MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Accepted 11 February 2002
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SUMMARY |
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Key words: Drosophila, Brain, Patterning, Foregut, Mesoderm
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INTRODUCTION |
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In contrast to the situation in deuterostomes, the induction of brain patterning in protostomes has been little studied. With the fruit fly Drosophila, there are a variety of genetic tools now available that allow us to address the question of whether the protostome brain is patterned by induction. By systematically removing tissues adjacent to the embryonic Drosophila brain, I have found that the foregut and the mesoderm are involved in establishing and refining brain pattern by influencing brain size and apoptosis. Furthermore, I have investigated the role of the Hedgehog signaling pathway in Drosophila brain patterning. The results argue that the protostome embryonic brain pattern is influenced by induction as it is in deuterostomes, and that Hedgehog signaling has a conserved role in this effect among these groups.
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MATERIALS AND METHODS |
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Laser ablations
Dechorionated cellularized-blastula stage embryos were lined up on glass slides after staging on a dissecting microscope in Voltalef 3S oil. All staging of embryos was carried out using standard guidelines (Campos-Ortega and Hartenstein, 1997). The cells of the foregut anlage were ablated using a Laser Sciences VSL-337ND-S nitrogen laser. Cells were exposed to 1-second bursts at 10 Hz, and the area around where the laser was focused was checked for blebbing, thereby indicating cell death. I destroyed what appeared to be most of the cells within the foregut anlage (see Hartenstein and Campos-Ortega, 1985
). Subsequent to laser treatment, embryos were placed at 25°C until they reached embryonic stage (ES) 15, as determined using a dissecting microscope. Approximately 60% of the laser-treated embryos clearly lacked a foregut; these were the embryos that were used for subsequent experiments.
Immunohistochemistry and microscopy
Embryos were collected, fixed and immunostained according to standard procedures (Patel, 1994). The following primary antibodies were used: BP102 [mouse, 1:200, Developmental Studies Hybridoma Bank (DSHB)], anti-Fasciclin II (Bastiani et al., 1987
) (mouse, 1:20), anti-Elav (rat, 1:100, DSHB), anti-Repo (Halter et al., 1995
) (rabbit, 1:750), anti-GFP (rabbit, 1:1000, Molecular Probes), anti-ß-galactosidase (rabbit, 1:1500, Cappel), anti-Hunchback (Struhl et al., 1989
) (rat, 1:2000), anti-Prospero (mouse, 1:100, DSHB), anti-Fasciclin III (mouse, 1:100, DSHB) and anti-Hedgehog (Tabata and Kornberg, 1994
) (rabbit, 1:1500). For light microscopy, biotin-conjugated secondary antibodies were used (1:200, Jackson). Embryos were viewed and photographed on a Zeiss Axiophot microscope. Secondary antibodies used for laser confocal microscopy were Texas Red-conjugated anti-mouse (1:200, Jackson), FITC-conjugated anti-rabbit (1:200, Jackson), and Cy5-conjugated anti-Rat (1:200, Jackson). Images were collected on BioRad MRC 1024 or Radiance confocal microscopes, and were processed using Adobe PhotoShop and Illustrator.
Acridine Orange staining
Staining for apoptosis using Acridine Orange was done according to standard procedure (Abrams et al., 1993). Briefly, embryos were dechorionated, then shaken for 5 minutes in a 1:1 mixture of 5 µg/ml Acridine Orange in PBS and heptane, and then mounted in Voltalev 10S oil. Embryos at late ES13 were examined on a BioRad Radiance confocal microscope for staining at the level of the pre-oral brain commissure at the dorsal midline of b1.
Cell counting
To quantify brain phenotypes, stained embryos were examined in the following ways:
(1) To estimate the area occupied by neuronal nuclei and the number of glia in b1, confocal sections were collected from at the dorsal most region of the ES15 brain, progressing ventrally through the brain at 2.5 µm increments for 35 µm (which is a level slightly ventral to the preoral brain commissure in the wild-type brain), using a 60x oil immersion objective on a BioRad Radiance confocal microscope. These sections were then projected into one image using BioRad software. The number of glia present was counted directly and the area occupied by neurons was measured by tracing the outline of the anti-Elav marked brain using Adobe PhotoShop and obtaining the total pixel area within the outlined image. The pixel area was then multiplied by the conversion factor 0.154 µm2/pixel (this value was obtained by dividing the area of a scanning box provided by the BioRad confocal software by the number of pixels in such a box as measured in Adobe PhotoShop) to obtain an estimate of the area occupied by neuronal nuclei.
(2) To estimate the number of glia and the area occupied by neurons in b2-S3, a procedure similar to that used in b1 was used except that optical sectioning began at the lateral edge of the foregut (or 5µm from the midline when the foregut was ablated) and progressed medially at 2.5 µm increments for 25 µm. Images were then projected and analyzed as described above. The boundaries of b2-S3 were estimated based on the positions of commissures.
(3) For statistical analysis of the difference in cell counts between wild-type and mutant brains, Students t-test was used (Sokal and Rohlf, 1997).
It is important to note that the data provided are not intended to represent the absolute area or number of cells in the brains of embryos, but rather a relative comparison of brain size and glia number between groups of embryos as measured using consistent techniques.
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RESULTS |
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Ablation of the foregut and mesoderm results in changes in the pattern of brain apoptosis at the dorsal midline
Why were there excess cells at the dorsal midline in foregut- and mesoderm-ablated embryos? During normal brain development, more neurons are born than will be present in the adult brain and apoptosis eliminates the excess cells (for a review, see Hutchins and Barger, 1998). Defects in apoptosis could contribute to the observed defects in brain patterning by failing to remove excess cells. To see if apoptosis was perturbed when the foregut and mesoderm were ablated, Acridine Orange staining, which labels apoptotic cells (Abrams et al., 1993
), was carried out. In forkhead loss-of-function embryos, the pattern of apoptosis in the brain at the level of the preoral brain commissure was clearly different from wild type at late ES13. In the wild-type b1 neuromere, there were groups of apoptotic cells at the dorsomedial edges of the hemispheres (Fig. 4A). This correlates with previous observations regarding the expression of the apoptosis regulatory protein Reaper (Nassif et al., 1998b
). In forkhead loss-of function embryos, there was a clear reduction in the number of these cells (Fig. 4B). Examination of tinman loss-of-function embryos showed that removal of mesoderm results in a similar reduction in the number of apoptotic cells at the dorsal midline (Fig. 4C), thus suggesting that the mesoderm and possibly the foregut have an influence on the normal pattern of apoptosis in brain development.
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Loss of Hedgehog function results in changes in apoptosis
To see if changes in apoptosis underlie the b1 dorsal midline defects seen in Hedgehog null embryos, Acridine Orange staining was carried out. This showed that there was a decrease in the number of apoptotic cells at the dorsal midline of the brain at late ES13 (Fig. 5H), suggesting that the defects in this region in Hedgehog null embryos were due to a disruption in the normal pattern of apoptosis.
Blocking Hedgehog signaling in neural cells influences brain size, but not apoptosis at the dorsal midline
To see if Hedgehog signaling is directly required in brain cells, I expressed a form of Patched that is insensitive to Hedgehog (Briscoe et al., 2001) specifically in neural tissues using the 1407-GAL4 line (Sweeney et al., 1995
). In these embryos, the size of the brain was decreased; however, excess cells did not appear to be present at the dorsal midline (Fig. 5I). This suggests that Hedgehog signaling has a direct function in influencing brain size; however, its function in inducing apoptosis in the dorsal midline of b1 may be indirect.
Influence of the foregut and mesoderm on the formation of neural precursors
When do the inductive influences of the foregut and mesoderm exert their effect on brain development? To answer this question, I stained embryos lacking foregut (Forkhead null) or mesoderm (Tinman null) with anti-Hunchback antibody, which stains neuroblasts in the brain (Kambadur et al., 1998). At ES10, after the brain neuroblasts have started to delaminate, the pattern of Hunchback-expressing neuroblasts appeared to be the same as wild type in all these embryos (Fig. 6A-C). This suggested that the initial formation of brain neuroblasts was normal in the absence of a foregut or mesoderm. Next, I examined the pattern of ganglionic mother cells (GMCs) using the marker anti-Prospero (Doe et al., 1991
). In this case, the number of brain GMCs in embryos lacking a foregut was clearly decreased at ES11 when compared with wild type (Fig. 6D,E). By contrast, in embryos lacking mesoderm, the pattern of GMCs did not appear to be affected (Fig. 6F). This suggested that there are two inductive events: an earlier event that is mediated by signals originating from the foregut, in which neural precursor cells of the brain receive a signal that influences the formation of GMCs. That Hedgehog may be involved in this signaling event is suggested by the fact that in a Hedgehog null background, the number of Prospero-expressing GMCs is reduced (Fig. 6G). The later event appears to be mediated by the visceral mesoderm and seems to influence the survival of late GMCs or postmitotic cells.
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DISCUSSION |
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Involvement of Hedgehog in the induction of brain pattern
When function of Hedgehog is lost, brain patterning defects occur that resemble those seen in foregut ablated embryos, including a fusion of brain hemispheres in b1, a reduction in the size of the brain and abnormal defasciculation of the preoral brain commissure. Importantly, Hedgehog is expressed in the foregut adjacent to the brain, and patched, which encodes a putative receptor for Hedgehog, is expressed in brain cells surrounding the foregut. Loss of Hedgehog function causes a reduction in brain size that resembles that seen when the foregut is ablated, thus suggesting that the foregut source of Hedgehog influences brain size. Hedgehog loss of function also results in a disruption of apoptosis that resembles what is seen when the mesoderm is removed; however, hedgehog does not appear to be expressed in the visceral mesoderm surrounding the foregut. This suggests that Hedgehog from the foregut may be received by the mesoderm (where patched is upregulated), which then responds by producing another signal that influences apoptosis at the dorsal midline. This hypothesis is supported by the observation that inhibiting Hedgehog signaling specifically in neural tissue using a mutant form of Patched results in a decrease in b1 size, but not in an excess of cells, at the dorsal midline.
Significance for understanding disease and evolution
Considering the reduction in the number of brain cells and the joining of the brain hemispheres, the foregut ablation brain phenotype resembled aspects of human HPE. Importantly, in the vertebrate nervous system, Sonic hedgehog is involved in the formation of oligodendrocytes and motoneurons, and in regulating apoptosis (Ericson et al., 1995; Nery et al., 2001
). Disruption of Hedgehog signaling causes HPE; correspondingly, in Drosophila Hedgehog mutants, a HPE-like phenotype occurs, including a reduction in the number of glia and Fasciclin II expressing motoneurons. The recapitulation of aspects of the human HPE phenotype in Drosophila i.e. the loss of brain cells and the defects in hemispheric separation means that fly embryos might have use for understanding some mechanisms of this disease.
Furthermore, this work demonstrates that brain patterning via induction by the foregut and mesoderm appears to be a mechanism that is shared between protostomes and deuterostomes. This finding supports the hypothesis that the ground plan for the brain was established in the last common ancestor of bilaterally symmetric animals (Arendt and Nubler-Jung, 1996; Arendt and Nubler-Jung, 1999
; Dohrn, 1875
; Leydig, 1864
). This also suggests how the brain of the much-debated last common ancestor of Bilateria may have developed. I hypothesize that this animal had a brain that formed in close association with the foregut, and molecules such as Hedgehog expressed in the foregut patterned this brain. As regards the origins of the brain in evolution, if the foregut is assumed to be more ancient than the brain, then the possibility arises that the ventral neural mass of a bilaterian ancestor that lacked a brain could have expanded dorsally, using the foregut (which would probably have already been expressing patterning molecules such as Hedgehog) as a scaffolding, thus forming a brain.
The apparent homology between the foreguts of protostomes and deuterostomes raises a problem in nomenclature: the foregut of protostomes is considered ectodermal in origin, while the foregut of deuterostomes is considered endodermal. However, there appears to be homology in function, blastodermal fate map position (Arendt and Nubler-Jung, 1997), gene expression (Arendt et al., 2001
) and induction between tissues derived from what have been traditionally regarded as distinct germ layers. Perhaps the assignment of the protostome foregut to the ectoderm and the deuterostome foregut to the endoderm should be reconsidered, as there are no absolute criteria for these assignments.
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ACKNOWLEDGMENTS |
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