C. elegans Hunchback homolog

A Caenorhabditis elegans homolog of the Drosophila gap gene hunchback has been cloned and designated hbl-1 (hunchback-like). hbl-1 encodes a predicted 982 amino acid protein, containing two putative zinc finger domains similar to those of Drosophila Hunchback. The 3' UTR consists of 1405 bases. The N terminal Zn finger domains of HBL-1 contains three Zn-finger repeats and shows a high degree of similarity to Drosophila Hb (72% identical), as well as to the products of hb homolog Lzf2 from the leach Helobdella triserialis (80%). The C terminal domain of HBL-1 contains two Zn finger repeats and shares a lower degree of similarity with the corresponding regions in these proteins (25%, 29% and 33% identical, respectively). Little further similarity is observed between Drosophila Hb and HBL-1 with the exception of a stretch of 35 residues directly preceding the N terminal Zn finger domains of both proteins. The gene is transcribed embryonically, but unlike the maternally expressed Drosophila hb, its mRNA is not detected in C. elegans oocytes. A hbl-1;gfp reporter is expressed primarily in ectodermal cells during embryonic and larval development. During embryogenesis,GFP is detected in the majority of pharyngeal cells as well as in neurons, with no discernible expression in cells of the hypodermis. In larvae, FGP expression is observed predominantly in neurons. Staining in the ventral nerve cord (VNS) is first detected in L1 larvae prior to the migration of P-cells and is later observed in many cells of the VNC from L2 to L4 stages. In addition, staining is detected in the hermaphrodite-specific neurons beginning around the L2 stage and in the canal-associated neurons beginning around the L3 stage. Staining is also observed in many neurons of the anterior nerve ring, the dorsal nerve cord, and several neurons in the tail. Double-stranded RNA-interference (RNAi) was used to indicate hbl-1 loss-of-function phenotypes. Progeny of hbl-1(RNAi) hermaphrodites exhibit a range of defects: the most severely affected progeny arrest as partially elongated embryos or as hatching, misshapen L1 larvae. Animals that survive to adulthood exhibit a variety of aptly named phenotypes [dumpy (Dpy), uncoordinated (Unc), and egg-laying defective (Egl)] as well as defects in vulval morphology (Pvl). Unc phenotype varies from complete paralysis to moderately impaired movement.The hypodermis is a single layer of epithelial cells that encloses the animal and is responsible for secretion of the protective cuticle. Abnormal organization of hypodermal cells and expression of a hypodermal marker in hbl-1(RNAi) animals suggests that Dpy and misshapen phenotypes could be due to improper specification of hypodermal cells. The pattern of hbl-1 expression is similar to that reported for the leech hunchback homolog, Lzf-2, suggesting that these proteins may have similar biological functions in diverse species with cellular embryos (Fay, 1999).

Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. lin-57 has been identified as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 has been identified as hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback (hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3'UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs (miRNAs), including let-7 (see Drosophila let-7). Indeed, hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3'UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation (Abrahante, 2003).

The C. elegans hunchback-like gene hbl-1 regulates postembryonic developmental time. In ve18 mutants, vulval cell divisions occur abnormally early during the L2 stage; fusion of lateral epidermal cells, termed seam cells, and adult cuticle synthesis occur precociously during the L3 molt, and seam cell nuclei divide inappropriately during the L4 stage. Removal of hbl-1 activity postembryonically through RNAi causes similar defects, and, in addition, proliferative seam cell divisions are sometimes skipped during the L1 molt. These observations indicate that the worm hunchback ortholog functions at multiple times during postembryonic development and plays key roles in specifying temporal cell fates in the vulva and hypodermis (Abrahante, 2003).

Because the L1 molt seam cell division phenotype correlates with seam cell expression of hbl-1::gfp during the L1 stage, hbl-1 may function cell autonomously in the seam to control this early division pattern. However, hbl-1::gfp is not detected in the seam after the L1 stage, nor is it detected in the vulval precursor cells, suggesting that hbl-1 may function non-cell autonomously to time cell divisions at later stages in these tissues. One candidate tissue for producing an hbl-1-dependent signal is the syncytial hypodermis (termed hyp7) that surrounds the seam, abuts the VPCs, and is a major contributor to cuticle synthesis. The roller phenotype used in genetic screens is likely to result from hyp7-mediated ultrastructural defects in the cuticle, and, therefore, identification of ve18 as a precocious roller suggests that temporal identity of hyp7 is also affected in hbl-1 mutants. Thus, similar to the seam, hyp7 initiates its adult program, possibly including precocious signaling to surrounding tissues. There is precedent for signaling from hyp7: lin-15(+) is required in hyp7 for repression of cell divisions in VPCs that do not normally contribute to vulva formation. Thus, one possibility is that hbl-1 function in hyp7 is required for correct timing of seam and vulval cell fates (Abrahante, 2003).

In flies, hunchback (hb) is best known for its crucial role in spatial patterning of the embryo, as a member of the gap class of segmentation genes. Null mutations in hb cause deletion of specific sets of segments along the anterior-posterior axis. Similar to the regulation of hbl-1 during temporal patterning of the nematode, the hb 3'UTR plays a key role during spatial patterning in the early fly embryo. Pumilio, Nanos, and Brat form a quaternary complex on a pair of conserved 32 nt elements in the hb 3'UTR, called Nanos response elements (NREs), and repress hb translation in the posterior of the embryo, contributing to the formation of an hb protein gradient emanating from the anterior. The hbl-1 3'UTR does not contain canonical fly NREs, although there are three 'B box' sequences (AUUGUA), two of which are conserved in C. briggsae and C. remanei. The C. elegans genome does contain candidate homologs of the key fly hb regulatory proteins, and the evolutionary maintenance of these components raises the possibility that additional aspects of hb regulation are conserved between these species, perhaps acting through divergent 3'UTR sequences (Abrahante, 2003).

These Hb-meditated temporal phenotypes seen in the Drosophila CNS are reminiscent of those reported in this study for hbl-1 loss-of-function. Loss of hbl-1 activity causes the adult seam cell fate to be executed one stage too early, whereas overexpression of hbl-1 can cause failure of seam cell terminal differentiation, likely as a result of reiteration of L4 fates in the adult. Together, these observations suggest that the general involvement of hunchback proteins in programming temporal identity within specific cell lineages has been evolutionarily conserved (Abrahante, 2003).

There is a dramatic difference in seam cell versus neuroblast cell cycle time. Seam cells are scheduled to divide once per molt, at approximately 7 hr intervals, whereas fly neuroblasts undergo rapid cell divisions in the embryo, on the order of 40 min. How the rapid transitions in transcription factor expression are controlled in fly neuroblasts is unknown, but it is tempting to speculate that miRNAs could play a role in facilitating expeditious translational downregulation of hb and other transcription factors in this developmental context (Abrahante, 2003).

Indeed, the hb 3'UTR contains potential fly microRNA binding sites in evolutionarily conserved regions. Of particular note, mir-184, mir-4, and mir-13a are predicted to duplex with sequences overlapping the two NREs (NRE1 at nt 46-78 of the 3'UTR and NRE2 at nt 98-129), suggesting involvement of these miRNAs in fundamental aspects of hb regulation. Whether the predicted mir-3 binding sites are conserved in D. virilis is unknown, but the two D. melanogaster sites share high identity (13 out of 18 nt). Moreover, mir-3 and mir-4 are expressed during embryogenesis, but not thereafter (Lagos-Quintana, 2001), consistent with an early regulatory role. In summary, this work extends the similarities between the worm and fly hunchback genes beyond the level of sequence conservation. Both genes are deployed to temporally specify cell fates, and their expression patterns rely on 3'UTR sequences likely to be modulated through the action of microRNAs (Abrahante, 2003).

Postembryonic temporal downregulation of hbl-1 in the worm nervous system and hypodermis is programmed, at least in part, through its 3'UTR, which contains multiple putative let-7 binding sites that are evolutionarily conserved. In the nervous system, an hbl-1::gfp::hbl-1 reporter construct is temporally deregulated in a let-7 mutant background; enhanced expression is observed in the ventral nerve cord and anterior nerve ring of adults. Together, these results imply that the hbl-1 3'UTR is a direct target of the let-7 miRNA (Abrahante, 2003).

The extent of hbl-1::gfp::hbl-1 misexpression in let-7 mutants is less than might be expected if let-7 acts alone to downregulate neuronal expression and suggests that additional factors, perhaps other microRNAs, act together with let-7. Indeed, a large and diverse family of miRNAs has been discovered in C. elegans. Among the worm miRNAs reported, three (mir-84, mir-48, and mir-241) share sequence identity with let-7 RNA and are expressed with the same temporal specificity as let-7. The sequence conservation among these miRNAs, particularly between mir-84 and let-7 (81% identical), suggests that they may have target sites in common. Thus, complete temporal deregulation of the hbl-1 reporter may require simultaneous inactivation of multiple miRNAs (Abrahante, 2003).

The role of let-7 in control of hbl-1 in the hypodermis is less clear. The simplest way to interpret let-7 suppression by hbl-1, together with let-7 binding sites in the hbl-1 3'UTR, is that hbl-1 is a direct target of the let-7 miRNA. However, hypodermal hbl-1::gfp expression begins to subside in the L2 and disappears in the early L3, prior to let-7 accumulation in the mid to late L3 stage. Assuming that the hbl-1::gfp construct (which contains a 6.4 kb 5' flanking sequence through the first three introns) contains all relevant enhancer regions, this implies that 3'UTR-mediated downregulation of hbl-1 in hyp7 is controlled by other factors, perhaps including earlier-acting miRNAs (Abrahante, 2003).

let-7 could add to the repression of hbl-1 mRNA from the mid L3 stage onward, ensuring its silence at late developmental stages. However, consistent hbl-1::gfp::hbl-1 misexpression was not detected in the hypodermis of let-7 mutants, suggesting only a minor role for let-7 or redundant action by let-7-related genes. Alternatively, a low threshold level of the HBL-1 presumed transcription factor (not detectable by gfp assay) may be required for hypodermal function. Thus, small changes in HBL-1 level could lead to major developmental consequences through deregulation of target genes (Abrahante, 2003).

Temporal regulation of hbl-1 differs from that of lin-41, the other known let-7 target. lin-41::gfp is expressed in both neurons and hypodermis but is temporally downregulated only in the hypodermis. The discordant patterns of regulation suggest inherent differences between the hbl-1 and lin-41 3'UTRs and the assembled factors that orchestrate their function (Abrahante, 2003).

Reduction of hbl-1 activity by mutation or RNAi does not fully suppress let-7 null mutations. Explanations for this partial epistasis include incomplete loss of hbl-1 function, misexpression of let-7 targets, or redundancy at the hbl-1 step in the pathway. This work suggests that the let-7 target, lin-41, is at least part of the answer. Simultaneous removal of hbl-1 and lin-41 activities produces stronger suppression of the let-7 phenotype than does single depletion of either gene. In let-7(+) animals, depletion of hbl-1 and lin-41 activities produces a fully penetrant L3 molt phenotype and can cause terminal differentiation at the L2 molt, one stage earlier than in either single mutant. Together, these results indicate that let-7 acts through both hbl-1 and lin-41 and that these genes function with partial redundancy to inhibit premature activation of the adult hypodermal program at the L2 and L3 molts in wild-type animals (Abrahante, 2003).

These findings extend the intriguing parallels between the early and late timers of the heterochronic gene pathway, which together mediate stage-specific temporal identities. Each timer is initiated by a microRNA that has two known targets; in the early timer, lin-4 downregulates lin-14 and lin-28, and, in the late timer, let-7 acts through hbl-1 and lin-41. In each case, one target encodes a transcription factor (LIN-14 and HBL-1), and the other encodes a protein with hallmarks of a translational regulator (LIN-28 and LIN-41). Since loss-of-function for each pair of targets causes enhanced precocious phenotypes, it appears that both transcriptional and translational controls are necessarily integrated into both timers to ensure proper timing of cell fate specification (Abrahante, 2003).

Previous studies have generally supported a linear pathway of heterochronic genes, with lin-4 acting as the most upstream and global regulator. These analyses suggest that the pathway is branched. Concomitant loss of hbl-1 and lin-41 activities suppresses the let-7 mutant phenotype more completely than that of lin-4. Loss of hbl-1 and lin-41 activities only weakly restores alae synthesis at the L4 molt in lin-4 mutants, whereas it leads to essentially complete execution of the adult seam cell program at the L3 molt in a let-7 mutant background. These observations indicate that either lin-4 or the genes it regulates have additional targets that time the adult hypodermal program independently of hbl-1 and lin-41. Thus, multiple temporal inputs converge upon the transcription factor LIN-29, indicating that a branched pathway functions to ensure proper timing of seam cell terminal differentiation. Elaboration of these proposed branches will require searches for additional components of the heterochronic gene pathway (Abrahante, 2003).

hbl-1, the C. elegans hunchback ortholog, also controls temporal patterning. Furthermore, hbl-1 is a probable target of microRNA regulation through its 3'UTR. hbl-1 loss-of-function causes the precocious expression of adult seam cell fates. This phenotype is similar to loss-of-function of lin-41, a known target of the let-7 microRNA. Like lin-41 mutations, hbl-1 loss-of-function partially suppresses a let-7 mutation. The hbl-1 3'UTR is both necessary and sufficient to downregulate a reporter gene during development, and the let-7 and lin-4 microRNAs are both required for HBL-1/GFP downregulation. Multiple elements in the hbl-1 3'UTR show complementarity to regulatory microRNAs, suggesting that microRNAs directly control hbl-1. MicroRNAs may likewise function to regulate Drosophila hunchback during temporal patterning of the nervous system (Lin, 2003).

HBL-1/GFP is expressed strongly in hypodermal cells, including the embryonic seam cell precursors, and in neurons like those of the ventral nerve cord (VNC) during postembryonic stages. HBL-1/GFP expression was reexamined, focusing on hypodermal and VNC expression at postembryonic C. elegans developmental stages. Strain BW1932 contains an integrated array with the hbl-1 promoter, the first 133 amino acids of HBL-1 fused to GFP, and the hbl-1 3'UTR. During the L1 stage, HBL-1/GFP expression is observed in the hypodermal syncitial cells (e.g., hyp7), in the ventral hypodermal cells (P cells), and weakly in the lateral hypodermal seam cells (H, V, and T cells). By the L2 stage, HBL-1/GFP was no longer expressed in the seam cells but was still observed in P cell descendants and weakly in the non-seam cell hypodermis. By the L3 stage, HBL-1/GFP was virtually absent in the hypodermis and Pn.p cell descendants, but was still highly expressed in the ventral nerve cord (generated from Pn.a cells) and other unidentified neurons. Early L4 animals express high HBL-1/GFP levels in the VNC, while late L4 and adult animals express HBL-1/GFP very weakly in the VNC. In some adult VNCs, expression is undetectable. As judged by this HBL-1/GFP fusion, HBL-1 expression is downregulated during the course of postembryonic development, with highest expression in L1 animals and lowest expression in adults (Lin, 2003).

let-7 RNA is expressed predominantly in the L4 and adult stages. HBL-1/GFP expression in the VNC is downregulated during the L4 and adult stages by a 3'UTR-dependent mechanism. The similar timing of these two events suggest that let-7 might be involved in downregulation of hbl-1 in the VNC. Indeed, it was found that while 45% of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% of wild-type animals did the same. lin-4 RNA is also present in the L4 stage. Intense HBL-1/GFP expression is seen in the VNC of 100% of lin-4(e912) adult animals. Thus, both wild-type let-7 and lin-4 RNAs are required for proper hbl-1 downregulation in the VNC (Lin, 2003).

The let-7 microRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans: their target is the homolog of Drosophila hunchback

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. Doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the 3′ UTR of the hunchback homolog hbl-1 and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions (Abbott, 2005).

The C. elegans genome encodes at least 19 microRNA gene families containing from 2 to 8 members with significant sequence conservation within the ~22 nt microRNA sequence. Sequence conservation among family members is strongest near the 5' end of the microRNA in the region known as the 'seed',which has been proposed to reflect a potential for family members to direct the repression of shared target genes. Because mir-48, mir-84, and mir-241 display complete sequence conservation in the seed region at the 5′ end, it is possible that they repress a common set of targets and hence may be functionally equivalent. The current findings suggest that let-7, mir-48, mir-84, and mir-241 may all act to repress a shared target, hbl-1. let-60 RAS also has been proposed to be a target of mir-84 based on overexpression experiments. Elevated levels of let-60 RAS expression lead to a multivulva phenotype; however, no multivulva phenotype was observed in mir-84 single mutants nor in mir-48 mir-241; mir-84 triple mutants (Abbott, 2005).

The results leave open the possibility that mir-48, mir-84, and mir-241 are not functionally equivalent in all respects. Sequence differences in the 3′ end of the let-7 family microRNAs may direct the repression of some distinct sets of targets, the repression of which could function coordinately to regulate developmental timing. Target sites that lack strong complementarity at the microRNA 5′ end can direct repression if there is extensive compensatory pairing at the 3′ end, thus allowing for distinct activities of microRNA family members. Indeed, let-7 complementary sites in the lin-41 mRNA have extensive complementarity to the let-7 3′ region, along with imperfect pairing to the let-7 5′ seed region. The specificity imparted by compensatory 3′ pairing may function to enable repression of lin-41 by let-7 and not allow for the repression of lin-41 by mir-48, mir-84, or mir-241. Similarly, extensive 3′ pairing to one of the other three let-7 family members might compensate for a lack of strong 5′ pairing and therefore could restrict the repression of specific targets to individual let-7 family members (Abbott, 2005).

The findings suggest that the four let-7 family microRNAs may all act to repress hbl-1. Reduction of hbl-1 activity can suppress the heterochronic defects observed in both mir-48 mir-241; mir-84 and let-7 mutant animals, indicating that hbl-1 functions downstream of the let-7 family microRNAs. Moreover, the failure to appropriately downregulate hbl-1 can be detected in the hypodermis of mir-48 mir-241; mir-84 mutants and in neuronal cells of let-7 mutants. The hbl-1 3′ UTR contains eight let-7 complementary sites. Because these potential binding sites differ in sequence, each may be able to bind the individual let-7 family microRNAs with differing efficacies. The relative contribution of individual let-7 family microRNAs to the repression of hbl-1 activity remains to be tested (Abbott, 2005).

Previous studies showed a role for hbl-1 activity in controlling the L4-to-adult transition. The current findings indicate that hbl-1 also controls the L2-to-L3 transition in the hypodermis. This early role for hbl-1 is consistent with the observation that reduction of hbl-1 activity by RNAi results in a decreased number of seam cells in L2-stage animals. A reduced number of seam cells likely reflects a partial omission of the L2-stage proliferative program. This precocious phenotype is relatively weak in comparison to that of lin-28(lf) mutants, in which all seam cells generated from the V lineage fail to execute the L2-stage program. This weak phenotype may be a consequence of residual hbl-1 activity of the partial loss-of-function allele, ve18. It is possible that complete loss of hbl-1 activity would result in a stronger precocious L2-omission phenotype similar to that seen in lin-28(lf) mutants (Abbott, 2005).

An important regulator of the L2-to-L3 transition is lin-28, yet multiple lines of evidence suggest that the control of the L2-to-L3 transition by mir-48, mir-84, and mir-241 does not occur through regulation of lin-28 activity. (1) A lin-28::gfp::lin-28 reporter transgene that recapitulates the wild-type temporal regulation of LIN-28 protein and that rescues the phenotype of lin-28(lf) worms is not derepressed in mir-48 mir-241; mir-84 triple mutants. (2) It was found that the level of endogenous LIN-28 protein was not significantly elevated in mir-48 mir-241; mir-84 triple mutants, whereas, in lin-4 retarded mutants, LIN-28 protein is abnormally abundant at later larval stages. (3) Two alleles of lin-58 that contain mutations upstream of mir-48, and hence lead to the misexpression of mir-48, enhance the precocious phenotype of a lin-28 null mutant, indicating that mir-48 does not act exclusively through lin-28. (4) It was found that the L2 reiteration phenotype of mir-48 mir-241; mir-84 triple mutants could occur independently of lin-28 activity. These data together indicate that mir-48, mir-84, and mir-241 control the L2-to-L3 transition primarily through downstream effectors other than lin-28, even though the lin-28 3′ UTR contains a let-7 complementary site. It is possible that the let-7 family microRNAs may contribute to the repression of lin-28 expression, but to a degree undetectable by the assays used (Abbott, 2005).

Genetic epistasis analysis indicates that mir-48, mir-84, and mir-241 function in parallel with the lin-28 and lin-46 pathway to downregulate hbl-1 activity and hence control the L2-to-L3 transition. One model to account for this convergence of pathways on hbl-1 would be that LIN-46, in its putative role as a scaffolding protein, could control assembly of a protein complex that directly interacts with HBL-1 protein to inhibit its activity in parallel with the repression of hbl-1 mRNA translation exerted by mir-48, mir-84, and mir-241. Alternatively, LIN-46 could interact with RNA binding protein(s) and directly potentiate the activity of the mir-48, mir-84, and mir-241 microRNAs (Abbott, 2005).

These data suggest that mir-48, mir-84, and mir-241 control developmental timing in two physically associated but distinct cell types in the hypodermis: the postmitotic main body hypodermal syncytial cell, hyp7, and the proliferative seam cells. Two lines of evidence point to a role in hyp7 for mir-48, mir-84, and mir-241 to repress hbl-1 and control hyp7 temporal behavior. (1) mir-48; mir-84 mutant worms displayed heterochronic defects in hyp7; the expression of the adult-specific transgene col-19::gfp was retarded in hyp7 but was regulated normally in the seam cells. Thus, the supernumerary molt observed in mir-48; mir-84 double mutants may be a consequence of a heterochronic defect in hyp7. (2) The data indicate that mir-48, mir-84, and mir-241 act in hyp7 to repress hbl-1 activity. In mir-48 mir-241; mir-84 worms, hbl-1::gfp::hbl-1 was misregulated in hyp7. Thus, the 3′ UTR-dependent downregulation of hbl-1::gfp::hbl-1 in hyp7 that occurs in wild-type animals can be accounted for largely by the regulation of hbl-1 by mir-48, mir-84, and mir-241 (Abbott, 2005).

mir-48, mir-84, and mir-241 may also function in the hypodermal seam cells to control developmental timing. Reduction of hbl-1 activity genetically or by hbl-1 RNAi affected stage-specific behavior of seam cells, resulting in suppression of the retarded seam cell and alae phenotypes of mir-48 mir-241; mir-84 worms. This could be a consequence of the repression of hbl-1 by mir-48, mir-84, and mir-241 in the seam cells. Interestingly, hbl-1::gfp::hbl-1 cannot be detected in the seam cells after the L1 stage, suggesting that, at the time of the L2-to-L3 transition, the amount of hbl-1 expression in seam cells is relatively low. Thus, mir-48, mir-84, and mir-241 may function cell autonomously in the seam cells at the L3 stage to downregulate hbl-1, albeit beginning from a level already below the threshold of detection by the assays. Alternatively, since repression of hbl-1::gfp::hbl-1 is readily observed at the L2-to-L3 transition in hyp7 (the main body hypodermal syncytial cell), it is conceivable that the stage-specific behavior of seam cells may be controlled non-cell autonomously by a hbl-1-regulated signal from hyp7. Non-cell autonomous signaling from hyp7 to neighboring cells has been proposed in the pathway to specify the fates of vulval precursor cells (VPCs). Mosaic analyses suggest that the sites of action of the multivulva (Muv) gene locus lin-15 and of the synthetic Muv genes lin-37 and lin-35 are in hyp7. One model is that hyp7 generates a signal to neighboring VPCs to inhibit vulval cell fate specification. Similarly, a signal from hyp7 to the lateral hypodermal seam cells may regulate the temporal behavior of seam cells and thereby help coordinate developmental timing throughout the hypodermis (Abbott, 2005).

In summary, the results presented in this study demonstrate a role for the let-7 family microRNA genes mir-48, mir-84, and mir-241 in the heterochronic pathway to control the L2-to-L3 cell fate transitions in the hypodermis. Proper progression through the L1 and L2 larval stages requires downregulation of lin-14 and lin-28, primarily through the action of the microRNA lin-4. These findings extend the involvement of microRNAs in the regulation of C. elegans developmental timing to include a requirement for the downregulation of hbl-1 by the combined action of the three let-7 family microRNAs, mir-48, mir-84, and mir-241, in the hypodermis. The L2-to-L3 transition is controlled by complex genetic mechanisms involving two microRNA-regulated pathways that converge on hbl-1: the lin-4, lin-28, lin-46 pathway and the mir-48, mir-84, mir-241 pathway. These parallel inputs to hbl-1 may serve to couple hbl-1 downregulation to distinct upstream temporal signals. Further, the functional redundancy among mir-48, mir-84, and mir-241 could reflect alternative mechanisms for triggering the L2-to-L3 transition throughout the hypodermis. mir-48, mir-84, and mir-241 seem to have more minor roles, compared to let-7, at the L4-to-adult transition in the hypodermis, indicating that different microRNA family members can be deployed for distinct roles, perhaps through differences in temporal or spatial expression patterns and/or differences in target specificity. These findings suggest analogous forms of genetic redundancy and regulatory complexity may be expected in pathways involving other families of related microRNAs (Abbott, 2005).

Insect Hunchback homologs

While the function of hb in AP patterning of the body axis appears to be conserved across insect orders, there are elements of the hb spatiotemporal expression domain that differ from one order to the next. For example, differences are seen in the hb expression in insect extraembryonic tissue, structures analogous to the hb expressing provisional epithelium in leeches. In the flour beetle Tribolium castaneum and the mothmidge Clogmia albipunctata Hb protein is expressed early in embryogenesis in the serosa, an extraembryonic, squamous epithelium that eventually covers the entire embryo but is never directly attached to it. Their other extraembryonic tissue, the amnion, does not express Hb and forms later during development and remains attached to the embryo until dorsal closure is complete. In contrast, at the time of gastrulation Drosophila does not express Hb in their single extraembryonic tissue, the amnioserosa; this is a tissue that stretches over the entire dorsal surface of the embryo and is attached to the embryo until its degeneration following dorsal closure. However, following gastrulation, Hb expression is observed in amnioserosa in Drosophila. It is believed that the serosa had been lost in Drosophila in the transition from the ancestral dual amnion and serosa to the single fused amnioserosa of flies, which may explain the lack of Hb protein expression in Drosophila extraembryonic tissues during gastrulation (Iwasa, 2000 and references therein).

However, while fusion to the developing embryo and Hb expression patterns alone suggest that the Drosophila amnioserosa is simply a derivative of the amnion found in Tribolium and Clogmia, other data suggest complementarity to the serosa. For instance, the Tribolium homologs of the zerknüllt (zen) gene, which in Drosophila specifies the extraembryonic structures of the amnioserosa is expressed in serosa cells but not in the amnion. These data indicate that the amnioserosa, rather than simply being a derivative of the amnion, incorporates the functions of both the amnion and the serosa into one (Iwasa, 2000).

Although the function of the serosa is not known beyond its secretion of the embryonic cuticle, there is genetic evidence suggesting that the amnioserosa in Drosophila functions during gastrulation. Two sets of mutants that affect amnioserosa formation result in dramatic gastrulation defects in flies. In mutants of decapentaplegic, a protein involved in patterning the dorsal side of the developing embryo, the amnioserosa fails to differentiate and germ band extension and retraction are inhibited. In mutants of the U-shaped-group class of genes, the amnioserosa is specified normally, but after germ band extension the cells of the amnioserosa undergo premature apoptosis resulting in the failure of the germ band to retract. These data provide genetic evidence pointing to a role for surrounding epithelial tissues, the amnioserosa in Drosophila, and through evolutionary relatedness the serosa in ancestral insects and the provisional integument in leeches in coordinating morphogenetic movements during gastrulation. Whether this function is homologous or convergent, however, represents an important unanswered question (Iwasa, 2000).

The role for epithelial tissues is further supported by recent studies of a hb homolog in the nematode Caenhorhabditis elegans where the homolog, hunchback-like 1 (hbl-1) has been shown to exhibit an expression pattern strikingly similar to that found in leeches. Analogous to the finding for LZF2, hbl-1 is expressed in the hypodermal precursor cells and later in the CNS but does not appear to function in AP patterning. The postembryonic hypodermal cells give rise to the hypodermis, a single layer of epithelial cells that surround the embryo. RNA-mediated interference (RNA-i) experiments inhibit hbl-1 function and result in the failure of the embryos to elongate and hatch. These nonelongation gastrulation defects appear to result largely from incomplete or improper differentiation of epithelial hypodermal cells (Iwasa, 2000).

It seems therefore that the ancestral hb gene in the last common ancestor of annelids, nematodes, and arthropods functioned early in embryogenesis in the epidermal and extraembryonic tissues and later in the CNS. The role of hb in AP pattern formation may be a relatively recent innovation in the insect lineage, but further investigations into hb function in other arthropods are required to address this issue (Iwasa, 2000).

In Drosophila, the morphogen Bicoid organizes anterior patterning in a concentration-dependent manner by activating the transcription of target genes such as orthodenticle (otd) and hunchback (hb), and by repressing the translation of caudal. Homologs of the bicoid gene have not been isolated in any organism apart from the higher Dipterans. In fact, head and thorax formation in other insects is poorly understood. To elucidate this process in a short-germband insect, the functions of the conserved genes orthodenticle-1 (otd-1) and hb were analyzed in the flour beetle Tribolium castaneum. In contrast to Drosophila, Tribolium otd-1 messenger RNA is maternally inherited by the embryo. Reduction of Tribolium otd-1 levels by RNA interference (RNAi) results in headless embryos. This shows that otd-1 is required for anterior patterning in Tribolium. As in Drosophila, Tribolium hb specifies posterior gnathal and thoracic segments. The head, thorax and the anterior abdomen fail to develop in otd-1/hb double-RNAi embryos. This phenotype is similar to that of strong bicoid mutants in Drosophila. It is suggested that otd-1 and hb are part of an ancestral anterior patterning system (Schröder, 2003).

In Drosophila, bicoid is the central gene in the anterior patterning system. Embryos that do not express Bicoid protein fail to develop a head or thorax, and form a second telson at the anterior pole instead. Binding specificity of Bicoid to its DNA and RNA targets is mediated by a lysine (K) at position 50 of its homeodomain (K50HD). Despite its pivotal role in defining anterior pattern in Drosophila, bicoid orthologs have been isolated only from closely related higher Dipterans. In Drosophila and other higher Dipterans, a bicoid ortholog is located in the Hox gene complex close to the zerknüllt/Hox3 locus -- between proboscipedia/ Hox2 and Deformed/Hox4. A bicoid ortholog is not present in this genomic interval in the beetle Tribolium. This finding corroborates the hypothesis that bicoid evolved recently, probably through the divergence of a Hox3 paralog during evolution of the higher Dipterans, and is not part of an ancient anterior system common to more basal insects (Schröder, 2003).

How is anterior patterning in the embryo organized in the absence of bicoid? In Drosophila bicoid mutants, high levels of bicoid-independent zygotic hb are able to direct the formation of a partial thorax. However, since hb cannot induce formation of head structures on its own, the conserved protein Otd, which like Bicoid contains a K50 homeodomain, has been considered to be an ancestral head determinant. This hypothesis has been tested in an insect that develops as a short-germband embryo, and therefore shows a more general type of embryogenesis. Initially, the function of otd-1 was analyzed in the beetle Tribolium (Schröder, 2003).

In Tribolium and Drosophila, zygotic otd seems to act as a head gap gene. otd-1 is also involved in the formation of the anterior-most region of the egg -- the extraembryonic serosa. In otd-1pRNAi embryos, the nuclei of the serosa are irregularly arranged or, in more severe cases, reduced in number. hb, which is strongly expressed in the serosa of the wild-type embryos, is also still detectable in the nuclei of the serosa in otd-1pRNAi embryos. otd-1 is therefore not required to activate hb expression in the serosa. Nevertheless, otd-1 could assist other factors in activating hb expression, such as zerknüllt, which is also expressed in this tissue (Schröder, 2003).

The fact that the otd-1RNAi phenotype only incompletely mimics the Drosophila bicoid mutant phenotype implies that otd-1 is only a partial functional equivalent of bicoid in Tribolium. hb expression was disrupted by pRNAi to determine whether this would result in a phenotype overlapping that of otd-1RNAi. Disruption of hb mRNA results in a lack of the maxillary, the labial and all three thoracic segments in 50% of the analyzed embryos, whereas the anteriormost head segments all developed normally. Drosophila embryos that lack maternally derived and zygotic hb have the same phenotype (Schröder, 2003).

This indicates that, in Drosophila and Tribolium, hb acts as a canonical head gap gene. In the wasp Nasonia, the hb mutant phenotype is more severe: the complete head (except for the anterior-most labrum) and the thorax are missing. It appears that the function of hb as a gap gene has been conserved throughout evolution. The head defects observed in the otd-1RNAi experiments indicate that otd-1 and hb are both involved in regulating the development of gnathal segments. To evaluate the extent of their overlapping functions, embryos were generated in which both otd-1 and hb expression were disrupted by pRNAi. These embryos were called otd-1/hbpRNAi double phenocopies. Forty out of 61 (65.5%) of these embryos developed a headless phenotype, indicating that otd-1 and hb function together to regulate head development (Schröder, 2003).

Analysis of the Engrailed expression pattern in the otd-1/hbpRNAi embryos reveals that only two to six abdominal segments of normal polarity develop in otd-1/hbpRNAi embryos. This shows that otd-1 and hb not only direct the development of head and thorax, but also act synergistically during the segmentation process of the anterior abdomen, possibly by regulating posterior gap and/or homeotic genes. A synergistic relationship between bicoid and hb has been described previously in Drosophila, and might represent an evolutionary principle to confer robustness to the segmentation process. In Tribolium, Otd-1 and Hb might substitute for the function of Bicoid as a transcriptional activator in Drosophila (Schröder, 2003).

In Drosophila, caudal is translationally repressed in the anterior half of the egg by Bicoid. Since no ectopic posterior structures form at the anterior pole of otd-1/hbpRNAi embryos, Otd-1 might not serve as a translational repressor in Tribolium, as Bicoid does in Drosophila. In Drosophila, the RNA-binding activity of Bicoid, and therefore its ability to inhibit caudal translation, requires the presence of an arginine at residue 54 of its homeodomain. This amino acid is replaced by an alanine in the homologous position of Tribolium Otd-1, so its RNA-binding specificity (but not DNA-binding specificity) might be lost. A repressor of Tribolium caudal is therefore likely to exist in the beetle, and serves as a further component of the anterior development system. Such a repressor has yet to be identified, but different mechanisms of caudal regulation have been described in other systems. During Dipteran evolution, the function of Bicoid seems to have replaced the function of maternally inherited otd-1 as well as that of a repressor of caudal (Schröder, 2003).

The expression of otd-1 might be controlled early in embryogenesis by repression, and during later stages by autoactivation. Maternal otd-1 mRNA translation could be repressed at the posterior pole, in a manner similar to that of maternal hb in Drosophila. In Drosophila, and similarly in the grasshopper Schistocerca, hb expression is repressed by the Pumilio/Nanos complex. A predicted Nanos-response element (NRE), identified by the sequence TgGTTGTattatAATTGTAggTA (position 1429-1451, capital letters indicating identity to the NRE of hb in Drosophila and Musca), is present in the 3' untranslated region of Tribolium otd-1. Although no ortholog of pumilio or nanos has been isolated from Tribolium, their function in Tribolium has already been implicated by the downregulation of maternal hb at the posterior pole of the blastoderm embryo19. Maternal otd-1 could serve to activate the expression of zygotic otd-1 only in the embryonic region, at the border of the serosa. The zygotic expression could then be maintained by an autoregulatory loop (Schröder, 2003).

To test this possibility, otd-1-binding sites in its regulatory region will have to be identified, and the effects of disrupting this site will have to be tested in vitro and in vivo. bicoid might have evolved from a duplicated zerknüllt-like gene, which was converted by mutation into a gene that codes for a K50HD-containing protein -- thus adopting the same DNA-binding specificity as Otd-1. During evolution of the higher Diptera, otd expression came under the regulation of the maternal gene bicoid, so that maternal Otd-1 function was not longer required. Thus, otd was restricted to become a gap gene in insects derived from this lineage (Schröder, 2003).

The Drosophila gene bicoid functions at the beginning of a gene cascade that specifies anterior structures in the embryo. Its transcripts are localized at the anterior pole of the oocyte, giving rise to a Bicoid protein gradient, which regulates the spatially restricted expression of target genes along the anterior-posterior axis of the embryo in a concentration-dependent manner. The morphogen function of Bicoid requires the coactivity of the zinc finger transcription factor Hunchback, which is expressed in a Bicoid-dependent fashion in the anterior half of the embryo. Whereas hunchback is conserved throughout insects, bicoid homologs are known only from cyclorrhaphan flies. Thus far, identification of hunchback and bicoid homologs rests only on sequence comparison. In this study, double-stranded RNA interference (RNAi) was used to address the function of bicoid and hunchback homologs in embryos of the lower cyclorrhaphan fly Megaselia abdita (Phoridae). Megaselia-hunchback RNAi causes hunchback-like phenotypes as observed in Drosophila, but Megaselia-bicoid RNAi causes phenotypes different from corresponding RNAi experiments in Drosophila bicoid mutant embryos. Megaselia-bicoid is required not only for the head and thorax but also for the development of four abdominal segments. This difference between Megaselia and Drosophila suggests that the range of functional bicoid activity has been reduced in higher flies. These results indicate that Ma-bcd RNAi in Megaselia embryos causes the specific deletion of the anterior abdominal segments, which is not observed in the corresponding RNAi experiments with Drosophila or with bicoid- or hunchback-deficient Drosophila embryos. It is important to note, however, that in Drosophila, a symmetrical bicaudal-like phenotype had been observed when the combined activities of bicoid and hunchback are repressed in the anterior half of the embryos, indicating synergistic effects of both genes. It was therefore asked whether coinjection of Ma-bcd and Ma-hb dsRNAs into Megaselia embryos results in a more than additive extension of anterior deletions as compared with single dsRNA injections. The phenotypes obtained after combined Ma-bcd and Ma-hb dsRNA injections are similar to the sum of effects observed in independent Ma-bcd dsRNA and Ma-hb dsRNA injections. In addition, the ventral nerve cord is more disorganized and interrupted between the symmetrical abdominal halves, and the gut is reduced in size. These results suggest only a weak synergistic effect of Ma-bcd and Ma-hb in the abdomen (Stauber, 2000).

Insect axis formation is best understood in Drosophila, where rapid anteroposterior patterning of zygotic determinants is directed by maternal gene products. The earliest zygotic control is by gap genes, which determine regions of several contiguous segments and are largely conserved in insects. Isolation of mutations has been used to approach a genetic question: do early zygotic patterning genes control similar anteroposterior domains in the parasitoid wasp Nasonia vitripennis as in Drosophila? Nasonia is advantageous for identifying and studying recessive zygotic lethal mutations because unfertilized eggs develop as males while fertilized eggs develop as females. On first consideration, the Hymenopteran Nasonia and the Dipteran Drosophila appear very similar in their embryonic development, though the Hymenoptera diverged from the Diptera >200 million years ago. Embryos of both species produce larvae in about 1 day at 25°C. In Nasonia, the fertilized egg gives rise to an embryo that undergoes syncytial and cellular blastoderm stages morphologically similar to those of Drosophila. Both Nasonia and Drosophila undergo the long germband mode of embryonic development. Despite these similarities, two observations suggest that the relative importance of maternal versus zygotic patterning functions may differ in the two insects. (1) Although postgastrulation events proceed with very similar timing, the time for early development differs substantially - at 25°C: the events preceding gastrulation take only about 3 hours in Drosophila but almost 10 hours in Nasonia. This difference in timing may allow for greater zygotic control of patterning in Nasonia than in Drosophila. (2) Among the relatives of Nasonia, a polyembryonic mode of development has evolved in which a single fertilized egg gives rise to hundreds or thousands of progeny. Polyembryonic development is likely to rely heavily on zygotic control of patterning. Polyembryony has arisen several times in the Hymenoptera, and the polyembryonic Copidosoma floridanum is in the same superfamily as Nasonia. These considerations pose the following question -- is early development substantially controlled by the zygotic genome in Hymenopterans? This question may be approached genetically, by isolating zygotic mutations that disrupt early anteroposterior patterning in Nasonia. Recessive zygotic mutations have identified three Nasonia genes: head only mutant embryos have posterior defects, resembling loss of both maternal and zygotic Drosophila caudal function; headless mutant embryos have anterior and posterior gap defects, resembling loss of both maternal and zygotic Drosophila hunchback function, and squiggy mutant embryos develop only four full trunk segments, a phenotype more severe than those caused by lack of Drosophila maternal or zygotic terminal gene functions. head only mutant embryos lack all segmentation posterior to the head, in the strongest manifestation of the phenotype, and have only a narrow domain of Ubx-Abd-A expression. head only differs from Drosophila gap genes with respect to the extent of pattern deleted and effects on Ubx-Abd-A. In Drosophila, neither Krüppel nor knirps affects a domain as large as that of head only. Moreover, the wild-type functions of Krüppel and knirps are not required for the positive regulation of Ubx or abd-A in Drosophila (Pultz, 1999).

headless is similar to Drosophila hunchback in controlling the patterning of both anterior and posterior embryonic regions. Drosophila hunchback is expressed zygotically in both anterior and posterior embryonic domains; in addition, maternal Hunchback mRNA is translationally repressed by nanos in the posterior, generating a maternal gradient of Hunchback protein. hunchback is evolutionarily conserved in insects. In both headless and hunchback mutants, posterior abdominal segments are deleted. In Drosophila hunchback, the posterior deletion spans from the posterior seventh through the eighth and last full abdominal segment. In Nasonia headless, the deletion spans from the posterior seventh through the tenth and last abdominal segment, and terminalia are also defective. In both headless and hunchback mutants, the anterior gap domain includes the three thoracic segments, plus part of the head. In Drosophila embryos lacking zygotic hunchback function, the anterior pattern deletion extends only into the labial segment. In contrast, in Nasonia headless mutant embryos, the deletion extends further to the anterior, through the gnathal and antennal segments, though the most anterior labral cuticular derivatives are present. In Nasonia headless as in Drosophila hunchback, the trunk pattern elements remaining include the denticle belts of the first through seventh abdominal segments and the trunk En stripes anterior to each of those denticle belts. For both headless and zygotic hunchback, the remaining trunk pattern spans parasegments six through twelve. The effects of headless on Ubx-Abd-A bear out the interpretation that headless is comparable to Drosophila hunchback, except that more anterior regions of the embryo are affected in headless than in fly embryos lacking zygotic hunchback function. In headless mutant embryos, Ubx-Abd-A expression is expanded anteriorly through and beyond the region that would develop into the gnathal head segments in wild-type embryos. In fly embryos lacking zygotic hunchback, the Ubx-Abd-A domain shows a more limited anterior expansion that extends only slightly into the gnathal region of the embryo. In both headless and hunchback mutant embryos, the Ubx-Abd-A domain also expands posteriorly. The zygotic headless phenotype best resembles that of Drosophila embryos lacking both maternal and zygotic hunchback. The simplest interpretation is that headless may be a mutation in the Nasonia hunchback gene, which controls functions zygotically that are jointly controlled by both maternal and zygotic hunchback in Drosophila (Pultz, 1999).

squiggy mutant embryos have severe defects both anteriorly and posteriorly, leaving only four consistently developed trunk segments. This cuticular phenotype differs substantially from the phenotypes of maternal terminal group genes in Drosophila, such as torso, in which loss-of-function maternal-effect mutations delete pattern elements from both ends of the embryo. The terminal structures deleted in torso embryos are anterior to the gnathal segments and posterior to the seventh abdominal segment, and are thus limited compared to those of the zygotic squiggy mutant embryos. The extensive zygotic control of terminal development by squiggy appears to be a departure from Drosophila developmental mechanisms. The Drosophila maternal terminal gene patterning system is not known to be widely conserved, and the follicle cell types that express torsolike do not appear to be conserved even in the lower Diptera. Terminal patterning in insects may therefore be subject to considerable evolutionary flexibility. Zygotic control of early patterning in head only, headless and squiggy mutants share a common theme: the zygotic Nasonia phenotypes are more extreme than those of Drosophila gap genes and all three genes appear to control processes zygotically that are partially or fully subject to maternal control in the fly. These results indicate greater dependence on the zygotic genome to control early patterning in Nasonia than in the fly (Pultz, 1999).

To obtain a clearer understanding of the evolutionary transition between short- and long-germ modes of embryogenesis in insects, the expression of two gap genes hunchback (hb) and Kruppel (Kr) as well as the pair-rule gene even-skipped (eve) were studied in the dipteran Clogmia albipunctata (Nematocera, Psychodidae). Embryogenesis in this species has features of both short- and long-germ modes of development. In Clogmia, hb expression deviates from that known in Drosophila in two main respects: (1) it shows an extended dorsal domain that is linked to the large serosa anlage, and (2) it shows a terminal expression in the proctodeal region. These expression patterns are reminiscent of the hb expression pattern in the beetle Tribolium, which has a short germ mode of embryogenesis (see Tribolium early embryonic development). However, Kruppel expression is rather similar to the Drosophila expression, both at early and late stages. eve expression starts with six stripes formed at blastoderm stage, while the seventh is only formed after the onset of gastrulation and germband extension. Surprisingly, no segmental secondary Eve stripes could be observed in Clogmia although such segmental stripes are known from higher dipterans, beetles and hymenopterans. Another nematoceran, Coboldia, was therefore studied to address this question and it was found that some segmental stripes form by intercalation as in Drosophila, although belatedly. These results suggest that Clogmia embryogenesis, both with respect to morphological and molecular characteristics represents an intermediate between the long-germ mode known from higher dipterans such as Drosophila, and the short-germ mode found in more ancestral insects (Rohr, 1999).

In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, a long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb), were investigated. Focused was placed on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, the following was found: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of these results with the results for other derived species reveals that the canonical hb function may have evolved from the non-canonical hb functions during evolution (Mito, 2005).

In Gryllus the caudal gene (Gb'cad), instead of bcd, organizes the gap domains of Gb'hb and Gb'Kr. Gb'hb RNAi analysis suggested that Gb'hb activates Gb'Kr, directly or indirectly, although more data are needed to establish this regulatory interaction. Furthermore, Gb'hb directly or indirectly regulates the Gb'eve expression in the prospective thoracic region of the embryo, suggesting that the hierarchical relationship between hb and eve is conserved between Gryllus and Drosophila. The results also suggested that the Gb'abdA expression is suppressed by Gb'hb and Gb'Kr, directly or indirectly, in the anterior region, as in Drosophila. Thus, although the maternal morphogenetic organizer in the regulatory network of segmentation may have switched from cad to bcd during insect evolution, its downstream relationship in the segmentation and Hox genes appears to have been mostly conserved between Gryllus and Drosophila (Mito, 2005).

In spite of these conserved aspects of the putative regulatory network, it was found that Gb'hb functions differ considerably from those of Drosophila hb in segment formation. Although more segments might be deleted in Gryllus, as observed in the Drosophila hb mutant, it is likely that Gb'hb plays the role of a gap gene that patterns fewer segments than in Drosophila. Interestingly, in the hb RNAi phenotypes of Oncopeltus, no segmentation gap was observed in the gnathal and thoracic regions, suggesting that the requirement of the hb gap function in this species is minimal, if at all present. On the contrary, Tribolium hb was reported to have the anterior gap function similar to Drosophila. In Tribolium, (a short germ insect) some of the anterior segments are patterned under syncytial conditions. These lines of evidence suggest that the number of anterior segments regulated by hb increased during evolution from cellular to syncytial segmentation, or that the canonical function of hb may have evolved from the non-canonical functions of an ancestral hb during insect evolution. Although the anterior hb-expression domain appears to be fundamentally conserved in insects, the number of pair-rule stripes of the pair-rule genes regulated by hb in the anterior region may increase during evolution, probably owing to modification of the cis-regulatory elements of the pair-rule genes. Comparative analyses of the cis-regulatory elements of the pair-rule genes would shed some light on this issue (Mito, 2005).

In short/intermediate germ insects, the posterior segments form sequentially from the posterior growth zone during germband elongation. Gb'hb is required in the formation of at least seven posterior segments. In addition to this, Oncopeltus Of'hb was found to be expressed in the posterior growth zone and involved in growth and segmentation. In contrast, in Drosophila, hb is reported to be required only for the formation of the A7 and A8 segments. These facts suggest that the hb function in the posterior region may have been reduced during evolution from the short/intermediate to long germ embryogenesis. Since hb is similarly expressed in the prospective A7 to A9 segments in Gryllus and Drosophila, the hb functions in these segments may have been conserved in both short/intermediate and long germ embryogenesis. Recently, it has been proposed that the posterior sequential segmentation in short/intermediate germ insects is controlled by a Notch signaling-dependent segmentation clock. If it is the case, Gb'hb might be involved in regulating segmentation clock. It is also possible that Gb'hb controls posterior growth through regulation of the morphogenesis of the germband, because the shape of the elongating posterior region was affected by Gb'hb RNAi depletion. Further precise analyses of posterior segmentation in short/intermediate germ insects will be required to elucidate its mechanisms (Mito, 2005).

Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. This study focuses a comparative analysis of Nasonia hunchback function and expression. A lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, the gene is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. The maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Nasonia hunchback does not have a canonical Nanos response element (NRE) such as is found in Schistocerca, Locusta, Tribolium and Drosophila hunchback mRNAs; however, Nasonia hunchback does have candidate NREs that are similar in structure to the Drosophila melanogaster cyclin B1 NRE, which is translationally regulated by Pumilio and Nanos in the germline. Finally, the comparisons of Nasonia and Drosophila hunchback mutant phenotypes are extended, and it is proposed that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry (Pultz, 2005).

In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi was investigated by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (1) L. migratoria manilensis hb (Lmm’hb) controls germ band morphogenesis and segmentation in the anterior region; (2) Lmm’hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (3) Lmm’hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila (He, 2006).

Evidence for a composite anterior determinant in the hover fly Episyrphus balteatus (Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage

Most insect embryos develop from a monolayer of cells around the yolk, but only part of this blastoderm forms the embryonic rudiment. Another part forms extra-embryonic serosa. Size and position of the serosa anlage vary between species, and previous work raises the issue of whether such differences co-evolve with the mechanisms that establish anteroposterior (AP) polarity of the embryo. AP polarity of the Drosophila embryo depends on bicoid, which is necessary and sufficient to determine the anterior body plan. Orthologs of bicoid have been identified in various cyclorrhaphan flies and their occurrence seems to correlate with a mid-dorsal serosa or amnioserosa anlage. This paper introduces Episyrphus balteatus (Syrphidae), a cyclorrhaphan model for embryonic AP axis specification that features an anterodorsal serosa anlage. Current phylogenies place Episyrphus within the clade that uses bicoid mRNA as anterior determinant, but no bicoid-like sequence could be identified in this species. Using RNA interference (RNAi) and ectopic mRNA injection, evidence was obtained that pattern formation along the entire AP axis of the Episyrphus embryo relies heavily on the precise regulation of caudal, and that anterior pattern formation in particular depends on two localized factors rather than one. Early zygotic activation of orthodenticle is separated from anterior repression of caudal, two distinct functions which in Drosophila are performed jointly by bicoid, whereas hunchback appears to be regulated by both factors. Furthermore, it was found that overexpression of orthodenticle is sufficient to confine the serosa anlage of Episyrphus to dorsal blastoderm. These findings are discussed in a phylogenetic context, and it is proposed that Episyrphus employs a primitive cyclorrhaphan mechanism of AP axis specification (Lemke, 2009).

This study found that AP axis specification in Episyrphus is strongly dependent on Eba-cad. Throughout the embryo, ectopic Eba-cad expression interferes with segmentation and differentiation, whereas loss of Eba-cad activity interferes with the formation of all but the anterior head segments. In Drosophila, ectopic translation of the ubiquitous maternal caudal mRNA causes temperature-dependent head involution defects. Ubiquitous expression of a caudal transgene in the syncytial blastoderm also causes head involution defects and, in addition, leads to variable fusions of adjacent segment pairs along the entire embryo. The much stronger gain-of-function phenotype of caudal in Episyrphus could reflect differences in the experimental designs that were employed. However, loss-of-function experiments also suggest that embryonic development in Episyrphus relies more heavily on Eba-cad than embryonic development in Drosophila does on caudal. In Episyrphus, Eba-cad RNAi suppresses the formation of all but one of the seven Eba-eve stripes and severely affects or deletes most postoral segments, whereas caudal-deficient Drosophila embryos form four out of the seven even-skipped stripes and show segmentation in the head, thorax and even parts of the abdomen. The comparatively weak dependence of AP axis specification in Drosophila on caudal can be explained by compensatory input from the anterior gradients of bicoid and maternal hunchback. In turn, the high caudal-dependence of AP axis-specification in Episyrphus, which is similarly observed in species that lack the bicoid gene such as Nasonia and the cricket Gryllus, might reflect the absence of maternal hunchback and/or bicoid activities in this species (Lemke, 2009).

Although endogenous Eba-nos appeared to be dispensable for AP axis specification, ectopic Eba-nos expression in gain-of-function experiments could be used as a functional tool to reveal differences in anterior pattern formation between Episyrphus and Drosophila. Drosophila embryos that ectopically express nanos at the anterior pole develop a mirror-image duplication of the posterior abdomen. This effect is due to the translational repression of maternal bicoid and hunchback mRNAs, which control all aspects of anterior development. Both genes contain functionally important Nanos regulatory elements (NREs), although in wild-type embryos Nanos appears to be irrelevant for the regulation of bicoid. In Episyrphus, no trace was observed of abdominal development at the anterior pole after ectopic expression of Eba-nos, although the activity was high enough to completely suppress the formation of all but the most posterior segments (A6-A8). This phenotype would be expected if at least two independent factors determine anterior development in Episyrphus, only one of which is targeted by ectopic anterior Eba-nos activity, whereas the second factor prevents the formation of ectopic posterior structures. It is proposed that the first factor (Factor 1) consists of an anteriorly enriched NRE-containing mRNA that encodes a protein for the early zygotic activation of Eba-otd and Eba-hb, and that the second factor (Factor 2), which is not repressed by ectopic Eba-nos activity, mediates the repression of Eba-cad and part of the anterior Eba-hb activation. Factor 2 appears to function independently of the terminal system, as neither Eba-cad nor Eba-hb display altered anterior expression domains following RNAi against the putative torso homolog of Episyrphus. Candidate genes for Factor 1 could possibly be identified by searching for NRE-containing sequences in an early embryonic Episyrphus EST database (Lemke, 2009).

In summary, AP polarity of the Episyrphus embryo appears to be determined by two distinct factors at the anterior pole. It cannot be excluded that one of these factors shares homology with bicoid, but in any case the model differs significantly from AP axis specification in Drosophila, where a single protein, Bicoid, activates orthodenticle and hunchback, and represses caudal. Furthermore, the Episyrphus model differs from the Nasonia model in that the transcripts of Eba-otd and Eba-gt (the putative Episyrphus ortholog of giant) are of zygotic origin and not localized (Lemke, 2009).

Episyrphus shares various traits of early embryonic development with non-cyclorrhaphan rather than other cyclorrhaphan flies. It features an anterodorsal serosa anlage, strong influence of caudal on the AP axis, a (nearly) ubiquitous early zygotic activation of hunchback, as well as hunchback expression in the serosa anlage, which has been reported for non-cyclorrhaphan insects and is absent in Drosophila, Musca and Megaselia. During late embryonic development, Engrailed expression in the hindgut of Episyrphus embryos is narrow and ring-shaped similar to some non-cyclorrhaphan insects, whereas Engrailed expression in the hindgut of other cyclorrhaphans is much broader and restricted to the dorsal half. Based on the primitive features of Episyrphus development, it is speculated that the ancestral cyclorrhaphan mechanism of AP axis specification was retained in the Episyrphus lineage. The restriction of the serosa anlage to dorsal blastoderm in response to increased Eba-otd activity might therefore indicate the evolutionary mechanism that altered the position of the serosa anlage (Lemke, 2009).

Maternal activation of gap genes in the hover fly Episyrphus

The metameric organization of the insect body plan is initiated with the activation of gap genes, a set of transcription-factor-encoding genes that are zygotically expressed in broad and partially overlapping domains along the anteroposterior (AP) axis of the early embryo. The spatial pattern of gap gene expression domains along the AP axis is generally conserved, but the maternal genes that regulate their expression are not. Building on the comprehensive knowledge of maternal gap gene activation in Drosophila, loss- and gain-of-function experiments were used in the hover fly Episyrphus balteatus (Syrphidae) to address the question of how the maternal regulation of gap genes evolved. It was found that, in Episyrphus, a highly diverged bicoid ortholog is solely responsible for the AP polarity of the embryo. Episyrphus bicoid represses anterior zygotic expression of caudal and activates the anterior and central gap genes orthodenticle, hunchback and Krüppel. In bicoid-deficient Episyrphus embryos, nanos is insufficient to generate morphological asymmetry along the AP axis. Furthermore, torso transiently regulates anterior repression of caudal and is required for the activation of orthodenticle, whereas all posterior gap gene domains of knirps, giant, hunchback, tailless and huckebein depend on caudal. It is conclude that all maternal coordinate genes have altered their specific functions during the radiation of higher flies (Cyclorrhapha) (Lemke, 2010).

Therefore, Episyrphus and other lower cyclorrhaphan flies establish global AP polarity only through bicoid and lack sizable input of nanos, although endogenous nanos activity in these species might stabilize the AP axis by repressing anterior development. Despite the absence of a redundant maternal system to generate global AP polarity, Eba-bcd appears to be a less potent transcriptional activator than Bicoid. In contrast to Drosophila, gap gene activation at the anterior pole of the Episyrphus embryo requires a strong contribution of the terminal system, whereas the posterior domains of knirps and giant are strictly dependent on caudal and do not appear to receive a significant activating input by Eba-bcd. Thus, rather than a strong activation potential, the exclusive control of the central Eba-Kr domain by Eba-bcd appears to be the crucial difference to Drosophila, which renders AP polarity in the Episyrphus embryo entirely dependent on bicoid (Lemke, 2010).

Diversity in insect axis formation: two orthodenticle genes and hunchback act in anterior patterning and influence dorsoventral organization in the honeybee (Apis mellifera)

Axis formation is a key step in development, but studies indicate that genes involved in insect axis formation are relatively fast evolving. Orthodenticle genes have conserved roles, often with hunchback, in maternal anterior patterning in several insect species. Two orthodenticle genes, otd1 and otd2, and hunchback act as maternal anterior patterning genes in the honeybee (Apis mellifera) but, unlike other insects, act to pattern the majority of the anteroposterior axis. These genes regulate the expression domains of anterior, central and posterior gap genes and may directly regulate the anterior gap gene giant. It was shown otd1 and hunchback also influence dorsoventral patterning by regulating zerknült (zen) as they do in Tribolium, but zen does not regulate the expression of honeybee gap genes. This suggests that interactions between anteroposterior and dorsal-ventral patterning are ancestral in holometabolous insects. Honeybee axis formation, and the function of the conserved anterior patterning gene orthodenticle, displays unique characters that indicate that, even when conserved genes pattern the axis, their regulatory interactions differ within orders of insects, consistent with relatively fast evolution in axis formation pathways (Wilson, 2011).

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods

Segmentation, i.e., the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. This study presents the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods (Janssen, 2011).

A gradient of the Bcd protein controls the expression of genes in anterior cap domains with different posterior borders in the Drosophila embryo. But what controls the anterior expression of these genes in arthropods that do not possess a bcd gene? For a number of reasons such as that it is provided maternally, that it forms an anterior to posterior gradient and that it shares biochemical characters with the bcd gene, otd was proposed to be one of the anterior determinants substituting for bcd in basal holometabolous insects such as the beetle Tribolium and the wasp Nasonia. The other factor that is apparently involved in substituting for bcd function is hb, that acts synergistically with otd. Contradictory data on hb in Tribolium states that its function is rather that of a regulator of the trunk gap genes than that of a gap gene like it is in Drosophila (Janssen, 2011).

Although in other insects such as the mosquito Anopheles or the pea aphid Acyrthosiphon, and the spider Achaearanea otd is not contributed maternally, the gene is transcribed soon after fertilization of the egg and localizes in an anterior domain in the developing embryo. Like in Drosophila also in other insects and the spider Achaearanea hunchback is expressed maternally. This study found that in Glomeris hb is a maternally provided factor as well and that otd is expressed immediately after fertilization. Furthermore, both genes are expressed in anterior caps in blastoderm stage embryos. How the anterior localization of these genes is controlled in the absence of bcd is however still unclear. One possibility is that other unknown anterior determinants restrict otd and hb transcripts to their anterior domains in the early embryo, as has been proposed for Tribolium. Another possibility is that a posterior factor represses, directly or indirectly, the expression in the posterior part of the embryo and thus restricts the expression to an anterior domain. Nanos is a candidate for such a posterior factor. Nanos acts as a posterior translational repressor of hb in Drosophila and presumably also in the grasshopper Schistocerca americana. Consistent with this is the presence of a putative Nanos-Reponse-Element (NRE) in the 3′UTR of arthropod otd and hb orthologs including those of Glomeris, suggesting that the anterior localization of these transcripts may be initiated by translational repression at the posterior. A combination of such posterior repression with auto-regulation then could result in anterior-only expression domains. Indeed, auto-regulation of the hb gene has been described in Drosophila embryos. And in the spider Achaearanea, hb as well as otd, appear to be involved in their own regulation as well. Thus despite some differences in the way otd and hb transcripts become localized anteriorly in different arthropods, in all analyzed species the outcome is consistent with a role as anterior patterning genes. Whether such implied function is rather that of gap genes or that of general anterior morphogens with similar functions as bcd has in Drosophila is unclear (Janssen, 2011).

Recent studies actually suggest that in the beetle Tribolium the function of otd is rather that of a head gap gene than that of a general anterior determinant (Janssen, 2011).

In Tribolium the anterior function of hb was suggested to be homeotic only rather than that of a gap gene. Similar data come from hemimetabolous insects where RNAi experiments with hb amongst other effects also result in homeotic transformations. It is however important to keep in mind that also in Drosophila weaker alleles of gap gene mutants often result in homeotic transformation and that only in strong mutants segments are missing. In the spider Achaearanea the function of hb is that of a gap gene supporting the hypothesis that one of the ancestral functions of hb in arthropods is indeed that of a gap gene. In the spider hb-RNAi experiments cause the loss of walking leg segments one, two and four (homologous to mandibular, maxillary and first thoracic segments in insects) (Janssen, 2011).

The broad gap-gene like expression domain in the developing gnathal segments in Glomeris is similar to the ones found in insects and the spider. This conserved expression domain in Glomeris and other arthropods therefore indicates that hb may act as a gap gene in Glomeris development as well (Janssen, 2011).

Another conserved function of early hb activity in Drosophila is the regulation of Hox genes as reflected by the homeotic transformations in insects. While for example hb represses the transcription of the homeotic gene Antennapedia (Antp), Krüppel (Kr), another gap gene, activates Antp. The expression patterns of Kr and hb display this function in Drosophila and other insects where the early expression domains are largely complementary with no or only little overlap. Interestingly, in Glomeris the early expression domains of hb and Kr are also broadly complementary, although overlapping in the postmaxillary segment. This is in agreement with a possible regulatory function of hb and Kr on Antp expression; Antp is only expressed in non hb-expressing tissue and Kr is expressed in all Antp expressing segments. Kr may thus act as an activator, and hb as a repressor of Antp. A similar scenario is shown later during development when the CNS expresses hb and Kr: again the expression patterns of both genes are complementary with now Kr acting in the anterior CNS and hb being restricted to the posterior CNS (Janssen, 2011).

The early expression of both Gm-otd and Gm-hb is consistent with a role of these genes in anterior patterning. To obtain additional evidence for conservation of anterior patterning Glomeris orthologs of genes that in Drosophila are direct or indirect targets of bcd, otd and hb were recovered and analyzed, i.e. the Drosophila head patterning genes buttonhead (btd), cap-n-collar (cnc), collier (col), and crocodile (croc). Glomeris orthologs were found to be expressed in surprisingly similar domains like their fly counterparts. The Drosophila head gap gene btd is expressed in and required for the antennal, intercalary, and mandibular segments, and Gm-btdl is expressed in the corresponding segments in the millipede, suggesting conserved regulation of btd in these segments. In Tribolium, however, btd expression is restricted to the mandibular segment, which is significantly different from the expression domains in Drosophila and Glomeris, where btd orthologs cover more than one head segment. In Drosophila and Tribolium the col gene is expressed in parasegment 0 that corresponds to the posterior part of the intercalary segment and the anterior portion of the mandibular segment. This expression pattern is also conserved in myriapods. Drosophila col acts downstream of the head gap genes in the regulation network that patterns the anterior part of the head, with btd being essential for col activation (Janssen, 2011).

The Gm-cnc gene is expressed in two domains, the most anterior one is in the region of the anlage of the stomodaeum; this domain corresponds to the 'cap' expression domain of insects such as Drosophila, Tribolium, Oncopeltus and Thermobia cnc in the 'labral' region of the foregut, while the second domain of Gm-cnc expression is in a stripe in the mandibular segment, that corresponds to the 'collar' stripe of expression in the mandibular segment of insects. The anterior 'labral' expression in Drosophila requires bcd, while the regulation of the mandibular stripe depends on btd and otd (Janssen, 2011).

The Gm-croc gene is initially expressed around the presumptive anlage of the stomodaeum, that intercalates between the two hemisegments of the ocular segment. In insects croc is expressed in a comparable anterior area, the clypeolabrum, and at least in Drosophila it requires bcd activity (Janssen, 2011).

Taken together head patterning genes that in Drosophila are directly or indirectly controlled by the anterior determinant bcd, and of which expression is conserved also in Tribolium, show conserved expression patterns also in Glomeris. A very recent paper describing the early expression of otd, col, cnc and croc in the milkweed bug Oncopeltus fasciatus confirms these findings. The four investigated anterior head patterning genes are expressed in comparable domains in Oncopeltus and Glomeris. It is worth mentioning that the expression patterns of the later regulatory genes (i.e. croc, cnc and col, but also otd) are indeed highly conserved, whereas the expression domains of the primary regulators hb and btd appear to show more divergence in their early expression domains. Nevertheless the current data further suggest that major components of anterior patterning system are conserved in myriapods and insects (Janssen, 2011).

Because of technical limitations it is not possible to perform functional studies in any myriapod species, which would possibly answer the question whether or not the function of anterior patterning gene orthologs is conserved in insects and myriapods. To further investigate the assumption that anterior patterning genes may play conserved roles, it would be interesting to examine expression and if possible function of these genes in onychophorans or tardigrades, the sister groups of the arthropods (Janssen, 2011).

Gene expression and functional data support the hypothesis that hb and otd are involved in anterior patterning in all hitherto examined arthropods. However some of the putative target genes of anterior patterning, i.e. the anterior patterning genes col and cnc and possibly also btd (or related gene orthologs) are expressed in conserved patterns only in insects and myriapods. Data on croc and cnc expression are not available from crustaceans; data on croc and cnc expression in spiders suggest that the early role of croc, but not that of cnc may be conserved in this group of arthropods as well. The absence of early function of col and cnc in chelicerates could be explained by the different head morphology. Insects, myriapods and crustaceans are historically grouped as Mandibulata based on e.g. the possession of mandibles. Chelicerates lack mandibles and evolved a pair of walking legs on the homologous segment. However the lack of early col expression in crustaceans could either be interpreted as support for the traditional Atelocerata theory, or as the result of convergent evolution of anterior col-expression in insects and myriapods. If the role of cnc in anterior patterning is plesiomorphic for Atelocerata, Mandibulata or Arthropoda is unclear until studies of this gene in Crustacea and Onychophora will possibly reveal the ancestral state of its expression (Janssen, 2011).


A quantitative atlas of Even-skipped and Hunchback expression in Clogmia albipunctata (Diptera: Psychodidae) blastoderm embryos

Comparative studies of developmental processes are one of the main approaches to evolutionary developmental biology (evo-devo). Over recent years, there has been a shift of focus from the comparative study of particular regulatory genes to the level of whole gene networks. Reverse-engineering methods can be used to computationally reconstitute and analyze the function and dynamics of such networks. These methods require quantitative spatio-temporal expression data for model fitting. Obtaining such data in non-model organisms remains a major technical challenge, impeding the wider application of data-driven mathematical modeling to evo-devo. Antibodies were raised against four segmentation gene products in the moth midge Clogmia albipunctata, a non-drosophilid dipteran species. These antibodies were used to create a quantitative atlas of protein expression patterns for the gap gene hunchback (hb), and the pair-rule gene even-skipped (eve). The data reveal differences in the dynamics of Hb boundary positioning and Eve stripe formation between C. albipunctata and Drosophila melanogaster. Despite these differences, the overall relative spatial arrangement of Hb and Eve domains is remarkably conserved between these two distantly related dipteran species. This study has provided a proof of principle that it is possible to acquire quantitative gene expression data at high accuracy and spatio-temporal resolution in non-model organisms. The quantitative data extend earlier qualitative studies of segmentation gene expression in C. albipunctata, and provide a starting point for comparative reverse-engineering studies of the evolutionary and developmental dynamics of the segmentation gene system (Janssens, 2014).

Other invertebrate Hunchback homologs

Leech zinc finger genes Lzf1 and Lzf2 are orthologs to hunchback. At the time of segmental pattern formation, Lzf2 mRNA is expressed uniformly along the length of the segmented trunk in both the ectodermal and mesodermal tissues. This is in contrast to the restricted anterior gradient of HB RNA, critical to anterior/posterior patterning in the fly. During organogenesis Lzf2 is expressed in segmentally restricted patterns in the central nervous system, the gut, and epidermally derived structures. This suggests that hb may have acquired gap gene function in insects after their phyletic separation from annelids (Savage, 1996).

The Drosophila segmentation gene hunchback is critical for the proper anteroposterior development of the fly embryo, but its function outside the diptera is currently unknown. The protein expression pattern of Leech Zinc Finger II (LZF2), a leech ortholog of hb has been characterized. In early embryogenesis, LZF2 protein is expressed in a subset of micromeres and is later expressed in the micromere-derived epithelium of the provisional epithelium and prostomium. LZF2 protein is detected in the ventral nerve cord during organogenesis, first in interganglionic muscle cells and later in subsets of neurons in each neuromere of the CNS. The location of immunoreactive cells during development and the similarity of the expression pattern of LZF2 to the expression of the C. elegans hb homolog hbl-1 suggests that LZF2 plays a role in the morphogenetic movements of leech gastrulation and later in CNS specification but not in anteroposterior pattern formation (Iwasa, 2000).

Although LZF2 transcripts have been detected in the ectodermal and mesodermal precursors of the segmented trunk, immunostaining reveals an absence of LZF2 protein in these tissues until relatively late in the process of organogenesis. This discrepancy between protein and transcript expression could be due to the presence of a nanos responselike element located in the 3' untranslated region of the LZF2 transcript. It is the responselike element that mediates the translational repression of hb in the posterior half of the fly embryo, and therefore it is possible that a similar repression system may be operating in the leech to prevent early LZF2 protein expression in the segmental tissues. If this is the case, LZF2 function during segmental pattern formation in the leech differs significantly from that of hb function in Drosophila (Iwasa, 2000).

The first characterization of a segmentation gene homolog in the basal polychaete Capitella capitata is reported, using a pan-annelid cross-species antibody to the hunchback-like gene product. In flies, the gap segmentation gene hunchback encodes a C2H2 zinc-finger transcription factor that plays a pivotal role in patterning the anterior region of the fly body plan. The hb ortholog in Capitella (Cc-hb) is expressed maternally and in all micromere and macromere cells throughout cleavage. At gastrulation, nuclear Cc-hb protein is expressed in the micromere-derived surface epithelium that undergoes epiboly and in the large vegetal blastomeres that gradually become internalized. During organogensis, Cc-hb is expressed in the developing gut epithelium, the prostomial and pygidial epithelium, and in a subset of differentiated neurons in the adult central nervous system. Cc-hb is not expressed in the segmental precursor cells in the trunk. The Cc-hb expression domains in Capitella are similar to those reported for the leech hb ortholog (LZF2), and many of the observed differences between the annelid classes correlates with changes in life history. The lack of detectable annelid hb protein in the trunk at the time of AP pattern formation in leech and in polychaete suggests that the anterior organizing function of hb in flies originated in the arthropod or insect lineage (Werbrock, 2001).

The pattern of annelid hb expression in the temporary epithelium prior to organogenesis and later in the ventral nerve cord correlate with a subset of zygotic expression patterns of the hb gene in insects and the hbl-1 gene in nematodes. In all three phyla, the hb orthologs are expressed in the developing CNS as are most, if not all, of the segmentation gene orthologs studied to date. Fly hb is also expressed in the extraembryonic epithelium called the amnioserosa and in the evolutionary related serosa of more basal insects. The function of hb in the amnioserosa or the serosa is currently unknown, but the amnioserosa tissue has been shown to play a role in fly morphogenesis. Similarly the nematode hbl-1 gene is expressed in the epithelial (hypodermal) precursor cells before organogenesis and when hbl-1 transcripts are removed from the hypodermal precursor cells via RNAi, the embryo fails to elongate. In all three phyla, hb orthologs are expressed in temporary epithelium at the time important morphogenetic events are taking place, but only in nematodes is there a direct correlation of hbl-1 function and normal morphogenesis. Functional studies of hb function in the amnioserosa in flies, and its orthologs in the serosa of nondipteran insects like Tribolium, and in the temporary (larval) epithelium of annelids are required to determine what role temporary epithelial cells play, if any, in body plan morphogenesis (Werbrock, 2001).

Axial patterning is a fundamental event in early development, and molecules involved in determining the body axes provide a coordinate system for subsequent patterning. While orthologs of Drosophila bicoid and nanos play a conserved role in anteroposterior (AP) patterning within at least a subset of Diptera, conservation of this process has not yet been demonstrated outside of the flies. Indeed, it has been argued that bicoid, an instrumental 'anterior' factor in Drosophila melanogaster, acquired this role during the evolution of more-derived dipterans. Interestingly, the interaction of Drosophila maternal nanos and maternal hunchback provides a system for patterning the AP axis that is partially redundant to the anterior system. Previous studies in grasshoppers suggest that hunchback may play a conserved role in axial patterning in this insect, but this function may be supplied solely by the zygotic component of hunchback expression. Evidence suggests that the early pattern of zygotic grasshopper Hunchback expression is achieved through translational repression that may be mediated through the action of grasshopper nanos. This is consistent with the notion that an anterior gradient system is not necessary in all insects and that the posterior pole probably conveys longitudinal polarity on the ensuing germ anlage (Lall, 2003)

The results indicate that nanos mRNA and protein are expressed asymmetrically at several stages of development. Within the germarium, Nanos protein is asymmetrically distributed within the developing oocytes. During early oogenesis, hunchback mRNA and protein are expressed in the same pattern of cells, suggesting that there is no translational repression of hunchback at this stage (there is also little or no Nanos protein in the Hunchback-expressing stage oocytes). Later in oogenesis, and in newly laid eggs, nanos mRNA is localized to the posterior pole of the egg. When cellularization begins, Nanos protein is found in cells toward the posterior (but not anterior) end of the egg. While this superficially resembles the asymmetry of Nanos protein in syncytial Drosophila embryos, it is important to remember that it is not possible, at least with the current data, to correlate this expression pattern to the future AP axis of the grasshopper embryo. It should be noted that the seeming lack of correspondence of the AP egg axis with the AP embryo axis may be a derived situation in the grasshopper, since the correlation is obvious in most other insects (Lall, 2003)

Nevertheless analysis of grasshopper nanos expression in the germ anlage indicates that this phase of asymmetric expression may underlie formation of the embryonic AP axis and posterior patterning of the embryo via Hunchback regulation. This suggests that an axial patterning mechanism involving translational repression of hb mRNA may be an ancestral feature of insect pattern formation (at least as far back as the common ancestor of Schistocerca and Drosophila). However, since maternal S. americana Hb is provided as protein, the target of translational repression in grasshopper would appear to be zygotic hunchback mRNA and not maternal hunchback mRNA as in Drosophila. It is currently unclear whether S. americana Nanos is acting as a switch that specifies some cells as posterior or whether it is acting in a graded fashion to permit the differentiation of different posterior identities. It is also interesting to note that work in Tribolium suggests that caudal may act as an activator of hunchback transcription and that S. gregaria caudal is expressed during condensation of the germ disc and in the early germ anlage. On the basis of these data, it is suggested that grasshopper caudal (and, possibly, maternally inherited Hunchback protein) could act to promote zygotic hunchback transcription throughout the entire embryonic primordium and that nanos acts to prevent translation of zygotic hunchback mRNA in the posterior of the grasshopper embryo (Lall, 2003)

Drosophila nanos also has a well-studied role in germline development, and it has been suggested that the ancestral role of nanos in metazoans was in germline function. Data presented in the current paper indicate, however, that the role of nanos in both axial patterning and germline development is probably ancestral to at least the insects. Furthermore, Cnnos2 is expressed in a manner consistent with a role in axial patterning of the growing buds and regenerating head, but not foot, of the cnidarian, H. magnipapillata. Thus, the function of nanos in both axial patterning (not necessarily via hb regulation) and germline development may be ancient. Indeed nanos may function in situations where a specific set of cells must be set aside and protected from patterning factors. This is entirely consistent with the role of nanos in germline specification as well as its role in protecting cells from anterior patterning factors, such as hunchback, within the insects (Lall, 2003)

hunchback is required for suppression of abdominal identity, and for proper germband growth and segmentation in the intermediate germband insect Oncopeltus fasciatus

Insects such as Drosophila melanogaster undergo a derived form of segmentation, termed long germband segmentation. In long germband insects, all of the body regions are specified by the blastoderm stage. Thus, the entire body plan is proportionally represented on the blastoderm. This is in contrast to short and intermediate germband insects where only the most anterior body regions are specified by the blastoderm stage. Posterior segments are specified later in embryogenesis during a period of germband elongation. Although much is known about Drosophila segmentation, very little is known about how the blastoderm of short and intermediate germband insects is allocated into only the anterior segments, and how the remaining posterior segments are produced. In order to gain insight into this type of embryogenesis, the expression and function of the homolog of the Drosophila gap gene hunchback was investigated in an intermediate germ insect, the milkweed bug, Oncopeltus fasciatus. Oncopeltus hunchback (Of'hb) is expressed in two phases, first in a gap-like domain in the blastoderm and later in the posterior growth zone during germband elongation. In order to determine the genetic function of Of'hb, a method of parental RNAi was developed in the milkweed bug. Using this technique, it was found that Oncopeltus hunchback has two roles in anterior-posterior axis specification. First, Of'hb is required to suppress abdominal identity in the gnathal and thoracic regions. Subsequently, it is then required for proper germband growth and segmentation. In milkweed bug embryos depleted for hunchback, these two effects result in animals in which a relatively normal head is followed by several segments with abdominal identity. This phenotype is reminiscent of that found in Drosophila hunchback mutants, but in Oncopeltus is generated through the combination of the two separate defects (Liu, 2004).

Oncopeltus embryogenesis can be divided into two distinct phases -- a blastoderm phase, which in some ways is similar to that of Drosophila, and a germband growth phase which it shares with other short germ insects. Oncopeltus embryogenesis begins with the first nuclear divisions occurring synchronously within the yolk mass without concomitant cellular divisions. After several such divisions, the resulting cleavage nuclei migrate to the egg cortex. By fifteen hours after egg lay, they reach the surface of the egg and after an additional two hours, the formation of cell membranes is complete. At this stage, the large ovoid blastoderm superficially appears very Drosophila like. However, blastoderm cells of a 36- to 40-hour-old embryo are not evenly arranged around the yolk but are concentrated in two broad lateral domains on either side of the yolk mass that are similar to the 'lateral plates' seen in Rhodnius (Liu, 2004).

In addition to observing the cellular movements in the blastoderm, the number of segments to have been specified at this stage in embryogenesis was ascertained. Since the segment polarity gene engrailed (en) is expressed in the posterior compartments of segments in many arthropods, including Oncopeltus, it serves as a convenient molecular segmental marker. In situ hybridization of 36- to 40-hour-old embryos with Oncopeltus engrailed (Of'en) probe revealed a total of six vertical stripes on the blastoderm surface. This shows that by this stage, the blastoderm has already been allocated into six segments. The migration of these stripes was followed throughout embryogenesis to deduce their segmental affinities; these six initial stripes correspond to the mandibular through third thoracic segments. That engrailed is expressed at this stage is somewhat surprising, and shows that anterior patterning has occurred all the way to the segment polarity level long before the posterior body regions even exist. This reinforces the idea that although the Oncopeltus blastoderm may in some ways superficially resemble the Drosophila blastoderm, the milkweed bug blastoderm is subdivided in a distinctly different way (Liu, 2004).

Oncopeltus embryos are of the 'invaginating' type: this refers to the cell movements that give rise to the germband. Shortly after the formation of the blastoderm lateral plates, the germband begins to form when the cells at the posterior end of the blastoderm dive into the center of the yolk mass. The early site of invagination is marked by a small pit at the posterior pole of the late blastoderm. The cells of the blastoderm surface migrate toward the posterior, while the leading tip of the elongating germband dives into the interior of the yolk mass, toward the anterior pole of the egg. In order to visualize these movements, it is instructive to imagine the blastoderm as an inflated balloon, with invagination occurring as if a finger is poked into the interior of the balloon. Thus, the cells on the outside of the blastoderm move toward the posterior of the egg, dive into the yolk and migrate toward the anterior of the egg. As germband invagination continues, the tip of the germband eventually reaches the anterior pole of the egg and the resulting germband stage embryo ends up with its head at the posterior of the egg (the embryo does eventually right itself through later embryonic movements). Since these embryonic movements can potentially lead to confusion, when discussing the blastoderm the anteroposterior and dorsoventral axis is referred to in regards to the fate maps of the tissues (Liu, 2004).

During the germband stage, the remaining posterior body segments that were not specified during blastoderm stage are now produced through elongation of the posterior portion of the germband coupled with progressive anterior to posterior segmental specification. First, the abdominal region is generated through rearrangement and growth of the posterior growth zone and then engrailed stripes appear one by one in an anterior to posterior direction. This is similar to other short germband insects such as Thermobia domestica, Schistocerca americana and Tribolium castaneum. Thus, it is clear that in Oncopeltus, as in other short and intermediate germband insects, posterior segments arise during a secondary growth phase during which the posterior germband undergoes great elongation with specification of abdominal segments occurring sequentially and in an anterior to posterior direction (Liu, 2004).

Oncopeltus hunchback expression and function reflect the biphasic nature of milkweed bug embryogenesis. hb is expressed in two broad stripes during the blastoderm stage. The stronger band spans the posterior maxillary, labial and anterior first thoracic segments. Since hb is not expressed in these segments during the germband stage (except in the mesoderm and a neural-like domain), its region-specifying function in these segments is attributed to its expression domain in the blastoderm. This abdomen-repression function in the labial and thoracic segments may occur either through direct suppression of abd-A in the anterior, or may occur indirectly through regulation of a downstream gene responsible for specifying abdominal regional identity. Since the ectopic domain of abd-A in hb RNAi animals was not detected at the blastoderm stage but only later during the germband stage and the region of homeosis in hb RNAi animals is much larger than the hb blastoderm expression domain, it is most likely that hb indirectly regulates abd-A. Indeed, normal abd-A expression in the abdomen appears long after hb expression in the growth zone has already faded. The observation that hunchback in milkweed bugs serves to repress abdominal identity is not without precedence. In Drosophila, certain hypomorphic alleles of hunchback, class V alleles, also produce homeotic transformations of the gnathal or thoracic segments. However, unlike Oncopeltus, these transformations are superimposed on a deletion phenotype (Liu, 2004).

As in flies, hunchback RNAi depletion in the red flour beetle, Tribolium castaneum, also produces the gnathal and thoracic gap phenotype. In this light, it is interesting that Oncopeltus hunchback knockdowns do not show the canonical gap phenotype but rather a transformation. This may reflect either incomplete RNAi knockdown of the Of'hb gene product or may reflect differences in anterior-posterior patterning in milkweed bugs. It is possible that if further depletion were possible, the anterior homeotic phenotype would be replaced by deletion of those segments. However, this would be in contrast to the case in Drosophila because weak alleles of hunchback in flies are not associated with transformations but rather yield small deletions while stronger alleles serve to increase the size of the deleted region. The Oncopeltus hunchback phenotype reported here seems to reflect a strong depletion of the hunchback gene product because of the large domain of the homeotic defect from the labium to the third thoracic segment in the severe phenotypic classes. Transformation of the third thoracic segment is indicative of strong hb RNAi yet in these same animals, the labial segment is present and without segmental defects. If deletion of the labial segment were merely an issue of sensitivity, it would be expected that animals with transformed third thoracic segments also show segmental defects in the labial segment. Therefore, if hunchback has a gap function in this animal, its requirement must be minimal (Liu, 2004).

Therefore, hunchback transcript is provided maternally in Oncopeltus and it is a formal possibility that the protein is as well. If this were the case, it is possible that maternal hunchback serves to specify the presence of the gnathal and thoracic segments, while zygotic activity functions to suppress abdominal identity in these regions. Although maternal loading of hunchback-encoded protein has not been reported in either flies or beetles, the protein is provided maternally in grasshoppers. In grasshoppers however, axial patterning by hunchback appears to be performed entirely by zygotic function whereas maternal hunchback activity in this animal may serve to distinguish embryonic from extra-embryonic cells (Liu, 2004).

In strongly affected Oncopeltus hunchback RNAi animals, the abdomen is severely compacted and segmentation is defective. hunchback function in the developing germband probably reflects its expression in the posterior 'growth zone'. Therefore it is proposed that Of'hb is required for proper growth and segmentation of the posterior germband. It is possilble to speculate on the nature of this requirement. It may be that Of'hb is directly involved in the generation of segments as the posterior germband grows. However, it may also be that Of'hb is merely required for posterior elongation of the germband while the actual patterning of segments occurs relatively independently of growth. Thus, the segmental defects seen in developing germbands may be a consequence of improper elongation. Alternatively, hunchback may be required for proper functioning of the growth zone itself (Liu, 2004).

hunchback has also been examined in Schistocerca and Tribolium, two other short germband insects. Schistocerca hunchback is not expressed continuously in the growth zone as it is in both milkweed bugs and beetles but rather arises in abdominal patches corresponding to the A4/A5 and A7-A9 segments. Therefore the continuous expression in the growth zone may represent a derived pattern in the insects. As noted, Tribolium and Oncopeltus hunchback are expressed in identical patterns in the growth zone. However, it has not yet been reported that hb RNAi in Tribolium leads to posterior compaction, rather the phenotype reported is the canonical hunchback gap phenotype. With such disparate expression and functional data from these three insects, it is difficult to determine the ancestral function of hunchback in the insect growth zone and it is clearly imperative that wider taxonomic sampling needs to be done (Liu, 2004).

This brings the discussion to examinations of the very nature of the insect growth zone itself. Almost nothing is known about how this special region of the germband develops and ultimately gives rise to the posterior segments. There are no overt morphological features that distinguish it. However, the growth zone must be special because several segmentation genes such as even-skipped, caudal and hunchback are expressed there. Given that some form of short germband development is ancestral in insects, this mode of development must be understoood in order to understand the evolutionary transition from short to long germ segmentation. Moreover, since other arthropods undergo embryogenesis in a manner similar to insect short germband segmentation, functional studies in Oncopeltus and other short germband insects may in fact shed light on all the arthropods (Liu, 2004).

Ikaros family of genes

The Ikaros gene, an essential regulator of lymphocyte differentiation, encodes, by means of differential splicing, protein isoforms with a distinct number of Kruppel-type zinc fingers organized in two domains. Deletion of the N-terminal zinc finger domain, responsible for the sequence-specific DNA binding of the Ikaros proteins, results in an early and complete arrest in lymphocyte development in homozygous mutant mice. In sharp contrast, heterozygotes reliably develop T cell leukemias and lymphomas. The C-terminal zinc finger domain present in all of the Ikaros wild-type and mutant isoforms, is responsible for their stable interactions off DNA; this domain plays a pivotal role in determining the overall isoform activity. The double-finger domain is related to a domain of the Drosophila Hunchback protein. There is 43% identity between the second finger cluster of Ikaros and that of Hunchback. Mutations in the C-terminal zinc fingers, which ablate Ikaros protein interactions, have a dramatic effect on the ability of these proteins to bind DNA and activate transcription. Therefore, interactions between Ikaros isoforms with an intact DNA binding domain are essential for their function. In contrast, interactions between isoforms with and without a DNA binding domain result in Ikaros complexes that do not bind DNA and, as a consequence, cannot activate transcription. Dominant-negative Ikaros isoforms are generated in smaller amounts by the wild-type Ikaros gene but, are produced exclusively by the N-terminally deleted Ikaros locus. Given these data, it is proposed that interactions between Ikaros isoforms are essential for normal progression through the lymphoid pathways. Mutations in the Ikaros gene that prevent Ikaros protein interactions or which change the relative ratio of DNA to non-DNA binding isoforms have profound effects in both lymphoid specification and homeostasis (Sun, 1997).

Development of the lymphoid system is dependent on the activity of zinc finger transcription factors encoded by the Ikaros gene. Differences between the phenotypes resulting from a dominant-negative and a null mutation in this gene suggest that Ikaros proteins act in concert with another factor with which they form heterodimers. Aiolos, a gene which encodes an Ikaros homolog, codes for a protein that heterodimerizes with Ikaros proteins. In contrast to Ikaros--which is expressed from the pluripotent stem cell to the mature lymphocyte--Aiolos is first detected in more committed progenitors with a lymphoid potential and is strongly up-regulated as these differentiate into pre-T and pre-B cell precursors. The expression patterns of Aiolos and Ikaros, the relative transcriptional activity of their homo- and heteromeric complexes, and the dominant interfering effect of mutant Ikaros isoforms on Aiolos activity all strongly suggest that Aiolos acts in concert with Ikaros during lymphocyte development. It is therefore proposed that increasing levels of Ikaros and Aiolos homo- and heteromeric complexes in differentiating lymphocytes are essential for normal progression to a mature and immunocompetent state (Morgan, 1997).

Ikaros proteins are required for normal T, B, and NK cell development and are postulated to activate lymphocyte-specific gene expression. Ikaros distribution in the nucleus of B lymphocytes was examined using confocal microscopy and a novel immunofluorescence in situ hybridization (immuno-FISH) approach. Unexpectedly, Ikaros localizes to discrete heterochromatin-containing foci in interphase nuclei, which comprise clusters of centromeric DNA as defined by gamma-satellite sequences and the abundance of heterochromatin protein-1 (HP-1). Using locus-specific probes for CD2, CD4, CD8alpha, CD19, CD45, and lambda5 genes, it has been shown that transcriptionally inactive but not transcriptionally active genes associate with Ikaros-heterochromatin foci. These findings support a model of organization of the nucleus in which repressed genes are selectively recruited into centromeric domains (Brown, 1997).

The mammalian Ikaros gene encodes multiple protein isoforms that contribute critical functions during the development of lymphocytes and other hematopoietic cell types. The intracellular functions of Ikaros are not known, although recent studies have shown that Ikaros proteins colocalize with inactive genes and centromeric heterochromatin. Ikaros proteins are found to be components of highly stable complexes. The complexes from an immature T cell line were purified, revealing associated proteins of 70 and 30 kD. The p70 gene, named Helios, encodes two protein isoforms with zinc finger domains, exhibiting considerable homology to those within Ikaros proteins. Helios and Ikaros recognize similar DNA sequences and, when overexpressed, Helios associates indiscriminately with the various Ikaros isoforms. Although Ikaros is present in most hematopoietic cells, Helios is found primarily in T cells. The relevance of the Ikaros-Helios interaction in T cells is supported by the quantitative association of Helios with a fraction of Ikaros. Interestingly, the Ikaros-Helios complexes localize to the centromeric regions of T cell nuclei, similar to the Ikaros localization previously observed in B cells. Unlike the B cell results, however, only a fraction of the Ikaros (presumably the fraction associated with Helios) exhibits centromeric localization in T cells. These results establish immunoaffinity chromatography as a useful method for identifying parners of Ikaros and suggest that Helios is a limiting regulatory subunit for Ikaros within centromeric heterochromatin (Hahm, 1998).

The Ikaros gene family encodes zinc finger DNA-binding proteins essential for lineage determination and control of proliferation in the lymphoid system. In the nucleus of a T cell, a major fraction of Ikaros and Aiolos proteins associate with the DNA-dependent ATPase Mi-2 (see Drosophila Mi-2) and histone deacetylases, in a 2 MD complex. This Ikaros-NURD complex is active in chromatin remodeling and histone deacetylation. Upon T cell activation, Ikaros recruits Mi-2/HDAC to regions of heterochromatin. These studies reveal that Ikaros proteins are capable of targeting chromatin remodeling and deacetylation complexes in vivo. It is proposed that the restructuring of chromatin is a key aspect of Ikaros function in lymphocyte differentiation (Kim, 1999).

Ikaros is a sequence-specific DNA-binding protein that is essential for lymphocyte development. Little is known about the molecular function of Ikaros, although recent results have led to the hypothesis that it recruits genes destined for heritable inactivation to foci containing pericentromeric heterochromatin. To gain further insight into the functions of Ikaros, the mechanism by which it is targeted to centromeric foci was examined. Efficient targeting of Ikaros was observed upon ectopic expression in 3T3 fibroblasts, demonstrating that lymphocyte-specific proteins and a lymphoid nuclear architecture are not required. Pericentromeric targeting did not result from an interaction with the Mi-2 remodeling factor, since only a small percentage of Mi-2 localized to centromeric foci in 3T3 cells. Rather, targeting is dependent on the amino-terminal DNA-binding zinc finger domain and carboxy-terminal dimerization domain of Ikaros. The carboxy-terminal domain is required only for homodimerization, because targeting is restored when this domain is replaced with a leucine zipper. Surprisingly, a detailed substitution mutant analysis of the amino-terminal domain reveals a close correlation between DNA-binding and pericentromeric targeting. These results show that DNA binding is essential for the pericentromeric localization of Ikaros, perhaps consistent with the presence of Ikaros binding sites within centromeric DNA repeats (Cobb, 2000).

Members of the Ikaros family of transcription factors, Ikaros, Aiolos, and Helios, are expressed in lymphocytes and have been implicated in controlling lymphoid development. These proteins contain two characteristic clusters of zinc fingers, an N-terminal domain important for DNA recognition, and a C-terminal domain that mediates homo- and heterotypic associations between family members. The conservation of these domains is such that all three proteins recognize related DNA sequences, and all are capable of dimerizing with other family members. Two additional Ikaros family proteins, Eos and Pegasus, are described. Eos is most highly related to Helios and shares its DNA binding and protein association properties. Pegasus is related to other Ikaros proteins in its C-terminal dimerization domain but contains a divergent N-terminal zinc finger domain. Pegasus self-associates and binds to other family members but recognizes distinct DNA-binding sites. Eos and Pegasus repress the expression of reporter genes containing their recognition elements. These results suggest that these proteins may associate with previously described Ikaros family proteins in lymphoid cells and play additional roles in other tissues (Perdomo, 2000).

Members of the Ikaros multigene family of zinc finger proteins are expressed in a tissue-specific manner and most are critical determinants in the development of both the B and T lymphocytes as well as NK and dendritic APC lineages. A PCR amplification strategy that is based on regions of shared sequence identity in Ikaros multigene family members found in mammals and several other vertebrates has led to the recovery of cDNAs that represent the orthologs of Ikaros, Aiolos, Helios, and Eos in Raja eglanteria (clearnose skate), a cartilaginous fish that is representative of an early divergence event in the phylogenetic diversification of the vertebrates. The tissue-specific patterns of expression for at least two of the four Ikaros family members in skate resemble the patterns observed in mammals, i.e., in hematopoietic tissues. Prominent expression of Ikaros in skate also is found in the lymphoid Leydig organ and epigonal tissues, which are unique to cartilaginous fish. An Ikaros-related gene has been identified in Petromyzon marinus (sea lamprey), a jawless vertebrate species, in which neither Ig nor TCRs have been identified. In addition to establishing a high degree of evolutionary conservation of the Ikaros multigene family from cartilaginous fish through mammals, these studies define a possible link between factors that regulate the differentiation of immune-type cells in the jawed vertebrates and related factors of unknown function in jawless vertebrates (Haire, 2000).

Ikaros family members play critical roles in hematopoietic development, yet molecules regulated by Ikaros proteins remain incompletely characterized. To determine the requirements for functional Ikaros proteins, Ik7, a dominant negative Ikaros protein, was overexpressed in human cell lines and hematopoietic progenitor cells. Ik7 is known to block the normal function of other Ikaros family members in human and mouse cells. Retroviral-mediated overexpression of Ik7 affects two distinct, migratory properties of the CEM T-cell line. Ik7 down-regulates L-selectin cell-surface expression, an effect not a result of increased shedding but of a decrease in L-selectin mRNA levels. Ik7 also reduces the spontaneous migration of CEM T cells in 3-D collagen gels. A reduction in L-selectin, cell-surface expression was also induced by Ik7 in CD34+ hematopoietic progenitor cells. In contrast, the Reh B cell line showed an up-regulation of L-selectin, cell-surface levels when expressing Ik7. For the first time, this study defines an effect of Ikaros proteins in the control of migration-related properties and shows that intact Ikaros proteins are important in a cell type-specific manner for the normal regulation of L-selectin expression (Christopherson, 2001).

Ikaros family proteins and chromatin

The lymphoid lineage-determining factors Ikaros and Aiolos can function as strong transcriptional repressors. This function is mediated through two repression domains and is dependent upon the promoter context and cell type. Repression by Ikaros proteins correlates with hypo-acetylation of core histones at promoter sites and is relieved by histone deacetylase inhibitors. Consistent with these findings, Ikaros and its repression domains can interact in vivo and in vitro with the mSin3 family of co-repressors, which bind to histone deacetylases. Based on these and the recent findings of associations between Ikaros and Mi-2-HDAC, it is proposed that Ikaros family members modulate gene expression during lymphocyte development by recruiting distinct histone deacetylase complexes to specific promoters (Koipally, 1999).

Ikaros can repress transcription through the recruitment of histone deacetylase complexes. Ikaros can also repress transcription through its interactions with the co-repressor, C-terminal binding protein (CtBP). CtBP interacts with Ikaros isoforms through a PEDLS motif present at the N terminus of these proteins but not with homologs like Aiolos, which lack this motif. Mutations in Ikaros that prevent CtBP interactions reduce its ability to repress transcription. CtBP interacts with Sin3A (see Drosophila Sin3A) but not with the Mi-2 co-repressor and it represses transcription in a manner that is independent of histone deacetylase activity. These data strongly suggest that CtBP contributes to a histone deacetylase activity independent mechanism of repression by Ikaros. The viral oncoprotein E1A, which also binds to CtBP, shows a strong association with Ikaros. This Ikaros-E1A interaction may underlie Ikaros's decreased ability to repress transcription in E1A transformed cells (Koipally, 2000).

A SWI/SNF-related protein complex (PYR complex) is restricted to definitive (adult-type) hematopoietic cells and specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human beta-globin locus construct results in delayed human gamma- to beta-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and antibodies to Ikaros also supershift the complex. NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. These results suggest that Ikaros targets two types of chromatin-remodeling factors -- activators (SWI/SNF) and repressors (NuRD) -- in a single complex (PYR complex) to the beta-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress gamma-globin gene expression and facilitate gamma- to beta-globin switching (O'Neill, 2000).

Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromatin and has been implicated in heritable gene inactivation. Binding and competition experiments demonstrate that Ikaros dimers compete with an Ets activator for occupancy of the lymphocyte-specific TdT promoter. Mutations that selectively disrupt Ikaros binding to an integrated TdT promoter have no effect on promoter function in a CD4(+)CD8(+) thymocyte line. However, these mutations abolish down-regulation upon differentiation, providing evidence that Ikaros plays a direct role in repression. Reduced access to restriction enzyme cleavage suggested that chromatin alterations accompany down-regulation. The Ikaros-dependent down-regulation event and the observed chromatin alterations appear to precede pericentromeric repositioning. Current models propose that the functions of Ikaros should be disrupted by a small isoform that retains the dimerization domain and lacks the DNA-binding domain. Surprisingly, in the CD4(+)CD8(+) thymocyte line, overexpression of a small Ikaros isoform has no effect on differentiation or on the pericentromeric targeting and DNA-binding properties of Ikaros. Rather, the small isoform assembles into multimeric complexes with DNA-bound Ikaros at the pericentromeric foci. The capacity for in vivo multimer formation suggests that interactions between Ikaros dimers bound to the TdT promoter and those bound to pericentromeric repeat sequences may contribute to the pericentromeric repositioning of the inactive gene (Trinh, 2001).

The Ikaros family of proteins are DNA binding factors required for correct development of B and T lymphocytes. Cytogenetic studies have shown that these proteins form complexes with pericentromeric heterochromatin in B cells, and the colocalization of transcriptionally silent genes with these complexes suggests that Ikaros could silence transcription by recruiting genes to heterochromatin. A site in the lambda5 promoter that binds Ikaros and Aiolos is required for silencing of lambda5 expression in activated mature B cells. Analysis of methylation and nuclease accessibility indicates that the silenced lambda5 gene is not heterochromatinized in B cells, despite being associated with pericentromeric heterochromatin clusters. A promoter mutation, which affects Ikaros-mediated silencing of lambda5 expression, is not rescued in a transgenic line that has the gene integrated into pericentromeric heterochromatin. These results indicate that the Ikaros proteins initiate silencing of lambda5 expression through a direct effect on the promoter with localization to pericentromeric heterochromatin likely to affect the action of Ikaros on regulatory sequences rather than causing heterochromatinization of the gene (Sabbattini, 2001).

The mouse kappa opioid receptor (KOR) gene is constitutively expressed in mouse embryonal carcinoma P19 stem cells and suppressed by retinoic acid (RA) in cells undergoing neuronal differentiation. A negative regulatory element is located within intron 1 of the KOR gene, which contains an Ikaros (Ik)-binding site (GGGAAgGGGAT). This sequence is an Ik-1 responsive, functionally negative element as demonstrated in the context of both natural KOR and heterologous promoters. Two G residues of the second half-site are critical for Ik-1 binding and Ik-mediated repression of the KOR gene. RA induces Ik-1 expression within 1 day of treatment and suppresses KOR expression between 2 and 3 days. Overexpression of Ik-1 in P19 suppresses endogenous KOR gene expression, accompanied by increased binding of Ik-1 to the Ik-binding site and chromatin histone deacetylation on KOR promoters. It is proposed that in an RA-induced P19 differentiation model, RA elevates Ik-1 expression, which recruits histone deacetylase to intron 1 of the KOR gene and silences KOR gene promoters (Hu, 2001).

Ikaros SUMOylation: Switching out of repression

Ikaros plays a key role in lymphocyte development and homeostasis by both potentiating and repressing gene expression. Ikaros interacts with components of the SUMO pathway and is SUMOylated in vivo (see Drosophila SUMO). Two SUMOylation sites have been identified on Ikaros whose simultaneous modification results in a loss of the Ikaros repression function. Ikaros SUMOylation disrupts its participation in both histone deacetylase (HDAC)-dependent and HDAC-independent repression but does not influence its nuclear localization into pericentromeric heterochromatin. These studies reveal a new dynamic way by which Ikaros-mediated gene repression is controlled by SUMOylation (Gomez-del Arco, 2005).

How does SUMOylation of Ikaros interfere with its interactions with HDAC-dependent and -independent corepressors? SUMOylation of Ikaros may simply block access to their binding sites. For example, the Ikaros K58 SUMOylation site lies next to the CtBP interaction motif (amino acids 34 to 38) and may be responsible for the more severe disruption of Ikaros-CtBP interactions relative to the other corepressors. The proteins Mi-2ß and Sin3 share binding domains located at the N- and C-terminal regions of the Ikaros proteins, and accessibility to these common interaction domains is likely to be similarly affected by SUMOylation. SUMOylation may affect Ikaros-corepressor associations by inducing conformational changes that alter the Ikaaros interaction interface (Gomez-del Arco, 2005).

Studies with primary thymocytes and cycling T cells show that a small but significant fraction of the total Ikaros protein is SUMOylated. Nonetheless, mutation of the SUMOylation sites of Ikaros has a strong effect on its activity as a repressor. Given the dynamic and transient nature of SUMOylation, it may be required to initiate the disassembly of Ikaros-repressor complexes but not to maintain them in a separate state. DeSUMOylated Ikaros may then be preferentially retained in a different type of protein complex, i.e., Swi/Snf, that is not greatly affected by SUMOylation. SUMO modifications regulating the disassembly of septin ring structures during mitosis have been reported in yeast (Gomez-del Arco, 2005).

Ikaros confers early temporal competence to mouse retinal progenitor cells

In the developing mouse retina, multipotent retinal progenitor cells (RPCs) give rise to specific retinal cell types at different times, but the molecular mechanisms regulating how RPCs change over time remain unclear. In the Drosophila neuroblast lineage, the zinc finger transcription factor Hunchback (Hb) is both necessary and sufficient to specify early-born neuronal identity. This study shows that Ikaros, a mouse ortholog of Hb, is expressed in all early embryonic RPCs, which then give rise to Ikaros-negative RPCs at later stages in the lineage. Remarkably, misexpression of Ikaros in late RPCs is sufficient to confer competence to generate early-born neurons. Conversely, Ikaros mutant mice have reduced numbers of early-born cell types, whereas late-born cell types are not affected. These results suggest a model in which Ikaros expression is both necessary and sufficient to confer early temporal competence to RPCs and raise the possibility that a similar strategy might be used to control the sequential order of cell birth in other parts of the nervous system (Elliott, 2008).

Attempts were made to determine how Ikaros might induce early-born cell type production when misexpressed in late RPCs. One possibility was that it could function as a positive transcriptional regulator of genes required for early-born cell-type specification. To test this hypothesis, a bicistronic retroviral vector was used to express Ikaros together with GFP in P0 RPCs. After 48 hr, the GFP-positive cells were purified by FACS, mRNA was extracted, and semiquantitative RT-PCR was performed. Interestingly, it was found that the expression of Prox-1, a homeodomain protein expressed in dividing RPCs that is both necessary and sufficient for horizontal cell specification, was drastically increased following Ikaros misexpression in late RPCs. In contrast, the expression of Math5 and Islet-1, which are not expressed in proliferating RPCs but operate in postmitotic cells to promote the ganglion cell fate, did not change 48 hr after Ikaros misexpression. These results indicate that Ikaros functions to promote the expression of Prox-1 in postnatal RPCs, thereby conferring competence to these cells to acquire the horizontal cell fate. This result suggests that Ikaros might function as a specific inducer of genes involved in early-born cell type specification (Elliott, 2008).

These findings support the hypothesis that Ikaros functions to confer early temporal competence to RPCs, rather than to instruct specific cell-fate decisions. Cell-fate determinants, such as bHLH transcription factors, generally act instructively to promote a particular cell fate. In contrast, Ikaros misexpression in late RPCs does not promote the production of one specific cell type but instead causes a general increase in the production of early-born cell types. Ikaros could still be a cell-fate determinant factor that acts instructively to specify three different fates that just happen to be early-born cell types. The results, however, do not appear to be consistent with this possibility. Misexpression of Ikaros in late RPCs does not completely prevent the generation of late-born cell types, and some 2-cell clones can even contain an early-born cell type, together with a late-born cell type, indicating that the RPC that gave rise to this type of clone was competent to produce both early and late-born cell types at the same time. This type of clone is most likely due to late RPCs expressing the normal late temporal competence factor(s) that allow production of late-born cell types, but the reintroduction of Ikaros adds another set of possible fates to these cells by conferring early temporal competence, thereby producing an RPC that can generate both early- and late-born cell types. These results indicate that Ikaros increases, but does not reset, the developmental potential of late RPCs, supporting a model in which the presence of Ikaros is not instructive but is instead permissive for the production of early-born cell types (Elliott, 2008).

The mode of action of Ikaros appears to be unique compared to other previously identified so-called competence factors. For example, the bHLH transcription factor Math5 is thought to determine the competence state of retinal ganglion cell precursors. In contrast to Ikaros, Math5 is not expressed in RPCs, but acts in postmitotic RGC precursors to activate the expression of transcription factors involved in RGC differentiation. In addition, Math5 expression alone is not sufficient to induce RGC generation from late RPCs, and Math5-expressing cells have been shown to give rise to other cell types that are generated at both early (amacrine and horizontal cells) and late (photoreceptor cells) stages of retinogenesis. Since Math5 expression is significantly reduced in ikaros−/− retinas, the results suggest that Ikaros might act upstream of Math5. Recently, SOX2 was proposed to act as a regulator of RPC competence. In contrast to Ikaros, however, SOX2 appears to regulate the general competence of RPC to proliferate and differentiate rather than to control temporal competence to generate a class of cell types produced at a specific time in development. Another recently identified competence factor is GDF11, a member of the TGF-β family of signaling molecules. Unlike Ikaros, GDF11 is a secreted protein and apparently acts by regulating the time window that RPCs express certain transcription factors involved in RGC genesis such as Math5, without affecting RPC proliferation. Thus, GDF11 is a negative feedback signal that prevents prolonged RGC production, whereas Ikaros is a positive, cell-intrinsic regulator of early temporal competence in RPCs. But could GDF11 control the timing of Ikaros expression? This possibility appears unlikely as the number of horizontal cells is unchanged in the gdf11 mutant mice, whereas horizontal cells are reduced in ikaros mutant mice. It remains possible, however, that GDF11 acts in concert with another factor to regulate Ikaros expression (Elliott, 2008).

Consistent with Ikaros regulating early temporal competence, the number of Ikaros-positive RPCs gradually decreases over time during retinal development. By P2, less than 1% of RPCs express Ikaros, whereas at P4 no RPCs express Ikaros. Because a few amacrine cells are still generated at P2, it is proposed that the rare RPCs expressing Ikaros in the postnatal retina are those that are still competent to give rise to amacrine cells. Interestingly, it was found that Ikaros-positive RPCs actually give rise to Ikaros-negative RPCs at later stages in the same lineage. These results indicate that, much like Hunchback in Drosophila neuroblast lineage, Ikaros expression constitutes a temporal stage of all RPCs, rather than a separate pool of cells that expand specifically during early stages of retinogenesis. Although Ikaros apparently confers the competence to generate early-born cell types, one exception appears to be the cone photoreceptors. In the mature retina, Ikaros is not expressed in rod or cone photoreceptor cells, and it was found that the number of cone photoreceptor cells is not affected in the ikaros−/− retinas. This suggests that the mechanisms regulating temporal competence to generate cone photoreceptors do not depend on Ikaros (Elliott, 2008).

Perhaps one of the most interesting findings in this study is that misexpression of Ikaros alone in late RPCs is sufficient to confer competence to generate early-born neurons. This result is strikingly similar to experiments in Drosophila in which Hunchback was reintroduced in older neuroblasts, which then resumed the generation of early-born GMCs. Another similarity between the Ikaros misexpression results in late RPCs and the misexpression experiments of Hunchback in Drosophila neuroblasts is that temporal competence appears to be independent of proliferation capacity. In both cases, misexpression of Ikaros or Hunchback increased early-born cell type production at the expense of late-born cell types, without increasing clone size significantly. Despite these similarities with Hunchback misexpression experiments, there are some differences. In Drosophila, young neuroblasts that are forced to continually express Hunchback generate exclusively early-born GMCs. In contrast, early E13 RPCs that are forced to continually express Ikaros give rise to more early-born neurons but can still progress to generate some late-born neurons such as bipolar cells. These results suggest that downregulation of Ikaros expression is not required for RPCs to progress to a late temporal competence stage in which they can generate bipolar cells. An attractive possibility to explain this result is that the expression of a late temporal competence regulator is required for production of late-born neurons, and this factor can function even in the presence of Ikaros. Interestingly, however, continuous expression of Ikaros from E13 RPCs or misexpression of Ikaros in P1 RPCs either dramatically reduces, or completely abolishes, Müller cell production. These results are very similar to Hunchback overexpression experiments in flies and suggest that downregulation of Ikaros is required for RPCs to progress to the competence stage in which they can generate Müller cells. Thus, although downregulation of Ikaros is not required for production of all late-born retinal cell types, its downregulation appears to be a requirement for RPCs to progress to the last temporal competence stage in which they can generate Müller cells (Elliott, 2008).

The mechanism by which Ikaros regulates temporal competence in RPCs remains unclear. During hematopoiesis, Ikaros is thought to act as a transcriptional repressor by associating with the histone deacetylase (HDAC)-containing complexes such as NuRD and Sin3. However, Ikaros can also function as a potentiator of gene expression in T cell development, apparently by recruiting Swi/Snf chromatin-remodeling complexes to specific cell-fate-decision genes (Harker, 2002; Koipally, 2002). A recent study has shown that a switch in subunit composition of neural SWI/SNF-like chromatin remodeling complexes accompanies the developmental transition from neural stem cells to committed neuronal lineages. It will be interesting to determine whether Ikaros might be involved in this switch. Based on these previous studies, it is tempting to speculate that Ikaros might function to regulate chromatin conformation around genes involved in cell-fate specification and differentiation in the retina. Interestingly, a recent bioinformatics study identified a potential functional role for an Ikaros motif in retina-specific genes (Nelander, 2005), and future experiments should help determine whether these genes are under the control of Ikaros in RPCs and in differentiated retinal cell types. If Ikaros acts as a regulator of gene expression in RPCs, it could either repress the expression of genes required for late-born cell type production or promote the expression of genes required for early-born cell type production. The results are consistent with the latter possibility. Indeed, it was found that Ikaros misexpression in late RPCs increases expression of Prox-1, a homeodomain transcription factor that is necessary and sufficient for horizontal cell specification. The finding that Ikaros misexpression does not affect Math5 and Islet-1 expression is not surprising, as these genes are thought to function in postmitotic cells, whereas Ikaros apparently functions in dividing RPCs to confer early temporal competence. The induction of Prox-1 expression by Ikaros is consistent with this idea, as Prox-1 is expressed and functions in dividing RPCs to control horizontal cell genesis. It will be interesting to determine whether other genes are induced by Ikaros misexpression in late RPCs, as they would constitute prime candidates to instruct dividing RPCs to take on early-born cell fates (Elliott, 2008).

The finding that the number of proliferating cells is reduced transiently at E13 in ikaros−/− retinas suggests that RPCs either slow down or stop their cell cycle during the period when Ikaros is required to specify temporal competence, but then resume normal proliferation once the RPCs no longer require Ikaros. This is an interesting observation as it suggests that temporal competence is specified independently of cell-cycle progression, since the progression to late temporal competence stage still occurs normally in the ikaros−/− retinas, despite reduced proliferation in early RPCs. Drosophila neuroblasts can also apparently specify temporal identity independently of cell-cycle progression. In the neuroblast lineage, although the Hunchback-to-Krüppel transition requires cytokinesis, the Krüppel-to-Pdm1-to-Castor transitions occur normally in G2-arrested neuroblasts. A similar cell-cycle independent differentiation timing mechanism has been proposed for the oligodendrocyte lineage, which differentiate faster, after fewer divisions, when their cell cycle is slowed down by cooling to 33°C. It will be interesting to directly determine whether the transition from Ikaros-positive to Ikaros-negative RPCs is dependent on cell-cycle progression (Elliott, 2008).

Because other Ikaros family members are also expressed in the developing retina, the possibility cannot be excluded that other members act together with Ikaros to regulate temporal competence. Because mouse knockouts of eos and pegasus have not yet been generated, it remains unknown whether these genes can compensate for Ikaros loss of function. As early-born cell types are not completely missing in the ikaros−/− retina, it appears that this is a likely possibility. Alternatively, Ikaros might function together with another yet unidentified factor that can partially compensate for Ikaros inactivation. Some combinatorial code of Ikaros family member expression in subtypes of RPCs might also function to regulate temporal competence more precisely. At this time, however, it remains unknown whether a particular RPC can express different combination of Ikaros family members. The generation of specific antibodies should help resolve this issue (Elliott, 2008).

Are all RPCs born equal and then 'learn' their temporal competence from their environment, or are they intrinsically preprogrammed to change their competence to generate particular cell types over time? Several lines of evidence now support the latter possibility. (1) RPCs are heterogeneous and do not necessarily have the same temporal competence at any specific time. For example, at mid to late embryonic stages, some RPCs are still competent to generate early-born cell types, whereas others have lost this competence and acquired the competence to generate late-born retinal cell types. Based on these observations, it is difficult to imagine how a single environmental signal could provide the temporal cues for a population of heterogeneous RPCs. The finding that Ikaros is expressed in only a sub-population of RPCs at mid stages of retinogenesis suggests a potential molecular explanation for this heterogeneous temporal competence of RPCs. (2) Even when isolated in clonal-density cultures, RPCs can give rise to clones that contain the same number of cells and the same proportion of cell types as clones that develop in vivo. As the in vitro and in vivo environments are very different, this suggests an important role for intrinsic developmental programs in the regulation of temporal competence. (3) There is now evidence for reproducible lineages giving rise to specific combinations of cell types in the vertebrate retina, suggesting that lineage-dependent fate decisions contribute to changes in temporal competence. Together, these observations suggest that cell-intrinsic mechanisms act in RPCs to control temporal competence, and the data indicate that Ikaros is one of these potential intrinsic regulators. Based on these results, the following model is proposed. In large multipotent lineages, Ikaros-positive RPCs constitute the beginning of the lineage and give rise to Ikaros-negative RPCs at the end of the lineage. Expression of Ikaros in early RPCs confers the competence to generate early-born cell types by inducing expression of genes involved in early-born cell fate specification, whereas Ikaros-negative RPCs loose this early temporal competence (Elliott, 2008).

In conclusion, these findings point to Ikaros as a key regulator of early temporal competence, suggesting a general strategy for the regulation of chronological order of cell birth from Drosophila to mammals. The results also have potential implications for cell replacement therapies, since it has been shown that re-expression of a single gene into late, lineage-restricted RPCs, can turn them back into RPCs with the differentiation potential of early multipotent RPCs, without resetting their proliferation potential. This suggests that, in theory, lineage-restricted neural progenitors could be manipulated to behave more like 'stem cells' as far as their differentiation potential is concerned, but without the inconvenience of indefinite cell proliferation, which can lead to tumor formation (Elliott, 2008).

Ikaros promotes early-born neuronal fates in the cerebral cortex

During cerebral cortex development, a series of projection neuron types is generated in a fixed temporal order. In Drosophila neuroblasts, the transcription factor hunchback encodes first-born identity within neural lineages. One of its mammalian homologs, Ikaros, was recently reported to play an equivalent role in retinal progenitor cells, raising the question as to whether Ikaros/Hunchback proteins could be general factors regulating the development of early-born fates throughout the nervous system. Ikaros is also expressed in progenitor cells of the mouse cerebral cortex, and this expression is highest during the early stages of neurogenesis and thereafter decreases over time. Transgenic mice with sustained Ikaros expression in cortical progenitor cells and neurons have developmental defects, including displaced progenitor cells within the cortical plate, increased early neural differentiation, and disrupted cortical lamination. Sustained Ikaros expression results in a prolonged period of generation of deep layer neurons into the stages when, normally, only late-born upper layer neurons are generated, as well as a delayed production of late-born neurons. Consequently, more early-born and fewer late-born neurons are present in the cortex of these mice at birth. This phenotype was observed in all parts of the cortex, including those with minimal structural defects, demonstrating that it is not secondary to abnormalities in cortical morphogenesis. These data suggest that Ikaros plays a similar role in regulating early temporal fates in the mammalian cerebral cortex as Ikaros/Hunchback proteins do in the Drosophila nerve cord (Alsiö, 2013).

hunchback: Biological Overview | Regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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