BIOLOGICAL OVERVIEW

Two closely linked genes, sloppy paired 1 and 2, share the combined characteristics of gap, pair rule and segment polarity genes. Because slp2 is dispensible, particularly in the head, slp1 will be the major concern in this discussion, and will be referred to as sloppy paired (slp). The two genes are first expressed in the head where they act like gap genes, but their action in the trunk is more like pair rule and segment polarity genes.

Expression of slp1 in the head is independent of the pair rule genes, which regulate segment polarity genes in the trunk. Combinatorial inputs from gap genes establish the domains of segment polarity genes in the head. The gap genes, in combination with sloppy paired , define seven regions. Thus gap genes subdivide the head into the various segments which define the adult head stucture (Grossniklaus, 1994).

In the trunk, sloppy paired is expressed in the posterior cells of each parasegment, acting like a segment polarity gene. This expression abuts cells expressing engrailed. Sloppy paired activates wingless and represses engrailed. This function is critical for the sharp boundary of expression between en and wg (Cadigan, 1994a).

Sloppy paired and Even-skipped are involved in cell fate determination and segmentation in the Drosophila mesoderm. In wild-type embryos, slp1 first appears during gastrulation (stage 6) in a pattern of 7 stripes. slp is expressed in both ectoderm and mesoderm. A second set of stripes appears between the first 7 such that a regular 14-stipe pattern is generated by stage 7. The primordia for heart, fat body, and visceral and somatic muscles arise in specific areas of each segment in the Drosophila mesoderm. The primordium of the somatic muscles, which expresses high levels of twist, a crucial factor of somatic muscle determination, is lost in sloppy-paired mutants. The effect of slp on Twist levels is probably partly, but not completely mediated by wingless. wg mutant embryos show a premature and ectopic decay of Twist, but not to the same degree as seen in slp embryos. Whereas patches of cells expressing high levels of Twist are initially established in wg mutant embryos, no Twist is seen in the trunk region of slp mutant embryos after stage 11. At the same time that twist expression is lost in slp mutants, the primordium of the visceral muscles is expanded (Riechmann, 1997).

bagpipe and serpent expressing mesodermal domains corresponding to the ectodermal even-skipped domains, alternate with the sloppy-paired expressing high-twist mesodermal domains. Ectodermal even-skipped is thought to act through engrailed and subsequently hedgehog to promote bagpipe expression in cardiac and dorsal muscle and serpent in the fat body (Azpiazu, 1996). Ectodermal Dpp is required for the maintenance of mesodermal tinman, which in turn activates bap expression in the eve domain. The visceral muscle and fat body primordia require even-skipped for their development and the mesoderm is thought to be unsegmented in even-skipped mutants. However, it has been found that even-skipped mutants retain the segmental modulation of the expression of twist. Both the domain of even-skipped function and the level of twist expression are regulated by sloppy-paired; eve serves reciprocally to regulate the slp domain. sloppy-paired thus controls segmental allocation of mesodermal cells to different fates (Riechmann, 1997).

Wingless (Wg) and other Wnt proteins play a crucial role in a number of developmental decisions in a variety of organisms. In the ventral nerve cord of the Drosophila embryo, Wg, signaling from row 5 is non-autonomously required for the formation and specification of a neuronal precursor cell, NB4-2. NB4-2 gives rise to a well-studied neuronal lineage, the RP2/sib lineage. While the various components of the Wg-signaling pathway are also required for generating NB4-2, the target gene(s) of this pathway in the signal-receiving cell is not known. In this paper, it is shown that sloppy paired 1 and sloppy paired 2 function as the downstream targets of the Wg signaling to generate the NB4-2 cell. Thus, while the loss-of-function mutations in wg and slp have the same NB4-2 formation and specification defects, these defects in wg mutants can be rescued by expressing slp genes from a heterologous promoter. The fact that slp genes function downstream of the Wg signaling is also indicated by the result that expression of slp genes is lost from the neuroectoderm in wg mutants and that ectopic expression of wg induces ectopic expression of slp. Finally, Gooseberry (Gsb) prevents Wg from specifying NB4-2 identity to the wg-expressing NB5-3. In this paper, it is shown that gsb interacts with slp and prevents Slp from specifying NB4-2 identity in NB5.3. Overexpression of slp overcomes this antagonistic interaction and respecifies NB5-3 as NB4-2. This respecification, however, can be suppressed by a simultaneous overexpression of gsb at high levels. This mechanism appears to be responsible for specifying NB5-3 identity to a row 5 neuroblast and preventing Wg from specifying NB4-2 identity to that neuroblast (Bhat, 2000).

NB4-2 is delaminated from an equivalence group of 4-6 neuroectodermal cells during the second wave of neuroblast delamination in mid stage 9 (approximately 4.5 hours old) of embryogenesis. It is located in the 4th column along the anterior-posterior axis and 2nd row along the medio-lateral axis within each hemisegment. The NB4-2 undergoes its first asymmetric division approximately 1.5 hours after formation to self renew and to generate its first GMC, GMC-1 (this GMC-1 is also called GMC4-2a: the first GMC generated from NB4-2). The GMC-1 divides about 1.5 hours later to generate two cells, the larger RP2 and the smaller sib. The RP2 cell migrates to its specific position within the anterior commissure and projects its axon antero-ipsilaterally to the intersegmental nerve bundle (ISN) and innervates muscle #2 on the dorsal musculature. The sib cell migrates to a position posterior and more dorsal to RP2. NB4-2, GMC-1, RP2 and RP2-sib cells can be reliably identified by their gene expression pattern, physical sizes and position within the half-segment (Bhat, 2000).

In the epidermis, mutation in slp genes result in a fusion of abdominal segments A1-A2, A3-A4, A5-A6 and A7-A8 (characteristic of pair-rule mutants) and replacement of naked cuticle by denticle belts, a wg-type of segment polarity phenotype. During the patterning of the epidermis, slp genes function upstream of wg to maintain wg expression (Cadigan, 1994a and b). Since wg is also required for the formation and specification of NB4-2 identity, it is possible that the effect of loss of slp genes on NB4-2 is mediated via its effect on wg expression. Therefore, to determine the precise temporal requirement of slp for maintaining wg expression during neurogenesis, the expression of wg in slp mutant embryos was first examined. In slp mutants the wg expression begins to fade from the neuroectoderm initially in even-numbered parasegments approximately 3.75 hours of development (stage 7, early germ band extension). This fading is particularly prominent in abdominal segments. By approximately 4.5 hours of development (early stage 9), wg expression in these parasegments is completely lost. By contrast, in odd numbered parasegments, wg expression is nearly as high as in wild type during early stage 9 (approximately 4.5 hours of development), and is only lost by approximately 6-6.5 hours of development (stage 10). These results are consistent with the previous findings (Cadigan, 1994a and b) and show that slp genes function upstream of wg and positively regulate wg expression (Bhat, 2000).

Studies using a temperature-sensitive allele of wg have revealed that Wg activity is required for the specification of NB4-2 identity at approximately 4 hours of development (between early to mid-stage 8, at 22°C). However, in slp mutants the expression of wg is still high in the odd-numbered parasegments around the time of NB4-2 specification and the expression of wg is lost in these parasegments only by approximately 6.5 hours of development (stage 10), nearly 2.5 hours after the specification of NB4-2 identity. Thus the loss of Wg expression from odd-numbered parasegments is well past the temporal requirement of wg for NB4-2 specification. Therefore, at least in the odd-numbered parasegments, the specification of NB4-2 identity in slp mutants must occur earlier than the decay of wg expression. Therefore, it is concluded that the loss of NB4-2 identity in slp mutants is unlikely due to the loss of wg expression, at the least in the odd-numbered parasegments, and possibly in the even-numbered parasegments as well (Bhat, 2000).

While the evidence to support the conclusion that slp genes regulate expression of wg in the epidermis is quite strong (Cadigan, 1994a and b), the evidence that the wg-signaling controls the expression of slp in the CNS is also equally strong. (1) The slp genes are expressed not only in the wg-expressing row 5 cells but also in the Wg-negative, but Wg-receiving row 4 cells. (2) The expression of slp is affected in wg mutant embryos. That is, staining of wg mutant embryos show that the expression of slp is lost from the Wg-receiving row 4 neuroectodermal cells. This result is also supported by the western analysis of embryo extract from wg mutants in which the level of Slp protein is found to be greatly reduced. (3) Consistent with the above result, the ectopic expression of wg induces ectopic expression of slp in the neuroectoderm. (4) In slp mutants, just as in wg mutants, the formation and identity specification of a well-studied neuronal precursor cell, NB4-2, is affected; this defect in wg mutants can be rescued by the expression of slp genes from a heterologous promoter. Moreover, a similar relationship also appears to exist between slp and wg during mesoderm specification. For instance, in both wg and slp mutants, the specification of heart cells (derived from mesoderm) is affected and this defect in wg mutants can be rescued by expressing slp genes from a heterologous promoter. These results therefore indicate that wg is a positive regulator of slp expression not only during neurogenesis but also in other processes such as mesoderm specification (Bhat, 2000).

An intriguing aspect of regulation of slp genes by wg is the finding that this regulation is restricted primarily to the neuroectoderm but not extended to the neuroblasts that are derived from these neuroectodermal cells, with one exception: the NB4-2. Thus, while row 4 neuroectodermal cells in wg mutant are missing slp1 expression, row 4 neuroblasts other than NB4-2 have slp1 expression. The induction/maintenance of slp1 expression in these neuroblasts must, therefore, necessarily be under the control of some other pathway. Alternatively, the Wg-signaling pathway is redundant in these neuroblasts. These results are consistent with the finding that the induction of an ectopic gsb-stripe by gain-of-function wg occurs only in the neuroectodermal cells but not in the underneath neuroblasts. In summary, these results reveal a hitherto unsuspected relationship between wg and slp in the CNS that is the opposite of their relationship in the epidermis (Bhat, 2000).

Using a temperature-sensitive allele of wg, it has been shown that the requirement of Wg in the CNS for NB4-2 formation and specification is between late stage 7 and early stage 8 and precedes Wg requirement for epidermal patterning. Moreover, it is the neuroectodermal expression of wg that regulates NB4-2 formation and identity specification. Thus, while the timing of decay of wg expression in slp mutants in even-numbered parasegments coincides with the requirements of wg for NB4-2 formation and specification, it is not so in the odd-numbered parasegments. Thus, the odd-numbered parasegments in slp mutants have wg expression during the time Wg is required for NB4-2 formation and specification. Since the loss of NB4-2 lineage in slp mutants is not parasegment-specific and the expression of slp in NB4-2 and its precursor neuroectodermal cells is lost in wg mutants, it must be that slp genes are downstream of wg in these CNS cells (Bhat, 2000).

The Wg signal regulates the specification of NB4-2 identity via Armadillo and Pangolin. The Arm-Pan signaling complex must activate certain downstream target gene(s), presumably transcription factors, and these transcription factors then initiate a program that mediates the formation and specification of NB4-2. slp genes function as downstream targets of the wg signaling, regulating both the NB4-2 formation as well as the identity specification during neurogenesis. This conclusion is based on the following facts: (1) the loss of function effect for slp genes has the same effect as the loss of function for wg on NB4-2 lineage; (2) the loss of wg activity in row 5 cells in the CNS leads to a loss of slp expression from the Wg-receiving NB4-2 and its precursor cell; (3) the loss of NB4-2 in wg mutants can be rescued by the expression of slp genes from a heterologous promoter during the time when wg is known to be required for the process. It is acknowledged that the slp genes might be either the direct targets of the Wg-signaling pathway (i.e. Arm-Pan complex directly activating slp genes), or instead there may be additional genes in between pan and the slp genes. While this issue has not been resolved here, the rescue of the NB4-2 lineage defect in wg mutants by expressing slp genes from a heterologous promoter reveals that the Wg-signaling pathway must ultimately activate slp genes, and the slp genes then regulate the formation and specification of NB4-2 (Bhat, 2000).

GENE STRUCTURE

Bases in 5' UTR -107 for slp1; 301 for slp2

Bases in 3' UTR - 399 for slp1; 267 for slp2

PROTEIN STRUCTURE

Amino Acids - 322 for slp1; 445 for slp2

Structural Domains and Evolutionary Homologs

Slp1 and Slp2 are structurally and functionally related. They belong to a novel class of putative transcription factors containing a fork head domain, also found in mammalian hepatocyte transcription factors (Grossniklaus, 1992). The FKH domains of SLP-1 and SLP-2 are more closely related to each other than they are to Forkhead or the mammalian homolog.

Drosophila sloppy-paired 1 and 2 both have Forkhead domains (Hacker, 1992). Sloppy paired belongs to a different class of forkhead domain proteins than does Forkhead itself and HNF-3beta. Sloppy paired's closest homolog is BF-1. Another forkhead homolog in Drosophila has been discovered, the crocodile gene, required for the establishment of head structures. Crocodile's closest mammalian homolog is FD1 belonging to a different class of forkhead domain proteins than does Forkhead. Forkhead belongs to the same class as HNF-3alpha, HNF-3beta, HNF-3gamma, XFKH1/XFD1, and XFD1/pintallavis (Sasaki, 1993 and Hacker, 1995).

date revised: 30 January 2000

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