1 Program in Neuroscience, Washington University School of Medicine, 4566 Scott
Avenue, St. Louis, MO 63110, USA
2 Department of Genetics, Washington University School of Medicine, 4566 Scott
Avenue, St. Louis, MO 63110, USA
3 Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS
66506, USA
* Author for correspondence (e-mail: jskeath{at}genetics.wustl.edu)
Accepted 4 June 2003
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SUMMARY |
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Key words: achaete-scute, Tribolium castaneum, Drosophila melanogaster, Central nervous system
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INTRODUCTION |
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During Drosophila nervous system development, the proneural
ac/sc genes are first deployed in stereotyped patterns of ectodermal
cell clusters (proneural clusters). Proneural ac/sc gene expression
confers upon naive ectodermal cells the ability to acquire the neural
precursor fate. Within each proneural cluster, one or more cells retain
proneural ac/sc gene expression and commit to the neural precursor
fate, while the remaining cells in the cluster take on an epidermal fate. Once
formed, neural precursors activate expression of neural precursor genes and
rapidly extinguish proneural ac/sc gene expression. Neural precursor
genes are expressed in all neural precursors and appear to promote the
division and differentiation of these cells
(Dominguez and Campuzano,
1993; Jarman et al.,
1993
; Wallace et al.,
2000
).
Pioneering genetic and molecular studies led to the identification and
characterization of the four Drosophila ac/sc genes. These genes
exist in a complex spanning 100 kb at the distal tip of the X-chromosome.
Three of the genes, achaete (ac), scute
(sc) and lethal of scute (l'sc), were found to
promote neural precursor formation and were therefore termed `proneural
genes'; the fourth gene, asense (ase), is expressed only in
neural precursors and is thus termed a neural precursor gene. Genetic studies
established the essential role of ac, sc and l'sc in
promoting the initial decision of ectodermal cells to acquire the neural
precursor fate (Garcia-Bellido and
Santamaria, 1978
; Balcells et
al., 1988
). Expression studies showed that ac and
sc are expressed in identical patterns of proneural clusters during
central (CNS) and peripheral nervous system (PNS) development, while
l'sc is expressed in a broader and mostly complementary pattern of
proneural clusters in the CNS and only minimally in the PNS
(Cubas et al., 1991
;
Martin-Bermudo et al., 1991
;
Skeath and Carroll, 1991
;
Skeath and Carroll, 1992
). In
the CNS, the composite expression patterns of ac, sc and
l'sc mark all proneural clusters and their associated neural
precursors. In addition to their role in neural precursor formation,
ac and sc play a separate role in specifying the individual
fate of neural precursors. While ac, sc, and l'sc are
functionally interchangeable with respect to neural precursor formation, only
ac and sc can promote the proper gene expression profile and
cell division pattern of the MP2 precursor
(Parras et al., 1996
;
Skeath and Doe, 1996
). Thus in
the Drosophila CNS, ac/sc genes can regulate both the
formation and individual fate specification of neural precursors.
Owing to their central role in the formation of the Drosophila CNS
and PNS the ac/sc genes have become a focal point for understanding
the evolution of nervous system pattern in arthropods
(Skaer et al., 2002a). To
date, ac/sc genes have been identified in the medfly Ceratitis
capitata (Wulbeck and Simpson,
2000
), the blowflies Calliphora vicina and Phormia
terranovae (Pistillo et al.,
2002
; Skaer et al.,
2002b
), the malarial mosquito Anopheles gambiae
(Wulbeck and Simpson, 2002
),
and the spider Cupiennius salei
(Stollewerk et al., 2001
). The
number of insect proneural ac/sc genes differs, with three in
Drosophila, two in Ceratitis and Calliphora and one
in Anopheles, while each species has a single asense gene.
Thus, the basic subdivision of ac/sc genes into proneural and
asense genes and the functional roles these classes play in nervous
system development appear well conserved. In support of the conservation of
proneural ac/sc and asense function within arthropods, RNAi
studies in Cupiennius suggest that one of the ac/sc genes
carries out a proneural-like function (Cs-ASH1) while the other
(Cs-ASH2) carries out an asense-like function
(Stollewerk et al., 2001
).
To explore further the roles ac/sc genes play during arthropod
nervous system development and evolution we have focused on the red flour
beetle Tribolium castaneum (Coleoptera), a species 300 million
years diverged from Drosophila. Here we present the isolation of the
Tribolium ac/sc genes, and the characterization of their genomic
organization, expression and function. We have identified two Tribolium
ac/sc genes, achaete-scute homolog (Tc-ASH) and
asense (Tc-ase), and determined that these genes reside 55
kb apart from each other and thus define the Tribolium ac/sc complex.
Gene expression studies demonstrate that Tc-ASH is a proneural gene
expressed in all proneural clusters and transiently in all neural precursors.
Functional studies indicate that Tc-ASH is necessary for neural
precursor formation in Tribolium and sufficient for neural precursor
formation in Drosophila. These studies, however, do not support a
role for Tc-ASH in specifying the individual fate of neural
precursors, suggesting that the ability of ac and sc to
regulate this process may represent a recent evolutionary specialization
within the Diptera. We also show that Tc-ase, like other arthropod
asense genes, is expressed in all neural precursors. Thus, these
studies indicate significant plasticity in ac/sc gene number,
expression and function since the divergence of Tribolium and
Drosophila.
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MATERIALS AND METHODS |
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BAC library screening and BAC sequencing
An arrayed Tribolium BAC library
(Brown et al., 2002) was
screened independently with Tc-ASH and Tc-ase resulting in
25 hybridization-positive BAC clones, 16 clones hybridized to Tc-ase,
7 clones hybridized to Tc-ASH, and 2 clones hybridized to both genes.
A shotgun library was made from one of the BACs positive for both genes and
1820 paired sequencing reads were generated (SeqWright, Houston, TX). Reads
were assembled into a single 128 kb contig using PHRED, PHRAP and Consed
(Ewing and Green, 1998
;
Ewing et al., 1998
;
Gordon et al., 1998
). The
accuracy of the assembly was verified using paired read analysis as well as
comparison of virtual and actual restriction digests.
Rearing and preparation of Tribolium castaneum
Tribolium castaneum were purchased from Carolina Biological
(Burlington, NC) and maintained at 30°C on white flour supplemented with
2% yeast. Embryos were collected by size exclusion on a standard testing sieve
(Fisher Scientific, Chicago, IL) and fixed using standard protocols
(Mitchison and Sedat,
1983).
Immunohistochemistry and RNA in situ hybridization of whole mount
embryos
Immunohistochemistry and RNA in situ analyses were performed essentially as
described previously (Skeath,
1998). Mouse 4D9 anti-Engrailed/Invected was used at a 1:5
dilution (Patel et al.,
1989
).
Germline transformation and RNAi
A full-length cDNA of Tc-ASH was subcloned into the pUAST vector
(Brand and Perrimon, 1993) and
five independent transgenic Drosophila lines were established by
standard germline transformation protocols
(Rubin and Spradling, 1982
).
Tribolium RNAi was performed as previously described
(Brown et al., 1999
) except
that after injection embryos were incubated at 30°C for 18-28 hours
without an oil overlay, in a humid box and then fixed.
Phylogenetic analysis
Phylogenetic analysis was performed using programs from the PHYLIP package
(Felsentein, 1993). Protein
sequence corresponding to the basic, first helix, and second helix of the bHLH
region, aligned using CLUSTALW, was used in either distance (PROTDIST) or
parsimony (PROTPARS) methods.
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RESULTS |
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In phylogenetic analysis, both distance and parsimony methods group all of the insect Ac/Sc proteins consistently into proneural and Asense clades (Fig. 1C). In these analyses, Tc-ASH always groups in the proneural clade and Tc-Ase in the Asense clade (Fig. 1C). However, the conserved bHLH region is short (just 48 amino acids) and contains a high degree of amino acid identity obscuring the phylogenetic relationships between individual Ac/Sc proteins within the proneural and Asense clades.
The Tribolium ac/sc genes exist in a complex
In Drosophila, the ac/sc genes exist in a complex,
probably because they share regulatory elements
(Gomez-Skarmeta et al., 1995).
However, despite the identification of ac/sc genes in several species
we know little about the genomic organization of ac/sc genes outside
of the Diptera. To determine if Tc-ASH and Tc-ase exist in a
complex we independently screened a Tribolium BAC library
(Brown et al., 2002
) with each
Tribolium ac/sc gene. We identified 25 clones, two of which
hybridized to both Tc-ASH and Tc-ase indicating that the two
Tribolium ac/sc genes are linked.
To determine the precise molecular nature of the Tribolium ac/sc complex we sequenced one of the BAC clones that contained both Tc-ASH and Tc-ase. Sequence analysis of this 128 kb region revealed that Tc-ASH and Tc-ase reside 55.7 kb apart and are transcribed in the same orientation (Fig. 2). Within the sequenced region, there are no predicted genes upstream of Tc-ASH or between Tc-ASH and Tc-ase. In fact, the only other gene in the region is cytochrome P450 (cyt P450), lying 8.5 kb downstream of Tc-ase. In Drosophila, cyt P450 lies 5 kb downstream of ase suggesting a high degree of conservation in the genomic structure that surrounds asense in these species (Fig. 2). These data firmly establish the existence of the Tribolium ac/sc complex.
|
Anopheles is a basal dipteran
(Simpson et al., 1999) that
provides an important comparative link between the Diptera and other insect
groups. Like Tribolium, Anopheles contains a single proneural
ac/sc gene, Ag-ASH, and an asense homolog,
Ag-ase (Holt et al.,
2002
; Wulbeck and Simpson,
2002
). Analysis of the completed Anopheles genome
sequence identified that Ag-ASH and Ag-ase lie 22 kb apart
and are transcribed in the same orientation thus forming the Anopheles
ac/sc complex [Fig. 2; see
also Skaer et al. (Skaer et al.,
2002a
)]. As in Drosophila and Tribolium, cyt
P450 resides near the Anopheles ac/sc complex
(Fig. 2). However, unlike
Drosophila and Tribolium where cyt P450 lies
downstream of ase, Anopheles cyt P450 resides between the
ac/sc genes suggesting that a complicated rearrangement of this
region occurred within the Anopheles lineage. The existence of
ac/sc complexes in Drosophila, Anopheles and
Tribolium suggests that the organization of ac/sc genes
within gene complexes is a general feature of insects and perhaps all
arthropods.
Tc-ASH has a proneural expression pattern and
Tc-ase expression is restricted to neural precursors
We used RNA in situ hybridization to visualize the expression domains of
Tc-ASH and Tc-ase during Tribolium CNS development.
We find that Tc-ASH expression initiates prior to that of
Tc-ase in ectodermal cell clusters throughout the CNS (compare arrows
in Fig. 3A,C). Within each
cluster Tc-ASH expression is progressively restricted to the
presumptive neural precursor (Fig.
3A,B; arrowhead). The neural precursor then segregates into the
interior of the embryo and shortly thereafter extinguishes Tc-ASH
expression. These expression dynamics mirror those of all known insect
proneural ac/sc genes confirming our initial identification of
Tc-ASH as a proneural gene. Careful analysis of Tc-ASH
expression throughout CNS development reveals that all neural precursors arise
from Tc-ASH-expressing cell clusters. These results indicate that
additional proneural genes are not required for neural precursor formation
consistent with the idea that Tc-ASH is the only proneural
ac/sc gene in Tribolium.
|
In contrast to Tc-ASH, in situ hybridization shows that Tcase expression is restricted to neural precursors and is not present in the ectodermal cell clusters from which these cells segregate. We find that all morphologically identifiable neural precursors activate Tc-ase after segregating from Tc-ASH-expressing cell clusters (Fig. 3C,D) and that these precursors maintain Tc-ase expression throughout embryogenesis. The restriction of Tc-ase expression to neural precursors mirrors the expression pattern of all known insect asense genes confirming our identification of Tc-ase as an asense homolog. The dynamics of Tc-ase expression also demonstrate that sequential waves of neural precursor formation form a grid-like pattern of seven anteroposterior rows and three dorsoventral columns of neural precursors, a pattern essentially identical to that observed in the Drosophila CNS.
Tc-ASH regulates neural precursor formation
Genetic studies indicate that the Drosophila proneural
ac/sc genes promote neural precursor formation in the CNS and PNS
(Garcia-Bellido and Santamaria,
1978; Garcia-Bellido,
1979
; Dambly-Chaudiere and
Ghysen, 1987
; Jimenez and
Campos-Ortega, 1990
). To examine whether Tc-ASH promotes
neural precursor formation in the Tribolium CNS we used
double-stranded RNA interference (RNAi) to remove Tc-ASH function in
early Tribolium embryos and then assayed neural precursor formation
molecularly, by following Tc-ase expression, and by morphological
examination. In Tc-ASH RNAi-treated embryos we observed complete loss
of Tc-ase expression in 39.7% of embryos (n=23/58;
Fig. 4B), partial loss in 43.1%
of embryos (n=25/58; Fig.
4C) and wild-type Tc-ase expression in 17.2% of embryos
(n=10/58). 96% of buffer-injected control embryos displayed wild-type
Tc-ase expression (n=24/25,
Fig. 4A). In these experiments,
we observed near perfect correlation between loss of Tc-ase
expression and the absence of morphologically identifiably neural precursors.
These data indicate that Tc-ASH is necessary for neural precursor
formation in the Tribolium CNS.
|
We next tested whether Tc-ASH is sufficient to promote neural
precursor formation. In Drosophila, misexpression of proneural
ac/sc genes in the developing notum leads to the formation of ectopic
neural precursors that produce ectopic sensory bristles
(Rodriguez et al., 1990). This
assay is commonly used to test the proneural capabilities of ac/sc
genes (Brand et al., 1993
;
Dominguez and Campuzano, 1993
;
Hinz et al., 1994
;
Grens et al., 1995
;
Wulbeck and Simpson, 2002
). To
assay the proneural potential of Tc-ASH in Drosophila we
used the Gal4-UAS system and the apterous-Gal4 driver line to
misexpress Tc-ASH throughout the Drosophila notum
(Brand and Perrimon, 1993
;
Calleja et al., 1996
). In such
flies, we observe the formation of many ectopic sensory bristles, a phenotype
essentially identical to that observed when we misexpress Drosophila
sc under identical conditions (compare
Fig. 4E and F). These results
demonstrate that Tc-ASH is sufficient to promote neural precursor
formation in Drosophila and, together with our RNAi experiments,
indicate that Tc-ASH is both necessary and sufficient to promote
neural precursor formation.
Divergence of proneural ac/sc gene function between
Tribolium and Drosophila
In the Drosophila CNS, ac, sc and l'sc exhibit
essentially identical abilities to promote neural precursor formation,
however, ac and sc carry out functions distinct from
l'sc in the fate specification of the MP2 neural precursor
(Parras et al., 1996;
Skeath and Doe, 1996
). MP2
develops from a proneural cluster that expresses ac and sc
but not l'sc. In In(1) y3PL sc8R
embryos, ac and sc are not expressed in the MP2 cluster and
a neural precursor forms in this position 17% of the time - roughly half of
these precursors exhibit MP2-specific traits while the other half exhibit
traits characteristic of other neural precursors
(Table 1) (Parras et al., 1996
;
Skeath and Doe, 1996
). In this
background, expression of either ac or sc in the MP2
proneural cluster rescues both MP2 formation and fate specification to
essentially wild-type levels (Table
1) (Parras et al.,
1996
; Skeath et al., 1996). In contrast, while l'sc
expression rescues neural precursor formation almost completely, only 53% of
these precursors exhibit MP2-specific traits
(Table 1)
(Parras et al., 1996
; Skeath
et al., 1996).
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DISCUSSION |
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Conservation and plasticity in ac/sc gene number in
Arthropoda
Homologs of ac/sc genes have been described in a number of insect
and non-insect species. These data, together with our own, support and augment
the model proposed by Skaer et al., (Skaer
et al., 2002a) in which the last common ancestor of arthropods
contained a single prototypical ac/sc gene that carried out both
proneural and asense functions. In support of this model, the sole
Hydra ac/sc gene, CnASH, does not group with either the
proneural or asense genes in phylogenetic analysis and contains
motifs indicative of both the proneural and asense genes
(Grens et al., 1995
;
Skaer et al., 2002a
). In
addition, phylogenetic analysis of the two ac/sc genes found in the
chelicerate Cupiennius salei indicates these genes are more closely
related to each other than any other ac/sc genes
(Fig. 1C)
(Stollewerk et al., 2001
;
Skaer et al., 2002a
). These
data raise the possibility that a single ancestral ac/sc gene
underwent independent duplication events in chelicerates and insects
(Fig. 5; duplications a and b).
Given this possibility, it is interesting that one of the Cupiennius
ac/sc genes, Cs-ASH1, exhibits a proneural-like expression
pattern and appears to carry out a proneural-like function and the other,
Cs-ASH2, exhibits an asense-like expression pattern and
appears to carry out an asense-like function
(Stollewerk et al., 2001
).
These data suggest that independent duplications of an ancestral
ac/sc gene have independently given rise to proneural-like and
asense-like functions in the chelicerate and insect groups.
Alternatively, phylogenetic analysis may inappropriately partition chelicerate
ac/sc genes from insect ac/sc genes because of evolutionary
selection for species-specific amino acid changes in chelicerate as compared
to insect proteins.
|
In contrast to the plasticity in proneural ac/sc genes within insects, asense genes appear to be well conserved. A single asense gene exists in Tribolium and Anopheles as well as in the derived dipteran species Ceratitis and Drosophila. In addition, Cupiennius contains a single non-orthologous ac/sc gene with asense-like properties (Cs-ASH2). Thus, the potential that the asense function evolved independently in insects and chelicerates suggests an important role for the asense function in arthropod neural development.
The existence of ac/sc genes in complexes in Drosophila,
Anopheles and Tribolium suggests that this genomic arrangement
has been conserved in most if not all holometabolous insects. Shared
cis-regulatory regions probably explain why proneural ac/sc genes
remain linked in insects and perhaps other species. However, this does not
explain why asense is retained in the ac/sc complex as the
regulation of asense expression is distinct from that of the
proneural ac/sc genes. This phenomenon may be explained by the
presence of proneural ac/sc gene cis-regulatory regions surrounding
the asense gene. In this model, chromosomal rearrangements that
separate asense from the ac/sc complex would probably
disrupt proneural ac/sc gene expression and neural precursor
formation, thus leading to decreased viability. Consistent with this idea,
cis-regulatory regions that drive proneural ac/sc gene expression in
the Drosophila PNS appear to flank the ase gene
(Dambly-Chaudiere and Ghysen,
1987; Gomez-Skarmeta et al.,
1995
). Thus, the modular cis-regulatory regions that control
proneural ac/sc gene expression may also be responsible for the
evolutionary conservation of the ac/sc complex. Alternatively, other
as yet unidentified genomic forces may preserve the linkage between
asense and proneural ac/sc genes.
These findings raise a number of interesting points. First, they highlight
the potential for evolutionary plasticity of ac/sc genes. Significant
changes in ac/sc gene number and expression have occurred over
relatively short evolutionary distances and have been correlated with
modifications to neural pattern and/or gene function. For example, alterations
to ac/sc gene expression in Diptera appear to account for the
different patterns of sensory organs found on dipteran species
(Wulbeck and Simpson, 2000;
Skaer et al., 2002b
). In
addition, our data on the role of proneural genes in MP2 fate specification
suggest that the increase in ac/sc gene number in Drosophila
appears to have facilitated the evolution of new developmental roles for
ac and sc in this lineage. Second, the possibility that
independent duplication events in chelicerates and insects each gave rise to
proneural-like and asense-like genes, indicates that dividing these
genetic functions between two genes may be developmentally advantageous.
Third, the hypothesis that the last common ancestor of all arthropods
contained a single ancestral ac/sc gene suggests it may be possible
to identify direct descendants of the prototypical ac/sc gene in
extant basal members of each arthropod group. The recent emphasis on the
development of genomic resources in non-model organisms should greatly aid
progress along this line of inquiry. Thus, continued analysis of
ac/sc gene expression, organization and function in arthropods should
provide additional insight into the genetic basis of the development and
evolution of nervous system pattern.
Conservation and plasticity in ac/sc gene function and
expression
The work presented in this paper together with studies on ac/sc
gene function in Drosophila provide strong evidence that serial
duplications of proneural ac/sc genes in the dipteran lineage led to
the diversification of proneural ac/sc gene function in
Drosophila. Our work and that of others demonstrate that in
Drosophila, ac and sc carry out functions distinct from
l'sc in specifying the individual fate of the MP2 precursor
(Parras et al., 1996;
Skeath and Doe, 1996
). We show
that Tc-ASH can function in Drosophila as a proneural gene
but like Drosophila l'sc fails to specify efficiently the MP2 fate in
the CNS. Together these results suggest the ability of ac and
sc to specify MP2 fate in Drosophila arose after the
divergence of Drosophila and Tribolium. These data provide
an example whereby a subset of duplicated genes has evolved a new genetic
function while the entire set of duplicate genes has retained the ancestral
function.
In addition to functional changes, the generation of multiple proneural ac/sc genes in the insects was paralleled by modifications to the expression profiles of these genes. In Anopheles, a basal dipteran, and Tribolium a single proneural ac/sc gene is expressed in all CNS proneural clusters. In more derived Diptera the presence of multiple ac/sc genes allows for more complex proneural ac/sc gene expression patterns. For example, Ceratitis contains two proneural ac/sc genes, l'sc and sc; l'sc is expressed in all CNS proneural clusters while sc is expressed in a subset of these clusters. In Drosophila, ac and sc are expressed in the identical pattern of proneural clusters and their expression is largely complementary to that of l'sc. The sum of proneural ac/sc expression in each species then marks all CNS proneural clusters despite differences in the expression pattern of individual proneural ac/sc genes. Thus, in Drosophila, the complete expression pattern of proneural ac/sc genes is divided between the largely complementary expression profiles of ac and sc relative to l'sc. The division of labor between proneural ac/sc genes in Drosophila has resulted in mutually exclusive expression patterns for ac and sc relative to l'sc in proneural clusters like MP2. This spatial separation of proneural gene expression probably facilitated the potential for ac and sc to acquire developmental functions distinct from l'sc.
Together our work and that of others on arthropod ac/sc genes highlights the utility of studying ac/sc genes in elucidating the genetic basis of the development and evolution of arthropod nervous system pattern. These studies illustrate the dynamic nature of ac/sc gene number, expression and function over a relatively short evolutionary time. Based on this, future work on ac/sc genes in additional arthropod species should continue to provide insight into the molecular basis of the evolution of arthropod nervous system development.
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ACKNOWLEDGMENTS |
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