Temporal patterns of broad isoform expression during the development of neuronal lineages in Drosophila

Abstract

Background: During the development of the central nervous system (CNS) of Drosophila, neuronal stem cells, the neuroblasts (NBs), first generate a set of highly diverse neurons, the primary neurons that mature to control larval behavior, and then more homogeneous sets of neurons that show delayed maturation and are primarily used in the adult. These latter, 'secondary' neurons show a complex pattern of expression of broad, which encodes a transcription factor usually associated with metamorphosis, where it acts as a key regulator in the transitions from larva and pupa.

Results: The Broad-Z3 (Br-Z3) isoform appears transiently in most central neurons during embryogenesis, but persists in a subset of these cells through most of larval growth. Some of the latter are embryonic-born secondary neurons, whose development is arrested until the start of metamorphosis. However, the vast bulk of the secondary neurons are generated during larval growth and bromodeoxyuridine incorporation shows that they begin expressing Br-Z3 about 7 hours after their birth, approximately the time that they have finished outgrowth to their initial targets. By the start of metamorphosis, the oldest secondary neurons have turned off Br-Z3 expression, while the remainder, with the exception of the very youngest, maintain Br-Z3 while they are interacting with potential partners in preparation for neurite elaboration. That Br-Z3 may be involved in early sprouting is suggested by ectopically expressing this isoform in remodeling primary neurons, which do not normally express Br-Z3. These cells now sprout into ectopic locations. The expression of Br-Z3 is transient and seen in all interneurons, but two other isoforms, Br-Z4 and Br-Z1, show a more selective expression. Analysis of MARCM clones shows that the Br-Z4 isoform is expressed by neurons in virtually all lineages, but only in those cells born during a window during the transition from the second to the third larval instar. Br-Z4 expression is then maintained in this temporal cohort of cells into the adult.

Conclusion: These data show the potential for diverse functions of Broad within the developing CNS. The Br-Z3 isoform appears in all interneurons, but not motoneurons, when they first begin to interact with potential targets. Its function during this early sorting phase needs to be defined. Two other Broad isoforms, by contrast, are stably expressed in cohorts of neurons in all lineages and are the first examples of persisting molecular 'time-stamps' for Drosophila postembryonic neurons.

Figure 1

Immunocytochemistry showing staining with a MAb directed against the core region of the Broad transcription factor. (A, B) Whole mounts of Drosophila (A) and Manduca (B) embryos just prior to hatching. Broad expression is confined to the brain (b) and ventral CNS in both; mh, mouth hooks. (C) Confocal optical section through the thoracic and abdominal neuromeres of the CNS of a newly hatched larva of Drosophila that is double stained for the neuron-specific marker, Elav (magenta) and Broad (BR, green). All of the Broad-positive cells coincide with cells that also express Elav. Anterior is up; the CNS is slightly twisted so that only the right halves of the abdominal neuromeres are in the section. (D, E) Lateral views of Drosophila embryos at late stage 12 and stage 15, respectively, showing the expression of Broad confined to the developing CNS. (F) The CNS of a newly hatched larva lacking broad function (br^npr3 homozygote) showing no Broad immunostaining.

Figure 2

Examples of early embryonic induced MARCM clones and showing embryonic-born secondary neurons and their state of Broad expression. Left: Z-projections of green fluorescent protein (GFP)-labeled clones. Right: individual confocal sections at various levels through the clone (section number) showing GFP-labeled neurons (green) and either BrdU or Br-Z3 (magenta). (A) Early embryonic induced neuroblast clone of thoracic lineage 9 from a third instar larva that had fed on a BrdU diet from the time of hatching. The large basal neurons in the clone lack BrdU incorporation, showing that they were born before hatching. The red-framed area is represented in the individual sections. (B) Three neuroblast clones in the lateral thorax at the start of the third larval instar (approximately 72 h after egg laying), showing that the basal neurons express Br-Z3 at this time. NB, neuroblast.

Figure 3

The time-course of Br-Z3 expression in neuroblast MARCM clones induced after hatching. (A) Three examples of thoracic neuroblast clones for lineage 3 showing the change in Br-Z3 expression in neuronal populations through larval growth. Left: Z-projection of the clones showing the increase in overall size and morphology at 72 and 96 h AEL, and at puparium formation (P0). Right: sequential sections through each clone showing the distribution of Br-Z3+ neurons in the cluster. At 72 h AEL, Br-Z3 is barely detectable in larval born neurons; by 96 h AEL the basal cells (left) are Br-Z3+ but the more apical cells are negative. At P0 the basal cells (left) are now negative while more apical cells are Br-Z3+ except for those nearest the neuroblast (NB). (B, C) Use of BrdU pulses at 96 h AEL to establish the time of Br-Z3 expression relative to the end of DNA synthesis in the parent GMC. (B) Confocal sections through neuroblast clones for lineage 3 from larvae fed on a BrdU diet for 8 (top) or 16 h (bottom) prior to sacrifice. Br-Z3- and BrdU-labeled cells did not overlap after the 8 h pulse but did so after the 16 h pulse. Arrow points to the same nucleus in each panel. (C) The frequency of clones that had co-labeled cells as a function of the duration of the BrdU pulse. Numbers above each bar are the numbers of clones examined for each treatment.

Figure 4

Dynamics of Broad isoform expression during metamorphosis. (A) Z-projection of the ventral CNS showing the distribution of nuclei expressing Br-Z1, -Z3 and -Z4 at various times after puparium formation (APF). The neuropil staining for Br-Z1 at 94 h APF is likely due to a cross-reacting epitope rather than Broad. (B) Confocal sections showing Broad immunostaining in secondary neurons in the apical layers of the thoracic neuromeres at puparium formation. Br-Z4+ neurons are a subset of neurons that show Broad-core immunostaining. The Br-Z3+ neurons completely overlap the Broad-core immunostained neurons. The large nuclei of glia cells show no Br-Z3 and reduced Br-Z4 (arrows). (C) Cross-section in the T2 region of the CNS from a wandering stage larva that carried green fluorescent protein (GFP)-labeled MARCM clones and was immunostained for Br-Z4. Br-Z4+ cells were located at an intermediate layer of the cellular rind; the dotted line shows the outline of the CNS. (D, E) Comparison of immunostaining for Br-Z4 and Br-Z1 with that of Broad-core in the pharate adult nervous system. (D) Ventral view of the T1-T2 neuromeres showing that essentially all of the Broad-core+ neurons also show Br-Z4 immunostaining. By contrast, only a moderate number of Broad-core neurons also show the Br-Z1 epitope. (E) The brain shows a cluster of neurons that express BR-Z1 but not Br-Z4 (arrow) near the junction with the optic lobes (OL).

Figure 5

Br-Z3 expression in specialized brain regions. (A, B) Expression of Br-Z3 in neurons of the mushroom bodies. (A) A projected confocal z-stack showing an embryonically induced MARCM clone in the mushroom bodies of a mid third instar larva. Two individual sections from the red-framed area are on the right and show the expression of Br-Z3 (magenta) in the green fluorescent protein (GFP)+ (green) cells of the clone. Only the neuroblast (NB) and its most recently born progeny lack Br-Z3 expression. (B) A projected confocal Z-stack of a pharate adult brain (95 h APF) showing the four small clusters of Br-Z3 expressing neurons in the mushroom bodies (arrows). OL, optic lobe. (C, D) The changes of Br-Z3 expression in the optic lobe through early metamorphosis. (C) The progression of Br-Z3 in the neurons of the optic lobe. The confocal images at late wandering show a complete Z-projection (W(P)) and a confocal single slice (W). Br-Z3 expression is strong in the lamina (la) and medulla (m) but only the neurons in the most medial columns of medulla express Br-Z3. Subsequent sections show recruitment of more lateral medulla columns (arrows) in older prepupae (hours APF) with the inclusion of all of the medulla neurons by 14 h APF. 14(P): a full Z-projection of the 14 h preparation. In the sections, the arc indicates the extent of Br-Z3 expression in the medulla. (D) Br-Z3 expression in a confocal Z-projection at 27 h APF. Br-Z3 expression is still present in neurons of the medulla and lobula and lobula plate (lo/lp) even though it has disappeared from all other regions of the CNS except the mushroom bodies.

Figure 6

Time of birth and distribution of Br-Z4+ neurons in the thoracic lineages. (A) Schematic, showing the timing of heat-shocks (hs; arrows) to induce MARCM clones during larval neurogenesis and the time of analysis of the clones at pupariation [B, D, F] or just before adult emergence [C, E]. The cartoons show the predicted relationship of clone boundaries (green) to Br-Z4 expression (magenta), if the Br-Z4+ neurons were born during a discrete phase of larval life. P0, puparium formation. (B) Z-projections of lineage 14 neuroblast clones that were examined at pupariation but induced by heat shocks at 24 h or 76 h AEL. Left: anatomy of the whole clone. Right: sections from the basal (b) and middle (m) portions of the clone. The basal cells in early induced clones are Br-Z4 negative with the Br-Z4+ cells clustered in the middle layers. In the 76 h-induced clone, the oldest (basal) cells included only a few Br-Z4+ neurons (arrows) but positive neurons were outside the clone. (C) Examples of two-cell GMC clones from the adult CNS; examples from heat-shocks at 77 h AEL show both sibs expressing Br-Z4 or one sib with weak expression (arrow) and the other negative. Both sibs are negative after the late heat-shock induction. (D) Summary of neuroblast MARCM clones that were examined around the time of pupariation. Only clones induced at 99 h AEL were completely devoid of Br-Z4+ neurons. (E) Summary of GMC clones that were examined just prior to adult emergence. Br-Z4+ neurons were most commonly seen in clones induced at 77 h AEL. For (C, D), the numbers above each bar are the numbers of clones examined. (F) Counts of the number of Br-Z4+ neurons in neuroblast MARCM clones that were induced at 24 h (black dots) and 76 h (red dots) AEL. The neuroblast of origin was identified by the morphology of the axon bundles that exited the clone. Interneuron-generating lineages were grouped by whether they produced one or two classes of interneurons. Some neuroblasts (NBs 1 and 19) were in both categories because of segment-specific loss of one class of interneurons. The black and red bars give the averages for the two groups. MN, motor neuron lineages.

Figure 7

Ectopic expression of Br-Z3 in all neurons in stage 17 embryos as revealed by immunostaining with the Broad-core MAb. (A) Wild-type Broad expression seen in the CNS of a late embryo carrying a UAS-Br-Z3 transgene (line UAS-Br-Z3^216-1). Ventral expression is primarily in the thoracic and terminal abdominal neuromeres. (B) Broad expression in a late embryo of the genotype elav^C155; UAS-Br-Z3^216-1. Broad is expressed throughout the CNS as well as in the peripheral nervous system (arrowheads).

Figure 8

Projected confocal z-stacks of adult brains showing the effect of targeted expression of Br-Z3 on the metamorphic growth of the pigment dispersing factor-containing neurons that project to the dorsal protocerebrum. Images are frontal views of pigment dispersing factor (PDF)-stained neurons. (A, B) The medial projections of the small pdf neurons in pdf-GAL4 (A) and UAS-Br-Z3 (B) resembled that of wild-type, showing a simple termination well before the midline. (C) The pdf neurons of a pdf-GAL4; UAS-Br-Z3 individual showing the extensive branching of their terminals towards the midline. (D) Confocal section from a pdf-GAL4; UAS-Br-Z3 adult showing strong ectopic expression of Br-Z3 (magenta) in the PDF-expressing neurons (green).

Figure 9

Summary of the time of the expression of the various Broad isoforms in the secondary neurons of the thoracic segmental lineages. AEL, hours after egg lay; GMC, ganglion mother cell; NB, neuroblast; P, pupariation.