The CPEB Protein Orb2 Has Multiple Functions during Spermatogenesis in Drosophila melanogaster

Cytoplasmic Polyadenylation Element Binding (CPEB) proteins are translational regulators that can either activate or repress translation depending on the target mRNA and the specific biological context. There are two CPEB subfamilies and most animals have one or more genes from each. Drosophila has a single CPEB gene, orb and orb2, from each subfamily. orb expression is only detected at high levels in the germline and has critical functions in oogenesis but not spermatogenesis. By contrast, orb2 is broadly expressed in the soma; and previous studies have revealed important functions in asymmetric cell division, viability, motor function, learning, and memory. Here we show that orb2 is also expressed in the adult male germline and that it has essential functions in programming the progression of spermatogenesis from meiosis through differentiation. Like the translational regulators boule (bol) and off-schedule (ofs), orb2 is required for meiosis and orb2 mutant spermatocytes undergo a prolonged arrest during the meiotic G2-M transition. However, orb2 differs from boule and off-schedule in that this arrest occurs at a later step in meiotic progression after the synthesis of the meiotic regulator twine. orb2 is also required for the orderly differentiation of the spermatids after meiosis is complete. The differentiation defects in orb2 mutants include abnormal elongation of the spermatid flagellar axonemes, a failure in individualization and improper post-meiotic gene expression. Amongst the orb2 differentiation targets are orb and two other mRNAs, which are transcribed post-meiotically and localized to the tip of the flagellar axonemes. Additionally, analysis of a partial loss of function orb2 mutant suggests that the orb2 differentiation phenotypes are independent of the earlier arrest in meiosis

Ig Superfamily Ligand and Receptor Pairs Expressed in Synaptic Partners in Drosophila

Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Here, we sought to identify candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity

A study of Drosophila CPEB protein Orb2: expression, functions and regulatory mechanisms

Translational control plays an essential role in regulating gene expression. Members of the CPEB (Cytoplasm Polyadenylation Element Binding Protein) family of proteins have been shown to bind to the 3' UTR of the target mRNAs and regulate their translation. While the classic CPEB (the CPEB1 subfamily) members have conserved roles in germline development, the newly discovered CPEB2 subfamily members are enriched in the nervous system. Earlier studies in Drosophila revealed that orb (a CPEB1 subfamily member) is required for the specification and also the establishment of proper axes of the oocyte. However, Orb is only found in the germline, with no roles in other tissue. My thesis has focused on the newly discovered CPEB2 subfamily member orb2. Our analysis on Orb2 expression and function has revealed that Orb2 is involved in multiple processes including embryogenesis, spermatogenesis, and adult locomotion control. We discovered that Orb2 is highly expressed in embryonic CNS and its presence is required for the proper asymmetric division of neural and some somatic precursor cells. In the neuroblasts (NB), loss-of-function mutations in orb2 result in the disruption of correct mitotic spindle orientation and also influence the apical cortical localization of aPKC, a critical factor in determining the asymmetric neuroblast (NB) division plane. Orb2 also functions in the germline during spermatogenesis. Specifically, Orb2 is required for meiosis progression in spermatocytes, and differentiation of spermatids (meiotic division product). The orb236 null allele displays an over-accumulation of Twine-lacZ (Twine is a meiotic CDC25), and the mutant spermatocytes arrest at the Meiosis I G2-M transition. Analyzing a hypomorphic allele of orb2 revealed another function of Orb2 during subsequent differentiation steps: the proper asymmetric elongation of the spermatids. Orb2 accumulates preferentially in the apical termini of elongating spermatids. Moreover, like Orb2, aPKC - a critical factor involved in defining apical-basal polarity in many other cell types - is also found concentrated in the apical termini of elongating spermatids. In orb2 mutants we find that the elongation of the spermatids is randomized with respect to the testes apical-terminal axis, correlated with the loss of localized aPKC in the orb2 mutants. As the same thing was observed in dividing neuroblasts, it suggests that orb2 has a conserved role in localizing aPKC, and neuroblast asymmetric division regulators may be conserved in regulating spermatids asymmetric elongation. To better understand the mechanism of Orb2 mediated translational control, we used immunoprecipitation to pull down Orb2 associated proteins in vivo and analyzed their identities by large-scale mass spectrometry. The results support the idea that Orb2 functions as a classic CPEB in an mRNP complex to regulate polyadenylation of its target mRNAs. Analysis of the data also suggests that Orb2 can physically bind to other asymmetric division regulators such as Bazooka. Further studies will help us understand how Orb2, a novel member of the CPEB2 subfamily proteins, executes its role during translational regulation of target mRNAs

Spermatid Cyst Polarization in <i>Drosophila</i> Depends upon <i>apkc</i> and the CPEB Family Translational Regulator <i>orb2</i>

<div><p>Mature <i>Drosophila</i> sperm are highly polarized cells—on one side is a nearly 2 mm long flagellar tail that comprises most of the cell, while on the other is the sperm head, which carries the gamete's genetic information. The polarization of the sperm cells commences after meiosis is complete and the 64-cell spermatid cyst begins the process of differentiation. The spermatid nuclei cluster to one side of the cyst, while the flagellar axonemes grows from the other. The elongating spermatid bundles are also polarized with respect to the main axis of the testis; the sperm heads are always oriented basally, while the growing tails extend apically. This orientation within the testes is important for transferring the mature sperm into the seminal vesicles. We show here that orienting cyst polarization with respect to the main axis of the testis depends upon atypical Protein Kinase C (aPKC), a factor implicated in polarity decisions in many different biological contexts. When <i>apkc</i> activity is compromised in the male germline, the direction of cyst polarization within this organ is randomized. Significantly, the mechanisms used to spatially restrict <i>apkc</i> activity to the apical side of the spermatid cyst are different from the canonical cross-regulatory interactions between this kinase and other cell polarity proteins that normally orchestrate polarization. We show that the asymmetric accumulation of aPKC protein in the cyst depends on an mRNA localization pathway that is regulated by the <i>Drosophila</i> CPEB protein Orb2. <i>orb2</i> is required to properly localize and activate the translation of <i>apkc</i> mRNAs in polarizing spermatid cysts. We also show that <i>orb2</i> functions not only in orienting cyst polarization with respect to the apical-basal axis of the testis, but also in the process of polarization itself. One of the <i>orb2</i> targets in this process is its own mRNA. Moreover, the proper execution of this <i>orb2</i> autoregulatory pathway depends upon <i>apkc</i>.</p></div

<i>orb2</i> is required for cyst polarization.

<p>A and B) Green, phalloidin labeled Actin; Blue, DNA; Red, Orb2. A) Clustered nuclei of spermatid cysts (arrowheads) that have completed elongation and just assembled individualization complexes (ICs) (arrows) are found in the spermatogonial/early spermatocyte region of <i>orb2^ΔQ</i> testis. B) White arrows point to two ICs in an <i>orb2^ΔQ</i> testis that are moving in opposite directions in the middle of the testis (yellow arrows indicate directions of motion). C–E) Green, Bol; blue, DNA. C) Like Orb2, the translation factor Bol is localized a comet pattern during flagellar axoneme elongation in wild type testes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004380#pgen.1004380-Xu1" target="_blank">[29]</a>. D) A bi-polar <i>orb2³⁶</i> spermatid cyst that has Bol concentrated at both elongating ends, while the nuclei are in the middle of the cyst (arrows). E) A partially elongated <i>orb2³⁶</i> spermatid cyst in which spermatid nuclei and Bol protein are scattered randomly. Dotted line outlines one among four spermatid cysts in the figure. Yellow arrows point out a few of the scattered nuclei. All images are orientated with apical side of the testes to the left and basal to the right. Scale bar: 50 µm.</p

<i>orb2</i> autoregulates the localization and translation of <i>orb2</i> mRNA.

<p>A) Wild type spermatid cyst showing that Orb2 protein (red) and <i>orb2</i> mRNA (green) are distributed in the characteristic comet pattern and display extensive co-localization, particularly in the comet head. B) In <i>orb2^ΔQ</i> spermatids that elongate in the incorrect direction, Orb2^ΔQ protein (red) and <i>orb2^ΔQ</i> mRNA (green) are distributed uniformly in the cyst and there is no evidence of a comet head (expected position is indicated by arrow). All images are orientated with apical to the left and basal to the right. Scale bar: 20 µm.</p

<i>apkc-RA</i> and <i>orb2</i> mRNAs associate with Orb2 <i>in vivo</i>.

<p>A) The <i>apkc</i> transcription unit as annotated in <a href="http://flybase.org/reports/FBgn0261854.html" target="_blank">http://flybase.org/reports/FBgn0261854.html</a>. Transcripts expressed from four <i>apkc</i> promoters (P1–P4) plus a collection of alternatively spliced exons and UTRs are predicted to generate more than ten mRNAs. Green bar, protein coding exon; grey bar, UTR; solid black line, intron. <i>apkc-RA</i> CPE sites are labeled in red. Positions of primers used for RT-PCR experiments to identify different <i>apkc</i> mRNA species are marked in dark blue. The two dsRNA sequences used in the <i>apkc</i> RNAi knockdown experiments are marked in pink. Probes for the <i>apkc</i> Orb2 EMSAs, com-GS and RA-GS, are indicated above the gene in brown. B) <i>apkc</i> transcripts expressed in testes were detected by RT-PCR using the primer pairs as indicated. Not all of the primer pairs are specific to one of the four promoters, but some are specific to particular splice forms. C) <i>apkc-RA</i> mRNA can be immunoprecipitated with Orb2 antibodies from WT and <i>orb2^ΔQ</i> testis extracts. β-Gal antibody (a negative control) didn't immunoprecipitate <i>apkc-RA</i> mRNA. D) Orb2 antibody immunoprecipitated <i>orb2</i> mRNA from wild type testes while the control β-Gal antibody did not.</p