Integrative and Comparative Biology Advance Access originally published online on June 28, 2006
Integrative and Comparative Biology 2006 46(4):519-532; doi:10.1093/icb/icj054
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
The divergent roles of the segmentation gene hunchback
* Biology Department Williams College Williamstown, MA, USA
Molecular, Cellular and Developmental Biology, Yale University New Haven, CT, USA
Department of Organismal Biology and Anatomy, University of Chicago Chicago, IL, USA
Correspondence: 1E-mail: rsavage{at}williams.edu
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The hunchback (hb) gene is a member of the gap class of segmentation genes first identified in the dipteran insect Drosophila melanogastor. The hb gene encodes a C2H2 zinc finger transcription factor whose primary function is to regulate the expression of its target genes along the anteroposterior (AP) axis based on its distribution in the blastoderm embryo. The loss of zygotic hb in Drosophila results in a "gap" in anterior pattern elements that include the loss of labial and thoracic segments in addition to the fusion of the abdominal segments 7 and 8. The hunchback protein is also expressed in the extraembryonic epithelial tissues and the developing nervous system in the zygote. Although the role of hunchback in AP patterning is likely to be an ancestral trait to the insect clade, higher order comparisons of hunchback orthologs suggest that it is a derived trait specific to the arthropod and/or insect lineage. This view is supported by a combination of comparative gene expression data, phylogenetic analyses, and an examination of the evolution of structural domains in the hb protein isolated from annelids, nematodes, and insects. The 3 independent lines of comparative data strongly support the idea that the anterior organizing function of hb originated in the arthropod and/or insect lineage and that its roles in epithelial and CNS patterning are likely to be broadly conserved within protostomes.
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Comparative studies are an integral approach to understanding most aspects of biological processes because they provide a framework from which hypotheses are generated and subsequently tested. The study of the evolution of gene function is one area that draws heavily on this approach because functional studies are often unavailable or poorly developed in understudied systems. This article examines both the shared and specialized roles that the segmentation gene hunchback plays in diverse patterning events in metazoan embryos using available expression data, sequence identity, and domain structure comparisons between members of the hunchback family of zinc finger transcription factors. The hunchback (hb) gene was first characterized as a "gap" segmentation gene in Drosophila melanogastor (Tautz and others 1987
; Bender and others 1988) and was shown to play a central role in anterior spatial patterning in insects (Lehman and Nüsslein-Volhard 1987; Hülskamp and others 1990). The hunchback protein regulates target genes according to its graded distribution along the anteroposterior (AP) axis: highest in the prospective anterior region of the embryo while tapering off posteriorly (Wu and others 2001). Hunchback targets include other segmentation genes operating at all levels of the segmentation gene hierarchy as well as the HOM genes (Jäckle and others 1986; White and Lehmann 1986; Stanojevic and others 1991; Zhang and Bienz 1992).
hb belongs to a large family of C2H2 zinc finger proteins whose "finger" motif was first described in the protein Transcription Factor IIIA (TF111A) in Xenopus laevis (Brown and others 1985; Miller and others 1985; Rosenberg and others 1986). Structural studies have shown that the 2 cystidine and 2 histidine residues coordinate the central position of the zinc ion within each finger and that this coordination is required for proper folding of the 30 amino acid finger motif and for sequence-specific DNA binding (Berg 1988; Lee and others 1989; Pavletich and Pabo 1991). C2H2 finger proteins are known for their ability to bind both DNA and RNA, but they also have been shown to interact with other proteins (MacKay and Crossley 1998). Recent studies of the Ikaros protein (the hb homologue found in mammals [Georgopoulos and others 1992]) and of the fly hunchback protein have shown that the most C-terminal zinc fingers are involved in homotypic protein interactions (Hahm and others 1994; McCarty and others 2003). The versatility of hb protein interactions with nucleic acids and proteins may help to explain the prevalence of zinc finger proteins in eukaryotic genomes (Clark and Berg 1998; Venter and others 2001; Stein and others 2003). There are over 5000 C2H2 zinc finger proteins encoded by the human genome (McCarty and others 2003) and zinc finger genes represent more than 1.4% of the nematode genome (Stein and others 2003). Furthermore the ability of zinc fingers to bind other proteins offers a reasonable explanation for the large number of fingers in this family of proteins, many of which do not appear to bind to DNA (MacKay and others 1998).
A number of hb orthologs have been characterized in both the "higher" and "lower" insects (Sommer and Tautz 1991; Kraft and Jäckle 1994; Wolff and others 1995; Maderspacher and others 1998; Rohr and others 1999; Pultz and others 2000; Stauber and others 2000; McGregor and others 2001; Liu and Kaufman 2003). These studies show that hunchback's role as an anterior gap gene is highly conserved across diverse insect orders (Lehmnan and Nüsslein-Volhard 1987; Tautz and others 1987; Patel and others 2001; Liu and Kaufman 2003; Lynch and Desplan 2003; Schröder 2003). These comparative expression data suggest that hunchback protein is a key member of an ancestral AP patterning system basal to all insects (Schröder 2003). Hunchback protein is also expressed in other embryonic and larval tissues, which suggests that it plays additional developmental roles. hb is expressed in the extraembryonic epithelial membranes, the serosal primordium in lower insects and the amnioserosa in dipterans, and in the neuroblasts of the central nervous system in all insects that have been examined (Tautz and others 1987; Wolff and others 1995; Tautz and Nigro 1998; Rohr and others 1999; Patel and others 2001; Lynch and Desplan 2003).
To what extent are the hunchback expression elements in insects conserved in metazoans? To address this question, one effective approach is to infer relationships based on existing comparative data. The examination of hb expression patterns in distantly related animal groups provides key insights into the evolutionary history of this gene's function. In this article we have combined phylogenetic analyses, changes in the individual structural domains of the hb protein, and the accumulation of hb mRNA/protein expression patterns to infer the evolutionary roles of hunchback gene products. The 3 independent lines of comparative data, together, suggest that the anterior "gap" function of hunchback in insects is an evolutionary novelty that arose independently within the arthropod lineage. Moreover the ancestral function of hb gene products in nematodes, arthropods, and annelids is likely to have been in extraembryonic epithelium and/or CNS patterning in protostome embryos.
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Materials and methods |
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Embryos
Capitella capitata sp. I embryos were reared at 18°C in glass finger bowls containing artificial seawater and fed frozen-thawed marine sediment collected from Woods Hole, MA. Capitella embryos were collected as described in Werbrock and colleagues (2001)
.
Templates and polymerase chain reaction
Capitella embryos and trochophores were isolated from brood tubes and transferred to a dish containing artificial seawater. The embryos were frozen in liquid nitrogen and stored at –80°C. Total RNA was extracted using Trizol reagent (Gibco-BRL) followed by the isolation of mRNA from eggs and embryos using the MicroPoly (A) Pure mRNA Isolation KitTM according to manufacturer's instructions (Ambion). cDNA was generated by the SMARTTM RACE cDNA Amplification Kit using manufacturer's instructions (BD Biosciences Clontech). The Advantage® 2 PCR kit from BD Biosciences was used in the amplifications from cDNA templates. Two 6-fold degenerate PCR primers, HB3 and Caphb3'zfdrev, were synthesized to amplify a 1521 nucleotide fragment that included the highly conserved MF domain but not the complete CF zinc finger domain. Primer sequences were as follows: Hb3, 5'-TGYGTNGAYAAARTCMATG-3'; Caphb31zfdrev, 5'-AICYRTGRTANCCCATRTG-3'. Amplifications were performed in 100 ng Capitella cDNA, 500 ng of each primer, 200 µm Takara dNTPs, 1x Takara ExTaqTM Reaction Buffer, and 1 unit of Takara-TaqTM polymerase (Chemicon International). Cycling was 4 min at 95°C, 35 cycles of 30 s at 95°C, 30 s at 50°C, and 1 min at 72°C. The fragment was excised from gel, subcloned into Bluscript KS + (Stratagene), and sequenced commercially (Keck Sequencing Facility, Yale University).
An additional 1125 bp Cchb gene fragment was obtained via 5' RACE using the SmartTM RACE Kit from BD Biosciences using the same set of amplification and subcloning conditions as described above. The sequence of the reverse gene specific primer Caphb2 is as follows: 5'-TCGGTGGGCAAGCTGCCATCCGGGTTG-3'. The Cchb fragment was sequenced at the University of Maine Sequencing Center (Orono, ME).
Library screening
A 1400 bp LZF2 fragment obtained from Helobdella triserialis (Savage and Shankland 1996) was the template used to screen 4 x 105 recombinant clones from a Helobdella robusta Stage 7–10 
ZAP cDNA library provided by D. Weisblat (UC Berkeley). The probe was made using the ECLTM direct nucleic acid labeling and detection systems following manufacturer's instructions (Amersham Pharmacia Biotech). The probe was stored in 50 glycerol at –20°C. For prehybridization, nitrocellulose filters were placed in ECLTM Gold hybridization buffer with 0.5 M NaCl and 5% (w/v) blocking agent for 1 h at 42°C with gentle agitation. An amount of 10 ng labeled probe per ml of hybridization solution was added to the filters and were allowed to hybridize overnight at 42°C with constant agitation. Filters were washed in 0.1% SDS/2x SSC solution at 42°C for 1 h. The filters were then washed at room temperature for 20 min in 2x SSC, followed by a 0.1x SSC wash for 20 min at room temperature. A single positive clone was detected and was subsequently purified through a tertiary screen. The plasmid was excised according to manufacturer's instructions (Stratagene). The 1.9 kb cDNA fragment was sequenced (Keck Sequencing Facility, Yale) and named Helobdella robusta hunchback (Hrohb).
Phylogenetic analysis
A molecular phylogenetic tree was constructed based on the comparison of 200 highly conserved amino acid residues, which include the most highly conserved MF1–4 domain composed of 4 zinc fingers, using Bayesian analysis (Mr Bayes v3.0B4). The parameters of the analysis are as follows: 10 000 000 generations, burn in 7500, sampling frequency 100, 4 chains, under a mixed model of evolution. The protein and accession numbers are as follows: H. robusta hb (DQ023500
[GenBank]
), C. capitata hb (DQ023501
[GenBank]
), H. triserialis hb1 or LZF1 (91396), H. triserialis hb2 or LZF2 (91395), Oncopeltus fasciatus hb (AY460341
[GenBank]
), Bombyx mori hb (D38487), Clogmia albipunctata hb (AJ131041
[GenBank]
, Schistocerca americana hb (AY040606
[GenBank]
), Tribolium casteneum hb (X91618), Musca domestica hb (Y13050), Megaselia abdita hb (AJ295635
[GenBank]
), Nematode hb or hbl-1 (AF097737
[GenBank]
), Drosophila virilis hb (X15359), Drosophila seychelii hb (AJ005374
[GenBank]
), Manduca sexta hb (Z30281), Strigamia maritima hb (AY995115
[GenBank]
), Ikaros (Y11833), Locusta migratoria hb (DQ510870
[GenBank]
), Drosophila orena hb (AJ005375
[GenBank]
), D. melanogastor hb (Y00274), Lucilia sericata hb (AJ301662
[GenBank]
).
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Sequence comparisons and orthology assignment
The hallmark features that distinguish hunchback protein members from other families of zinc finger proteins are the central position of the middle finger (MF1–4) domain, the most C-terminal position of 2 zinc fingers (CF1–2), and the high amino acid sequence identity shared between these 2 finger domains. In all hb homologues to date, a stretch of poorly conserved amino acids (average of 240 amino acids in length) separates the 2 most highly conserved finger domains despite the fact that the length of the hb ORF varies between 438 amino acids in the dipteran insect Anopheles gambia to 982 amino acids in Caenorhabditis elegans (Fig. 4A). Our amino acid sequence and domain comparisons from newly identified annelid hb genes show that they also possess these family-specific traits, which suggest that they are hb orthologs (Figs 1, 2, and 4). Blast scores obtained of GenBank and Drosophila genome databases support the orthology assignment.
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The 3 annelid hb genes identified in 2 leech Helobdella species and 1 polychaete species (Capitella) have similar e-values (
1e–56) compared with the hunchback gene sequence using the Drosophila genome database. Non-dipteran insects such as O. fasciatus (hemipteran) and the nematode C. elegans hb (hbl-1) received values of 3e–71 and 1e–45, respectively. The e-values obtained from comparisons between annelid hb sequences to the Drosophila hunchback gene were as similar to those obtained from comparisons of non-dipteran insects and nematode hunchback orthologs to Drosophila hunchback (not shown). The second hb gene known as LZF1 in Helobdella received a blast score of 1e–28. The low but significant e-value is likely due to the lack of available C-terminal sequence information obtained from the genomic clone. Finally the transcription factor and gap gene Krüppel is the most closely related zinc finger protein to hb family members and serves as a representative outgroup for zinc finger proteins as a whole. The annelid and insect hb orthologs including the Drosophila hb gene have similar low e-values 1e–10) when compared with the Krüppel gene sequence. The high degree of sequence identity shared inferred from the e-values and the number of fingers and position of the MF1–4 and CF1–2 domains (see below) within the open reading frame suggest that the annelid genes are hunchback orthologs.
In this article we have isolated hunchback homologues from cDNA templates from the polychaete C. capitata sp I (Cchb) and the leech H. robusta (Hrohb) using standard cDNA library screening methods, PCR amplification using degenerate oligonucleotides and 5' RACE (see Materials and methods). The Cchb and Hrohb cDNA sequences have been deposited in the GenBank database under accession nos DQ023501
[GenBank]
and XDQ023500, respectively. The LZF1 (91396) and LZF2 (91395) genomic sequences were previously isolated from the leech H. triserialis (Savage and Shankland 1996). To date, a total of 4 hb homologues have been isolated from 3 annelid species and 1 ortholog from a basal polychaete and 3 from leeches. Based on ORFs obtained from cDNAs, the annelid hb genes encodes a protein that contains a minimum of 8 zinc fingers designated N-terminal finger (NF1) region, middle finger (MF1–4) region, extra finger (ExF) region, and C-terminal finger (CF1–2) region as determined by Patel and colleagues (2001).
The 2 leech hb orthologs named Hrohb from H. robusta and LZF2 from H. triserialis share 95% amino acid identity in the region between the middle fingers (MF1–4) and the extra finger (ExF) and 92% sequence identity between the ExF and the C-terminal finger domain (CF1–2) (not shown). Yet there is 100% amino acid identity shared within every zinc finger motif. The high degree of shared sequence identity is expected because the comparisons occur within the same genus. However the interclass and interphyletic comparisons of the zinc finger domains share a similar degree of shared amino acid identity. For example, the polychaete (C. capitata) and the grasshopper (S. americana) hunchback MF1 and MF4 domains each share 46% and 64% amino acid identity, respectively, with the corresponding zinc finger domains in the Helobdella leech (Fig. 1). Many of the conserved elements in the hb ORF shared a similar degree of sequence identity between phyla and within their respective phyla. Yet the zinc finger domains also demonstrate lineage-specific modifications in their primary amino acid sequence. The MF2 and MF3 zinc fingers in leeches and polychaete share significantly higher sequence identity (75 and 93%, respectively) compared with the corresponding finger domains in hb orthologs in insects (grasshopper 68 and 71%, respectively) (Fig. 1). These data suggest that individual fingers have been modified in a lineage-dependent manner.
The second major class of conserved structural elements present in the hunchback family of proteins is the box domains first described in insects (Tautz and others 1987; Wolff and others 1995). Tautz and colleagues (1987) identified small stretches (5–7 amino acids) of amino acid sequences after they compared the hunchback protein with a second zinc finger protein called Krüppel. The analyses of other insect hunchback sequences have identified additional box domains that range in size between 7 and 54 amino acids (Kraft and Jäckle 1994; Wolff and others 1995; Rohr and others 1999). We have shown that annelids possess many of the box domains first described in insects with a few notable exceptions. The A, E, C, and F box domains in the annelid hb protein ORF are arranged similarly in nematode and insect hb homologues. However the B and D box domains appear to be insect-specific and the G and H box domains appear to be annelid-specific (Fig. 4). Therefore the presence or absence of specific box domains appears to correlate with specific lineages.
Our sequence comparisons also show that the 4 annelid hb homologues share significant sequence similarity within each of the zinc fingers and the box domains (Figs 1 and 2). Based on the shared sequence identity, the number and location of the zinc finger and box domains within their respective ORFs (see Fig. 4), the Hrohb gene from H. robusta, the LZF2 gene from H. triserialis, and the Cchb gene from C. capitata are orthologs to a single hunchback gene in insects. The LZF1 gene identified is thought to be a paralog whose evolutionary history in annelids is unknown (Savage and Shankland 1996). The LZF1 gene is not expressed during embryogenesis based on in situ hybridization and RT–PCR analyses and the absence of the C-terminal sequence prevents a thorough sequence comparison analysis.
Based on available sequence information, the annelid hb gene is likely to possess a minimum of 8 zinc finger motifs; however, the data obtained from leech genomic clones using canonical splice junctions suggest that leeches may possess a ninth zinc finger domain similar to what is found in the nematode hb gene (hbl-1). This is not an unusual finding because the total number of zinc finger motifs also appears to be a lineage-specific trait. For example in insects the total number of zinc finger motifs varies from 6 fingers found in D. melanogastor hb and other holometabolous species to 8 fingers found in hemimetabolous insects such as grasshoppers (Fig. 4).
The presence or absence of individual domains in hb orthologs obtained from insects, annelids, and nematodes allows one to draw reasonable inferences regarding the structural characteristics of the ancestral hunchback gene that predated the diversification of protostome phyla. The zinc fingers were likely to have been arranged in a 2-4-1-2 pattern in which the A and E box domains were located before the NF1–2 domain, the C box followed directly after the MF1–4 domain, and the F box was located between the ExF and CF1–2 domains (Fig. 4B). Assuming that the presence and order of the specific domains are representative of the ancestral hb gene, one can identify gains/losses of structural elements that occur in a lineage-specific manner. Such an approach can be used to identify potentially significant changes in the hb ORF in a given lineage that then could be targeted for functional studies without having to rely on a genome mutagenesis screens typically used to characterize functional domains.
Molecular phylogeny
We constructed a gene tree using Bayesian methods based on the alignment of 200 conserved amino acid residues, which include the most highly conserved MF1–4 domain composed of 4 zinc fingers. As shown in Figure 3, the annelid hb genes constitute an independent clade to that of the insect hb genes, which is strongly supported by posterior probability. The hb phylogeny also divides the insects into 2 major groups: the holometabola insects that include the cyclorraphous dipterans, brachyceran dipterans, lepidopterans, and the coleopterans orders and the basal hemimetabola insects (orthopterans and hemipterans). The holometabola insects undergo complete metamorphosis and the hemimetabola are a group of basal insects that have an incomplete or gradual metamorphosis. The polytomy for the locust is due to a lack of available data. Finally the positions of the remaining hb orthologs is difficult to interpret given the absence of the C-terminal sequence for the centipede S. maritima hunchback and LZF1 gene from H. triserialis and given the high sequence divergence observed in both the vertebrate and nematode hunchback ORFs. The chicken Ikaros gene is the closest hb homologue in vertebrates and contains 2 zinc finger regions, the MF1–4 and the CF1–2, similar to the arrangement found in the hunchback ORF from higher dipteran insects.
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Hunchback expression domains
A similar comparative strategy can be used to infer the ancestral protostome hb expression pattern from data obtained from insects, annelids, and nematodes. The hunchback gene products, like most important developmental regulatory genes, serve multiple roles in development and as a result the hb spatiotemporal expression profile is complex in the protostome embryos that have been examined to date. The hb expression patterns can be divided into 4 distinct pattern elements (Table 1). First, a maternal expression domain is shared between insects and annelids suggesting a maternal hb function in these 2 groups. Second, hb gene products are expressed in the extraembryonic/temporary epithelial tissues in all 3 groups. The temporary or extraembryonic epithelium in embryos is known by different names in insects (serosa/amnion), nematodes (hypodermis), and annelids (provisional epithelium) but they all serve to wrap the embryo at gastrulation and all have been implicated to play a role in morphogenesis. In most insect orders, there are 2 distinct extraembryonic membranes called the serosa and the amnion. The serosa is the epithelium that surrounds the embryo and the yolk but is not directly connected to the embryo. The amnionic epithelium develops from the margins of the germ band but in most insects covers the ventral side of the embryo and in the cylorrhaphan lineages (higher dipterans) the 2 extraembryonic membranes have been reduced (Falciani and others 1996
; Schmidt-Ott 2000). The third and fourth expression domains occur during segmental pattern formation (anterior gap) and CNS development (neuroblast and post-mitotic patterns), respectively. The comparative data compiled in Table 1 suggest that the ancestral hb pattern elements present in the protostome ancestor were restricted to the oocyte, the temporary epithelial tissues in the embryo, and the CNS but the anterior gap expression of hb arose within the arthropod/insect lineage.
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This article combines molecular phylogenetic data, comparative expression analyses, and an examination of the structural elements in the hunchback class of proteins identified from 3 major protostome groups: insects, nematodes, and annelids. Together the 3 independent lines of comparative data have been used to generate testable hypotheses concerning the evolutionary roles of the hunchback protein in ecdysozoans and lophotrochozoans. The value of this approach centers on its ability to identify structural elements in the hb ORF that can be targeted for functional analyses that have only been conducted in Drosophila. This approach also provides a means to examine both the shared and derived roles of the hb gene within a phylogenetic context. We have found both shared and derived hb expression patterns in the animal groups studied and we have identified presence/absence of specific structural elements that have occurred in the evolution of a given hb ORF. The available comparative data regarding hb expression and function are consistent with the view that changes in the coding region and/or in the regulatory sequences of a gene are likely to represent 2 important mechanisms of evolutionary change that may affect gene function over time.
The topology of the molecular tree in Figure 3 shows strong support for division of the hb protein family into 2 clusters, the insects and the annelids. In addition to distinctive amino acid signatures between insects and annelids, there are differences in both the number and type of individual domains between hb protein family members. The domain-level comparisons of insect, nematode, and annelid hb coding regions provided the basis from which the structural elements of the ancestral hunchback ORF could be inferred. The ancestral hb gene likely possessed 9 zinc fingers arranged in a 2-4-1-2 pattern as described by Patel and colleagues (2001). In this article, we have added 4 conserved box domains to the ancestral configuration of zinc finger domains. The A and E box domains are located in the N-terminus before the NF1–2 zinc finger domain, the C box domain immediately follows the MF1–4 zinc finger region, and the F box domain is positioned between the ExF domain and the CF1–2 fingers (Fig. 4). Assuming that the number and position of these domains are likely to represent the ancestral structure of the hb gene, the lineage-specific gain or loss of domains can be superimposed onto an animal phylogeny (Fig. 5). The ability to identify potentially significant changes in protein structure allows one to generate specific and testable hypotheses concerning the evolutionary roles of the gene.
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As proof of concept, the gain of the D box domain in long-germ dipteran insects suggests that it may participate in an arthropod and/or insect-specific patterning function such as the anterior gap function. The hypothesis is supported by data obtained by Hülskamp and colleagues (1994)
, who analyzed the function of the D box domain in hb alleles generated from a mutagenesis screen conducted in D. melanogastor. Two of the 11 alleles (piPST and p
PST) possessed a deletion or an insertion of 108 nucleotides in the D box domain (Hülskamp and others 1994). The 2 mutant alleles resulted in a hypomorphic "gap-like" phenotype in which the second and third thoracic segments were absent but the labium and first thoracic segments were normal. At the molecular level, they were also able to analyze the effect of the mutants on 3 known hb target genes: hunchback, Krüppel, and knirps. They showed that the D box domain was involved in the regulating the expression of hb and Krüppel gene products, but there was no effect on knirps gene expression. Amorphic hb alleles mutations that disrupted the coordination of the zinc finger motif within the MF1–4 region resulted in a loss of function in 3 target genes (Hülskamp and others 1994). These data show that individual protein domains of hunchback have differential effects on their target genes and that the D box domain is likely to play a regulatory role in establishment and/or maintenance of the anterior gap function of hunchback in long-germ dipteran insects.
To date the emphasis of hb function has been placed on its role of binding nucleic acid (Tautz and others 1987; Hülskamp and others 1994; McKay and Crossley 1998) but there is strong support that it interacts with other proteins as well. The C-terminus of the Drosophila hb protein, which includes the conserved box domains (D domain and possibly the F domain) and the terminal CF1–2 zinc finger domains, has been shown to mediate heterotypic and homotypic interactions based on genetic evidence, yeast 2-hybrid screens, and mutagenesis studies (Hülskamp and others 1994; Kehle and others 1998; McCarty and others 2003). In a few cases, the interacting protein has been identified. Kehle and colleagues (1998) have shown that the C-terminus of hb binds heterotypically to a protein called dMI-2 and together the complex interacts with the polycomb group proteins to repress homeotic gene expression. In vertebrates, the hb homologue called Ikaros functions as a lymphoid cell-specific transcription factor and it is also involved in protein–protein interactions (Georgopoulos and others 1992; Sun and others 1996; Kelley and others 1998). The Ikaros ORF contains a 4–2 arrangement of zinc finger domains similar to what is found in Drosophila hb protein (Fig. 4), and studies have shown that the MF1–4 domain binds DNA and the CF1–2 domain binds other proteins in the Ikaros family members (Hahm and others 1994; Molnár and Georgopoulos 1994; Sun and others 1996; Hahm and others 1998). Using yeast 2-hybrid screens and coimmunoprecipitation assays, these authors demonstrated that the CF1–2 domain is essential to generate homodimer complexes in which the Ikaros proteins have been shown to bind themselves or to other Ikaros family members (Sun and others 1996; Hahm and others 1998; Perdomo and Crossley 2002). Recently McCarty and colleagues (2003) showed that the CF1–2 domains in both Ikaros and hunchback proteins function as a highly selective dimerization zinc finger (DZF) domain. Interestingly the selectivity of protein–protein binding of the DZF domain is determined by the same region of amino acids that mediates base recognition in the DNA-binding region of the fingers in the MF1–4 domain (Wolfe and others 2000; McCarty and others 2003). Based on the striking functional similarities in zinc finger domains between hb and Ikaros, it has been suggested that the DZF domain originally emerged in a hunchback ancestor as a result of duplication of the C2H2 DNA-binding zinc fingers followed by the necessary modifications to create the dimerization domain (McCarty and others 2003). It is reasonable to assume that the ability of the C2H2 zinc finger family of proteins, including the hb protein family, to bind to DNA, RNA, and other proteins may help to explain both the expansion of the number of zinc finger genes in eukaryotic genomes (Lander and others 2001; Venter and others 2001; Stein and others 2003) and the presence of non-DNA-binding zinc finger domains within a given zinc finger gene (MacKay and Crossley 1998).
Evolution of the anterior gap function in insects
Insights into the evolutionary roles of the hb protein can be inferred from the comparative expression data from hb orthologs characterized in insects, nematodes, and annelids (Table 1). Our data suggest that the anterior gap function originated within the arthropods and is not likely to be a pleisiomorphic trait of the ecdysozoa but specific to arthropods and/or insects. This conclusion is further supported by studies examining the function of the hunchback ortholog (hbl-1) gene in nematodes. hbl-1 RNAi experiments generated embryos defective in morphogenesis in which the embryos failed to elongate and showed no detectable defects in AP patterning (Fay and others 1999). A central question centers on the role of hunchback gene products in other arthropods. The characterization of hb orthologs in crustaceans is required before one can infer the origin of the anterior gap function in the arthropod phylum.
The compiled comparative expression data suggest that ancestral hb function can be divided into at least 3 expression domains: maternal, post-mitotic differentiation of neurons in the CNS, and extraembryonic/temporary epithelial membranes. The hb gene products are expressed in oogenesis in insects and annelids but its maternal function has been largely unexplored in animals outside of insects. Based on the current available data, the maternal hb function is likely to have predated the split between insects and annelids based on the shared maternal expression patterns in the 2 groups. The comparative data show stronger support for hb's role in CNS development, specifically its role in the differentiation of post-mitotic neurons in the ventral nerve cord of protostomes. In larval and juvenile ventral nerve cords of insects, nematodes, and annelids, hb protein is expressed in a subset of post-mitotic neurons in ganglia along the entire length of AP axis of the organism (Fay and others 1999; Iwasa and others 2000; Patel and others 2001; Werbrock and others 2001). The hb protein appears to play an additional role in CNS patterning. In nematodes and insects and possibly in all ecdysozoans, hb protein participates specification of neuroblast and/or neuroblast sublineage identity (Wolff and others 1995; Fay and others 1999; Isshiki and others 2001; McGregor and others 2001; Novotny and others 2002; Liu and Kaufman 2003; Pearson and Doe 2003; Grosskortenhaus and others 2005). Unlike protostome hb proteins, the Ikaros protein in mice is only expressed in the thymus and not in the CNS although hb's role in chordates and primitive chordates remains an open question (Georgopoulos and others 1992).
Our data suggests that the anterior "gap" function of the hb gene product is not likely to be a conserved pattern element between nematodes, insects, and annelids; however, products of hb orthologs are expressed in the temporary epithelial tissues that surrounds early stage embryos in all 3 protostome groups. The extraembryonic membranes called the serosa or amnioserosa in insects, the hypodermis in nematodes, and the provisional epithelium in annelids are a single layer of epithelial cells that encloses the embryo (Ho and Weisblat 1987; White 1988; Frank and Rushlow 1996; Fay and others 1999). In insects, there is a trend toward a reduction in the extraembryonic membranes moving from the hemimetabola, which are the group of insects that undergo incomplete or gradual metamorphosis, to the holometabola, which undergo complete morphogenesis (Schmidt-Ott 2000; Ziese and Dorn 2003). In most insects, the serosa and the amnion are separate extraembryonic epithelial membranes and it is in the cyclorrhaphan lineage the 2 membranes were thought fuse to form the amnioserosa tissue based on shared molecular expression patterns (Sommer and Tautz 1991; Wolff and others 1995; Falciani and others 1996; Rohr and others 1999; Dearden and others 2000; Schmidt-Ott 2000). Recent evidence suggests the reduction of the extraembryonic membranes observed in higher dipteran flies is because the 2 membranes overlap with one another but in fact do not fuse and remain distinct tissues (Nipam Patel, personal communication). A detailed analysis of hunchback protein in dipterans needs to be completed in order to clarify hb's role in the extraembryonic tissues. In insects, the primary function of the extraembyronic membranes is to secrete the embryonic cuticle, but these epithelial membranes may serve an additional role during morphogenesis. This hypothesis is supported by studies examining the function of the amnioserosa tissue in flies. Alleles from a number of genes such as decapentaplegic that controls the specification of the amnioserosa and the U-shaped-group of genes that control the differentiation of the aminoserosa generate mutant embryos that exhibit dramatic defects in germ band extension and retraction due to either a complete or partial loss of the amnioserosa tissue (Ray and others 1991; Arora and Nüsslein-Volhard 1992; Frank and Rushlow 1996; Lamka and Lipshitz 1999; Reed and others 2001). Experiments designed to disrupt hb function in the amnioserosa in dipteran flies or in the serosal tissue of lower insects have not been performed, but its expression at the time these morphogenetic events take place suggests that it may also play a direct or indirect role in the elongation of the embryo.
The possibility that the ancestral role of the hb protein may be involved in morphogenesis is supported by functional analysis of the hb ortholog called hbl-1 in nematodes (Fay and others 1999) and a series of annelid hb expression studies. Based on the data from a fusion hbl-1::GFP transgene (which may or may not be representative of the entire hbl-1 spatiotemporal expression profile), the nematode hb gene is expressed in the hypodermal precursor cells at the initiation of morphogenesis (500 cell stage). In a series of hb RNAi knock down experiments, Fay and colleagues (1999) showed that >90% of the embryos appeared to arrest during morphogenesis and in most cases the embryo failed to elongate. Similarly, the annelid hb orthologs are expressed in the single layer of epithelial cells that enclose the embryo at gastrulation, analogous to the hypodermis in nematodes and the serosa in most insects (Iwasa and others 2000; Werbrock and others 2001; Shimizu and Savage 2002). The few functional studies available in leeches rely on ablating precursor cells to the epithelium, which resulted in the disruption of integrity of the provisional epithelium and resulted in abnormal gastrulation (Ho and Weisblat 1987; Smith and others 1996).
Molecular phylogenetic data, comparative expression analyses, and an examination of individual domains within the hb protein family are 3 approaches, when taken together, that significantly strengthen the inference capabilities of comparative data and thus provides compelling insights to the study of evolutionary processes. The 3 sources of hb comparative data described in this article allowed us to infer ancestral developmental roles and structural properties of the hb protein in the protostome lineage leading to the lophotrochozoans and ecdysozoans. The analysis of the position and number of individual domains in hb orthologs based on intra- and interphyletic comparisons provided the opportunity to identify lineage-specific changes in the evolution of hb protein. This information can then be used to generate specific and testable predictions regarding hb function(s) in protostomes without having to rely on large-scale mutagenesis screens typically used to characterize the function of domains in a protein.
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This study was supported by the National Science Foundation grant IBN-0090378 and National Institute of Health grant GM071444 to RMS and by the HHMI grant awarded to Williams College. Funding to pay the open access publication charges for this article was provided by NIH GM071444.
Conflict of interest: None declared.
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From the symposium "WormNet: Recent Advances in Annelid Systematics, Development, and Evolution" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2005, at San Diego, California.
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