Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.

Dscam Proteins Direct Dendritic Targeting through Adhesion.

Cell recognition molecules are key regulators of neural circuit assembly. The Dscam family of recognition molecules in Drosophila has been shown to regulate interactions between neurons through homophilic repulsion. This is exemplified by Dscam1 and Dscam2, which together repel dendrites of lamina neurons, L1 and L2, in the visual system. By contrast, here we show that Dscam2 directs dendritic targeting of another lamina neuron, L4, through homophilic adhesion. Through live imaging and genetic mosaics to dissect interactions between specific cells, we show that Dscam2 is required in L4 and its target cells for correct dendritic targeting. In a genetic screen, we identified Dscam4 as another regulator of L4 targeting which acts with Dscam2 in the same pathway to regulate this process. This ensures tiling of the lamina neuropil through heterotypic interactions. Thus, different combinations of Dscam proteins act through distinct mechanisms in closely related neurons to pattern neural circuits.

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.

Multiple interactions control synaptic layer specificity in the Drosophila visual system.

How neurons form synapses within specific layers remains poorly understood. In the Drosophila medulla, neurons target to discrete layers in a precise fashion. Here we demonstrate that the targeting of L3 neurons to a specific layer occurs in two steps. Initially, L3 growth cones project to a common domain in the outer medulla, overlapping with the growth cones of other neurons destined for a different layer through the redundant functions of N-Cadherin (CadN) and Semaphorin-1a (Sema-1a). CadN mediates adhesion within the domain and Sema-1a mediates repulsion through Plexin A (PlexA) expressed in an adjacent region. Subsequently, L3 growth cones segregate from the domain into their target layer in part through Sema-1a/PlexA-dependent remodeling. Together, our results and recent studies argue that the early medulla is organized into common domains, comprising processes bound for different layers, and that discrete layers later emerge through successive interactions between processes within domains and developing layers.

The maternal-to-zygotic transition: a play in two acts.

All animal embryos pass through a stage during which developmental control is handed from maternally provided gene products to those synthesized from the zygotic genome. This maternal-to-zygotic transition (MZT) has been extensively studied in model organisms, including echinoderms, nematodes, insects, fish, amphibians and mammals. In all cases, the MZT can be subdivided into two interrelated processes: first, a subset of maternal mRNAs and proteins is eliminated; second, zygotic transcription is initiated. The timing and scale of these two events differ across species, as do the cellular and morphogenetic processes that sculpt their embryos. In this article, we discuss conserved and distinct features within the two component processes of the MZT.

An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition.

Genetic control of embryogenesis switches from the maternal to the zygotic genome during the maternal-to-zygotic transition (MZT), when maternal mRNAs are destroyed, high-level zygotic transcription is initiated, the replication checkpoint is activated and the cell cycle slows. The midblastula transition (MBT) is the first morphological event that requires zygotic gene expression. The Drosophila MBT is marked by blastoderm cellularization and follows 13 cleavage-stage divisions. The RNA-binding protein Smaug is required for cleavage-independent maternal transcript destruction during the Drosophila MZT. Here, we show that smaug mutants also disrupt syncytial blastoderm stage cell-cycle delays, DNA replication checkpoint activation, cellularization, and high-level zygotic expression of protein coding and micro RNA genes. We also show that Smaug protein levels increase through the cleavage divisions and peak when the checkpoint is activated and zygotic transcription initiates, and that transgenic expression of Smaug in an anterior-to-posterior gradient produces a concomitant gradient in the timing of maternal transcript destruction, cleavage cell cycle delays, zygotic gene transcription, cellularization and gastrulation. Smaug accumulation thus coordinates progression through the MZT.

The mother-to-child transition.

Recent genome-scale analyses have uncovered the magnitude of the changes in mRNA populations that occur during the maternal-to-zygotic transition in early Drosophila embryos as well as two of the key regulators of this process, SMAUG and bicoid stability factor (BSF).

SMAUG is a major regulator of maternal mRNA destabilization in Drosophila and its translation is activated by the PAN GU kinase.

In animals, egg activation triggers a cascade of posttranscriptional events that act on maternally synthesized RNAs. We show that, in Drosophila, the PAN GU (PNG) kinase sits near the top of this cascade, triggering translation of SMAUG (SMG), a multifunctional posttranscriptional regulator conserved from yeast to humans. Although PNG is required for cytoplasmic polyadenylation of smg mRNA, it regulates translation via mechanisms that are independent of its effects on the poly(A) tail. Analyses of mutants suggest that PNG relieves translational repression by PUMILIO (PUM) and one or more additional factors, which act in parallel through the smg mRNA's 3' untranslated region (UTR). Microarray-based gene expression profiling shows that SMG is a major regulator of maternal transcript destabilization. SMG-dependent mRNAs are enriched for gene ontology annotations for function in the cell cycle, suggesting a possible causal relationship between failure to eliminate these transcripts and the cell cycle defects in smg mutants.

Setting the stage for development: mRNA translation and stability during oocyte maturation and egg activation in Drosophila.

Early animal development is controlled by maternally encoded RNAs and proteins, which are loaded into the egg during oogenesis. Oocyte maturation and egg activation trigger changes in the translational status and the stability of specific maternal mRNAs. Whereas both maturation and activation have been studied in depth in amphibians and echinoderms, only recently have these processes begun to be dissected using the powerful genetic and molecular tools available in Drosophila. This review focuses on the mechanisms and functions of regulated maternal mRNA translation and stability in Drosophila--and compares these mechanisms with those elucidated in other animal models, particularly Xenopus--beginning late in oogenesis and continuing to the mid-blastula transition, when developmental control is transferred to zygotically synthesized transcripts.

Regulation of maternal transcript destabilization during egg activation in Drosophila.

In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation.