A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, the Drosophila sex determination master switch

The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. We show that Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci) the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched (Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely, overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci.     

An N-terminal truncation uncouples the sex-transforming and dosage compensation functions of sex-lethal

In Drosophila melanogaster, Sex-lethal (Sxl) controls autoregulation and sexual differentiation by alternative splicing but regulates dosage compensation by translational repression. To elucidate how Sxl functions in splicing and translational regulation, we have ectopically expressed a full-length Sxl protein (Sx.FL) and a protein lacking the N-terminal 40 amino acids (Sx-N). The Sx.FL protein recapitulates the activity of Sxl gain-of-function mutations, as it is both sex transforming and lethal in males. In contrast, the Sx-N protein unlinks the sex-transforming and male-lethal effects of Sxl. The Sx-N proteins are compromised in splicing functions required for sexual differentiation, displaying only partial autoregulatory activity and almost no sex-transforming activity. On the other hand, the Sx-N protein does retain substantial dosage compensation function and kills males almost as effectively as the Sx.FL protein. In the course of our analysis of the Sx.FL and Sx-N transgenes, we have also uncovered a novel, negative autoregulatory activity, in which Sxl proteins bind to the 3' untranslated region of Sxl mRNAs and decrease Sxl protein expression. This negative autoregulatory activity may be a homeostasis mechanism.     

Interaction of the sex-lethal RNA binding domains with RNA.

Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein. In part, Sxl functions by regulating the splicing of the transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts we analyzed the interaction of Sxl with RNA. The RNA binding activity of Sxl was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding showed that both RNA binding domains (RBDs) were required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract were identified using nuclear magnetic resonance techniques. In addition, our data suggest that multiple imino protons of the transformer polypyrimidine tract were involved in hydrogen bonding. Interestingly, in vitro Sxl bound with equal affinity to polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo. We discuss the implications of this finding for the mechanism through which Sxl may gain selectivity for particular polypyrimidine tracts in vivo.     

Histone acetylation and gene expression analysis of sex lethal mutants in Drosophila

The evolution of sex determination mechanisms is often accompanied by reduction in dosage of genes on a whole chromosome. Under these circumstances, negatively acting regulatory genes would tend to double the expression of the genome, which produces compensation of the single-sex chromosome and increases autosomal gene expression. Previous work has suggested that to reduce the autosomal expression to the female level, these dosage effects are modified by a chromatin complex specific to males, which sequesters a histone acetylase to the X. The reduced autosomal histone 4 lysine 16 (H4Lys16) acetylation results in lowered autosomal expression, while the higher acetylation on the X is mitigated by the male-specific lethal complex, preventing overexpression. In this report, we examine how mutations in the principal sex determination gene, Sex lethal (Sxl), impact the H4 acetylation and gene expression on both the X and autosomes. When Sxl expression is missing in females, we find that the sequestration occurs concordantly with reductions in autosomal H4Lys16 acetylation and gene expression on the whole. When Sxl is ectopically expressed in Sxl(M) mutant males, the sequestration is disrupted, leading to an increase in autosomal H4Lys16 acetylation and overall gene expression. In both cases we find relatively little effect upon X chromosomal gene expression.     

Sex determination in the germ line of Drosophila depends on genetic signals and inductive somatic factors

We have analyzed the mechanism of sex determination in the germ line of Drosophila by manipulating three parameters: (1) the ratio of X-chromosomes to sets of autosomes (X:A); (2) the state of activity of the gene Sex-lethal (Sxl), and (3) the sex of the gonadal soma. To this end, animals with a ratio of 2X:2A and 2X:3A were sexually transformed into pseudomales by mutations at the sex-determining genes Sxl (Sex-lethal), tra (transformer), tra-2 (transformer-2), or dsx (double-sex). Animals with the karyotype 2X;3A were also transformed into pseudofemales by the constitutive mutation SxlM1. The sexual phenotype of the gonads and of the germ cells was assessed by phase-contrast microscopy. Confirming the conclusions of Steinmann-Zwicky et al. (Cell 57, 157, 1989), we found that all three parameters affect sex determination in germ cells. In contrast to the soma in which sex determination is completely cell-autonomous, sex determination in the germ line has a non-autonomous component inasmuch as the sex of the soma can influence the sexual pathway of the germ cells. Somatic induction has a clear effect on 2X;2A germ cells that carry a Sxl+ allele. These cells, which form eggs in an ovary, can enter spermatogenesis in testes. Mutations that cause partial loss of function or gain of function of Sxl thwart somatic induction and, independently of the sex of the soma, dictate spermatogenesis or oogenesis, respectively. Somatic induction has a much weaker effect on 2X;3A germ cells. This ratio is essentially a male signal for germ cells which consistently enter spermatogenesis in testes, even when they carry SxlM1. In a female soma, however, SxlM1 enables the 2X;3A germ cells to form almost normal eggs. Our results show that sex determination in the germ line is more complex than in the soma. They provide further evidence that the state of Sxl, the key gene for sex determination and dosage compensation in the soma, also determines the sex of the germ cells, and that, in the germ line, the state of activity of Sxl is regulated not only by the X:A ratio, but also by somatic inductive stimuli.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.     

NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila sex-lethal protein with target RNA fragments with site-specific [3-15N]uridine substitutions.

It has been reported that a 183 residue fragment, consisting of the two RNA-binding domains (RBD1- RBD2) of the Drosophila melanogster Sex-lethal (Sxl) protein, strongly binds an oligonucleotide of the target RNA sequence (5'-GUUUUUUUUC-3') that regulates alternative splicing, and forms four or five hydrogen bonds with the imino groups of the RNA. In the present study, we used site-directed mutagenesis to improve the solubility of the didomain fragment of Sxl, and confirmed that this mutant fragment forms hydrogen bonds with the target RNA in the same manner as that of the wild-type fragment. The mutant fragment was shown to bind the cognate RNA sequences GUUUUUUUUC and AUUUUUUUUC more tightly than UUUUUUUUC. By using a [3-15N]uridine phosphoramidite, we synthesized a series of15N-labeled target RNAs, in which one of the uridine residues was specifically replaced by [3-15N]uridine. By observing the imino1H-15N coupling of the labeled uridine residue, we assigned all four of the hydrogen-bonded imino protons to U1, U2, U5 and U6, respectively, of the target RNA. The imino protons of U2 and U6 exhibited nuclear Overhauser effects with aliphatic protons of the protein. All these results indicate that the A/G, U1, U2, U5 and U6 residues in the target sequence of (G/A)UUUUUUUU are specifically recognized by the two RNA-binding domains of the Sxl protein.     

Sex determination in Drosophila melanogaster: X-linked genes involved in the initial step of Sex-lethal activation

Sex determination is the commitment of an embryo to either the female or the male developmental pathway. The ratio of X chromosomes to sets of autosomes is the primary genetic signal that determines sex in Drosophila, by triggering the functional state of the gene Sex-lethal: in females (2X;2A) Sxl will be ON, whereas in males (X;2A) Sxl will be OFF. Genetic and molecuar studies have defined a set of genes involved in the formation of the X:A signal, as well as other genes, with either maternal or zygotic effects, which are also involved in regulating the initial step of Sex-lethal activation. We review these data and present new data on two more regions of the X chromosome that define other genes needed for Sxl activation. In addition, we report on the interaction between some of the genes regulating Sxl activation. © 1994 Wiley-Liss, Inc.