Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila.

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.

Functional signal peptides bind a soluble N-terminal fragment of SecA and inhibit its ATPase activity.

The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is known about the requirements for recognition and the mode of binding of precursors to SecA. The major reason for this is the lack of a soluble system suitable for biophysical study of the SecA-precursor complex. Complicating the development of such a system is the likelihood that SecA interacts with the precursor in a high affinity, productive manner only when it is activated by binding to membrane and SecYEG. A critical aspect of the precursor/SecA interaction is that it is regulated by various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the release of the precursor, most likely in a stepwise fashion, for translocation. Several recent reports show that functions of SecA can be studied using separated domains. Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated version of SecA. We report here that this fragment, termed SecA64, binds signal peptides with significantly higher affinity than does SecA. Moreover, the ATPase activity of SecA64 is inhibited by signal peptides to an extent that correlates with the ability of these signal peptides to inhibit either SecA translocation ATPase or in vitro protein translocation, arguing that the interaction with SecA64 is functionally significant. Thus, SecA64 offers a soluble, well defined system to study the mode of recognition of signal peptides by SecA and the regulation of signal peptide release.

Control of dendritic field formation in Drosophila: the roles of flamingo and competition between homologous neurons.

Neurons elaborate dendrites with stereotypic branching patterns, thereby defining their receptive fields. These branching patterns may arise from properties intrinsic to the neurons or competition between neighboring neurons. Genetic and laser ablation studies reported here reveal that different multiple dendritic neurons in the same dorsal cluster in the Drosophila embryonic PNS do not compete with one another for dendritic fields. In contrast, when dendrites from homologous neurons in the two hemisegments meet at the dorsal midline in larval stages, they appear to repel each other. The formation of normal dendritic fields and the competition between dendrites of homologous neurons require the proper expression level of Flamingo, a G protein-coupled receptor-like protein, in embryonic neurons. Whereas Flamingo functions downstream of Frizzled in specifying planar polarity, Flamingo-dependent dendritic outgrowth is independent of Frizzled.

Genes regulating dendritic outgrowth, branching, and routing in Drosophila.

Signaling between neurons requires highly specialized subcellular structures, including dendrites and axons. Dendrites exhibit diverse morphologies yet little is known about the mechanisms controlling dendrite formation in vivo. We have developed methods to visualize the stereotyped dendritic morphogenesis in living Drosophila embryos. Dendrite development is altered in prospero mutants and in transgenic embryos expressing a constitutively active form of the small GTPase cdc42. From a genetic screen, we have identified several genes that control different aspects of dendrite development including dendritic outgrowth, branching, and routing. These genes include kakapo, a large cytoskeletal protein related to plectin and dystrophin; flamingo, a seven-transmembrane protein containing cadherin-like repeats; enabled, a substrate of the tyrosine kinase Abl; and nine potentially novel loci. These findings begin to reveal the molecular mechanisms controlling dendritic morphogenesis.

Cell number control and timing in animal development: the oligodendrocyte cell lineage.

Our studies of oligodendrocyte development in the rodent optic nerve provide clues as to how cell numbers and the timing of differentiation may be controlled during mammalian development. Both cell number and the timing of differentiation depend on intracellular programs and extracellular signals, which together control cell survival and cell division. As the cells seem to compete for limiting amounts of both survival signals and mitogens, the levels of these extracellular signals must be tightly regulated, but it is not known how this is achieved. The timing of cell-cycle exit, and therefore the onset of differentiation, seems to depend in part on the progressive accumulation of the intracellular Cdk inhibitor p27/Kip1, but it is still unclear how the level of this protein is controlled over time in the dividing cells. The timing of cell-cycle exit is also regulated by thyroid hormone, which, along with other hormones, seems to coordinate the timing of development in various organs, much as the timing of the multiple changes in metamorphosis in both vertebrates and invertebrates is coordinated by hormones. In this sense, one might think of mammalian development as a prolonged metamorphosis.

Cell-intrinsic timers and thyroid hormone regulate the probability of cell-cycle withdrawal and differentiation of oligodendrocyte precursor cells.

During vertebrate development many types of precursor cells divide a limited number of times before they stop dividing and terminally differentiate. It is unclear what causes the cells to stop dividing when they do. We have been studying this problem in the oligodendrocyte cell lineage, which is responsible for myelination in the vertebrate central nervous system. Here we show for the first time that in clonal cultures of oligodendrocyte precursor cells purified from embryonic day 18 (E18) rat optic nerves the first oligodendrocytes develop within 3-4 days, equivalent to the time they first differentiate in the nerve, and that this timely differentiation depends on the presence of thyroid hormone. These findings suggest that a cell-intrinsic, thyroid-hormone-regulated timer determines when the first oligodendrocytes develop. Whereas the first oligodendrocytes develop asynchronously within clones, the vast majority develop after the first week in culture and do so more synchronously within clones. We show that beta1 thyroid hormone receptors in the precursor cells increase in clonal cultures in the absence of thyroid hormone in parallel with the increasing sensitivity of the cells to the cell-cycle-arresting activity of thyroid hormone; moreover, the increase in beta1 receptors, like the timer itself, is accelerated at 33 degrees C compared to 37 degrees C, suggesting that the increase in receptors may be part of the intrinsic timer. Finally, we show that the precursor cells do not divide indefinitely when stimulated to divide extensively in the absence of thyroid hormone but, instead, eventually stop dividing and either die or differentiate.

Messenger RNAs in dendrites: localization, stability, and implications for neuronal function.

In the mammalian central nervous system (CNS), each neuron receives signals from other neurons through numerous synapses located on its cell body and dendrites. Molecules involved in the postsynaptic signaling pathways need to be targeted to the appropriate subcellular domains at the right time during both synaptogenesis and the maintenance of synaptic functions. The presence of messenger RNAs (mRNAs) in dendrites offers a mechanism for synthesizing the appropriate molecules at the right place in response to local extracellular stimuli. Several dendritic mRNAs have been identified, and the mechanisms controlling their localization are beginning to be understood. In many cell types, controls on mRNA stability play an important role in the regulation of gene expression, but it is unclear to what extent this type of control operates in dendrites. The regulation of protein synthesis and the control of mRNA stability in dendrites could have important implications for neuronal function.

Cell size control and a cell-intrinsic maturation program in proliferating oligodendrocyte precursor cells.

We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G1 proposed by "transition probability" models, may explain the random variability of cell cycle times seen within clonal cell lines in culture.

Oligodendrocyte precursor cells count time but not cell divisions before differentiation.

During vertebrate development, many types of precursor cell divide a limited number of times before they stop and terminally differentiate. It is unclear what limits cell proliferation and causes the cells to stop dividing when they do. The stopping mechanisms are important as they influence both the number of differentiated cells generated and the timing of differentiation. We have been studying the 'stopping' problem in the oligodendrocyte cell lineage [1] [2], which is responsible for myelination in the vertebrate central nervous system. Previous studies demonstrated that the proliferation of oligodendrocyte precursor cells isolated from the developing rat optic nerve is limited by an intrinsic 'clock' mechanism [3], which consists of two components: a counting mechanism that counts time or cell divisions, and an effector mechanism that arrests the cell cycle and initiates cell differentiation when the appropriate time is reached [4] [5]. In the present study, we address the question of whether the counting mechanism operates by counting cell divisions. We show that precursor cells cultured at 33 degrees C divide more slowly but stop dividing and differentiate sooner, after fewer cell divisions, than when they are cultured at 37 degrees C, indicating that the counting mechanism does not count cell divisions but measures time in some other way. In addition, we show that the levels of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) rise faster at 33 degrees C than at 37 degrees C, consistent with previous evidence [6] that the accumulation of p27 may be part of the counting mechanism.

Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation.

Many types of vertebrate precursor cells divide a limited number of times before they stop and terminally differentiate. In no case is it known what causes them to stop dividing. We have been studying this problem in the proliferating precursor cells that give rise to postmitotic oligodendrocytes, the cells that make myelin in the central nervous system. We show here that two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes. More importantly, we show that the cyclin-dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate and is present at high levels in oligodendrocytes. Our findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. The recent findings of others that p27-deficient mice have an increased number of cells in all of the organs examined suggest that this function of p27 is not restricted to the oligodendrocyte cell lineage.

Hel-N1/Hel-N2 proteins are bound to poly(A)+ mRNA in granular RNP structures and are implicated in neuronal differentiation.

Human proteins Hel-N1 and Hel-N2 contain three RNA recognition motifs (RRMs), and are members of a family of proteins highly homologous to Drosophila ELAV, which is essential for neuronal differentiation. Both proteins bind to A+U-rich 3' untranslated regions of a variety of growth-related mRNAs in vitro. Here we demonstrate that in medulloblastoma cells derived from childhood brain tumors, Hel-N1 and Hel-N2 are mainly expressed in the cytoplasm, but are detectable in the nucleus. Both proteins are associated with polysomes and can be UV-crosslinked to poly(A)+ mRNA in cell extracts. In the cytoplasm the Hel-N1 protein family resides in granular structures that may contain multiple protein molecules bound to each mRNA. Evidence supporting this multimeric ribonucleoprotein (RNP) model includes in vitro reconstitution and competition experiments in which addition of a single RRM (RRM3) can alter complex formation. As in medulloblastoma cells, the Hel-N1 protein family is present in granular particles in the soma and the proximal regions of dendrites of cultured neurons, and colocalizes with ribosomes. In addition, we demonstrate that expression of the Hel-N1 protein family is up-regulated during neuronal differentiation of embryonic carcinoma P19 cells. Our data suggest that the Hel-N1 protein family is associated with the translational apparatus and implicated in both mRNA metabolism and neuronal differentiation. Furthermore, our findings open the possibility that these proteins participate in mRNA homeostasis in the dendrites and soma of mature neurons.

Selection of a subset of mRNAs from combinatorial 3' untranslated region libraries using neuronal RNA-binding protein Hel-N1.

Hel-N1, a human RNA-binding protein, shares significant homology with Drosophila protein ELAV, which is essential for fly neuronal development. Hel-N1 has been shown to bind in vitro to 3' untranslated regions of mRNAs encoding c-myc, c-fos, granulocyte/macrophage colony-stimulating factor, and transcriptional repressor, Id. We report that Hel-N1 and a related form, Hel-N2, are expressed in human medulloblastoma cells, but their ratio differs significantly from that in adult brain and fetal brain. Selection of RNA targets from randomized combinatorial libraries yielded (A+U)-rich consensus sequences for both Hel-N1 and Hel-N2. As a means to identify cellular RNA targets for these proteins, we devised combinatorial shape libraries representing naturally derived 3' untranslated regions and were able to select a structurally related subset of transcripts that bound to Hel-N1. Approximately 10% of the proteins encoded by these subset mRNAs were identifiable in the data bases and most are implicated in cell growth regulation. This approach provides a means to gain access to novel genes expressed in various cell types by partitioning mRNAs containing common sequence elements using RNA-binding proteins.