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First published online 9 February 2005
doi: 10.1242/dev.01691

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Charlatan, a Zn-finger transcription factor, establishes a novel level of regulation of the proneural achaete/scute genes of Drosophila

Luis M. Escudero^1,*, Eva Caminero¹, Karen L. Schulze², Hugo J. Bellen² and Juan Modolell^1,

¹ Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Cantoblanco, 28049 Madrid, Spain
² HHMI, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (104K): [in a new window]   Fig. 1. Physical map of the chn locus and notum bristle phenotypes of the overexpression of chn and of its interaction with ac/sc. (A) The structure of the chn and bda transcripts, as deduced by sequence comparisons of cDNAs and the genomic DNA, are shown. Light rectangles indicate putative coding regions. The 5' end of chn cDNA is taken as the origin of coordinates and corresponds to position 231192 of scaffold AE003812 of the D. melanogaster genome sequence (release 3.1). Available cDNAs suggest a common 5' region in chn and bda transcripts (red). The possibility of a small intron (hatched region) in bda has not been ruled out. P elements (open triangles) are inserted at positions -230 (EPIL6) and -152 (42/18). Mutation chnECJ1 is associated with a deletion (open rectangle with uncertainty lines for its end points). The structure of the Chn protein with the position of the C2H2 Zn-finger motifs (red rectangles) is indicated. The sequence of three of these motifs is compared with the similar region of the sequence of the human putative Zf462 protein. (B) Notum of a wild-type fly. (C) Expression of the EP line EPIL6 with the MS1096-Gal4 driver (25°C) generates extra bristles. (D) A similar phenotype is observed by overexpressing UAS-chn with the C765-Gal4 driver at 18°C. At 25°C most individuals die before the pharate stage (see below). (E) Halving the genetic dose of ac/sc largely reduces the effect of overexpressing chn (C765-Gal4 driver, 18°C), and removing ac/sc renders overexpression of chn inactive in bristle formation (F). (G-I) UAS-sc and UAS-chn interacted synergistically in the formation of extra bristles. Flies were cultured at 18°C except that, approximately from 48 to 0 hours before puparium formation, they were kept at 25°C. (G) UAS-sc; C765-Gal4. (H) UAS-chn; C765-Gal4. (I) UAS-sc/UAS-chn; C765-Gal4. Note in I, the large increase in macro- and mesochaetae on the anterior region of the notum (arrowheads).

View larger version (129K): [in a new window]   Fig. 5. Overexpression of UAS-chn stimulates the expression of sc and of constructs bearing ASC enhancers specific for individual proneural clusters. (A-C) Sc accumulation is shown in the green channel and that of Sens in the red one. (A) Wild-type late third instar disk shows the distribution of Sc in proneural clusters and that of Sens in SOPs and additional cells flanking the prospective wing margin (WM). Proneural clusters: T/TSM, twin sensilla of the WM; L, vein L3; D/DC, dorsocentral. (B) Overexpression in the wing pouch (MS1096-Gal4 driver) induces ectopic expression of sc and of sens, but largely eliminates expression of these genes at the prospective wing margin. (C) Overexpression in the medial and central notum territory (MS248-Gal4 driver) leads to strong, almost generalized expression of sc and the emergence of many ectopic SOPs. (D) Generalized UAS-chn expression (C765-Gal4 driver) stimulated Sc accumulation (green) at many sites of the wing disk, but it did not enhance atonal expression, which remained confined to its wild-type sites (Jarman et al., 1993b), like a few cells in the prospective ventral radius (red, arrowhead). (E-O) ß-galactosidase accumulation is shown in green. (E,F) Expression of the 2.3-lacZ construct (Culí, 1998), which bears the L3 + TSM enhancer, in a wild-type disk and in a disk overexpressing UAS-chn with the MS1096-Gal4 driver, respectively. (G,H) Expression of the AS1.4DC-lacZ=DC-lacZ construct, which bears the DC enhancer, in a wild-type disk and in a disk overexpressing UAS-chn with the C765-Gal4 driver. (I,J) DC-lacZ expression in disks devoid of functional ac and sc genes (In(1)sc10.1 allele). The construct is still expressed (I) and it is greatly stimulated by UAS-chn (J; C765-Gal4 driver). (K,L) Expression of the DC-lacZ construct in the presence of UAS-chn driven by pnr-Gal4 [a hypomorphic allele of pnr (Heitzler et al., 1996)] in the presence of a wild-type allele of pnr (K) or the null allele pnrVX6 (L). (M) This construct is not expressed in the pnr-Gal4/pnrVX6 genetic background (the green channel background has been enhanced to better appreciate the absence of expression). (N,O) Overexpression of UAS-chn with the C734-Gal4 driver, whose pattern of expression is revealed by the accumulation of Chn protein (red), stimulates expression of the SOP-specific enhancer SRV-lacZ (green) in a background wild-type for the ASC (N), but fails to do so in an In(1)sc10.1 mutant disk (O). The green channel is also shown separately.

View larger version (62K): [in a new window]   Fig. 4. Loss-of-function conditions of chn remove notum macrochaetae. (A,B) f-, chnECJ1 homozygous clones often lose macrochaetae (black arrowheads), although they can also develop them (white arrowheads). (C) Overexpression of UAS-chniS directed by MS248-Gal4 incompletely removes notum macrochaetae. The phenotype is similar to that of the chnECJ1 homozygous clones. (D) Interference with notal fusion (asterisk) and generation of extra macrochaetae (arrowheads) due to the overexpression of UAS-chn with the MS248-Gal4 driver. (E) These phenotypes are largely rescued by the coexpression of UAS-chniS. (F,G) The number of extra macrochaetae formed by overexpressing UAS-sc is sharply reduced by simultaneous expression of UAS-chniW (60 to 80 heminota were examined to quantify the number of bristles, as indicated in the text). No such reduction was observed when UAS-chniW was replaced by UAS-GFP. Note the essentially complete removal of extra macro- and mesochaetae from the anterior notum (arrowheads).

View larger version (96K): [in a new window]   Fig. 2. Expression of chn in embryos and imaginal disks. Expression was detected by in-situ hybridization with an antisense RNA chn probe labeled with DIG. (A,B) Views of a stage-5 embryo focusing at the lateral surface or internally at the invaginating mesoderm (arrowhead), respectively. Note the weak segmental pattern in A at the level of the ectoderm. (C,D) Lateral views of a stage-11 embryo showing (arrowheads) expression in the mesoderm and in patches of the lateral ectoderm located between the tracheal pits, respectively. (E) High magnification view of a similar embryo showing the position of the tracheal pits (arrowheads) and the hybridization in between them. (F) At stage 15, expression is almost exclusively found in the developing PNS (arrowheads) and in the CNS (arrow). (G) A high magnification view of the region under the red line in F shows that expression occurs in cells of the clusters of sensory neurons (compare with Fig. 3A). The image is centered on the dorsal and lateral clusters. (H) In third instar wing imaginal disks, chn expression occurs in proneural clusters (arrowheads) and in the posterior notum and wing hinge (arrows). (I) Expression of chn in proneural clusters depends on ac/sc, as it is absent in In(1)sc10.1 disks. (J) Overexpression of UAS-sc in the posterior compartment of the wing disk (en-Gal4 driver) causes ectopic expression of chn (arrowheads). (K) chn is expressed in a number of patches of the leg disks, which correspond to proneural clusters since they are absent in a In(1)sc10.1 mutant background (not shown), excepting for the femoral clusters, which express ato (Jarman et al., 1995). (L) Expression of chn in a third instar eye/antenna disk. Expression occurs ahead and/or at the morphogenetic furrow (arrow), at the presumptive head capsule (arrowheads) and at the second antennal segment (red arrowhead). This pattern is strongly reminiscent of that of the proneural gene atonal (Jarman et al., 1995).

View larger version (83K): [in a new window]   Fig. 3. chn is necessary for proper development of the embryonic PNS. Embryos were stained with 22c10 antibody (gray) and anti-ß-galactosidase (brown, a marker for the balancer chromosome) antibodies. Lateral views of segments T1 (at left) to A4 are shown. Insets show higher magnification views of the lateral A2 group of chordotonal organs (arrowed). (A) chnECJ1/+ embryo. Its nervous system appears wild type. d, l, v' and v indicate the dorsal, lateral, ventral' and ventral neuronal clusters. Black and white arrowheads point to the dbp and the v'chn1 neurons. (B) chnECJ1 homozygous embryo. Note the disorganized pattern, the reduced number of neurons compared with (A), the absence of dbp and v'chn1 neurons, and the altered morphology of the lateral group. (C) chnECJ1 embryo in which UAS-chn is expressed with the 69B-Gal4 driver. Note the substantial recovery of the wild-type phenotype.

View larger version (108K): [in a new window]   Fig. 6. Loss of chn function leads to decreased expression of sc and enhancer-lacZ constructs, and can impede SOP formation. All figures show parts of third instar wing disks. Except in D, clones homozygous for chnECJ1 are marked by the absence of green. Anti-Sens antibody marks emerged SOPs (blue channel). (A) Clone that includes part of the DC proneural cluster. The mutant cells accumulate less Sc protein (red channel) and give the clone a split appearance. The Sens marker has just started making discernable the SOP of the posterior DC macrochaetae (arrowhead). (B) A large mutant clone that includes the anterior part of a DC proneural cluster (arrowhead), as revealed by expression of the AS1.4DC-lacZ construct (red). (C) Higher magnification image of the same DC cluster, showing merged, green, and red plus blue channels. Most of the cells with strong accumulation of ß-galactosidase and the PDC SOP are in the heterozygous territory. The cluster appears roundish rather than elongated (Fig. 5A,F) because there is little accumulation of ß-galactosidase in the homozygous territory (arrowhead). (D) Cells of a DC proneural cluster that overexpress UAS-chniS (green) accumulate less Sc protein than neighboring cells (red channel). (E) An SOP has been singled out from an heterozygous chnECJ1 cell (arrowhead). (F) Clone that includes part of the L3 proneural cluster, as revealed by the expression of the L3-TSM-lacZ construct. Note the irregular shape of the cluster and the reduced expression within the homozygous territory (arrowhead). A control proneural cluster entirely within heterozygous territory has a roundish shape (G). (H) Merged and red plus blue channels views of the notum region of a late third instar wing disk harboring several clones of homozygous chnECJ1 cells. Sc (red) and Sens (blue) stainings reveal SOPs. The PSC SOP, which would have to develop within a clone (arrow) and is one of the earliest SOPs to emerge, is absent. Nomenclature for other SOPs is indicated. The presence of the ADC SOP confirms the late stage of the disk.

View larger version (50K): [in a new window]   Fig. 7. Chn can bind to the DC enhancer in vitro, and model for the genetic control of macrochaeta SOP singling out. (A) Scheme of the subfragments of the AS1.4DC enhancer (thin lines under the AS1.4DC thick line) that were assayed for binding by a polypeptide containing the five zinc fingers motifs of Chn (Chn5ZF). A, AvaII; B, BglII; P, PstI; S, SalI: Only the DC6 fragment (in red) was bound by Chn5ZF in an EMSA assay, as shown in (B). A polypeptide with the five zinc fingers motifs of Chn (Chn5ZF) binds to the 32P-labeled DC6 DNA probe in an EMSA assay. (1) 32P-labeled DC6 DNA probe alone; (2) labeled DC6 with Chn5ZF present; (3) labeled DC6 with Chn5ZF and an 8-fold molar ratio of cold DC6 added; (4) labeled DC6 with Chn5ZF and an 8-fold molar ratio of cold control DNA (subfragment DC1) added. (C) A 0.5 kb fragment of the DC enhancer (PB0.5DC, panel A) directs lacZ expression only in the posterior DC SOP (García-García et al., 1999). (D) Overexpression of UAS-chn (C765-Gal4) promotes expression in many cells of the posterior notum DC region. (E) A combination of prepattern factors (PFs) acting on an ASC proneural cluster enhancer (PE) activate sc expression. The Dl/N signaling pathway, activated by Sc in the proneural clusters, blocks the SOP-specific enhancer (Artavanis-Tsakonas et al., 1995; Culí and Modolell, 1998; Giagtzoglou et al., 2003). An idealized representation of Sc accumulation in the proneural cluster is shown at the bottom of the panel. (F) Sc activates chn. This activation might be direct and mediated by E-boxes present in the chn gene. Chn binds to the PE and further stimulates sc expression, leading to higher accumulation of this protein, and also of Ac (data not shown). The ac/sc-chn stimulatory loop is established. Dl/N signaling still blocks the SOP-specific enhancer. (G) In a poorly understood process, a cell with high levels of Sc accumulation, and helped by the EGFR signaling pathway that is also activated by Sc (Culí et al., 2001), establishes a Sc self-stimulatory loop that is mediated by the SOP-specific enhancer (Culí and Modolell, 1998; Giagtzoglou et al., 2003). This cell accumulates much Sc, Ac and Sens and becomes the SOP. The Dl/N pathway no longer blocks the SOP enhancer in this cell, but it does so in the neighboring cells (Artavanis-Tsakonas et al., 1995).

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