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Keywords:

  • fernández/distal antenna;
  • hernández/distal antenna related;
  • nervous system;
  • antenna;
  • eye

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The “pipsqueak” family is composed of proteins that contain a pipsqueak motif, a previously characterised DNA binding domain, and thus represents a new family of potential transcription factors. Previous functional characterisation of several Drosophila genes encoding pipsqueak domain-containing proteins has shown their crucial role in development. Here, I report the embryonic, larval, and pupal expression pattern of two Drosophila genes, fernández/distal antenna and hernández/distal antenna related, which encode protein members of the pipsqueak family with similar pipsqueak motifs. Furthermore, I show that, consistently with their expression pattern, these two genes are required in the nervous system during the embryonic development. Developmental Dynamics 230:361–365, 2004. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The “pipsqueak” family is a new family of eukaryotic proteins that contain a pipsqueak (psq) motif. This motif consists of a 50 amino acid sequence, which is structurally related to the helix-turn-helix domain, known for its role in DNA recognition. However, it lacks the conserved sequence signatures of the classic eukaryotic DNA-binding motifs, thus defining a new family of DNA-binding proteins and potential transcription factors (Lehmann et al., 1998). This family includes proteins from fungi, sea urchins, nematodes, insects, and vertebrates. Their role has been better studied in Drosophila, where 14 proteins, including a psq motif (psq proteins), have been identified. These proteins can be classified into three groups, depending on sequence homologies within the psq motif, the existence of different protein domains, and the position of the psq motif within the proteins (Siegmund and Lehmann, 2002). The first group is composed of members of the BTB (Broad-complex, Tramtrack, Bric à brac) protein family; the second group contains the Drosophila cell death regulator E93, with orthologues in sea urchins, nematodes, and humans; the third group includes the two Drosophila genes fernández (fer)/distal antenna (dan) and hernández (hern)/distal antenna related (danr), the human centromere protein B (CENP-B), a related predicted human protein (CAB66474), and transposases from phytopathogenetic fungi (Siegmund and Lehmann, 2002; Suzanne et al., 2003; Emerald et al., 2003). Among members of this protein group, the psq domain, located at the N-terminus of the protein, is particularly well conserved. Functional analysis of some of the Drosophila genes encoding psq proteins revealed their importance in a variety of fundamental processes: ribbon is required in epithelial migration of the tracheal system and in dorsal closure (Blake et al., 1998; Bradley and Andrew, 2001; Shim et al., 2001); pipsqueak in early oogenesis (Horowitz and Berg, 1996); bric à brac in appendage diversity, morphogenesis, and oogenesis (Godt et al., 1993; Godt and Laski, 1995; Chu et al., 2002); and finally, E93 in programmed cell death during metamorphosis (Lee et al., 2000).

Here, I describe the expression pattern of two of these genes, fer/dan and hern/danr, that belong to the CENP-B/transposase subfamily of psq proteins and have been characterised recently (Emerald et al., 2003; Suzanne et al., 2003). Their expression patterns, however, have only been described in third instar larvae. In the present work, I analyse in more detail their larval and pupal expression by using several Gal4 enhancer traps inserted into the fer and the hern genes and characterise their embryonic expression patterns. Finally, I show that they are required for the correct development of the embryonic nervous system.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

fer and hern Expression Is Very Similar During Larval and Pupal Stages

The expression pattern of fer and hern has been described in third instar larvae. Both genes are transcribed similarly in the antennal and eye primordia (Emerald et al., 2003; Suzanne et al., 2003). To complete this description and analyse the expression of these genes during pupal stages, I used four Gal4 enhancer trap lines: two inserted in the fer gene and two in the hern one. These lines reproduce the expression patterns of fer and hern genes in the third instar antennal primordium (in the third antennal segment and in the arista) and in the eye (see Fig. 1A), although the Gal4 signal in the differentiated eye is much stronger than the RNA expression (Suzanne et al., 2003). The restricted expression in antenna and eye primordia is maintained from prepupal stages to the adult (Fig. 1B,C). In addition, I noted a ubiquitous expression in the wing and a transient expression in the labium at the end of pupal stages (not shown).

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Figure 1. Expression pattern of a Gal4 enhancer trap insertion in the fer gene (the genotype is CES115 UAS-GFP/TM6B). Similar if not identical expression is detected with the two Gal4 enhancer trap insertions in the hern gene and the other Gal4 insert upstream of the fer gene (See Experimental Procedures section). In A and B, “a” indicates the antenna, and “e: the eye. A: Eye-antennal imaginal disc of a third instar larva showing a restricted expression in the antenna and the eye. B: Eye-antennal primordia at a prepupal stage, showing a similar expression in distal antenna and eye. C: Head of a pharate adult, showing the persistence of the expression of fer in the same regions. The arrowhead indicates the green fluorescent protein expression in the arista.

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fer and hern Expression During Embryogenesis

Neither fer transcript nor Fer protein could be detected at the syncytial blastoderm stage, suggesting that the fer gene is not maternally expressed (not shown). The RNA and protein expression is first observed at the cellular blastoderm stage (stage 5, see Campos-Ortega and Hartenstein, 1985), in a large central domain, being excluded from the most anterior, posterior, and ventral cells, corresponding respectively to the terminal parts of the embryo and to the mesoderm primordium (Fig. 2A,A′,A″,B). When the neuroectoderm becomes morphologically distinct, during the initial phase of germ band elongation in stage 8, fer expression becomes stronger in the most ventral region of the embryo (which corresponds to the presumptive neuroectoderm), while it progressively disappears from the most dorsal cells (Fig. 2D,E). The neurectoderm can be recognized at this stage by its large cells, compared with the smaller cells of the lateral and dorsal ectoderm (Fig. 2D′,E′). At stage 9, the neuroblasts start segregating from the neuroectoderm, proceeding in three pulses that give rise to three subpopulations of neuroblasts: SI, SII, and SIII. The segregating neuroblasts leave the outer layer and move internally to become located between the mesoderm and the ectoderm. At this stage, Fer protein is detected both in the segregating neuroblasts SI and in the neuroectoderm (Fig. 2G). At stage 10, Fer protein is detected in the two rows of SI and SII neuroblasts, as well as in the neuroectoderm (Fig. 2H,I). At late stage 11, the neuroblast segregation is finished and fer RNA as well as Fer protein are present in the ventral cord, whereas no expression is detected in the ventral ectoderm (Fig. 2J,K). There is also a transient but strong expression of the Fer protein as well as the fer RNA in the posterior part of the last abdominal segment during stages 10–11, in a group of epidermal cells surrounding the proctodeum (Fig. 2K, not shown). Finally, Fer expression (RNA and protein) is maintained in the neurons, in the ventral cord and the brain until the end of embryogenesis (see stage 15 in Fig. 2M,N).

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Figure 2. Expression pattern of fer and hern during embryonic development. Except in A′,A″,H,I,M–O, which are ventral views, anterior is to the left and dorsal up. A,A′,A″,D,J,M: fer RNA; B,E,G–I,K,N: Fer protein; and C,F,L,O: hern RNA expression pattern at different stages (st.) of the embryogenesis. A,A′,A″,B: In cellular blastoderms, fer RNA and protein are lower in the poles and the ventral region (arrow in A′) of the embryo. A″: Detail of the A′ image. C:hern expression is similar to that of fer except for the signal in the anterior region (arrow). D,E: At stage 8, fer RNA and protein are detected more strongly in the presumptive neurectoderm (bigger cells, arrows in D′ and E′) than in the lateral ectoderm (smaller cells, arrowheads in D′ and E′). Cell size is indicated by red circles in D′ and E′. Note the similarity with the hern RNA expression at this stage (F, compare with D and E). G: At stage 9, Fer/Dan is expressed in neuroblasts (arrow; one neuroblast is encircled) when they start to delaminate and in the ventral ectoderm (arrowhead). H: As formation of the nervous system continues, more rows of neuroblasts are delaminated (arrows), all of them expressing Fer. I: Different focal plane of the same embryo showed in H; note the presence of Fer expression in the neurectoderm at this stage. J–L: At stage 11, fer RNA (J), Fer protein (K), and hern RNA (L) disappear from the ectoderm (arrowheads) and persist in the ventral cord (arrows). Note also the strong Fer expression in cells surrounding the proctodeum (red arrowhead in K). M–O: Expression of fer and hern are finally restricted to the ventral cord at the end of embryogenesis (fer RNA in M, Fer protein in N, and hern RNA in O).

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During embryogenesis, hern RNA is expressed in a very similar pattern to that of the fer RNA and protein. In particular, it shows the same expression pattern in the neurectoderm being also restricted to the ventral cord at the end of embryogenesis (Fig. 2, compare A,D,J,M with C,F,L,O). However, at the blastoderm stage, hern RNA is detected in a group of cells in the anterior dorsal part of the embryo (Fig. 2C). This expression is specific of hern and is maintained until stage 11 (Fig. 2L).

fer and hern Embryonic Phenotype

To test if fer and hern are required during embryogenesis, both genes were inactivated by RNA interference. The ifer and ihern constructs (see Experimental Procedures section), making Gal4-induced double-stranded fer and hern RNA, respectively, have been shown previously to induce fer or hern RNA degradation (Suzanne et al., 2003). I induced the expression of the ifer and ihern constructs with a maternal Gal4 (mat-Gal4) driver to ensure early inactivation of the fer and hern function and visualised the general structure of the central nervous system by staining the embryos with an anti-FascII antibody (Vactor et al., 1993). In some embryos carrying both the Gal4 driver and either the UAS-ifer or the UAS-ihern constructs, the ventral nerve cord was disrupted. The phenotype ranges from a very weak defect to a total disorganisation of the nervous system (Fig. 3, compare B and C with the wild-type in A). This phenotype corresponds to around 31% of the mat-Gal4; UAS-ifer embryos and 47% of the mat-Gal4; UAS-ihern embryos. Similar defects were observed in 76% of embryos homozygous for a deficiency for the two genes (Df dan danrex56, Emerald et al., 2003). Although the deficiency has been described as homozygous viable (Emerald et al., 2003), only 50% of the homozygotes reach the adult stage, and the survivors live just for a few days. These results are consistent with a role of fer and hern in the formation of the central nervous system. Besides, embryos mat-Gal4; UAS-ihern, mat-Gal4;UAS-ifer, or homozygous for the deficiency dan danrex56, do not complete the retraction of the germ band (not shown).

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Figure 3. Inactivation of fer and hern by RNA interference showing that they are required for embryonic central nervous system development. A: Wild-type expression of FascII, which stains the brain, the ventral nerve cord and the peripheral nervous system. B: Embryos of ifer / +; T331-Gal4 / + genotype. C: Embryos of ihern/T331-Gal4 genotype. D: Embryos homozygous for the deficiency dan danrex56, which eliminates both the fer and hern genes. Note the similar disruption of the ventral cord in each of the three genotypes (arrows).

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It was previously reported that hern/danr and fer/dan are required for the correct development of the eye and the antenna (Emerald et al., 2003; Suzanne et al., 2003). I have shown here that, in addition, fer and hern are needed to form the central nervous system in the embryo; these results correlate with their precise expression in this tissue and suggest that they might be partly redundant.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Isolation of GAL4 Lines and Genetic Strains

The method used to isolate the GAL4 lines (Calleja et al., 1996) and the UAS-GFP (Ito et al., 1997) stock has been previously described. CES115 and AC116 are two Gal4 insertions inserted at 1,444 kb and 1,557 kb, respectively, upstream of the fer transcription start site; MD634 and CES75 are two Gal4 insertions located at 338 kb and 396 kb, respectively, upstream of the hern transcription start site (Suzanne et al., 2003). The four lines show a very similar or identical pattern of expression.

Immunostaining of Drosophila Embryos

The antibody staining was done as described previously (Azpiazu and Frasch, 1993), and embryos were mounted in glycerol 87%. The antibodies used are anti-Dan (Emerald et al., 2003) and anti-FascII (Vactor et al., 1993).

RNA Interference

The ifer and ihern constructs were obtained as described in Suzanne et al. (2003).

In Situ Hybridization

It was done according to Azpiazu and Frasch (1993). The fer and hern probes were synthesised with the T3 and T7 RNA polymerases, from bluescript vectors containing the fer and the hern cDNA, to generate the antisense and the sense (negative control) probes of both genes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

I thank E. Sánchez-Herrero for help, encouragement, and comments on the manuscript, S. Cohen for providing the Dan (Fer) antibody and the deficiency dan danrex56, A. Preiss for the pHIBS vector, D.S. Johnston for the Gal4 driver, and G. Morata for comments on the manuscript. This work has been supported by grants from the Comunidad Autónoma de Madrid (08.9/0003/98 and 08.1/0031/2001.1) to Ernesto Sánchez-Herrero, and an Institutional Grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa. I was supported by an European Marie Curie fellowship.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
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