The observed expression pattern of aly mRNA is consistent with the earliest known phenotype of aly mutant testes: failure to initiate transcription of a set of genes in early primary spermatocytes. In wild-type testes, the aly transcript is detected at high levels in early primary spermatocytes by RNA in situ hybridization using an antisense RNA probe generated from a cDNA clone. The level of expression decreases as the spermatocytes mature, and is undetectable in meiotic cysts. No staining was observed in the most apical region of the testis, which contains mitotically proliferating cells (White-Cooper, 2000).
The lin-9 gene of C. elegans has been identified as part of a genetic pathway that antagonizes the EGFR-Ras-MAPK-based induction of vulval cell fate during larval development. The lin-9 (SynMuvB) pathway is postulated to involve signalling between the hypodermis and the vulval precursor cells, based on mosaic analysis with different genes in the pathway. Many components of the pathway have now been shown to be nuclear; some ubiquitously expressed, e.g., TAM-1 and LIN-53, others restricted to the vulval precursor cells and not hypodermis, e.g. LIN-36, and still others restricted to the hypodermis and not vulval precursor cells, e.g. LIN-13. The location in the cell at which LIN-9 acts, or even in which cell the protein is required is still unknown. How the lin-9 pathway interacts with the EGFR-Ras-MAPK pathway also remains unknown. The lin-9 pathway may act directly by inhibiting one of the components of the EGFR-Ras-MAPK cascade, or in parallel, perhaps controlling transcription of downstream targets of MAPK activation. To explore how aly, and by analogy lin-9 and other aly homologs, may act, the subcellular distribution of Aly protein was examined (White-Cooper, 2000).
Indirect immunofluorescence using an antibody raised against a bacterially expressed Aly protein has revealed that Aly is nuclear, and mostly localized on the chromatin of primary spermatocytes, consistent with the predicted nuclear localization signals in the protein sequence. The protein is not detected in the nuclei of the somatic cyst cells that surround each cyst of 16 germ cells. This result was expected from Northern blot analysis, that indicated the aly transcript is germline dependent. The subcellular localization of Aly protein depends on the stage of spermatocyte growth. Staining of wild-type testes by whole-mount immunohistochemistry revealed that in very early primary spermatocytes Aly protein is both nuclear and cytoplasmic. When the cysts start to mature, Aly protein becomes predominantly nuclear and is concentrated on chromatin as seen by immunofluorescence staining of cells at this developmental stage in squashed preparations. Aly protein staining becomes weaker as the primary spermatocytes mature and is undetectable in cells undergoing the meiotic divisions and all stages thereafter. Aly protein is not detected at the very apical tip of the testis where spermatogonia are undergoing mitotic amplification divisions, as expected from the lack of aly mRNA in those cells (White-Cooper, 2000).
The shift of Aly protein from cytoplasm to nucleus in early primary spermatocytes in wild-type testes suggests that the nuclear localization of the protein may be regulated, perhaps in response to a signalling event. In testes from a loss-of-function allele, the defective Aly protein is exclusively cytoplasmic, both in early and later primary spermatocytes, supporting the possibility that the cytoplasmic form of Aly is inactive (White-Cooper, 2000).
The Aly protein migrates as a 68 kDa doublet in polyacrylamide gels. Western blot analysis shows that the 68 kDa doublet is present in wild-type, and in can, mia and sa mutant testes, but not in null alleles. The Aly doublet is detected in testes from alyz3-1393 homozygotes. The upper band of the doublet is consistently weaker than the faster migrating band, but the relative abundance of the two isoforms is somewhat variable. The slower migrating isoform is relatively more abundant in testes that contain arrested cells, suggesting that there may be a stage dependent modification of the protein. A second band is sometimes detected at about 90 kDa; this band does not represent the Aly protein since it is not consistently present in wild-type samples, and is also occasionally detected in transcript null allele aly5. Additionally this band was not detected by serum from the second rabbit immunized with the same fusion protein. The aly1 allele is somewhat temperature sensitive: whereas most cysts arrest as primary spermatocytes, a few cysts complete the meiotic divisions and proceed through some differentiation in homozygous males raised at 18°C. In contrast, homozygous males raised at 25°C show a spermatocyte arrest phenotype indistinguishable from that of the null alleles (Lin, 1996). The 68 kDa Aly doublet is present in testes from aly1 homozygotes raised at 18°C, but not in testes extracts from aly1 males raised at 25°C. Consistent with this Western blotting result, no staining above background levels was detected by whole-mount immunohistochemistry in testes of aly1 males reared at 25°C, whereas strong staining was observed in testes of aly1 males reared at 18°C. In testes from aly1 males reared at 18°C testes, as in wild type, Aly protein is first detected throughout the nucleus and cytoplasm in early primary spermatocytes. In slightly older spermatocytes, the aly1 protein shows strong localization on the chromatin, where it persists in the arrested cells that accumulate at 18°C. Aly protein is not detected in the occasional cysts where meiotic divisions have occurred. The localization of Aly protein in can1 mutant testes and in mia and sa1 is essentially the same as in aly1 reared at 18°C, except that no meiotic or post-meiotic cysts are present (White-Cooper, 2000).
Several of the aly mutant alleles have sequence alterations that alter the predicted protein, confirming that the transcription unit identified from the aly region is indeed the aly gene. In addition to the eight published alleles (Lin, 1996), four new EMS-induced aly alleles that fail to complement aly5 and one another, were generated in a large scale mutagenesis screen. The P-element hybrid dysgenesis-induced allele aly5 has an insertion of the transposon Hobo associated with a 10 bp duplication of the genomic DNA within the predicted open reading frame. The EMS induced alleles aly2 and alyz3-3504 have nonsense mutations that would truncate the predicted Aly protein at amino acid positions 414 and 189, respectively. In both alyz3-4302 and alyz3-4307 the final eight bases of the second intron were replaced with a different sequence of six bases, deleting the splice acceptor site. If this lesion results in failure to splice out the intron, the resulting mRNA would encode a truncated protein containing 92 amino acids of the normal protein, followed by a novel sequence of 36 amino acids. The EMS induced alyz3-1393 allele has a missense mutation such that Val150 is changed to glutamic acid. The four new EMS alleles were induced on a different background chromosome from the previously described alleles. They shared several silent polymorphisms and four polymorphisms that lead to conservative amino acid changes compared with the previously determined genomic sequence and the other alleles. These were N62K, D66E, N220K and E518D (White-Cooper, 2000).
The C. elegans homolog of aly, lin-9, antagonizes induction of vulval differentiation by the EGFR/Ras/MAPK pathway. To test whether aly might act to directly downregulate Ras pathway signalling in the Drosophila testis, the phosphorylation (and thus the activation) state of MAP kinase was examined in wild-type and aly mutant testes. Drosophila has a single homolog of the ERK MAP kinase, encoded by the rolled gene. The di-phosophorylated active form of this 44 kDa protein is recognized by the dp-ERK antibody raised against the phosphorylated activation loop of vertebrate ERK1 and ERK2. No differences in the overall level or phosphorylation state of ERK/Rolled protein were detected by Western blot analysis of testis extracts from aly mutant males compared with wild type. This suggests that aly, and by analogy lin-9, acts downstream of MAP kinase activation, or in a separate pathway, to control target gene transcription (White-Cooper, 2000).
aly is required for cyclin B and twine expression in primary spermatocytes. The level of cyclin B protein is reduced significantly in aly mutant testes compared to wild type. Although low levels are still detected by Western blotting of testis extracts, the three different aly alleles tested all cause a similar marked reduction in cyclin B protein level when compared to wild-type testis extracts. Cyclin B protein appears to be expressed at normal levels in can, mia and sa mutant testes, indicating that these genes might influence cell cycle progression through a biochemically distinct pathway from aly. aly is not required for the expression of all cell cycle genes, since both Cyclin A and Cdc2 proteins are expressed at high levels in aly testes (White-Cooper, 1998).
The wild-type function of aly is required for the transcription or accumulation of both cyclin B and twine mRNAs. In wild-type testes, cyclin B transcripts are detected by in situ hybridization at low levels in the mitotic cells at the apical tip, but are not detected at the position where cells undergo pre-meiotic S-phase. Cyclin B message is abundant throughout the primary spermatocyte stage, accumulating to very high levels in mature primary spermatocytes. Cyclin B mRNA is detected in meiotically dividing cells, but is absent from the post-meiotic stages. In aly mutant testes cyclin B transcript is detected in the mitotic cells at a level comparable to wild type. However cyclin B mRNA is not detectable in aly mutant primary spermatocytes. The expression pattern of twine mRNA in wild-type testes is similar to that of cyclin B, except that twine mRNA is not detected in the mitotic cells at the apical tip of the testis. In aly mutant testes twine mRNA is not detected by in situ hybridization. can, mia and sa mutant testes express both twine and cyclin B mRNA at normal levels (White-Cooper, 1998).
The lack of cyclin B and twine mRNAs in aly mutant spermatocytes is not due to a general defect in transcription, since cyclin A and other messages are abundant in the mutant spermatocytes. In wild-type testes cyclin A mRNA is detected at low levels in both mitotic and S-phase cells at the apical tip of the testis. Cyclin A shows high levels of expression throughout the primary spermatocyte stage, with the message disappearing during meiosis. In aly, can, mia and sa mutant testes cyclin A mRNA is expressed in mitotic and S-phase cells and spermatocytes as in wild type. However cyclin A transcript levels remain high in the arrested mature primary spermatocytes, only disappearing at the base of the testes where the cells finally degenerate, suggesting that the wild-type function of aly, can, mia and sa is required directly or indirectly for the normal shut down of transcription and/or turnover of cyclin A message at meiosis (White-Cooper, 1998).
The timing of entry into the meiotic divisions in wild type may be controlled by post-transcriptionally regulated accumulation of Cyclin B and Twine protein. Although Cyclin B mRNA is expressed at high levels in early spermatocytes, the accumulation of Cyclin B protein is delayed in wild-type testis until the late primary spermatocyte stage. Cyclin B protein begins to accumulate in the cytoplasm of late primary spermatocytes as chromosome condensation is initiated just before the entry into the first meiotic division and is present at high levels in pro-metaphase I cells. Cyclin B protein is degraded at the metaphase to anaphase transition of meiosis I, and reaccumulates in preparation for the second meiotic division. In aly mutant testis Cyclin B protein is detected in the mitotic cells at the apical tip, but does not accumulate in the mutant spermatocytes. Twine protein is likewise delayed until just before the entry into the first meiotic division, days after the transcript is first detected. Neither protein nor mRNA is detected in an aly mutant background (White-Cooper, 1998).
aly, can, mia and sa are required for accumulation of Twine protein but not twine transcript in late primary spermatocytes. These three meiotic arrest genes are required for the expression of fuzzy onions, whose product is required for mitochondrial fusion in early spermatids. Similarly, severe reductions in message level are observed for Male-specific RNA 87F (Mst87F), a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. gonadal, which is expressed as two differentially terminated variants in the testis, shows dramatic reduction of both variants in can, mia and sa mutant testis. It is proposed that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. It is also proposed that control of translation or protein stability regulates entry into the first meiotic division. It is suggested that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of Twine protein, thus coordinating meiotic division with onset of spermatid differentiation (White-Cooper, 1998).
aly, can, mia and sa are required for the transcription in primary spermatocytes of several genes involved in postmeiotic spermatid differentiation. The fuzzy onions (fzo) gene product is required for mitochondrial fusion in early haploid spermatids. fzo transcription initiates in early primary spermatocytes and the mRNA is present throughout the growing stages in wild type. fzo mRNA is greatly reduced in aly, can, mia and sa testes, despite the presence of primary spermatocytes in the mutant tissue. Message levels in mutant testes ranged from undetectable to low levels under conditions in which the in situ hybridization signal in wild type was strong, indicating that transcription may be reduced to a low basal level, but not entirely turned off. Similarly severe reductions in message level were observed for Mst87F, a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. Several other genes also showed dramatic reductions in transcript levels in meiotic arrest mutant testes when assayed by in situ hybridization. Reduced transcript levels in aly, can, mia and sa spermatocytes are not due to a general defect in transcription since a number of genes were transcribed at normal levels in mutant spermatocytes (White-Cooper, 1998).
Comparison of the effects of aly, can, mia and sa mutations on transcript levels suggests that genes normally transcribed in primary spermatocytes can be grouped into three classes. The transcription of the first (general) class of genes is independent of aly, can, mia and sa function. The second (meiotic) class of genes requires the normal function of aly, but not can, mia or sa. Expression of the third (spermiogenic) class of genes requires the wild-type activity of all four of the meiotic arrest genes (White-Cooper, 1998).
Although mutations in aly, can, mia and sa appear to cause arrest at the same point in the G2-M transition of meiosis I (Lin, 1996), the genes apparently control cell cycle progression by different biochemical mechanisms. aly, but not can, mia or sa, is required for the transcription of cyclin B and twine. The wild-type function of can, mia and sa instead appears to be required either to allow translation of twine message or to stabilize twine protein in mature primary spermatocytes. In either case aly, can, mia or sa mutations presumably cause cell cycle arrest at the same point in the G2-M transition, due to lack of active Cdc2/Cyclin B kinase complex. Cdc2 protein resolves into two distinct isoforms in Western blots. The slower migrating form, which is enriched compared to the faster migrating form in twine mutant testes, has been identified as a hyperphosphorylated, inactive form. The slower migrating form of Cdc2 also appears to be enriched compared to the faster migrating form in aly, can and sa. Production of Twine protein, but not Cyclin B, is dependent on can, mia and sa. Thus, although both Cyclin B and Twine protein accumulation are regulated posttranscriptionally in wild-type testes, the genetic control of their expression is different (White-Cooper, 1998).
It is proposed that can, mia, and sa act together or in a pathway to activate a tissue and stage-specific transcription program in primary spermatocytes, and that failure to initiate this program results in a global block in spermatid differentiation due to the lack of an array of gene products. The wild-type functions of can, mia and sa appear to be required for transcription in primary spermatocytes of a set of genes encoding products involved in post-meiotic spermatid differentiation. Transcription of these genes is initiated early in the primary spermatocyte stage, several days before the arrest point of the meiotic arrest mutants. Therefore the lack of transcription of this set of genes is likely to be a cause of the arrest rather than merely a downstream consequence (White-Cooper, 1998).
Of the eight genes identified so far that depend on can, mia and sa for transcription, some information about the function or time of action of the gene products is available for six. The product of the fzo gene is required for mitochondrial fusion, a post-meiotic event. Although fzo is transcribed in primary spermatocytes, the protein is not detected by immunofluorescence staining of testes until late in meiosis II. Expression of Mst87F, of four related genes at 84D and two related genes at 98C is regulated translationally. Although mRNAs are transcribed in primary spermatocytes, the proteins do not accumulate until days after the meiotic divisions. All of these genes encode proteins that are components of a structure in the sperm tail. Similarly the translation of janB and dj mRNAs is delayed until several days after the completion of meiosis. While the function of LanB is unknown, Dj is thought to serve a dual function; it is found in the sperm tail, but sequence comparisons suggest a possible role as a chromatin component (White-Cooper, 1998).
It is proposed that aly acts upstream of can, mia and sa, possibly to control expression or activation of components of the transcription machinery that drives expression of the spermatid differentiation genes. Wild-type function of aly is required for accumulation of at least three different mRNAs in primary spermatocytes that are not dependent on can, mia and sa, suggesting that aly is able to act independently of can, mia and sa. However aly mutations cause the same phenotype, and fail to express the same set of spermatid differentiation genes, as can, mia and sa mutations. This strongly suggests that aly might affect spermatid differentiation through an effect on expression or activity of either can, mia or sa. The block in meiotic cell cycle progression in can, mia and sa mutant testes could be due to a cross-regulatory mechanism that serves to coordinate meiosis and the spermatid differentiation program. It is proposed that a gene or genes transcribed in primary spermatocytes under the control of can, mia and sa encode(s) product(s) required either directly or indirectly to relieve the translational repression of twine message or to stabilise the Twine protein. Such a cross-regulatory mechanism between the pathways leading to spermatid differentiation and meiosis could serve in wild type to ensure that spermatocytes do not enter meiotic division until the proposed transcription program for post-meiotic spermatid differentiation genes has been successfully initiated. A late cross-regulatory mechanism may also explain why mutations that block spermatid differentiation but not meiotic cell cycle progression have not yet been isolated (White-Cooper, 1998).
The signal that activates the G2/M transition in male meiosis could be accumulation of the product of the proposed crossregulatory gene to a threshold sufficient to allow expression of twine protein. Alternatively, timing of the G2/M transition for meiosis I could be set via a less direct mechanism, involving the proposed cross regulatory gene, but not set directly by its level. For example accumulation of Twine protein may require an extrinsic signal received or transduced by a gene or genes controlled by the can, mia and sa transcription program. The degenerative spermatocyte (des) gene, encoding a novel protein that may be membrane associated, is a possible candidate for a component of such a signalling pathway (White-Cooper, 1998).
Mutations in des, like aly, can, mia and sa, cause a block in both meiotic cell cycle progression and the onset of spermatid differentiation. des mutations are also semi-lethal, suggesting a role for this gene outside the testis. Pole cell transplantation experiments also implicate extracellular signals in the regulation of meiotic progression and spermatid differentiation. Male (XY) germ cells transplanted into a female (XX) host initiate spermatogenesis in the host ovary. However the transplanted cells arrest as primary spermatocytes and fail to undergo the meiotic divisions or initiate spermatid differentiation. Part of the program of spermatid differentiation regulated by can, mia and sa could act to destabilize or turn off transcription of certain messages expressed in primary spermatocytes but not needed or deleterious after meiosis. In wild-type testes, cyclin A mRNA is present in primary spermatocytes but not detectable in post-meiotic cells. Loss of cyclin A mRNA could be an important mechanism to prevent DNA replication during meiosis II or in haploid spermatids. In wild-type testes Cyclin A protein is degraded at metaphase I and is not resynthesised for the second meiotic division. In males mutant for aly, can, mia or sa, cyclin A message and Cyclin A protein (Lin, 1996) persist in the arrested primary spermatocytes, suggesting that the wild-type function of the meiotic arrest genes and/or the transcription program they control is required directly or indirectly for disappearance of cyclin A message midway through spermatogenesis. A similar effect on message stability was seen for all of the other pre-meiotic genes tested (White-Cooper, 1998).
Yeast meiosis bears striking similarities to Drosophila spermatogenesis. In both cases S phase is followed by an extended G2 phase, characterized by high levels of transcription of genes required for meiosis and subsequent differentiation into spores or sperm. Many yeast mutants, including certain alleles of cdc2 in S. pombe, are analogous to twine, in that the mutant cells fail to complete one or both of the meiotic divisions, but still differentiate into spores. However meiosis and differentiation are coordinated, since mutations in some genes, mei4 in S. pombe or NDT80 in S. cerevisiae, like the meiotic arrest mutants of Drosophila, block both the meiotic division cycle and subsequent differentiation. The failure to accumulate both cell cycle and spermiogenesis mRNAs in aly mutants suggests that there may be parallels in the genetic control of animal spermatogenesis and yeast sporulation (White-Cooper, 1998 amd references therein).
Hybrid males resulting from crosses between closely related species of Drosophila are sterile. The F1 hybrid sterility phenotype is mainly due to defects occurring during late stages of development that relate to sperm individualization, and so genes controlling sperm development may have been subjected to selective diversification between species. It is also possible that genes of spermatogenesis experience selective constraints given their role in a developmental pathway. The molecular evolution was examined of three genes playing a role during the sperm developmental pathway in Drosophila at an early (bam), a mid (aly), and a late (don juan: dj) stage. The complete coding region of these genes was sequenced in different strains of Drosophila melanogaster and Drosophila simulans. All three genes showed rapid divergence between species, with larger numbers of nonsynonymous to synonymous differences between species than polymorphisms. Although this could be interpreted as evidence for positive selection at all three genes, formal tests of selection do not support such a conclusion. Departures from neutrality were detected only for dj and bam but not aly. The role played by selection is unique and determined by gene-specific characteristics rather than site of expression. In dj, the departure was due to a high proportion of neutral synonymous polymorphisms in D. simulans, and there was evidence of purifying selection maintaining a high lysine amino acid protein content that is characteristic of other DNA-binding proteins. The earliest spermatogenesis gene surveyed, which plays a role in both male and female gametogenesis, was bam, and its significant departure from neutrality was due to an excess of nonsynonymous substitutions between species. Bam is degraded at the end of mitosis, and rapid evolutionary changes among species might be a characteristic shared with other degradable transient proteins. However, the large number of nonsynonymous changes between D. melanogaster and D. simulans and a phylogenetic comparative analysis among species confirms evidence of positive selection driving the evolution of Bam and suggests an yet unknown germ cell line developmental adaptive change between these two species (Civetta, 2006).
Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported in this study aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. A collection of 97 recessive lethal P-lacZ enhancer trap lines were screened for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) were identified that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration (McClure, 2008).
There are three different mechanisms that organisms use to re-grow and replace lost or damaged body parts, and often, more than one mechanism can function within different tissues of the same organism. Muscle and bone, for example, repair themselves by activating a resident stem cell population, while the liver regenerates by compensatory proliferation of normally quiescent differentiated cells. Appendage/fin regeneration in lower vertebrates occurs by a process termed epimorphic regeneration, which proceeds in three distinct stages: (1) wound healing and migration of the surrounding epithelial cells to form the wound epidermis, (2) formation of the regeneration blastema -- a mass of undifferentiated and proliferating cells of mesenchymal origin and (3) regenerative outgrowth and pattern re-formation. Whether these diverse modes of regeneration share a common molecular and genetic basis is not known (McClure, 2008).
Regeneration in the Drosophila imaginal discs, the primordia of the adult fly appendages, closely parallels epimorphic limb/fin regeneration in lower vertebrates. Cells in the imaginal discs are rigidly determined to form specific adult structures (e.g., legs and wings) by the third larval instar. If the discs are fragmented at this time and cultured in vivo, they will regenerate. Disc regeneration begins 12 h after wounding, when transient heterotypic contacts are made between peripodial (squamous epithelium) and columnar cells (disc proper) near the cut edges of the wound. These initial contacts involve microvilli-like extensions and provide temporary wound closure. Then, approximately 24 h after wounding, homotypic cell contacts (between columnar or between squamous cells) are made involving the close apposition of cell membranes and cellular bridges, which eventually (48 h after wounding) restore the physical continuity of the disc. Before and during wound healing, cell division is randomly distributed throughout the disc. However, once completed (36-48 h after wounding), division is observed only in cells near the wound site. These cells are known as the regeneration blastema. Thus, like appendage regeneration in lower vertebrates, disc regeneration involves wound healing followed by blastema formation (McClure, 2008).
Blastema cells are responsible for the regeneration and repatterning of the entire missing disc fragment. Thus, these cells exhibit remarkable developmental plasticity. For example, in anterior- only leg disc fragments, some blastema cells will switch to posterior identity and establish a novel posterior compartment in the regenerate. This anterior/posterior conversion occurs during heterotypic wound healing, when hedgehog (hh)- expressing peripodial cells induce ectopic engrailed (en) expression in the apposing anterior columnar cells. In addition, the disc blastema, like its vertebrate counterpart, is able to form a normal regenerate (complete leg disc and adult leg) when isolated from the remaining disc fragment. Regenerative plasticity is also observed when a few blastema cells switch fate to that of another disc type (e.g., leg-to-wing), in a phenomenon known as transdetermination. Transdetermination events are closely associated with regenerative disc growth. Clonal analysis, for example, has shown that blastema cells first regenerate the missing disc structures, and only then, are they competent to transdetermine (McClure, 2008).
Little is known about how the regeneration blastema forms in the fragmented leg disc, although ectopic Wingless (Wg/Wnt1) expression is detected along the cut site, both prior to and during blastema formation. Wg is a developmental signal in many different tissues and animals; in flies Wg patterns all of the imaginal discs, functioning as both a morphogen and mitogen to regulate disc cell fate and growth. In lower vertebrates, Wnt ligands are key regulators of blastema formation during epimorphic regeneration. Thus, activation of Wg within the disc blastema is potentially important for regeneration. This idea is consistent with the observation that ubiquitous expression of wg during the second or third larval instars, in unfragmented leg discs, is sufficient to induce a regeneration blastema in the proximodorsal region of the disc, known as the weak point. Moreover, ubiquitous expression of wg mimics the pattern deviations associated with leg disc fragmentation and subsequent regeneration, including the duplication of ventral with concomitant loss of dorsal pattern elements and leg-to-wing transdetermination events. Thus, leg disc regeneration can be examined using two experimental protocols: fragmentation or ubiquitous wg expression. However, it is important to point out that only fragmentation-induced regeneration involves wound healing (McClure, 2008).
Precisely which molecules and signaling pathways are required for the process of regeneration remain poorly understood, partly because the organisms historically used to study regeneration (e.g., newts and salamanders) have been refractory to genetics and molecular manipulations. Recently, however, the use of new genetic techniques together with 'regeneration' model systems -- such as planarians, hydra and zebrafish have given researchers the opportunity to examine the mechanisms of regeneration and to identify the genes, proteins and signaling pathways that regulate different regenerative processes. For example, a large scale RNAi-based screen was performed to survey gene function in planarian tissue homeostasis and regeneration. Out of ~1000 genes examined, RNAi knock-down of 240 displayed regeneration-related phenotypes, including defects in wound healing, blastema formation and blastema cell differentiation. Despite these studies, however, it remains unclear whether regeneration requires only the modulation of genes expressed at the time of injury, the reactivation of earlier developmental genes and/or signaling pathways, or the activation of novel genes specific to the process of regeneration. Thus, a major interest in the field of regenerative biology is the identification of gene products that regulate blastema formation, blastema growth and regenerative cellular plasticity. A genetic screen, using wg-induced leg disc regeneration, aimed at identifying genes that regulate cellular plasticity and regeneration using Drosophila was carried out prothoracic leg discs. A collection of 97 recessive lethal P-element lacZ (PZ) insertion lines were screened for ectopic lacZ expression during wg-induced leg disc regeneration, and six genes were identified that function in wg-induced leg disc regeneration, including genes with functional ties to Wg signaling as well as chromatin remodeling proteins (McClure, 2008).
This study consisted of an enhancer trap screen designed to identify genes with changed gene expression during leg disc regeneration as well as required for regenerative proliferation and growth. The screen identified 19 genes that when heterozygous mutant (PZ/+), dominantly modify wg-induced leg-to-wing transdetermination, which serves as a functional assay for disc regeneration. Of the 19 genes, 37% are transcription factors or involved in transcriptional regulation (tai, Krh1, ken, jing, combgap (cg), rpd3 and Aly), 21% function in cell cycle regulation and growth (oho23B, S6k, polo and cycA), 10.5% play a role in protein secretion (Secβ61 and Syx13), and 31% are of other or unknown function [l(3)01629, CG30947, l(2)00248, l(3)05203, l(3)01344, Nup154]. The identification of transcription factors as the most frequent class of genes that modify wg-induced leg disc regeneration was similarly observed in a DNA microarray screen designed to identify genes enriched in leg disc cells that transdetermine to wing (Klebes, 2005). Together, these findings strongly suggest that transcription factors and their downstream targets play a prominent role in disc cell plasticity (McClure, 2008).
Using lacZ expression analyses, together with whole mount in situ hybridization experiments, the expression patterns of the 19 genes that modified wg-induced leg-to-wing transdetermination were verified. This analysis identified several different expression patterns upon wg-induced regeneration, including a loss of gene expression, ubiquitous expression and genes with expression limited to the regeneration blastema. Such observations indicate that a complex change of gene expression, both negative and positive, mediates the process of epimorphic regeneration. Six (jing, Alyi cg, rpd3, Kr-h1 and S6k) of the 19 modifiers displayed expression limited to the regeneration blastema, indicating that novel markers of regeneration and transdetermination have been identified. The blastema-specific expression patterns of jing, Aly, cg, Kr-h1, rpd3 and S6k raised the intriguing possibility that these genes may be functionally involved in the formation, cell proliferation or maintenance of the blastema during disc regeneration. Indeed, upon ubiquitous wg expression jing/+ animals rarely formed a regeneration blastema, indicating that two wild-type copies of jing are required for the initiation of the regenerative process. In contrast, Aly/+ and cg/+ animals formed a normal blastema, but only after a one-day delay. Therefore, two wild-type copies of the Aly and cg genes are required for the proper timing of regeneration. In addition, it was found that the frequency of blastema formation was reduced in rpd3/+ animals, implicating this gene in the process of regeneration. Interestingly, heterozygous mutations in all four of these genes (jing, Aly, cg and rpd3) strongly suppress wg-induced leg-to-wing transdetermination. It is speculated that the transdetermination frequency declines in these mutant animals because the initiation and/or timing of blastema formation is delayed. This idea is consistent with all previous work which has shown that blastema cells are only competent to transdetermine after they have regenerated the missing disc structures. Heterozygous mutations in Kr-h1 and S6k did not significantly alter the formation of the wg-induced regeneration blastema, however, these genes did affect regeneration-induced transdetermination. Such results suggest that Kr-h1 and S6k specifically function to modulate the cell fate changes that occur as a consequence of regeneration (McClure, 2008).
Investigations into the molecular basis of transdetermination have shown that inputs from the Wg, Decapentapelagic (Dpp) and Hedgehog (Hh) signaling pathways activate key selector genes out of their normal developmental context, such as ectopic Vg activation in the leg disc, which then drives cell-fate switches. Several of the genes identified in this screen have functional ties to Wg, Dpp and Hh signaling pathways. For example, Cg is a zinc-finger transcription factor that is required for proper transcriptional regulation of the Hh signaling effector gene Cubitus interruptus (Ci). In cg mutant wing and leg discs, Ci expression is lowered in the anterior compartment, resulting in the ectopic activation of wg and dpp and significant disc overgrowth. Another gene identified in this screen -- ken, functions in concert with Dpp to direct the development of the Drosophila terminalia. Further characterizations of whether these genes and other modifiers of transdetermination and regeneration affect Wg, Dpp and Hh expression and/or signaling may shed light on the regulation of regeneration and regeneration-induced proliferation and cell fate plasticity (McClure, 2008).
Ayyar, S., Jiang, J., Collu, A., White-Cooper, H. and White, R. A. H. (2003). Drosophila TGIF is essential for developmentally regulated transcription in spermatogenesis. Development 130: 2841-2852. 12756169
Beall, E. L., Manak, J. R., Zhou, S., Bell, M., Lipsick, J. S. and Botchan, M. R. (2002). Role for a Drosophila Myb-containing protein complex in site-specific DNA replication. Nature 420: 833-837. Medline abstract: 12490953
Beall, E. L., et al. (2004). Dm-myb mutant lethality in Drosophila is dependent upon mip130: positive and negative regulation of DNA replication. Genes Dev. 18: 1667-1680. Medline abstract: 15256498
Beall, E. L., Lewis, P. W., Bell, M., Rocha, M., Jones, D. L. and Botchen, M. R. (2007). Discovery of tMAC: a Drosophila testes-specific meiotic arrest complex paralogous to Myb-MuvB. Genes Dev. 21(8): 904-19. Medline abstract: 17403774
Beitel, G. J., Lambie, E. J., and Horvitz, H. R. (2000). The C. elegans gene lin-9, which acts in an Rb-related pathway, is required for gonadal sheath cell development and encodes a novel protein. Gene 254(1-2): 253-63. 10974557
Boxem, M., van den Heuvel, S. (2002). C. elegans class B synthetic multivulva genes act in G(1) regulation. Curr. Biol. 12(11): 906-11. 12062054
Cayirlioglu, P., Bonnette, P. C., Dickson, M. and Duronio, R. J. (2001). Drosophila E2f2 promotes the conversion from genomic DNA replication to gene amplification in ovarian follicle cells. Development 128: 5085-5098. Medline abstract: 11748144
Civetta, A., et al. (2006). Rapid evolution and gene-specific patterns of selection for three genes of spermatogenesis in Drosophila. Molec. Biol. Evol. 23(3): 655-662. 16357040
Ferguson, E. L., Horvitz, H. R. (1989). The multivulva phenotype of certain Caenorhabditis elegans mutants results from defects in two functionally redundant pathways. Genetics 123(1): 109-21. 2806880
Frolov, M. H., Huen, D. S., Stevaux, O., Dimova, D., Balczarek-Strang, K., Elsdon, M. and Dyson, N. (2001). Functional antagonism between E2F family members. Genes Dev. 15: 2146-2160. Medline abstract: 11511545
Gagrica, S., Hauser, S., Kolfschoten, I., Osterloh, L., Agami, R. and Gaubatz, S. (2004). Inhibition of oncogenic transformation by mammalian Lin-9, a pRB-associated protein. EMBO J. 23: 4627-4638. Medline abstract: 15538385
Harrison, M., Coel, C. J., Lu, X. and Horvitz, H. R. (2006). Some C. elegans class B synthetic multivulva proteins encode a conserved LIN-35 Rb-containing complex distinct from a NuRD-like complex. Proc. Natl. Acad. Sci. USA 103: 16782-16787. Medline abstract: 17075059
Hiller, M. A., Lin, T.-Y., Wood, C. and Fuller, M. T. (2001). Developmental regulation of transcription by a tissue-specific TAF homolog. Genes Dev. 15: 1021-1030. 11316795
Jiang, J. and White-Cooper, H. (2003). Transcriptional activation in Drosophila spermatogenesis involves the mutually dependent function of aly and a novel meiotic arrest gene cookie monster. Development 130: 563-573. 12490562
Jiang, J., et al. (2007). Tombola, a tesmin/TSO1-family protein, regulates transcriptional activation in the Drosophila male germline and physically interacts with Always early. Development 134: 1549-1559. Medline abstract: 17360778
Klebes, A., et al. (2005). Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes. Development 132: 3753-3765. PubMed citation: 16077094
Korenjak, M., Taylor-Harding, B., Binne, U. K., Satterlee, J. S., Stevaux, O., Aasland, R., White-Cooper, H., Dyson, N. and Brehm, A. (2004). Native E2F/RBF complexes contain Myb-interacting proteins and repress transcription of developmentally controlled E2F target genes. Cell 119: 181-193. Medline abstract: 15479636
Kornfeld, K. (1997). Vulval development in Caenorhabditis elegans. Trends Genet. 13: 55-61. 9055606
Lin, T.-Y., Viswanathan, S., Wood, C., Wilson, P. G., Wolf, N. and Fuller, M. T. (1996). Coordinate developmental control of the meiotic cell cycle and spermatid differentiation in Drosophila males. Development 122: 1331-1341. 8620860
Lu, X. W. and Horvitz, H. R. (1998). lin-35 and lin-53, two genes that antagonize a C-elegans Ras pathway, encode proteins similar to Rb and its binding protein RbAp48. Cell 95: 981-991. 9875852
Matsuura, T., Kawasaki, Y., Miwa, K., Sutou, S., Ohinata, Y., Yoshida, F. and Mitsui, Y. (2002). Germ cell-specific nucleocytoplasmic shuttling protein, tesmin, responsive to heavy metal stress in mouse testes. J. Inorg. Biochem. 88: 183-191. Medline abstract: 11803038
McClure, K. D. and Schubiger, G. (2008). A screen for genes that function in leg disc regeneration in Drosophila melanogaster. Mech. Dev. 125(1-2): 67-80. PubMed citation
Mikhaylova, L. M., Boutanaev, A. M. and Nurminsky, D. I. (2006). Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line. Proc. Natl. Acad. Sci. 103(32): 11975-80. Medline abstract: 16877538
Perezgazga, L., Jiang, J., Bolival, B., Hiller, M. A., Benson, E., Fuller, M. T. and White-Cooper, H. (2004). Regulation of transcription of meiotic cell cycle and terminal differentiation genes by the testis-specific Zn finger protein matotopetli. Development 131: 1691-1702. Medline abstract: 15084455
Solari, F. and Ahringer, J. (2000). NURD-complex genes antagonise Ras inducted vulval development in Caenorhabditis elegans. Curr. Biol. 10: 223-226. 10704416
Stevaux, O., Dimova, D., Ji, J.-Y., Moon, N. S., Frolov, M. and Dyson, N. (2005). Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target genes. Cell Cycle 4: 1272-1280. Medline abstract: 16082225
Sutou, S., Miwa, K., Matsuura, T., Kawasaki, Y., Ohinata, Y. and Mitsui, Y. (2003). Native tesmin is a 60-kilodalton protein that undergoes dynamic changes in its localisation during spermatogenesis in mice. Biol. Reprod. 68: 1861-1869. Medline abstract: 12606435
Wang, Z. and Mann, R. S. (2003). Requirement for two nearly identical TGIF-related homeobox genes in Drosophila spermatogenesis. Development 130: 2853-2865. 12756170
White-Cooper, H., Schafer, M. A., Alphey, L. S. and Fuller, M. T. (1998). Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila. Development 125: 125-134. 9389670
White-Cooper, H., Leroy, D., MacQueen, A. and Fuller, M. T. (2000). Transcription of meiotic cell cycle and terminal differentiation genes depends on a conserved chromatin associated protein, whose nuclear localization is regulated. Development 127: 5463-5473. 11076766
date revised: 30 July 2008
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.