, 3id and 11id in T3), suggest that the initial targets for the neurites in a bundle from one lineage may be bundles from other lineages. The map that is presented is the first attempt to identify the lineage-level rules that are used for establishing the initial connectivity map in the thoracic CNS. Whether these initial contacts are maintained and how they relate to secondary targets remains to be determined (Truman, 2004).

Preliminary observations of embryonic induced single and double cell clones in lineage 6 show that in single neuron clones there is a single neurite that is either in the 6cm or 6cd bundle. By contrast, two neuron clones (arising from a GMC) show a neurite in both bundles. This suggests that the two bundles are built up by each GMC producing two daughters, one that chooses one pathway and one that chooses the other. While it obviously needs to be tested, it is expected that this pattern will hold for all of the lineages that have bundles projecting to two initial targets. Interestingly, in the cases in which one bundle is lost in a given segment the cell cluster in that segment is markedly smaller that in other segments. A possible mechanism to explain the segmental difference is that cell death shapes the projection pattern by having the inappropriate daughter cell die after its birth. Studies of the median lineage in grasshopper embryos show the importance of divergent sibling fates and cell death in shaping features of that lineage (Truman, 2004).

The results from this study have developmental, behavioral and evolutionary implications. Previous studies on the ventral ganglia and the brain show that the neuroblasts express a striking diversity of transcription factors and signaling molecules. Some of these molecules are involved in the establishment of the unique identity of the neuroblasts and their early-born progeny. Others, though, may function later in directing patterns of connectivity. It has been difficult to determine the latter, however, because projection patterns and potential targets were unknown for the vast majority of neurons in the lineage. This study indicates that the first step in establishing the extreme complexity of CNS connections involves a rather simple set of rules, with the bulk of the neurons of a given lineage following one or two projection paths. At this time, it is not known if the 33 different projection trajectories seen in the thoracic neuropil are the product of just 33 individual neurons per hemineuromere that pioneer the track for the rest of their lineage or if all of the adult-specific neurons follow the same set of cues to their initial targets. Irrespective of how they navigate their path, the initial connectivity patterns suggest that neurons in one lineage use other lineages as their targets. This information should help gain an understanding of the roles of patterning genes such as wingless and hedgehog in establishing connectivity and neuronal properties within the CNS (Truman, 2004).

The elegant studies of the neural circuitry underlying sensory to motor coordination in the legs of grasshoppers showed that functionally related neurons were clustered, and some, indeed are siblings that come from the same neuroblast. The uniformity of initial projections that were seen within each of the adult-specific lineages led to a speculation that each neuroblast is devoted to making a very small number of functional neuronal types, with the noted exceptions of the early-born cells that have unique identities sculpted by the expression of hunchback, kruppel, etc. Changes in specific behavioral functions between species might then be reflected in selective alterations in the particular lineages whose neurons participated in that behavior. One possible illustration of this is in the shift from primitively wingless insects to those that can fly. This was accompanied with marked increase in neuronal progeny in only 14 out of the 31 thoracic lineages. Indeed, the later born neurons in some subsets of lineages may co-evolve because these cells are functionally connected. Although there have been only minor differences in the neuroblast arrays when one compares grasshoppers to Drosophila, some of the neuroblasts have changed the blend of transcription factors that they express. It will be interesting to determine if these changes do indeed reflect a change in identity of the neuroblast or whether it reflects an alteration in instructions as to how these neurons should connect (Truman, 2004).

Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila

Locomotion in adult Drosophila depends on motor neurons that target a set of multifibered muscles in the appendages. This study describes the development of motor neurons in adult Drosophila, focusing on those that target the legs. Leg motor neurons are born from at least 11 neuroblast lineages, but two lineages generate the majority of these cells. Using genetic single-cell labeling methods, the birth order, muscle targeting, and dendritic arbors for most of the leg motor neurons were analyze. The results reveal that each leg motor neuron is born at a characteristic time of development, from a specific lineage, and has a stereotyped dendritic architecture. Motor axons that target a particular leg segment or muscle have similar dendritic arbors but can derive from different lineages. Thus, although Drosophila uses a lineage-based method to generate leg motor neurons, individual lineages are not dedicated to generate neurons that target a single leg segment or muscle type (Baek, 2009).

To study the development of the Drosophila leg motor neurons, a clonal analysis was performed using a modified version of mosaic analysis with a repressible cell marker (MARCM) method. The Vglut-Gal4 (also called OK371-Gal4) driver was used to positively label clones. This Gal4 driver, which is inserted into the Vglut gene, is expressed in all neurons that use glutamate as a neurotransmitter, including all motor neurons. As can be seen in adult leg preparations in which Vglut-Gal4 was used to express a membrane-tagged version of green fluorescent protein (CD8GFP), motor neurons innervating all of the muscles in the coxa (co), trochanter (tr), femur (fe), and tibia (ti) were labeled by this driver. In addition, a subset of sensory neurons, whose cell bodies reside in the tibia and tarsal segments, were labeled by Vglut-Gal4. Except for the tarsus, each leg segment has a stereotyped set of multifibered muscles that are labeled by the MHC-tauGFP reporter gene. This reporter gene was used to identify each of the muscles innervated by the leg motor neurons. In the adult CNS, Vglut-Gal4 labeled groups of neurons in each thoracic hemisegment. In addition to motor neuron cell bodies, the dendritic arbors of these neurons were observed in densely packed neuropils in each thoracic hemisegment. This study focused on the motor neurons innervating the first thoracic (T1) legs. The axons of these motor neurons fasciculate and exit the CNS through a large nerve that extends into the ipsilateral leg (Baek, 2009).

Drosophila NBs are born during embryogenesis and undergo two waves of neurogenesis, one during embryogenesis and one during larval development. During the first, embryonic wave of NB divisions, the majority of the embryonically born neurons are dedicated to larval motor and sensory functions and die during metamorphosis. To determine how many independent NB lineages give rise to the leg motor neurons, positively labeled MARCM clones were induced during embryogenesis and analyzed in the adult. Because these clones were generated infrequently and early in development, entire NB lineages were labeled. These data revealed that the leg motor neurons are derived from at least 11 independent lineages. Strikingly, two of these lineages, Lin A and Lin B, give rise to the majority of the leg motor neurons. Embryonically induced clones of Lin A innervated the muscles of the femur and tibia but did not include any motor neurons that targeted the coxa or trochanter. Moreover, the tibia is only targeted by Lin A-derived motor neurons. Thus, Lin A motor neurons generally target distal, but not proximal, leg segments (Baek, 2009).

The second major lineage defined by these experiments is Lin B, which gives rise to seven leg motor neurons. In contrast to Lin A, Lin B motor neurons target the three most proximal leg segments, the coxa, trochanter, and femur, but does not generate any motor neurons that target the tibia. Thus, Lin B motor neurons generally target proximal, rather than distal, leg segments (Baek, 2009).

Embryonically induced MARCM clones revealed that another 12 Vglut-Gal4⁺ leg motor neurons are generated from nine additional lineages, Lin C to Lin K. These 12 motor neurons target the coxa (six), the trochanter (one), and the femur (five), but not the tibia. In contrast to Lin A and Lin B, these lineages give rise to only one or two Vglut-Gal4-expressing leg motor neurons. Lin E is distinctive because, in addition to generating a single motor neuron targeting the coxa, it also gives rise to ~25 Vglut-Gal4-expressing interneurons. Five of these lineages (C to G) were labeled frequently, by both embryonic and postembryonic heat shocks. In contrast, four of these lineages, Lin H to Lin K, were labeled infrequently and only by embryonic heat shocks. These findings suggest that these motor neurons, which target the coxa (one) and femur (five), are born during embryogenesis and persist to the adult stage in which they contribute to the adult leg nervous system (Baek, 2009).

In total, 53 neurons were identified, derived from 11 independent NBs, that innervate the T1 leg. Two of these lineages give rise to 35 of these 53 motor neurons. By characterizing individually labeled motor neurons, the birth dates, muscle targets, and dendritic arbors for most of these motor neurons were determined. These results show that, although each motor neuron is born from a specific lineage, and at a specific time during development, individual lineages give rise to motor neurons that target multiple leg segments and multiple muscles within these leg segments (Baek, 2009).

Accurate motor neuron development in the fly requires that axons target the correct muscles along the PD axis of the leg. This axis has several levels of refinement. The first level is the global PD axis of the leg. Lin A only generates motor neurons that target the two more distal leg segments, the tibia and the femur. In addition, Lin A is the only lineage that produces motor neurons that target the tibia. In contrast, the seven Lin B motor neurons target all leg segments except the tibia. Thus, there is a PD bias built into these lineages (Baek, 2009).

A second level of refinement within the PD axis is targeting the correct muscle in individual leg segments. Among the Lin A-derived motor neurons, a PD bias was observed within the tibia and within the femur that correlates with birth date: the first half of the motor neurons born from Lin A have a strong bias for targeting proximal positions in these segments, whereas the later-born half of the motor neurons target distal muscles in these segments (Baek, 2009).

Third, for muscles that are targeted by multiple motor neurons (e.g., ltm1 in the tibia), it was found that more distal projecting motor neurons are born before those that target more proximal positions in the same muscle. The differential targeting of axons to unique positions within the same muscle suggests the existence of high-resolution topographic maps that match specific motor neurons to specific muscle compartments, as has been observed in mouse skeletal muscles (Baek, 2009).

Most of the leg motor neurons are born within a narrow window of development. The NB that gives rise to Lin A, for example, switches into a phase that is dedicated to generating leg motor neurons at ~50 h AEL. At that time, this NB begins to produce its 28 motor neurons for the next ~40 h. Presumably, this NB gives rise to nonmotor neuron progeny before this time and possibly after it completes this motor neuron generating phase. This scenario shares some similarities with the lineages that give rise to postembryonic neurons in the fly brain. For example, the entire mushroom body of Drosophila, the portion of the fly brain used in olfactory learning and memory, is derived from only four NBs that each give rise to one of four nearly identical anatomical units. Interestingly, there is a temporal switch in the types of neurons that these NBs generate at specific times of development. Thus, like Lin A, mushroom body NBs switch the type of neuron they generate at specific times. However, unlike the leg motor neuron NBs, those that generate the mushroom body are dedicated to forming this brain structure. In contrast, it was found that functionally related leg motor neurons, for example those that target a specific leg segment, muscle, or muscle type, are often derived from several different NB lineages. This logic is reminiscent of that used to generate olfactory projection neurons in the fly, in which three neuroblasts each give rise to different numbers and types of projection neurons (Baek, 2009).

The temporal control of NB identity in Drosophila is directed by transcription factors that are sequentially expressed as NBs age. During embryogenesis, progeny postmitotic neurons inherit the transcription factor expressed in the NB at the time it was born. This temporal information works in combination with positional information that makes each NB unique, providing progeny neurons their individual identities. Although the specific factors are not yet known, a similar transcription factor code may exist for leg motor neurons. Two of the temporal control genes that are used during Drosophila embryogenesis, seven-up (svp) and castor (cas), are also important for controlling postembryonic neural fates. Interestingly, some NBs switch from expressing cas to svp at ~50 h AEL, similar to the time that the leg NBs begin to generate their leg motor neuron progeny. It will be interesting to determine whether this or other changes in transcription factors are responsible for initiating the production of leg motor neurons in the lineages defined here (Baek, 2009).

These results demonstrate that adult motor neurons in the fly come from identifiable lineages that give rise to stereotyped progeny with defined birth dates. Importantly, however, of the 11 lineages that give rise to leg motor neurons in the fly, only one of these, Lin A, appears to be dedicated to producing these neurons. Even this restriction only occurs during the ~50 to ~90 h AEL time window. Although most of the progeny produced by the other lineages were not marked in these experiments (except for Lin E, which generates ~25 Vglut-Gal4⁺ interneurons), it is likely that these lineages also produce nonmotor neuron progeny. Thus, although seemingly invariant lineages are used in the fly, the closest relatives for many leg motor neurons are not other leg motor neurons. This conclusion is similar to the picture that emerged from lineage analyses performed in the vertebrate spinal cord showing that cell lineages are not dedicated to the production of motor neurons. As in the fly, closely related cells in the spinal cord may have distinct fates. Conversely, although adult fly motor neurons are born from stereotyped lineages, position within the CNS determines NB identities and, consequently, the progeny they generate. Although C. elegans has a more extreme version of a lineage-based mechanism, even in this case cell-cell signaling plays an important role in specifying identities. These considerations blur the distinction between lineage and position-based mechanisms and suggest that both play a role in vertebrates and invertebrates (Baek, 2009).

Consistent with the idea that lineage may play a role in vertebrates, the transcription factor Coup-TF acts as a temporal switch between neurogenesis and gliogenesis in the vertebrate brain. Interestingly, Coup-TF is a relative of Drosophila svp, which encodes one of the temporal transcription factors used in postembryonic fly neuroblasts. The use of Coup-TF/Svp for executing a temporal switch in both flies and vertebrates suggests the existence of a conserved molecular mechanism for controlling developmental timing in neural lineages (Baek, 2009).

Because motor neurons receive complex inputs from interneurons and sensory neurons, the architecture of their dendritic arbors is critical for forming the circuitry that is required for locomotion. An initial analysis of the dendritic arbors of the leg motor neurons suggests that, as in other systems, they exhibit a functional organization in the thoracic neuromere. For example, nine leg motor neurons, targeting two different reductor muscles in different leg segments (coxa and femur), have overlapping dendritic arbors. That these nine motor neurons have similar dendritic architectures suggests that they share presynaptic inputs, perhaps allowing these two reductor muscles to contract in synchrony. Similarly, all eight motor neurons that have dendrites that cross the midline of the CNS, and thus probably make contacts with neurons in the contralateral neuromere, send their axons to one of two long tendon muscles, one in the tibia and one in the femur. These two examples suggest that the organization of motor neuron dendrites may be important for muscle synergies as described in vertebrate locomotion (Baek, 2009).

In vertebrate motor systems, motor neuron cell bodies are organized in columns and pools that correlate with their muscle targets. This organization implies that many of the presynaptic inputs into the motor neurons within individual pools will be similar. Consistently, in some cases, the dendritic arbors of motor neurons have been shown to correlate with motor neuron targeting. In these examples, the arborization patterns are controlled by the transcription factor Pea3, which requires a specific Hox code to be activated, but is only induced after motor axons invade the limb target. In contrast, the myotopic map exhibited by the dendrites of the fly larval motor neurons does not need target muscles to form. In the fly olfactory system, the dendrites of projection neurons form a map in the antennal lobe before the arrival of olfactory receptor neurons (ORNs), suggesting that this map forms independently of ORNs. It remains unclear whether the characteristic dendritic arbors of the fly's leg motor neurons require muscle targeting or whether they form independently of their targets using local cues in the CNS and the identities they acquire at birth (Baek, 2009).

Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity: Removing Pvr or disrupting Rac1 function inhibits CNS condensation

Condensation is a process whereby a tissue undergoes a coordinated decrease in size and increase in cellular density during development. Although it occurs in many developmental contexts, the mechanisms underlying this process are largely unknown. This study investigated condensation in the embryonic Drosophila ventral nerve cord (VNC). Two major events coincide with condensation during embryogenesis: the deposition of extracellular matrix by hemocytes, and the onset of central nervous system activity. Preventing hemocyte migration by removing the function of the Drosophila VEGF receptor homologue, Pvr, or by disrupting Rac1 function in these cells, inhibits condensation. In the absence of hemocytes migrating adjacent to the developing VNC, the extracellular matrix components Collagen IV, Viking and Peroxidasin are not deposited around this tissue. Blocking neural activity by targeted expression of tetanus toxin light chain or an inwardly rectifying potassium channel also inhibits condensation. Disrupting Rac1 function in either glia or neurons, including those located in the nerve cord, causes a similar phenotype. These data suggest that condensation of the VNC during Drosophila embryogenesis depends on both hemocyte-deposited extracellular matrix and neural activity, and suggest a mechanism whereby these processes work together to shape the developing central nervous system (Olofsson, 2005).

Thus, disrupting hemocyte migration inhibits VNC condensation in the embryo. Lack of hemocyte migration is associated with a severe reduction of ECM components (Collagen IV and Peroxidasin) throughout the embryo and more particularly a loss of these components around the VNC. This leads to a proposal that correct assembly of the ECM depends on hemocytes, and that basement membrane is required for condensation. Supporting a role for ECM in VNC condensation, defects are observed in loss-of-function mutants of integrins, which are ECM receptors and appear themselves to be required for correct assembly of basement membranes. Mutants in integrins or the ECM component Laminin A share at least one other phenotype with embryos in which hemocyte migration has been inhibited: gut morphogenesis is impaired. Thus, a dysfunctional ECM may explain several of the morphogenetic defects seen in embryos with defective hemocyte migration (Olofsson, 2005).

How might basement membrane contribute to VNC condensation? Basement membrane may serve as a substrate for cellular movements involved in condensation and/or regulate signaling events relevant to condensation. Basement membrane is also required for normal neuromuscular junction development, and might be part of the functional blood-brain barrier in Drosophila. Hence, neural function may be disrupted when basement membrane formation is inhibited. However, condensation phenotypes in embryos with impeded hemocyte migration are more severe than in embryos in which neural activity has been blocked. This argues that the condensation phenotype seen in hemocyte migration-blocked embryos cannot be explained simply by a loss of neural activity (Olofsson, 2005).

Although animals in which hemocyte migration is blocked fail to deposit Collagen IV appropriately, it has not been demonstrated that Collagen IV function is required for condensation. However, embryos expressing a dominant negative form of Collagen IV under the control of a heatshock promoter fail to condense their nerve cord. While these data point towards a functional role of Collagen IV in condensation, further studies will be necessary to clarify the specific role of Collagen IV during condensation (Olofsson, 2005).

This study has not investigated whether phagocytosis of cells within the VNC contributes to condensation. pvr mutants show a perdurance of unengulfed cells at the ventral surface of the CNS at stage 14. The majority of these cells seem to disappear later, possibly engulfed by epidermal cells. pvr mutants also maintain some very restricted points of attachment between the epidermis and the VNC. This phenotype is not observed when hemocyte migration is blocked using mutant Rac1 expressed by crq-GAL4. This likely reflects failure of hemocyte migration at a later stage, after the two tissues have separated (Olofsson, 2005).

The major cell type that engulfs apoptotic corpses within the CNS is the subperineural glia. In the absence of macrophages (in the Bic-D mutants), apoptotic cells are still expelled from the CNS but accumulate at the ventral surface, similar to the observations in the pvr mutant. Hemocytes are required for normal CNS morphogenesis: at stage 16, pvr mutants and Crq RNAi treated embryos have mispositioned glia and minor axon scaffolding defects. These data were interpreted to reflect a failure of engulfment of cell corpses. In the context of these findings, an additional cause for glial mispositioning in pvr mutant embryos could be a loss of basement membrane components and the failure to condense (Olofsson, 2005).

VNC condensation correlates with the onset of neural activity in the CNS, and it is found that expressing tetanus toxin light chain or the inwardly rectifying K+ channel Kir2.1 pan-neuronally impairs condensation. This suggests that neural activity influences normal condensation. Neural activity could contribute to condensation in multiple ways. It could directly regulate cellular events relevant to condensation, such as adhesion or actin-based motility, or activity could influence the transcription of genes relevant to such events. Alternatively, neural activity could maintain synaptic connectivity among cells necessary for condensation, rather than directing changes in cellular behavior leading to condensation. Some condensation occurs before neural activity begins, and the condensation phenotypes resulting from impeding hemocyte migration are more severe than those resulting from blocking neural activity. This suggests that there may be multiple stages of condensation, including an earlier activity-independent stage and a later stage that is influenced by activity (Olofsson, 2005).

VNC condensation can be inhibited by expressing mutant Rac1 in lateral glia or neurons. In glia, migration and ensheathing behaviors require cytoskeletal integrity. When mutant Rac1 is expressed in peripheral glia, the formation of cellular extensions is disrupted, and this is accompanied by glia migration and axon ensheathment defects. Similarly, ensheathment of longitudinal axon tracts by longitudinal glia is disrupted in htl loss of function embryos. The VNC condensation phenotype in these embryos is interpreted as indication that glia need dynamic actin cytoskeleton to generate a condensing force. Two types of VNC glia are particularly well placed to generate such a force: longitudinal glia associated with VNC longitudinal connectives, and perineural glia, which ensheath the cortex of the VNC. Cell-cell contacts and cell-ECM contacts among these cells accompanied by remodeling of extracellular matrix could help generate a condensing force within and across neuromeres through changes in cell shape, adhesion or migration. A similar process occurs during mesenchymal condensation (Olofsson, 2005).

In neurons, neurite extension requires normal Rac GTPase activity. Expressing mutant Rac1 in these cells causes defects in axonal outgrowth. In wild type animals, VNC axons are arranged into longitudinal connectives that extend along the length of the nerve cord, and these are well placed to generate an anteroposterior condensing force. This could happen through differential cell adhesion of neurites within the longitudinal connectives or overall shortening of the axons. The observation that axons in VNC longitudinal connectives loop out during condensation in metamorphic insects is consistent with this idea. It is interesting to note that condensation is inhibited in embryos in which mutant Rac1 is expressed in glia, but longitudinal axon tracts appear normal in these animals. This suggests that if axons help generate a condensing force, they likely do this with the help of glia, possibly using these cells as a substrate (Olofsson, 2005).

It is also possible that at least part of the force required for condensation may come from outside the VNC. Somatic muscles connect to the VNC during embryogenesis, and embryonic muscle activity toward the end of embryogenesis is well timed for generating such a force. Also, the methods used to manipulate glia or neuron development in this study may affect neuromuscular activity by disrupting blood-brain barrier formation, or by affecting the Rac-dependent formation of synaptic structures. However, the observation that the CNS can condense in mutants in which muscles do not form normally argues against a major contribution from muscle activity (Olofsson, 2005).

These data identify several areas for further investigation. By following the behavior of small populations of cells in the VNC it may possible to analyze in vivo changes associated with the condensation process and get insight into how changes in organ shape are generated and coordinated. It will also be interesting to examine the contributions made by components of the ECM to normal blood-brain barrier function. Finally, it may be possible to use VNC condensation in embryonic Drosophila to investigate the molecular and cellular basis of how neural activity is translated into a morphogenetic event (Olofsson, 2005).

Polarity and intracellular compartmentalization of Drosophila neurons

Proper neuronal function depends on forming three primary subcellular compartments: axons, dendrites, and soma. Each compartment has a specialized function (the axon to send information, dendrites to receive information, and the soma is where most cellular components are produced). In mammalian neurons, each primary compartment has distinctive molecular and morphological features, as well as smaller domains, such as the diffusion barrier marked axon initial segment, that have more specialized functions. How neuronal subcellular compartments are established and maintained is not well understood. Genetic studies in Drosophila have provided insight into other areas of neurobiology, but it is not known whether flies are a good system in which to study neuronal polarity because a comprehensive analysis of Drosophila neuronal subcellular organization has not been performed. This study used new and previously characterized markers to examine Drosophila neuronal compartments. This study found that; (1) axons and dendrites can accumulate different microtubule-binding proteins; (2) protein synthesis machinery is concentrated in the cell body; (3) pre- and post-synaptic sites localize to distinct regions of the neuron, and (4) specializations similar to the initial segment are present. In addition, EB1-GFP dynamics were tracked and it was determined that microtubules in axons and dendrites have opposite polarity. It is concluded that Drosophila will be a powerful system to study the establishment and maintenance of neuronal compartments (Rolls, 2007; full text of article).

To determine whether microtubule-binding proteins can be preferentially localized to axons and dendrites in flies, exogenous and endogenous microtubule-binding proteins were examined in the Drosophila larval brain. Two exogenous proteins were examined: tau-green fluorescent protein (tau-GFP) and nod-yellow fluorescent protein (nod-YFP). Some reports have suggested that tagged versions of the microtubule binding domain of bovine tau preferentially label axons in flies, although others have also reported dendrite localization. The distribution of one of these tagged bovine tau proteins, tau-myc-GFP (which is call tau-GFP for simplicity) was examined in mushroom body and projection neurons. In both mushroom body and projection neurons, tau-GFP was abundant in the main axon tracts. It was less abundant in distal axons and dendrites. For comparison, mCD8-GFP is present in all neuronal compartments. Thus, tau-GFP preferentially labels proximal axons. Fusion proteins that consist of the nod motor domain, kinesin coiled-coil, and a tag have previously been localized to dendrites. To confirm that nod fusions label dendrites specifically, nod-YFP was expressed in mushroom body and projection neurons. In both cases nod-YFP localized clearly to dendrites but not axons. Thus, two exogenous microtubule-binding proteins, tau-GFP and nod-YFP, localize to different neuronal compartments, indicating that axonal and dendritic microtubules have distinct features in Drosophila (Rolls, 2007).

The localization was examined of two Drosophila microtubule-binding proteins under control of their own promoters, GFP-Map205 and GFP-Jupiter. Since a difference in microtubule orientation in axons and dendrites is a fundamental aspect of vertebrate neuronal polarity, it was of interest to determine exactly how microtubules are arranged in fly neurons. The dendritic localization of nod fusion proteins, which are believed to act as minus-end directed motors, has been used to argue that Drosophila dendrites are likely to have minus-ends distal to the cell body like vertebrate dendrites. However, direct analysis of the orientation of individual microtubules has not been performed in any invertebrate neuron (Rolls, 2007).

Analysis of a microtubule plus-end tracking protein was used in mammalian neurons to confirm that axon and dendrite microtubules have different orientations. Since these proteins generally bind only to the growing plus ends of microtubules, microtubule orientation can be inferred from the direction of movement of a tagged plus-end binding protein. The peripheral nervous system of the Drosophila larva is well-suited to live imaging and has been used to study actin dynamics. The plus-end binding protein EB1-GFP was expressed throughout the nervous system using an elav-Gal4 driver, and time lapse imaging of the dorsal cluster of the peripheral nervous system was performed in live, whole, early L2 larvae. EB1-GFP dynamics were analyzed in axons and dendrites of dendritic arborization neurons, which have highly branched dendrites (Rolls, 2007).

EB1-GFP dots were clearly seen moving in the cell body, axons, and dendrites. Movements of EB1-GFP dots were consistent with the tagged protein binding only to growing microtubule plus ends: an individual dot that could be followed through multiple frames never changed direction, and after a dot tracked through a particular region of an axon or dendrite and disappeared, often a dot with a similar track appeared several frames later. All EB1-GFP dots in axons moved away from the cell body. In dendrites the movements were very different. The vast majority of dots moved toward the cell body. Occasionally, dendrites were observed that had dots moving in opposite directions (Rolls, 2007).

Thus, in fly dendritic arborization neurons, axonal microtubules were oriented with plus ends distal to the cell body. Most dendritic microtubules were oriented with minus ends distal to the cell body, although dendrite microtubules were sometimes mixed in orientation (Rolls, 2007).

In mammalian neurons, the bulk of protein synthesis takes place in the cell body. A tagged ribosomal protein, L10-YFP, was generated to determine where protein synthesis takes place in Drosophila neurons. When expressed in both mushroom body and projection neurons, L10-YFP was concentrated in the cell body, with only very faint signal present in neuropil. Within the cell body it was seen in the cytoplasm, and in some cells it was also present in the nucleus, where ribosomes are assembled. Faint signal in dendrites may represent ribosomes or free L10-YFP (Rolls, 2007).

To confirm that the L10-YFP marker represents the localization of endogenous protein synthesis machinery, its localization was compared to two proteins identified in a protein trap screen. One of the GFP insertions was in the belle gene. Bel is a DEAD-box protein that is likely to function as an RNA-binding protein with a role in translation initiation. The GFP transposon insertion was homozygous viable. Since bel is an essential gene, this means that the GFP-tagged protein that is generated from the insertion is likely to be functional, and thus the localization of the protein trap very likely represents that of the endogenous protein. Bel was broadly expressed, and in neurons it localized to the cell body. The other insertion analyzed was in the Protein disulfide isomerase gene. Pdi is a chaperone that resides in the endoplasmic reticulum (ER) lumen and is involved in processing newly synthesized membrane and secretory proteins. GFP-Pdi was also seen throughout the brain and was highly concentrated in the neuron cell body. Within the cell body, GFP-Pdi was brightest in the perinuclear region, which is consistent with localization to the ER. Thus, an exogenous protein synthesis protein, L10-YFP, and two endogenous ones, GFP-Bel and GFP-Pdi, were all concentrated in the neuronal cell body, suggesting that the bulk of protein synthesis takes place there (Rolls, 2007).

One of the longest recognized forms of neuronal compartmentalization is concentration of postsynaptic sites to dendrites and presynaptic sites to axons. To determine whether excitatory synaptic inputs are received in dendrites, the distribution of the postsynaptic marker homer-GFP was analyzed in mushroom body neurons. Mammalian homer proteins bind metabotropic glutamate receptors and Shank, which forms a complex with NMDA glutamate receptors. The Drosophila Homer protein also binds Shank and localizes to postsynaptic sites. Homer-GFP localizes similarly to endogenous Homer. In brains that expressed Homer-GFP at low levels in the mushroom body, fluorescence was confined to dendrites and dots in the cell body (likely to be the Golgi), and was not present in axons. The pattern of Homer-GFP fluorescence in the mushroom body dendrite region (calyx) was similar to the strongest regions of staining with anti-Dlg and anti-Scrib immunofluorescence; both of these proteins are concentrated at postsynaptic sites. Thus, a marker of excitatory postsynaptic sites was polarized to dendrites (Rolls, 2007).

Low expression level n-synaptobrevin-YFP (n-syb-YFP) transgenic flies were generatede to specifically label synaptic vesicles in the mushroom body. These transgenes had either one or two UAS sites upstream of the transcriptional start site, rather than the usual five. Spots of fluorescence were seen in the axons and dendrites; very little was present in the cell body. To confirm that the dots of fluorescence represented synaptic vesicles, brains expressing n-syb-YFP were stained in the mushroom body with cysteine string protein (CSP) and Scrib antibodies. CSP is a synaptic vesicle protein that is abundant in all presynaptic terminals, and Scrib is a synaptic protein that is concentrated in the postsynaptic terminal. Many of the n-syb-YFP dots in the calyx region of the brain, which contains mushroom body dendrites, overlapped with the CSP, but not the Scrib, pattern, indicating that n-syb-YFP is present in synaptic vesicles. Extrinsic neurons, such as olfactory projection neurons, synapse onto mushroom body dendrites in the calyx, and so it is expected that not all synaptic vesicles would be accounted for by n-syb-YFP expressed in mushroom body neurons. Having verified that the low expression n-syb-YFP marker colocalized with synaptic vesicles, it is concluded that synaptic vesicles are present in axons and dendrites of mushroom body neurons (Rolls, 2007).

Thus far, this study has concentrated on basic differences between axons, dendrites and the cell body. One of the most important further regional specializations is the axon initial segment, which contains specific arrangements of membrane and cytoskeletal proteins. In this survey of marker localization in Drosophila neurons, two proteins were identified that showed very distinctive localization to the proximal neurite and axon (Rolls, 2007).

NgCAM-YFP expressed at low levels was concentrated at the beginning of the neurite in mushroom body neurons. In projection neurons, NgCAM-YFP was clearly seen in the primary neurite before the dendrites branched off, and in the proximal axon beyond the dendrite branch point. Much fainter fluorescence was present in distal axons and dendrites. NgCAM is a chick neural cell adhesion molecule that is selectively localized to axons when expressed in cultured hippocampal neurons, and can be tethered by ankyrins in the initial segment (Rolls, 2007).

Another tagged protein, Apc2-GFP, was targeted to the proximal region of Drosophila axons. In both mushroom body and projection neurons, Apc2-GFP was present in the cell body and dendrites. In mushroom body neurons it localized to just one region of the axons, near the beginning of the peduncle. In projection neurons, Apc2-GFP also localized to the proximal axon, but the pattern was not quite as striking as in the mushroom body, probably because Apc2-GFP expression levels were lower in the projection neurons. The region of the proximal axon to which Apc2-GFP was localized in mushroom body neurons was just distal to the stretch of proximal neurites in which NgCAM-YFP was concentrated. Adenomatous polyposis coli (APC) proteins regulate wingless signaling, and they also bind a number of cytoskeletal proteins, including microtubules and the plus-end microtubule binding protein EB1. The localization of two cytoskeleton-interacting proteins to the proximal axon in flies indicates that the Drosophila axon is divided into domains with specialized cytoskeletal properties. It will be interesting to determine whether this region is functionally similar to the vertebrate axon initial segment (Rolls, 2007).

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Genes involved in organ development

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