Mechanisms of Development 103 (2001) 159±162
www.elsevier.com/locate/modo
Gene expression pattern
Drosophila regulatory factor X is an embryonic type I sensory neuron
marker also expressed in spermatids and in the brain of Drosophila
Camille Vandaele, Madeleine Coulon-Bublex, Pierre Couble, BeÂneÂdicte Durand*
Centre de GeÂneÂtique MoleÂculaire et Cellulaire U.M.R. 5534, Laboratoire des MeÂcanismes MoleÂculaires et Cellulaires du DeÂveloppement,
Universite Claude Bernard Lyon I, 43, Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France
Received 8 December 2000; received in revised form 1 February 2001; accepted 28 February 2001
Abstract
We report the expression pattern of a Drosophila transcription factor, Drosophila regulatory factor X (dRFX), which belongs to the RFX
winged-helix transcription factor family. dRFX is distributed in type I sensory neuron lineage of the peripheral nervous system throughout
Drosophila development and thus represents the ®rst described type I lineage characteristic marker in Drosophila. In addition, dRFX is also
detected in the brain throughout development and in spermatids in adult ¯ies. q 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Regulatory factor X; Drosophila regulatory factor X; Drosophila; Transcription factor; Peripheral nervous system; Type I neuron; Spermatid;
Spermatogenesis; Brain; Development; Chordotonal organ; External sense organ; Monodendritic; Multidendritic; Ciliated neuron
1. Results and discussion
Drosophila regulatory factor X (dRFX) belongs to a subfamily of winged-helix transcription factors characterized
by a conserved DNA binding domain (Durand et al.,
2000; Emery et al., 1996; Gajiwala et al., 2000). dRFX is
homologous to CeRFX/daf-19 in the nematode and to RFX1,
2 and 3 in mouse and human. daf-19 is necessary for ciliated
sensory neuron differentiation in the nematode (Swoboda et
al., 2000). No physiological functions are clearly assigned
to RFX1±3 in mammals even if some candidate target genes
have been proposed (Emery et al., 1996; Iwama et al.,
1999). As a ®rst step towards understanding dRFX function
in Drosophila, we investigated its expression pro®le during
development with a polyclonal antibody raised against a Cterminus peptide.
In embryos, dRFX is ®rst detected at the onset of segmentation in the sensory organ precursors (SOP) located in the
gnathal segment and in the two ®rst SOPs of each thoracic
and abdominal hemisegments described previously as the A
(anterior) and the P (posterior) SOPs (Ghysen and O'Kane,
1989) (Fig. 1A). At stages 12±14 of embryogenesis, when A
and P cells have generated new precursors by division or
recruitment (Fig. 1B,C), dRFX is detected in these secondary precursors. At stage 15, dRFX is found predominantly in
* Corresponding author. Tel.: 133-4-7244-8000 ext. 83383; fax: 133-47244-0555.
E-mail address: [email protected] (B. Durand).
nuclei of all chordotonal (ch) and external sensory (es)
organ neurons, and, at a lower level, in the accessory sister
cells resulting from the last asymmetric division of the
precursors (Fig. 1D,F). At stage 16 of embryogenesis,
dRFX progressively disappears in the accessory sister
cells (Fig. 1E) and is only maintained in neuron nuclei at
the end of embryogenesis (Fig. 1G,H). These results are in
agreement with dRFX mRNA expression described
previously (Durand et al., 2000) and provide new important
information on dRFX stability. Whereas dRFX messenger
RNAs disappear in all cells of the peripheral nervous system
except ch neurons at the end of embryogenesis, the protein
still persists in es neurons. Two types of sensory neurons can
be distinguished in Drosophila: (a) type I neurons that
innervate es and ch organs bear a ciliated sensory process
at the tip of a unique dendrite and are surrounded by specialized supporting cells; and (b) type II neurons are nonciliated multidendritic neurons (Zacharuk, 1985). In the
embryo, both types of neurons are also characterized by
their relative position and shape (Ghysen et al., 1986;
Bodmer and Jan, 1987). With an antibody which labels all
sensory neurons (mAb 22C10; Zipursky et al., 1984), we
clearly demonstrate that dRFX is restricted to type I neurons
of the thoracic and abdominal segments (Fig. 1H,I). In the
head, dRFX is detected in all sensory neurons, except the
Bolwig's organ, which represents the future larval visual
organ (Fig. 1J,K).
An accumulation of dRFX in the brain is also observed
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C. Vandaele et al. / Mechanisms of Development 103 (2001) 159±162
Fig. 1. Analysis of dRFX distribution during embryonic development. (A) Stage 11 embryo, ventrolateral view. Labelled SOPs are detected in gnathal
segments (black arrows) and in A and P cells. (B) Stage 12 embryo, lateral view. dRFX is in the SOPs of each segment. A faint expression is visible in the
procephalic region (black arrow). (C) Stage 14 embryo, lateral view. dRFX is strongly labelled in the procephalic region (black arrow). (D) Stage 15 embryo,
ventrolateral view. dRFX is present in ch and es neurons and in associated accessory-sister cells. (E) Stage 16 embryo, ventrolateral view. dRFX progressively
disappears from accessory sister cells. (F) Magni®cation of stage 16 embryo abdominal segments. The black arrow points to the expression of dRFX in
accessory sister cells. (G) Stage 17 embryo. Magni®cation of abdominal segments. dRFX antibody labels only neuron nuclei. (H) Double staining of a stage 17
embryo. Magni®cation of abdominal segments. mAb 22C10 labels all sensory neuron membranes (brown). dRFX is present only in type I es and ch neuron
nuclei (deep blue). In this view, the two most dorsal es neurons are not visible but are also stained by dRFX antibody. (I) Schematic representation of one
abdominal hemisegment of (H) (adapted from Brewster and Bodmer, 1995) Blue full-®lled circles and triangles correspond to dRFX staining in es and ch
neurons, respectively. Light brown empty squares correspond to multidendritic neurons not stained by dRFX antibody. (J) Stage 17 embryo. Magni®cation of
the cephalic region, double stained with mAb 22C10 and dRFX antibody. dRFX (deep blue) is present in gnathal sensory neurons (white brackets). First
thoracic sensory neurons are also visible (black brackets). dRFX is absent in the Bolwig's organ (black arrow). (K) Magni®cation of the Bolwig's organ
(brown) only stained by mAb22C10. dRFX is present in the sensory organ of the clypeolabrum (asterisk). Stages were determined according to Campos-Ortega
and Hartenstein (1985). In all views, anterior is to the left and dorsal is to the top.
starting from stage 12 of embryogenesis in two small bilateral cell clusters in the procephalic neurogenic region (Fig.
1B). From stage 14 to 17 of embryonic development, dRFX
persists in a small number of cells abutting the optic lobe
invagination (Fig. 2A±C). dRFX is detected in the brain
throughout larval and pupal development (Fig. 2D,F,G) in
a restricted number of cells in each brain lobe (Fig. 2E,H).
During larval and pupal life, we observed that dRFX is
absent in early SOPs of the imaginal discs (data not shown),
but appears later on in development, i.e. after puparium
formation (APF). dRFX was found in all adult type I sensory
organ lineage analyzed (antennae, leg and wing discs). No
expression was detected in the eye disc. Expression is ®rst
detected in the leg discs (Fig. 3A,B), at the beginning of
eversion, in the innermost SOPs of the femoral ch organ,
which are ready to differentiate (zur Lage and Jarman,
1999). Similarly, dRFX is detected in the Johnston organs
of the second antennal segment of the antennal discs only
once they are fully everted (around 24 h APF; Fig. 3C,D).
Finally, dRFX is very transiently present in adult males
C. Vandaele et al. / Mechanisms of Development 103 (2001) 159±162
161
Fig. 2. dRFX staining in the brain throughout development. (A) Stage 14 embryo. dRFX is present in a small number of cells (arrow) of the procephalic
neurogenic region. White arrows correspond to labelled SOPs of the gnathal segments. (B) Stage 15 embryo. dRFX is maintained in the same number of cells.
(C) Stage 17 embryo. dRFX is still present in the embryonic brain. (D) Third instar larval brain. Fluorescent detection of dRFX in red. A subset of brain cells
are labelled. (E) Schematic representation of a third instar larval brain. dRFX expression was reported on only one lobe according to (F). (F) Magni®cation of
dRFX expressing cells in a third instar larval brain. (G) Magni®cation of one half of a pupal brain (36 h APF). Staining of dRFX is still observed in a small
number of cells. (H) Schematic representation of a pupal brain (36 h APF) to localize dRFX expressing cells according to (G).
during spermatogenesis in syncitial bundles of 64 spermatids (Fig. 4A). According to their shape and position within
the testis lumen, these spermatids are in the elongation
phase of their ¯agellum (Lindsley and Tokuyasu, 1980).
The protein is not detectable in spherical spermatid nuclei
nor in late condensing spermatid nuclei (Fig. 4B1±3). dRFX
was not detected in the female germline and in other tissues
of whole stained dissected larvae.
2. Methods
2.1. Fly stocks
Canton-S was used as the wild type strain. A101 strain
(Bellen et al., 1989) expresses b-galactosidase (b-gal) in all
SOPs.
Fig. 3. dRFX distribution in imaginal SOPs. Double labelling of A101
imaginal discs which express b-gal in all SOPs. (A) Everted leg disc (6 h
APF). b-Gal antibody labels mature SOPs (green). The white arrow points
to mid-mature SOPs and the white arrowhead points to mature SOPs. (B)
Same view, dRFX in red, only in most mature SOPs (white arrowhead). (C)
Fully everted antennal disc (24 h APF). b-Gal antibody labels mature SOPs
(green). Strong yellow green signal corresponds to aspeci®c ¯uorescence of
lipid droplets. (D) Same view, dRFX in red. dRFX is also present in SOPs
and as in leg discs all SOPs do not express dRFX at the same level. dRFX
appears to be stronger in low b-gal expressing SOPs (as in leg disc, A,B).
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Fig. 4. (A) Whole testis. Anti-dRFX antibody marks the spermatid heads (brown) at late spermatogenesis. (B) Confocal microscopic imaging of testis double
labelled with dRFX in red (B1) and DNA in green (B2). The merged view in B3 shows that only the 64 elongated spermatid nuclei are stained by dRFX
(yellow). dRFX is not present in spherical spermatids (arrow) and disappears in condensing sperm heads (arrowhead).
2.2. Immunohistochemistry
Antibodies staining was performed with the Vectastain
ABC staining kit with the modi®cations of Patel et al.
(1994) for double labelling experiments. Primary antibodies
were: rabbit anti-dRFX (raised against a synthetic C-terminus peptide CSSASSGGDVGNEAKRLKQE, Covalab),
mouse anti-b-gal (Sigma Aldrich), and mouse anti-22C10
(Zipursky et al., 1984). All were used at a dilution of 1:5000.
Secondary antibodies diluted at 1:400 were goat anti-mouse
horseradish peroxidase-conjugated (Vectastain ABC staining kit), goat anti-rabbit alkaline phosphatase-conjugated
(Biorad), goat anti-mouse Alexa Fluor 488-conjugated
(Molecular Probes) and goat anti-rabbit Cy3-conjugated
(Amersham Pharmacia Biotech). In testis, DNA staining
was performed with YO-PRO-1 (Molecular Probes).
Conventional ¯uorescent microscopic images were made
on a Zeiss Axioskop. Only Figs. 2F and 4B1±3 are confocal
images acquired on a Zeiss LSM5.
Acknowledgements
This work was supported by the CNRS and by a grant form
the Association pour la Recherche sur le Cancer (ARC). C.V.
was supported by a fellowship from the French Ministry of
Education. We are grateful to C. Dambly-ChaudieÁre for the
A101 strain. S. Benzer kindly provided the mAb22C10. We
would like to thank J. Thomas and J.C. Prudhomme for careful reading of the manuscript. We also thank B. Loppin for
help with the confocal image acquisitions.
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