Wingless signaling directly regulates cyclin E expression in proliferating embryonic PNS precursor cells

Abstract

Cell proliferation and cell type specification are coordinately regulated during normal development. Cyclin E, a key G1/S cell cycle regulator, is regulated by multiple tissue-specific enhancers resulting in dynamic expression during Drosophila development. Here, we further characterized the enhancer that regulates cyclin E expression in the developing peripheral nervous system (PNS) and show that multiple sequence elements are required for the full cyclin E PNS enhancer activity. We further show that Wg signaling is important for the expression of cyclin E in the sensory organ precursor (SOP) cells through two conserved TCF binding sites. Blocking Wg signaling does not completely block SOP cell formation but does completely block SOP cell proliferation as well as the subsequent differentiation.

Fig 1. A diagram of the cyclin E genomic region and of the different cyclin E PNS enhancer constructs

cyclin E genomic fragments were used to generate a series of nuclear GFP (nGFP) reporter constructs. The numbers on top represent the locations of the cyclin E genomic sequence relative to the translation start of the type I cyclin E transcript. The location of the Kpn I and Nco I restriction sites, which defines the PNS enhancer in the work of Jones et al. (2000) are indicated. Asterisks indicate the locations of the two TCF-binding sites in the cyclin E PNS enhancer. The presence or absence of GFP expression is indicated by plus or minus signs, respectively. The number of plus signs indicates the level of GFP expression relative to the full-length 4.6-PNS enhancer.

Fig. 2. GFP reporter expression from different cyclin E PNS enhancer constructs

GFP reporter expression of the indicated cyclin E transgenic constructs at embryonic stages 11/12 (A, C, E, G, I, and K) or stages 12/13 (B, D, F, H, J, and L) are shown. Multiple insertions of each deletion construct were examined and no significant differences were observed between insertions of the same constructs. The level of GFP expression is summarized in Fig. 1.

Fig. 3. The effect of wg mutation on cyclin E expression in the embryonic PNS

(A, B) Expression of the GFP reporter from the 4.6-PNS constructs was significantly reduced in the wg mutant embryos (B) as compared to that in the control (wg/CyO) embryos (A). (C, D) Double labeling of 4.6-PNS GFP reporter expression and Sens revealed large number of Sens positive cells express GFP reporter in wg/CyO embryos (C) while very few Sens positive cells express GFP reporter in wg mutant embryos (D). (E, F) Whole-mount in situ hybridization showing expression of zygotic cyclin E in the developing PNS. (E), wg/CyO embryos show normal cyclin E expression in the PNS. (F), wg mutant embryos exhibit significantly reduced PNS cyclin E expression.

Fig. 4. Wg signaling is required for cell proliferation in the developing PNS and for the development of neurons and Pros expressing cells in the PNS

(A, B) Significant numbers of SOP cells were observed in wg mutant (B) embryos. PNS SOP cells were identified by Senseless staining shown in magenta. Homozygous wg mutant embryos were identified by the absence of β-gal expression from the ftz-lacZ construct on the CyO balancer shown in green. (C, D) wg mutation blocks cell proliferation in the developing PNS. BrdU incorporation was not observed in the PNS region of the wg mutant embryos (D) but was observed in the wg/CyO embryos (C). It should be noted that BrdU incorporation in the head and the ventral CNS region was still observed in wg mutant embryos. (E–H) wg mutation blocks the differentiation of neurons and Pros expression cells in the developing PNS. Sheath cells in the es and scolopale cells in the ch organs are the PNS cell types that accumulate high levels of Pros. Very few Pros-expressing cells were observed in wg mutants (F) as compared to wg/CyO embryos (E). Similarly, very few neurons identified by Elav staining were observed in wg mutant (H) as compared with that of the wg/CyO embryos (G). Homozygous wg mutant embryos were identified by the absence of β-gal expression from the ftz-lacZ construct on the CyO balancer shown in red in G and H. Arrows point to a PNS segment in the embryos.

Fig. 5. Two conserved TCF-binding sites in the 4.6-PNS enhancer

(A) Alignment of sequences around the conserved TCF-binding sites between D. melanogaster and D. pseudoobscura. The bold letters represent the TCF-binding sites in the 4.6-PNS enhancer. The sequences of the mutant TCF sites are shown below the binding sites and base pair changes are indicated by “*”. (B) Gel shift assay showing that the Drosophila TCF protein bound strongly and specifically to the wild type TCF1 (lanes 2 and 3) and TCF-2 (lanes 8, 9). In contrast, no binding was observed when the TCF sites were mutated (mut-1 and mut-2, lanes 5, 6 and 11, 12). No dTCF protein were added in lanes 1, 4, 7, and 10; half the amount of dTCF protein was added to lanes 2, 5, 8, 11 compared to lanes 3, 6, 9, and 12. An arrow points to the dTCF-DNA complex. The sequences of TCF and mutated TCF probes are shown in Materials and Methods.

Fig 6

(A) A diagram of the 4.6-PNS enhancer and the 4.6-PNS enhancers with each of the TCF binding sites mutated either alone or in combination. Filled circles represent the TCF binding sites, and crossed (x) circles indicate mutated TCF binding sites in the PNS enhancer. (B–E) Both TCF binding sites contribute to the full activity of the 4.6-PNS enhancer. Transgenic lines carrying the 4.6-PNS enhancer with mutated TCF-1 binding site (C) or the TCF-2 binding site alone (D) show decreased GFP reporter expression when compared to WT 4.6-PNS enhancer (B). Transgenic lines carrying the 4.6-PNS enhancer with mutations of both TCF binding sites only show background levels of GFP reporter expression (E).