Microtubule nucleation and organization in dendrites.

Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies.

Centrosomin represses dendrite branching by orienting microtubule nucleation.

Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization.

Knot/Collier and cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape.

In a complex nervous system, neuronal functional diversity is reflected in the wide variety of dendritic arbor shapes. Different neuronal classes are defined by class-specific transcription factor combinatorial codes. We show that the combination of the transcription factors Knot and Cut is particular to Drosophila class IV dendritic arborization (da) neurons. Knot and Cut control different aspects of the dendrite cytoskeleton, promoting microtubule- and actin-based dendritic arbors, respectively. Knot delineates class IV arbor morphology by simultaneously synergizing with Cut to promote complexity and repressing Cut-mediated promotion of dendritic filopodia/spikes. Knot increases dendritic arbor outgrowth through promoting the expression of Spastin, a microtubule-severing protein disrupted in autosomal dominant hereditary spastic paraplegia (AD-HSP). Knot and Cut may modulate cellular mechanisms that are conserved between Drosophila and vertebrates. Hence, this study gives significant general insight into how multiple transcription factors combine to control class-specific dendritic arbor morphology through controlling different aspects of the cytoskeleton.

Identification and origin of the germline stem cells as revealed by the expression of nanos-related gene in planarians.

The planarian's remarkable regenerative ability is thought to be supported by the stem cells (neoblasts) found throughout its body. Here we report the identification of a subpopulation of neoblasts, which was revealed by the expression of the nanos-related gene of the planarian Dugesia japonica, termed Djnos. Djnos-expressing cells in the asexual planarian were distributed to the prospective ovary or testes forming region in the sexual planarian. During sexualization, Djnos-expressing cells produce germ cells, suggesting that in the asexual state these cells were kept as germline stem cells for the oogonia and spermatogonia. Interestingly, the germline stem cells were indistinguishable from the neoblasts by morphology and X-ray sensitivity and did not seem to contribute to the regeneration at all. Germline stem cells initially appear in the growing infant planarian, suggesting that germline stem cells are separated from somatic stem cells in the planarian. Thus, planarian neoblasts can be classified into two groups; somatic stem cells for regeneration and tissue renewal, and germline stem cells for production of germ cells during sexualization. However, Djnos-positive cells appeared in the newly formed trunk region from the head piece, suggesting that somatic stem cells can convert to germline stem cells.

Role of mitochondrial ribosome-dependent translation in germline formation in Drosophila embryos.

In Drosophila, mitochondrially encoded ribosomal RNAs (mtrRNAs) form mitochondrial-type ribosomes on the polar granules, distinctive organelles of the germ plasm. Since a reduction in the amount of mtrRNA results in the failure of embryos to produce germline progenitors, or pole cells, it has been proposed that translation by mitochondrial-type ribosomes is required for germline formation. Here, we report that injection of kasugamycin (KA) and chloramphenicol (CH), inhibitors for prokaryotic-type translation, disrupted pole cell formation in early embryos. The number of mitochondrial-type ribosomes on polar granules was significantly decreased by KA treatment, as shown by electron microscopy. In contrast, ribosomes in the mitochondria and mitochondrial activity were unaffected by KA and CH. We further found that injection of KA and CH impairs production of Germ cell-less (Gcl) protein, which is required for pole cell formation. The above observations suggest that mitochondrial-type translation is required for pole cell formation, and Gcl is a probable candidate for the protein produced by this translation system.

Expression of the ftsY gene, encoding a homologue of the alpha subunit of mammalian signal recognition particle receptor, is controlled by different promoters in vegetative and sporulating cells of Bacillus subtilis.

Bacillus subtilis FtsY (Srb) is a homologue of the alpha subunit of the receptor for mammalian signal-recognition particle (SRP) and is essential for protein secretion and vegetative cell growth. The ftsY gene is expressed during both the exponential phase and sporulation. In vegetative cells, ftsY is transcribed with two upstream genes, rncS and smc, that are under the control of the major transcription factor sigma(A). During sporulation, Northern hybridization detected ftsY mRNA in wild-type cells, but not in sporulating cells of sigma(K) and gerE mutants. Therefore, ftsY is solely expressed during sporulation from a sigma(K)- and GerE-controlled promoter that is located immediately upstream of ftsY inside the smc gene. To examine the role of FtsY during sporulation, the B. subtilis strain ISR39 was constructed, a ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative state. Electron microscopy showed that the outer coat of ISR39 spores was not completely assembled and immunoelectron microscopy localized FtsY to the inner and outer coats of wild-type spores.

Changes in subcellular localization of mtlrRNA outside mitochondria in oogenesis and early embryogenesis of Drosophila melanogaster.

Mitochondrial large ribosomal RNA (mtlrRNA) has been identified as a cytoplasmic factor inducing pole cells in ultraviolet (UV)-sterilized Drosophila embryos. In situ hybridization studies have revealed that mtlrRNA is present outside mitochondria localized on the surface of polar granules during the cleavage stage. In the present study, we describe the developmental changes in extramitochondrial mtlrRNA distribution through early embryogenesis using in situ hybridization at the light and electron microscopic level. No mtlrRNA signal was discernible on polar granules in the mature oocyte, unless the oocyte was activated for development. mtlrRNA was localized on the surface of polar granules during a limited period of stages from oocyte activation to pole bud formation and disappeared as soon as being detached from polar granules without entering pole cells. These changes in the temporal and spatial distribution of mtlrRNA outside mitochondria are compatible with the idea that mtlrRNA is required for pole cell formation but not for the differentiation of pole cells as functional germ cells.

Nonradioactive In Situ Hybridization Methods for Drosophila Embryos Detecting Signals by Immunogold-Silver or Immunoperoxidase Method for Electron Microscopy: (electron microscopy/pre-embedding/transcripts/Drosophila embryo).

We present details of in situ hybridization methods for electron microscopy applicable for Drosophila embryos. Improvements upon the foregoing methods were made at 1) hybridization and visualization of signals were carried out with whole embryos that were then processed for electron microscopy, and 2) digoxigenin-labeled probes were detected by the immunogold silver enhancement method or by the immunoperoxidase method. Using these methods. we demonstrated the localization of fushi tarazu transcripts in the apical region of blastodermal cells. We also showed that mitochondrial large ribosomal RNA is associated with polar granules in pole plasm of cleavage embryos. These methods will make a useful tool to determine the precise subcellular distribution of specific transcripts in Drosophila embryos.