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First published online 21 April 2004
doi: 10.1242/dev.01109

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Ftz modulates Runt-dependent activation and repression of segment-polarity gene transcription

Department of Biochemistry and Cell Biology and The Center for Developmental Genetics, State University of New York at Stony Brook, Stony Brook, NY 11794-5140, USA

View larger version (102K): [in a new window]   Fig. 1. slp1 is a sensitive target of Runt. In situ hybridization reveals the segmentally repeated expression patterns of different segmentation genes in gastrula stage embryos. Embryos in this and other figures are shown anterior towards the left, dorsal side upwards. Each row shows expression of a different segmentation gene as labeled on the right. The wild-type expression patterns are shown in the left column of embryos. Embryos in the middle and right column of embryos have intermediate and high levels of NGT-driven ectopic Runt expression, respectively. Intermediate and high levels of ectopic Runt were obtained by mating females homozygous for NGT11 and NGT40, respectively to homozygous UAS-runt[232] males. These are the same combinations used to demonstrate that high, but not intermediate, levels of Runt alter expression of eve and ftz (Tracey et al., 2000). Not shown in this panel are the responses of the pair-rule genes hairy and odd-paired (opa). NGT-driven Runt expression leads to stripe-specific repression of hairy similar to that obtained in hs-runt embryos, but only at high levels of expression. The opa pattern is unique amongst the pair-rule genes and is expressed in a broad band spanning the presegmental region of the embryo, rather than in a series of stripes. This pattern is not altered by ectopic Runt.

View larger version (49K): [in a new window]   Fig. 2. Parasegment-specific activation and repression of slp1 by Runt. (A) Wild-type slp1 expression in a gastrula stage embryo as visualized by in situ hybridization. (B) The phasing of slp1 expression relative to the expression of different pair-rule transcription factors. A strip of cells along the anteroposterior axis is depicted across the bottom with slp1-expressing cells indicated by shading. The higher expression level of the even-numbered stripes is indicated by darker shading. The four-cell wide Runt stripes are depicted above this strip as a trapezoid, reflecting the higher expression levels in the center of the stripes. By contrast, Eve and Ftz stripes are depicted as triangles with peak expression in the most anterior cells, whereas the uniform expression of Opa is depicted as a broad rectangle that spans the presegmental region. The regulatory circuitry responsible for generating the slp1 pattern is also depicted. Activation by Runt + Opa is indicated with an arrowhead, whereas repression by either Eve, or the combination of Runt + Ftz, is indicated with a horizontal bar. (C) Transient elimination of runt activity in an embryo hemizygous for the temperature-sensitive runt[YP17] mutation leads to loss of odd-numbered slp1 stripes and expansion of some of the even-numbered stripes. These changes are interpreted to be due to loss of Runt-dependent activation and repression as indicated in D. (E) Double in situ hybridization showing the complementary expression of slp1 (blue) and ftz (brown) mRNAs in embryos with a high-level of NGT-driven Runt. This embryo was obtained by crossing homozygous NGT40 females with homozygous UAS-runt[232] males. As indicated in F, slp1 expression in these embryos fills the presumptive odd-numbered parasegments and is repressed throughout the even-numbered parasegments.

View larger version (35K): [in a new window]   Fig. 3. Opa is required for Runt-dependent activation. Expression of slp1 mRNA in embryos that are wild-type for runt (A,B), or that have high levels of NGT-driven Runt (D,E). Ectopic Runt in these embryos was obtained by crossing females heterozygous for both NGT40 and NGTA to homozygous UAS-Runt[232] males. The embryos in A and D are wild type for opa, whereas the embryos in B and E are homozygous for the opa[1] mutation. (C) Schematic interpretation of the response of slp1 to the loss of Opa in an embryo with normal Runt expression. (F) Schematic interpretation of the effects of loss of Opa in an embryo with uniform Runt expression.

View larger version (59K): [in a new window]   Fig. 4. Runt and Opa cooperate to activate slp1transcription. In situ hybridization showing the expression of slp1 (A-I) and ftz (J-L) mRNAs in embryos with varying levels of NGT-driven Runt and Opa. In all cases, ectopic expression was obtained in crosses using females homozygous for both NGT40 and NGTA. Variations in expression were obtained using different UAS-runt and UAS-opa lines. Embryos in the first row carry UAS-runt[14] in combination with (A) UAS-opa[36], (B) UAS-opa[12] and (C) UAS-opa[10], which are ordered in increasing strength from left to right. Embryos in the second row (D-F) carry UAS-runt[232] in combination with the same three UAS-opa transgenes. Embryos in the third and fourth rows (G-L) carry UAS-runt[15], also in combination with the same three UAS-opa transgenes. The different levels of ectopic Runt expression are organized in increasing strength from top to bottom, UAS-runt[232] and UAS-runt[15], giving approximately two- and fivefold increases, respectively, over the level obtained with UAS-runt[14] (Li and Gergen, 1999).

View larger version (53K): [in a new window]   Fig. 5. Ftz converts Runt from an activator to a repressor of slp1. (A) Embryos mutant for ftz express slp1 in the anterior half of the even-numbered parasegments, resulting in six-cell wide stripes. (B) The ftz mutant phenotype and the way in which this expanded expression is accounted for by Runt+Opa dependent activation. Note that run expression is not significantly altered in ftz mutant embryos at this stage (Klingler and Gergen, 1993). (C) Ectopic Runt expression in ftz mutant embryos activates slp1 throughout the presegmental region. This embryo was obtained from a cross between a female heterozygous for NGT40, NGTA and the ftz[11] mutation with a male homozygous for UAS-runt[232] and heterozygous for ftz[11]. The level of NGT-driven Runt obtained with this combination does not fully overcome Eve-dependent repression, resulting in a few thin stripes of cells with reduced slp1 expression. Runt+Opa-dependent activation (D) results in the slp1 pattern shown in C. The effects of NGT-driven Ftz on slp1 is shown in E and interpreted in F. The embryo in E is from a mating between homozygous NGT40 females and homozygous UAS-ftz[261] males. The effects of co-expressing Runt and Ftz are shown in G and interpreted in H. The embryo in G is from a mating between homozygous NGT40 females and males homozygous for both UAS-runt[232] and UAS-ftz[261].

View larger version (22K): [in a new window]   Fig. 6. The pair-rule to segment-polarity transition. The contrasting Runt-dependent activation and repression of slp1 in different cells within the pre-segmental region of the blastoderm embryo is fully explained by the overlapping expression of Ftz, as indicated in this diagram. Also depicted are the expression domains of the pair-rule transcription factors Odd and Prd, which overlap the slp1stripes (Morrissey et al., 1991; Mullen and DiNardo, 1995). Combinatorial regulation by Eve, Runt, Opa and Ftz accounts for all aspects of slp1 regulation, except for activation in even-numbered parasegments. The minimal spatial domain of activity of a Factor X that is proposed to be responsible for this aspect of slp1 expression is depicted in pale blue. Factor X-dependent activation may also contribute to the expanded slp1 expression obtained by transient elimination of eve or run. The possibility that Factor X is active in other cells within the pre-segmental region is indicated by the broken blue line. The strip of cells along the anteroposterior axis drawn at the bottom of this diagram shows the relationship of wg and en expression in each parasegment to that of slp1 and the pair-rule transcription factors.

View larger version (66K): [in a new window]   Fig. 7. Ftz modulates Runt-dependent activation and repression of wg and en. Expression of wg (A-D) and en (E-H) in response to different perturbations in Runt and Ftz activity. (A,E) Response of these two genes to transient elimination of run in a temperature-shifted run[YP17] embryo. (B,F) Response to NGT-driven Ftz. These embryos are from a mating of homozygous NGT40 females to homozygous UAS-ftz[261] males. (C,G) NGT-driven co-expression of Runt and Ftz blocks wg expression, while producing a pair-rule pattern of en expression. (D) Response of wg to NGT-driven co-expression of Runt and Opa. This embryo is from a mating of females homozygous for both NGT40 and NGTA to males homozygous for the UAS-runt[13] and UAS-opa[14] transgenes. This combination results in a slp1 pattern similar to the embryos in Fig. 4F,H. (H) Response of en to NGT-driven Runt in embryos that are also homozygous for the ftz[11] mutation. This embryo is from the same cross used to generate the embryo in Fig. 5C.

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