Orthopedia expression during Drosophila melanogaster nervous system development and its regulation by microRNA-252.

During brain development of Drosophila melanogaster many transcription factors are involved in regulating neural fate and morphogenesis. In our study we show that the transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, plays an important role in this process. Otp is expressed in a stable pattern in defined lineages from mid-embryonic stages into the adult brain and therefore a very stable marker for these lineages. We determined the abundance of the two different otp transcripts in the brain and hindgut during development using qPCR. CRISPR/Cas9 generated otp mutants of the longer protein form significantly affect the expression of Otp in specific areas. We generated an otp enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the complete expression of otp during development except the embryonic hindgut expression. Since in the embryo, the expression of Otp is posttranscriptionally regulated, we looked for putative miRNAs interacting with the otp 3'UTR, and identified microRNA-252 as a candidate. Further analyses with mutated and deleted forms of the microRNA-252 interacting sequence in the otp 3'UTR demonstrate an in vivo interaction of microRNA-252 with the otp 3'UTR. An effect of this interaction is seen in the adult brain, where Otp expression is partially abolished in a knockout strain of microRNA-252. Our results show that Otp is another important factor for brain development in Drosophila melanogaster.

Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development.

BACKGROUND: The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. RESULTS: In this study we conducted a detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. We generated a new hbnKOGal4 enhancer trap strain by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. We characterized several enhancers that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, we generated deletions of four of these enhancer regions through gene targeting and analysed their effects on the expression and function of hbn. CONCLUSION: The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.

Enhancer analysis of the Drosophila zinc finger transcription factor Earmuff by gene targeting.

BACKGROUND: Many transcription factors are involved in the formation of the brain during the development of Drosophila melanogaster. The transcription factor Earmuff (Erm), a member of the forebrain embryonic zinc finger family (Fezf), is one of these important factors for brain development. One major function of Earmuff is the regulation of proliferation within type II neuroblast lineages in the brain; here, Earmuff is expressed in intermediate neural progenitor cells (INPs) and balances neuronal differentiation versus stem cell maintenance. Erm expression during development is regulated by several enhancers. RESULTS: In this work we show a functional analysis of erm and some of its enhancers. We generated a new erm mutant allele by gene targeting and reintegrated Gal4 to make an erm enhancer trap strain that could also be used on an erm mutant background. The deletion of three of the previously analysed enhancers showing the most prominent expression patterns of erm by gene targeting resulted in specific temporal and spatial defects in defined brain structures. These defects were already known but here could be assigned to specific enhancer regions. CONCLUSION: This analysis is to our knowledge the first systematic analysis of several large enhancer deletions of a Drosophila gene by gene targeting and will enable deeper analysis of erm enhancer functions in the future.

Functional analysis of enhancer elements regulating the expression of the Drosophila homeodomain transcription factor DRx by gene targeting.

BACKGROUND: The Drosophila brain is an ideal model system to study stem cells, here called neuroblasts, and the generation of neural lineages. Many transcriptional activators are involved in formation of the brain during the development of Drosophila melanogaster. The transcription factor Drosophila Retinal homeobox (DRx), a member of the 57B homeobox gene cluster, is also one of these factors for brain development. RESULTS: In this study a detailed expression analysis of DRx in different developmental stages was conducted. We show that DRx is expressed in the embryonic brain in the protocerebrum, in the larval brain in the DM and DL lineages, the medulla and the lobula complex and in the central complex of the adult brain. We generated a DRx enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the endogenous expression of DRx. With the help of eight existing enhancer-Gal4 strains and one made by our group, we mapped various enhancers necessary for the expression of DRx during all stages of brain development from the embryo to the adult. We made an analysis of some larger enhancer regions by gene targeting. Deletion of three of these enhancers showing the most prominent expression patterns in the brain resulted in specific temporal and spatial loss of DRx expression in defined brain structures. CONCLUSION: Our data show that DRx is expressed in specific neuroblasts and defined neural lineages and suggest that DRx is another important factor for Drosophila brain development.

The homeodomain transcription factor Orthopedia is involved in development of the Drosophila hindgut.

BACKGROUND: The Drosophila hindgut is commonly used model for studying various aspects of organogenesis like primordium establishment, further specification, patterning, and morphogenesis. During embryonic development of Drosophila, many transcriptional activators are involved in the formation of the hindgut. The transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, is expressed in the hindgut and nervous system of developing Drosophila embryos, but due to the lack of mutants no functional analysis has been conducted yet. RESULTS: We show that two different otp transcripts, a hindgut-specific and a nervous system-specific form, are present in the Drosophila embryo. Using an Otp antibody, a detailed expression analysis during hindgut development was carried out. Otp was not only expressed in the embryonic hindgut, but also in the larval and adult hindgut. To analyse the function of otp, we generated the mutant otp allele otpGT by ends-out gene targeting. In addition, we isolated two EMS-induced otp alleles in a genetic screen for mutants of the 57B region. All three otp alleles showed embryonic lethality with a severe hindgut phenotype. Anal pads were reduced and the large intestine was completely missing. This phenotype is due to apoptosis in the hindgut primordium and the developing hindgut. CONCLUSION: Our data suggest that Otp is another important factor for hindgut development of Drosophila. As a downstream factor of byn Otp is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells.

Generation of Mutants from the 57B Region of Drosophila melanogaster.

The 57B region of Drosophila melanogaster includes a cluster of the three homeobox genes orthopedia (otp), Drosophila Retinal homeobox (DRx), and homeobrain (hbn). In an attempt to isolate mutants for these genes, we performed an EMS mutagenesis and isolated lethal mutants from the 57B region, among them mutants for otp, DRx, and hbn. With the help of two newly generated deletions from the 57B region, we mapped additional mutants to specific chromosomal intervals and identified several of these mutants from the 57B region molecularly. In addition, we generated mutants for CG15651 and RIC-3 by gene targeting and mutants for the genes CG9344, CG15649, CG15650, and ND-B14.7 using the CRISPR/Cas9 system. We determined the lethality period during development for most isolated mutants. In total, we analysed alleles from nine different genes from the 57B region of Drosophila, which could now be used to further explore the functions of the corresponding genes in the future.