BIOLOGICAL OVERVIEW

Dorsal closure [Images] of the Drosophila embryo provides a good example of cell differentiation and how this is usually coupled to morphogenetic events and movements that shape the embryo in the late stages of development. Half way through embryogenesis, the dorsal surface of the embryo is covered by an extraembryonic membrane, the amnioserosa, which contacts the epidermis. After cell proliferation stops, the epidermis stretches dorsally, and as it does, it encroaches upon the amnioserosa and covers, closes the existing gap. The successful completion of this event may be divided into three phases: the dorsal-ward movement of the epidermal cells; the anteroposterior stretching of the embryo, and the seaming of the dorsal epidermis over the amnioserosa. The completion of this process takes several hours and is associated with specialized behavior of the dorsal-most epidermal cells. These cells display planar polarity reflected in the arrangement of the cytoskeleton, which is essential for the normal process of dorsal closure. Dorsal cuticle puckering and dorsal holes are indicative of a defect in dorsal closure, probably dependent on cell shape changes. During dorsal closure, the epidermal cells in the dorsal region of the embryo change shape dramatically. Beginning with cells immediately flanking the amnioserosa, there is an elongation along the dorsoventral axis; this change in cell shape gradually spreads ventrally through the epidermis, causing the opposing sides of the lateral epidermis to stretch until they meet along the dorsal midline. In puckered mutants, dorsal closure takes place, but an abnormal organization of the cells at the leading edge of the epidermis results in a defective process and puckering, which gives the mutant its descriptive name (Ring, 1993).

There are several mutations that disrupt the process of dorsal closure. In basket (bsk) and hemipterous (hep) mutants, dorsal closure fails and a hole remains in the embryo's dorsal cuticle. hep encodes a Drosophila homolog of MKK7, a kinase that regulates Jun N-terminal kinase (JNK); bsk encodes a Drosophila homolog of JNK. The involvement of the JNK pathway in dorsal closure is further supported by the observation that mutants for Djun, a target of DJNK signaling, fail to close dorsally, and that ectopic expression of a dominant-negative form of Drac1 (DN-Drac1), the Drosophila homolog of Rac1, also leads to the same dorsal closure defects (Martin-Blanco, 1998 and references).

In bsk, hep, and Djun mutants, cell shape changes are disrupted. It is interesting that the expression of dominant negative Drac1 generates phenotypes that are similar to those of hep and bsk mutants, suggesting that Drac1 might initiate signaling through this cascade. Drac1 seems to be involved in the control of the cell cytoskeleton. In wild-type embryos, the onset of dorsal closure coincides with a specific subplasmalemmal accumulation of nonmuscle myosin at the leading edge of the dorsal-most epidermal cells. It is likely that nonmuscle myosin contributes to the elongation of these cells by participating with actin in forming a dorsal constriction. In DN-Drac1 embryos, nonmuscle myosin and actin in epidermal cells are strongly reduced. These changes in the cytoskeleton are also evident in Djun mutant embryos (Martin-Blanco, 1998 and references).

The effects of the absence of puc (pucE69) and its overexpression were compared on the levels and organization of the actin cytoskeleton and nonmuscle myosin. In pucE69 mutants, the expression of myosin and actin does not change dramatically in the periphery of the cells in lateral regions of the embryo, but these proteins fail to accumulate along the leading edge of the epidermis. Cell shape changes proceed almost normally. In contrast, epidermal cells of embryos overexpressing puc fail to change their shapes and accumulate low levels of spatially disorganized myosin at the leading edge. In these embryos, actin fails to be expressed in the amnioserosa and its levels are reduced in the epidermis. Actin and myosin tend to form clumps in these epidermal cells. Thus puc is an essential component in the control of the different steps of dorsal closure progression; it acts by modulating the apical accumulation of actin and myosin at the leading edge. These results correlate with those of the effects of overexpression of DN-Drac1 and Djun mutants, and further suggest a role for Puc in the control of JNK activity over the cytoskeleton (Martin-Blanco, 1998).

The expression of most members of the VH-1 family of PTPs is subject to tight transcriptional regulation. The same is likely to be true for puckered because it displays dynamic patterns of expression in the embryo and the adult. During and after germ band shortening, puc is expressed in the dorsal-most epidermal cells that play a leading role in the process of dorsal closure. In embryos mutant for the JNKK encoded by hemipterous or for the JNK encoded by basket, there is no puc expression in these cells, and dorsal closure fails in a manner similar to that produced by the overexpression of puc (Glise, 1995 and Riesgo-Escovar, 1996). These results suggest a model in which signaling through Hep and Bsk leads to the expression of effectors of dorsal closure and a regulator encoded by puc. The function of the latter is to exert a negative feedback on the signaling cascade of hep and bsk. Interestingly, in mutants for Djun (a likely target of JNK activity), puc expression is absent at the leading edge of the epidermis (N. Perrimon, pers. comm. to Martin-Blanco, 1998), suggesting a transcriptional link between the activity of the JNK encoded by bsk and the expression of puc. Thus, the activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities during dorsal closure. In this system, Puckered seems to act in a feedback loop. Puckered expression is upregulated by DJun and in turn, Puckered inactivates MAPK, whose function is the activation of DJun downstream of Rac signaling (Martin-Blanco, 1998).

puc plays a role in follicle cell morphogenesis during oogenesis. The follicle cells (FCs) form an epithelial sheet around a cyst of germ cells (the oocyte and 15 nurse cells) that then develops as an egg chamber unit. Late in oogenesis, specific groups of follicle cells become distinct. During stage 9, most follicle cells migrate posteriorly to cover the oocyte surface in a columnar epithelium. Approximately 50 cells -- the nurse cell FCs (NCFCs) -- flatten to form a squamous domain over the nurse cells. Subsequently, subgroups of the columnar FCs change shape and migrate. Beginning in stage 10B, the centripetally migrating FCs (CMFCs) move inward between the oocyte and nurse cells. These cells will create anterior eggshell structures such as the operculum. The main body FCs (MBFCs) stretch during stage 11, as the oocyte rapidly enlarges with the transfer of the nurse cell contents, a process called nurse cell dumping. During stages 12 through 14, two dorsal anterior groups of FCs migrate anteriorly to create the dorsal appendages. The posterior pole FCs (PPFCs) include cells that produce the aeropyle of the eggshell. The FCs that will create a specific eggshell structure are fated earlier in oogenesis, as revealed by specific patterns of gene expression (Dobens, 2001 and references therein).

PUC mRNA accumulates preferentially in the CMFSs and cells of the elongating dorsal appendages. Proper levels of Puc activity in the follicle cells are critical for the production of a normal egg: either reduced or increased Puc activity results in incomplete nurse cell dumping and aberrant dorsal appendages. Phenotypes associated with puc mutant follicle cells include altered DE-cadherin (Shotgun) expression in the follicle cells and a failure of nurse cell dumping to coordinate with dorsal appendage elongation, leading to the formation of cup-shaped egg chambers. The JNK pathway target A251-lacZ shows cell-type-specific differences in its regulation by puc and by the small GTPase Rac1. puc mutant cells display region-specific ectopic expression of the A251-lacZ enhancer trap whereas overexpression of a transgene encoding Puc is sufficient to suppress lacZ expression in a cell autonomous fashion. Strikingly, decreased or increased puc function leads to a corresponding increase or decrease, respectively, of Fos and Jun protein levels. Taken together, these data indicate that puc modulates gene expression responses by antagonizing a Rho GTPase signal transduction pathway that stabilizes the AP-1 transcription factor. Consistent with this, overexpression of a dominant negative Rac1 results in lower levels of Fos/Jun (Dobens, 2001).

Production of puc mutant clones in the ovary leads to the formation of small eggs with anterior eggshell defects. The junction between the operculum and ventral collar is aberrant, so that the eggshells are open in the anterior, and the dorsal appendages are short and broad. The presence of opercula and micropyles indicate that centripetal migration occurs, but the specific defects suggest that it does not proceed normally. Late-staged egg chambers from these females retain significant nurse cell material at the anterior after the eggshell is deposited, indicating that nurse cell dumping is not coordinated with follicle cell morphogenesis. Germline clonal analysis indicates that these puc mutant phenotypes derive solely from a requirement for puc in the soma. Overexpression of Puc in the FC also leads to production of small eggs with dorsal appendage defects. Because either increased or decreased puc function disrupts the coordination of follicle cell morphogenesis and nurse cell dumping, it is concluded that Puc activity must be poised at the proper level to coordinate anterior morphogenesis. These data parallel the subtly different defects in dorsal closure that result from either reduced or increased Puc activity, and point to a general role for Puc in coordinating epithelial sheet movements (Dobens, 2001).

Loss of puc in the follicle cells leads to aberrant cell shapes. Puc mutant dorsal appendage FCs accumulate elevated levels of Shotgun and fail to extend properly. Late in oogenesis the mainbody FCs are irregular in shape when mutant for puc. It is concluded that the level of Puc activity finely modulates the morphogenesis of the follicular epithelium. This requirement is most evident in the dorsal appendage FCs, but is also important for morphogenesis of the operculum boundary (Dobens, 2001).

Enhancer traps A251-lacZ and E69-lacZ are inserted into the same region of the puc second intron. Both exhibit leading edge cell expression during embryonic dorsal closure and nurse cell FC-specific expression during nurse cell dumping. In contrast, PUC mRNA expression is more widespread in both tissues. In the egg chamber, it accumulates at low levels throughout the FC and at high levels in the centripetally migrating FCs, dorsal appendage FCs and posterior pole FCs. This pattern is recapitulated by the B48-lacZ enhancer trap insertion located in puc intron 1. Consistent with this, the puc gene is required in more FCs than the limited expression of A251-lacZ would predict. Given the widespread expression of PUC RNA in the embryo, A251-lacZ reveals only a subset of the complete puc expression pattern in this tissue as well (Dobens, 2001).

These lacZ reporters have been useful to dissect the regulation of epithelial cell morphogenesis during dorsal closure in embryos. Reporter expression is positively regulated by components of the JNK pathway, and negatively regulated by Puc itself. It has not been reported whether PUC RNA levels are similarly regulated. A251-lacZ expression is also negatively regulated by Puc in the FC. (1) When PUC mRNA levels are low, before stage 10, there is a requirement for Puc activity to prevent A251-lacZ expression throughout the FC. (2) After stage 10, when clear FC domains are established, striking regional differences are observed in the requirement for Puc to regulate A251-lacZ expression. In the nurse cell FC, A251-lacZ expression is increased by reduced Puc function and repressed by overexpression of UAS-Puc. Because PUC mRNA levels are low in these cells at this stage, it is inferred that Puc activity is at an intermediate level so that Jun kinase-like signaling is only partially repressed. In the centripetally migrating FCs, low A251-lacZ levels are Puc dependent because reduction of Puc function results in ectopic A251-lacZ expression. It is concluded that Jun kinase-like signaling is active in these cells, but strongly suppressed by the high levels of Puc activity normally there. Consistent with this, high levels of PUC mRNA are seen in these cells. High PUC mRNA levels correlate with strong repression of A251-lacZ at stage 10 in the posterior pole FCs, as well. The strong requirement for Puc activity in anterior FCs correlates well with the puc mutant defects seen in anterior FC morphogenesis (Dobens, 2001).

Low levels of A251-lacZ in the main body FC are not dependent on puc: mutant clones show only a slight derepression of A251-lacZ at very late stages. Consistent with this, these cells express only low levels of PUC mRNA, and overexpression of Puc has no visible effect on the morphology of these cells. However, Puc is active in the main body FCs, for these cells accumulate Fos and Jun when mutant (Dobens, 2001).

The Rho family of small GTPases are important modulators of cell shape and epithelial cell reorganization. Specific Rho proteins can manifest specialized cellular functions. The cell-type specific effects extend to their abilities to stimulate the JNK pathway. The differing requirements for Puc in different regions of the follicular epithelium suggest that these regions show distinct sensitivities to the actions of Rho family GTPases. Because of evidence that Rac1 and Cdc42 regulate expression of A251-lacZ during embryogenesis, the ability of these small GTPases to regulate A251-lacZ expression in the FC was tested. Two opposing effects were found: activated Rac1 and dominant negative Cdc42 activate A251-lacZ expression in posterior cells; dominant negative Rac1 and activated Cdc42 repress A251-lacZ expression in anterior cells. Clones for activated Rac1 were not recovered in the anterior nurse cell FCs, suggesting that these cells are highly sensitive to increased Rac activity. These data suggest that a balance between these two small GTPases may define the level of A251-lacZ expression in the nurse cell FCs and the posterior pole FCs. This may reflect the shared origins of these distinct cell groups as terminal follicle cells (Dobens, 2001).

Neither Rac1 nor Cdc42 appeared to regulate A251-lacZ expression in the main body FCs or the centripetally migrating FCs. This latter observation is surprising, given the strong requirement for Puc activity in these cells. Perhaps a distinct Rho family GTPase is active in these cells, such as Rho1. Consistent with this, mutants in PAK kinase, which acts in parallel to JNK signaling during dorsal closure, have a variable defect in centripetal migration (Dobens, 2001).

Activated GTPase expression in the follicle cells results in additional phenotypes. Activated Rac1 has non-autonomous phenotypes in the posterior pole FCs. Similarly, activated Rac1 has non-autonomous effects on gene expression in the dorsal ectoderm of the embryo. This suggests that in both tissues, a short-range signal dependent on Rac1 signaling can upregulate expression of these intron II enhancer traps in adjacent cells. Dpp is a local signal to the dorsal ectoderm during dorsal closure; however, the data suggest that Dpp is not the key signal to induce A251-lacZ in the posterior FC. It is noted that loss of puc in marked clones never results in upregulation of A251-lacZ expression in adjacent cells, indicating that whatever its identity, the short range signal is not regulated by puc (Dobens, 2001).

Expression of activated Rac1 in the posterior pole FCs causes domain-specific disorganization of the epithelium. Dominant negative Cdc42 also causes this phenotype, similar to genetic loss of Cdc42 activity. Similar phenotype are seen for activated RhoL. Thus the balance of Rho GTPases in the posterior follicular epithelium may be important to maintain epithelial structure, similar to roles ascribed to Rac in metastasis (Dobens, 2001).

Based on data presented here, it is proposed that puc functions as a rheostat to modulate gene expression responses to Rho family GTPases. This modulation is critical to coordinate morphogenesis in distinct FC domains. Because Puc can act as a Jun kinase phosphatase in the embryo, it has been speculated that Puc modulates the activity of similar kinases that act downstream of the small GTPases at the FC termini. It is likely that puc modulates Jun kinase signal in the anterior FCs, since the Jun kinase kinase encoded by hemipterous is required for anterior A251-lacZ expression. However, puc is also required at the posterior, whereas late A251-lacZ expression is not dependent on hep. Components of the posterior pathway remain to be identified (Dobens, 2001).

The insensitivity of main body FCs to stimulation by Rac or Cdc42 might suggest that these cells are incompetent to respond to small GTPase activation during the late stages of oogenesis. However, responses are suppressed by an independent mechanism. The dynamic pattern of Jun and Fos and puc itself, suggests that FCs may vary greatly, both in time and in space, in their competence to respond to the JNK pathway. To complicate this picture, the data indicates that puc regulates levels of Fos and Jun, even in the main body FCs. Loss of puc leads to elevation of Fos/Jun protein levels. Conversely overexpression of either Puc or DN-Rac1 lowers Fos/Jun levels. Currently the direct mechanism for puc regulation of Fos/Jun levels is unknown, but these results recall observations that stability of Fos and Jun depends on phosphorylation. In cell culture, site-specific mutations in Fos or Jun that block phosphorylation confer instability; conversely, phosphate-mimetic mutations confer greater stability. It is concluded that proper levels of Puc phosphatase modulates Rho family GTPase signal output to coordinate follicle cell morphogenesis. These results are similar to the effect of both loss- and gain-of-function Puc in blocking dorsal closure and a role for puc in disc epithelial morphogenesis. The precise regulation of Puc activity levels is critical to coordinate epithelial cell sheet spreading in at least three tissues (Dobens, 2001 and references therein).

GENE STRUCTURE

Bases in 5' UTR - 405

Exons - 3

Bases in 3' UTR - 485

PROTEIN STRUCTURE

Amino Acids - 496

Structural Domains

The Puc ORF encodes a protein tyrosine phosphatase, with a catalytic domain between amino acids 214 and 226 that includes the invariant cysteine known to be required for phosphatase activity. The protein contains eight putative sites for phosphorylation by MAPK, distributed throughout the carboxy-terminal part of the protein. The predicted protein contains no clear hydrophobic sequences indicative of either signal sequence or transmembrane domain, which suggests that the Puc protein is neither a secreted nor an integral membrane protein. The phosphatase encoded by puc has high similarity to nonreceptor dual specificity phosphatases of the VH-1 subfamily. Phylogenetic analysis, indicates that its closest relative is the protein encoded by Caenorhabditis elegans(identified in the C. elegans genome project) that has 38% identical residues over a 158 amino acid overlap. When conservative residues are taken into account, the comparison yields 59.5% similarity between the two sequences. Very high similarities with other proteins of this family highlight the conservation of their catalytic sites, which are identical at 9-11 of 13 amino acids. A single copy of an internally repeated domain of unknown function (amino acids 238-312; amino acids 386-460) is present in all VH-1 family phosphatases (Martin-Blanco, 1998 and references).

date revised: 10 May 2001 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.