Serial electron microscopic reconstruction of the drosophila larval eye: Photoreceptors with a rudimentary rhabdomere of microvillar-like processes.

Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3 µm diameter) alternating with thin segments (>0.1 µm). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.

Structure and development of the subesophageal zone of the Drosophila brain. I. Segmental architecture, compartmentalization, and lineage anatomy.

The subesophageal zone (SEZ) of the Drosophila brain houses the circuitry underlying feeding behavior and is involved in many other aspects of sensory processing and locomotor control. Formed by the merging of four neuromeres, the internal architecture of the SEZ can be best understood by identifying segmentally reiterated landmarks emerging in the embryo and larva, and following the gradual changes by which these landmarks become integrated into the mature SEZ during metamorphosis. In previous works, the system of longitudinal fibers (connectives) and transverse axons (commissures) has been used as a scaffold that provides internal landmarks for the neuromeres of the larval ventral nerve cord. We have extended the analysis of this scaffold to the SEZ and, in addition, reconstructed the tracts formed by lineages and nerves in relationship to the connectives and commissures. As a result, we establish reliable criteria that define boundaries between the four neuromeres (tritocerebrum, mandibular neuromere, maxillary neuromere, labial neuromere) of the SEZ at all stages of development. Fascicles and lineage tracts also demarcate seven columnar neuropil domains (ventromedial, ventro-lateral, centromedial, central, centrolateral, dorsomedial, dorsolateral) identifiable throughout development. These anatomical subdivisions, presented in the form of an atlas including confocal sections and 3D digital models for the larval, pupal and adult stage, allowed us to describe the morphogenetic changes shaping the adult SEZ. Finally, we mapped MARCM-labeled clones of all secondary lineages of the SEZ to the newly established neuropil subdivisions. Our work will facilitate future studies of function and comparative anatomy of the SEZ.

Metamorphosis of the Drosophila visceral musculature and its role in intestinal morphogenesis and stem cell formation.

The visceral musculature of the Drosophila intestine plays important roles in digestion as well as development. Detailed studies investigating the embryonic development of the visceral muscle exist; comparatively little is known about postembryonic development and metamorphosis of this tissue. In this study we have combined the use of specific markers with electron microscopy to follow the formation of the adult visceral musculature and its involvement in gut development during metamorphosis. Unlike the adult somatic musculature, which is derived from a pool of undifferentiated myoblasts, the visceral musculature of the adult is a direct descendant of the larval fibers, as shown by activating a lineage tracing construct in the larval muscle and obtaining labeled visceral fibers in the adult. However, visceral muscles undergo a phase of remodeling that coincides with the metamorphosis of the intestinal epithelium. During the first day following puparium formation, both circular and longitudinal syncytial fibers dedifferentiate, losing their myofibrils and extracellular matrix, and dissociating into mononuclear cells ("secondary myoblasts"). Towards the end of the second day, this process is reversed, and between 48 and 72h after puparium formation, a structurally fully differentiated adult muscle layer has formed. We could not obtain evidence that cells apart from the dedifferentiated larval visceral muscle contributed to the adult muscle, nor does it appear that the number of adult fibers (or nuclei per fiber) is increased over that of the larva by proliferation. In contrast to the musculature, the intestinal epithelium is completely renewed during metamorphosis. The adult midgut epithelium rapidly expands over the larval layer during the first few hours after puparium formation; in case of the hindgut, replacement takes longer, and proceeds by the gradual caudad extension of a proliferating growth zone, the hindgut proliferation zone (HPZ). The subsequent elongation of the hindgut and midgut, as well as the establishment of a population of intestinal stem cells active in the adult midgut and hindgut, requires the presence of the visceral muscle layer, based on the finding that ablation of this layer causes a severe disruption of both processes.

Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells.

Proliferating intestinal stem cells (ISCs) generate all cell types of the Drosophila midgut, including enterocytes, endocrine cells, and gland cells (e.g., copper cells), throughout the lifetime of the animal. Among the signaling mechanisms controlling the balance between ISC self-renewal and the production of different cell types, Notch (N) plays a pivotal role. In this paper we investigated the emergence of ISCs during metamorphosis and the role of N in this process. Precursors of the Drosophila adult intestinal stem cells (pISCs) can be first detected within the pupal midgut during the first hours after onset of metamorphosis as motile mesenchymal cells. pISCs perform 2-3 rounds of parasynchronous divisions. The first mitosis yields only an increase in pISC number. During the following rounds of mitosis, dividing pISCs give rise to more pISCs, as well as the endocrine cells that populate the midgut of the eclosing fly. Enterocytes do not appear among the pISC progeny until around the time of eclosion. The "proendocrine" gene prospero (pros), expressed from mid-pupal stages onward in pISCs, is responsible to advance the endocrine fate in these cells; following removal of pros, pISCs continue to proliferate, but endocrine cells do not form. Conversely, the onset of N activity that occurs around the stage when pros comes on restricts pros expression among pISCs. Loss of N abrogates proliferation and switches on an endocrine fate among all pISCs. Our results suggest that a switch depending on the activity of N and pros acts at the level of the pISC to decide between continued proliferation and endocrine differentiation.

Patterns of growth and tract formation during the early development of secondary lineages in the Drosophila larval brain.

The Drosophila brain consists of a relatively small number of invariant, genetically determined lineages which provide a model to study the relationship between gene function and neuronal architecture. In following this long-term goal, we reconstruct the morphology (projection pattern and connectivity) and gene expression patterns of brain lineages throughout development. In this article, we focus on the secondary phase of lineage morphogenesis, from the reactivation of neuroblast proliferation in the first larval instar to the time when proliferation ends and secondary axon tracts have fully extended in the late third larval instar. We have reconstructed the location and projection of secondary lineages at close (4 h) intervals and produced a detailed map in the form of confocal z-projections and digital three-dimensional models of all lineages at successive larval stages. Based on these reconstructions, we could compare the spatio-temporal pattern of axon formation and morphogenetic movements of different lineages in normal brain development. In addition to wild type, we reconstructed lineage morphology in two mutant conditions. (1) Expressing the construct UAS-p35 which rescues programmed cell death we could systematically determine which lineages normally lose hemilineages to apoptosis. (2) so-Gal4-driven expression of dominant-negative EGFR ablated the optic lobe, which allowed us to conclude that the global centrifugal movement normally affecting the cell bodies of lateral lineages in the late larva is causally related to the expansion of the optic lobe, and that the central pattern of axonal projections of these lineages is independent of the presence or absence of the optic lobe.

Lineage-associated tracts defining the anatomy of the Drosophila first instar larval brain.

Fixed lineages derived from unique, genetically specified neuroblasts form the anatomical building blocks of the Drosophila brain. Neurons belonging to the same lineage project their axons in a common tract, which is labeled by neuronal markers. In this paper, we present a detailed atlas of the lineage-associated tracts forming the brain of the early Drosophila larva, based on the use of global markers (anti-Neuroglian, anti-Neurotactin, inscuteable-Gal4>UAS-chRFP-Tub) and lineage-specific reporters. We describe 68 discrete fiber bundles that contain axons of one lineage or pairs/small sets of adjacent lineages. Bundles enter the neuropil at invariant locations, the lineage tract entry portals. Within the neuropil, these fiber bundles form larger fascicles that can be classified, by their main orientation, into longitudinal, transverse, and vertical (ascending/descending) fascicles. We present 3D digital models of lineage tract entry portals and neuropil fascicles, set into relationship to commonly used, easily recognizable reference structures such as the mushroom body, the antennal lobe, the optic lobe, and the Fasciclin II-positive fiber bundles that connect the brain and ventral nerve cord. Correspondences and differences between early larval tract anatomy and the previously described late larval and adult lineage patterns are highlighted. Our L1 neuro-anatomical atlas of lineages constitutes an essential step towards following morphologically defined lineages to the neuroblasts of the early embryo, which will ultimately make it possible to link the structure and connectivity of a lineage to the expression of genes in the particular neuroblast that gives rise to that lineage. Furthermore, the L1 atlas will be important for a host of ongoing work that attempts to reconstruct neuronal connectivity at the level of resolution of single neurons and their synapses.

Migration of Drosophila intestinal stem cells across organ boundaries.

All components of the Drosophila intestinal tract, including the endodermal midgut and ectodermal hindgut/Malpighian tubules, maintain populations of dividing stem cells. In the midgut and hindgut, these stem cells originate from within larger populations of intestinal progenitors that proliferate during the larval stage and form the adult intestine during metamorphosis. The origin of stem cells found in the excretory Malpighian tubules ('renal stem cells') has not been established. In this paper, we investigate the migration patterns of intestinal progenitors that take place during metamorphosis. Our data demonstrate that a subset of adult midgut progenitors (AMPs) move posteriorly to form the adult ureters and, consecutively, the renal stem cells. Inhibiting cell migration by AMP-directed expression of a dominant-negative form of Rac1 protein results in the absence of stem cells in the Malpighian tubules. As the majority of the hindgut progenitor cells migrate posteriorly and differentiate into hindgut enterocytes, a group of the progenitor cells, unexpectedly, invades anteriorly into the midgut territory. Consequently, these progenitor cells differentiate into midgut enterocytes. The midgut determinant GATAe is required for the differentiation of midgut enterocytes derived from hindgut progenitors. Wingless signaling acts to balance the proportion of hindgut progenitors that differentiate as midgut versus hindgut enterocytes. Our findings indicate that a stable boundary between midgut and hindgut/Malpighian tubules is not established during early embryonic development; instead, pluripotent progenitor populations cross in between these organs in both directions, and are able to adopt the fate of the organ in which they come to reside.

A novel tissue in an established model system: the Drosophila pupal midgut.

The Drosophila larval and adult midguts are derived from two populations of endodermal progenitors that separate from each other in the early embryo. As larval midgut cells differentiate into an epithelial layer, adult midgut progenitors (AMPs) remain as small clusters of proliferating, undifferentiated cells attached to the basal surface of the larval gut epithelium. During the first few hours of metamorphosis, AMPs merge into a continuous epithelial tube that overgrows the larval layer and differentiates into the adult midgut; at the same time, the larval midgut degenerates. As shown in this paper, there is a second, transient pupal midgut that develops from the AMPs at the beginning of metamorphosis and that intercalates between the adult and larval midgut epithelia. Cells of the transient pupal midgut form a multilayered tube that exhibits signs of differentiation, in the form of septate junctions and rudimentary apical microvilli. Some cells of the pupal midgut develop as endocrine cells. The pupal midgut remains closely attached to the degenerating larval midgut cells. Along with these cells, pupal midgut cells are sequestered into the lumen where they form the compact "yellow body." The formation of a pupal midgut has been reported from several other species and may represent a general feature of intestinal metamorphosis in insects.

Development of the Drosophila entero-endocrine lineage and its specification by the Notch signaling pathway.

In this paper we have investigated the developmental-genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors. As in the embryo, activation of the Notch pathway triggers enterocyte differentiation and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells.

Lineage-based analysis of the development of the central complex of the Drosophila brain.

Most neurons of the central complex belong to 10 secondary (larvally produced) lineages. In the late larva, undifferentiated axon tracts of these lineages form a primordium in which all of the compartments of the central complex can be recognized as discrete entities. Four posterior lineages (DPMm1, DPMpm1, DPMpm2, and CM4) generate the classes of small-field neurons that interconnect the protocerebral bridge, fan-shaped body, noduli, and ellipsoid body. Three lineages located in the anterior brain, DALv2, BAmv1, and DALcl2, form the large-field neurons of the ellipsoid body and fan-shaped body, respectively. These lineages provide an input channel from the optic tubercle and connect the central complex with adjacent anterior brain compartments. Three lineages in the posterior cortex, CM3, CP2, and DPMpl2, connect the posterior brain neuropil with specific layers of the fan-shaped body. Even though all of the compartments of the central complex are prefigured in the late larval brain by the axon tracts of the above-mentioned lineages, the neuropil differentiates during the first 2 days of the pupal period when terminal branches and synapses of secondary neurons are formed. During this phase the initially straight horizontal layers of the central complex bend in the frontal plane, which produces the characteristic shape of the fan-shaped and ellipsoid body. Our analysis provides a comprehensive picture of the lineages that form the central complex, and will facilitate future studies that address the structure or function of the central complex at the single cell level.

Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain.

The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are "scaffolded" by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments.

The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling.

The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.

Embryonic origin of the Drosophila brain neuropile.

Neurons of the Drosophila larval brain are formed by a stereotyped set of neuroblasts. As differentiation sets in, neuroblast lineages produce axon bundles that initially form a scaffold of unbranched fibers in the center of the brain primordium. Subsequently, axons elaborate interlaced axonal and dendritic arbors, which, together with sheath-like processes formed by glial cells, establish the neuropile compartments of the larval brain. By using markers that visualize differentiating axons and glial cells, we have analyzed the formation of neuropile compartments and their relationship to neuroblast lineages. Neurons of each lineage extend their axons as a cohesive tract ("primary axon bundle"). We generated a map of the primary axon bundles that visualizes the location of the primary lineages in the brain cortex where the axon bundles originate, the trajectory of the axon bundles into the neuropile, and the relationship of these bundles to the early-formed scaffold of neuropile pioneer tracts (Nassif et al. [1998] J. Comp. Neurol. 402:10-31). The map further shows the growth of neuropile compartments at specific locations around the pioneer tracts. Following the time course of glial development reveals that glial processes, which form prominent septa around compartments in the larval brain, appear very late in the embryonic neuropile, clearly after the compartments themselves have crystallized. This suggests that spatial information residing within neurons, rather than glial cells, specifies the location and initial shape of neuropile compartments.

Early development of the Drosophila brain: IV. Larval neuropile compartments defined by glial septa.

In this study, we have analyzed the architecture of the brain neuropile of the Drosophila larva, which is formed by two main structural elements: long axon tracts and terminal axonal/dendritic arborizations carrying synapses. By using several molecular markers expressed in neurons and glial cells, we show that the early larval neuropile is subdivided by glial sheaths into numerous compartments. The three-dimensional layout of these compartments and their relationship to the pattern of long axon tracts described in the accompanying article (Nassif et al. [2003] J. Comp. Neurol 417-434) was modeled by using a three-dimensional illustration computer software. On the basis of their location relative to each other and to long axon tracts, larval brain compartments can be identified with compartments defined by structural and functional criteria for the adult fly brain. We find that small precursors of most of the compartments of the adult central brain can be identified in the early larva. Changes in brain compartmental organization occurring during larval growth are described. Neuropile compartments, representing easily identifiable landmark structures, will assist in future analyses of Drosophila brain development in which the exact location of neurons and their axonal trajectories is of importance.

Development of neural lineages derived from the sine oculis positive eye field of Drosophila.

The Anlage of the Drosophila visual system, called eye field, comprises a domain in the dorso-medial neurectoderm of the embryonic head and is defined by the expression of the early eye gene sine oculis (so). Beside the eye and optic lobe, the eye field gives rise to several neuroblasts that contribute their lineages to the central brain. Since so expression is only very short lived, the later development of these neuroblasts has so far been elusive. Using the P-element replacement technique [Genetics, 151 (1999) 1093] we generated a so-Gal4 line driving the reporter gene LacZ that perdures in the eye field derived cells throughout embryogenesis and into the larval period. This allowed us to reconstruct the morphogenetic movements of the eye field derived lineages, as well as the projection pattern of their neurons. The eye field produces a dorsal (Pc1/2) and a ventral (Pp3) group of three to four neuroblasts each. In addition, the target neurons of the larval eye, the optic lobe pioneers (OLPs) are derived from the eye field. The embryonically born (primary) neurons of the Pp3 lineages spread out at the inner surface of the optic lobe. Together with the OLPs, their axons project to the dorsal neuropile of the protocerebrum. Pp3 neuroblasts reassume expression of so-Gal4 in the larval period and produce secondary neurons whose axonal projection coincides with the pattern formed by the primary Pp3 neurons. Several other small clusters of neurons that originate from outside the eye field, but have axonal connections to the dorsal protocerebrum, also express so and are labeled by so-Gal4 driven LacZ. We discuss the dynamic pattern of the so-positive lineages as a tool to reconstruct the morphogenesis of the larval brain.

Embryonic origin and differentiation of the Drosophila heart.

We have followed the normal development of the different cell types associated with the Drosophila dorsal vessel, i.e. cardioblasts, pericardial cells, alary muscles, lymph gland and ring gland, by using several tissue-specific markers and transmission electron microscopy. Precursors of pericardial cells and cardioblasts split as two longitudinal rows of cells from the lateral mesoderm of segments T2-A7 ("cardiogenic region") during stage 12. The lymph gland and dorsal part of the ring gland (corpus allatum) originate from clusters of lateral mesodermal cells located in T3 and T1/dorsal ridge, respectively. Cardioblast precursors are strictly segmentally organized; each of T2-A6 gives rise to six cardioblasts. While moving dorsally during the stages leading up to dorsal closure, cardioblast precursors become flattened, polarized cells aligned in a regular longitudinal row. At dorsal closure, the leading edges of the cardioblast precursors meet their contralateral counterparts. The lumen of the dorsal vessel is formed when the trailing edges of the cardioblast precursors of either side bend around and contact each other. The amnioserosa invaginates during dorsal closure and is transiently attached to the cardioblasts; however, it does not contribute to the cells associated with the dorsal vessel and degenerates during late embryogenesis. We describe ultrastructural characteristics of cardioblast differentiation and discuss similarities between cardioblast development and capillary differentiation in vertebrates.

Embryonic origin of the imaginal discs of the head of Drosophila melanogaster.

The embryonic development of the primordia of the Drosophila head was studied by using an enhancer trap line expressed in these structures from embryonic stage 13 onward. Particular attention was given to the question of how the adult head primordia relate to the larval head segments. The clypeo-labral bud to the stage 13 embryo is located at a lateral position in the labrum adjacent to the labral sensory complex ("epiphysis"). Both clypeo-labral bud and sensory complex are located anterior to the engrailed-expression domain of the labrum. Throughout late embryogenesis and the larval period, the clypeo-labral bud forms integral part of the epithelium lining the roof of the atrium. The labial disc originates from the lateral labial segment adjacent to the labial sensory complex ("hypophysis"). It partially overlaps with the labial en-domain. After head involution, the labial disc forms a small pocket in the ventro-lateral wall of the atrium. The eye-antenna disc develops from a relatively large territory occupying the dorso-posterior part of the procephalic lobe, as well as parts of the dorsal gnathal segments. Cells in this territory are greatly reduced in number by cell death during stages 12-14. After head involution, the presumptive eye-antenna disc occupies a position in the lateral-posterior part of the dorsal pouch. Evagination of this tissue occurs during the first hours after hatching. In the embryo, no en-expression is present in the presumptive eye-antenna disc. en-expression starts in three separate regions in the third instar larva.