To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is masterminded by polarity complexes, which are thought to direct downstream events such as polarized membrane traffic, though this interconnection is not well understood. We report that Rab11a regulates apical traffic and lumen formation via the Rab GEF Rabin8, and its target Rab8a. Rab8a/11a act via the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation via the Cdc42 GEF, Tuba. These components assemble at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical exocytosis during lumen formation, revealing a novel interplay of the machineries of vesicular transport and polarization.Most internal epithelial organs consist of a monolayer of polarized epithelial cells surrounding a central lumen. Polarization requires the interaction of the signaling complexes and scaffolds that define cortical domains with the polarized membrane sorting machinery 1 . In yeast, traffic from the trans-Golgi network to the surface is regulated by Ypt32p and Sec4p 2 , homologs of mammalian Rab11 and Rab8, respectively. These are linked by Sec2p (homolog of mammalian Rabin8), a guanine nucleotide exchange factor (GEF) for Sec4p, which is recruited by Ypt32p. Sec2p and Sec4p in turn interact with the exocyst, which docks vesicles to the surface 3 .Definition of cortical domains in metazoa involves a complex of Par3, Par6, atypical PKC (aPKC), and the GTPase Cdc42 4 . This complex is a master regulator of polarity, conventionally depicted upstream of membrane trafficking machinery. How this complex interfaces with membrane transport is poorly understood.Here we show a molecular mechanism for lumen and apical surface formation, linking Rab8a/ 11a, exocyst, annexin2, Cdc42 and its GEF Tuba, and the Par3/aPKC complex. This novel NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 November 8. Published in final edited form as:Nat Cell Biol. 2010 November ; 12(11): 1035-1045. doi:10.1038/ncb2106. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript pathway shows how the membrane traffic and cortical polarity machineries cooperate to generate the apical surface and lumen. RESULTS Apical polarization during lumen formationUpon plating into 3D culture, individual MDCK cells proliferate and assemble into cyst structures -a polarized spherical monolayer surrounding a central lumen. Lumenogenesis requires the apical membrane determinant gp135/podocalyxin 5 (PCX in figures). Initially, MDCK aggregates have podocalyxin at the ECM-contacting surface (Fig. 1a, 12 h; Fig. S1a), before polarity inversion occurs, with β-catenin and Na/K-ATPase at cell-cell junctions and podocalyxin now at the lumen (Fig. 1a, 24-48 h, arrows; Fig. S1d) 6,7 . Early lumens occur at a site previously termed the "Pre-Apical Patch" (PAP), where opposing plasma membranes are separated, but the po...

A challenge in biology is to associate molecular differences among progenitor cells with their capacity to generate mature cell types. Here, we used expressed DNA barcodes to clonally trace transcriptomes over time and applied this to study fate determination in hematopoiesis. We identified states of primed fate potential and located them on a continuous transcriptional landscape. We identified two routes of monocyte differentiation that leave an imprint on mature cells. Analysis of sister cells also revealed cells to have intrinsic fate biases not detectable by single-cell RNA sequencing. Finally, we benchmarked computational methods of dynamic inference from single-cell snapshots, showing that fate choice occurs earlier than is detected by state-of the-art algorithms and that cells progress steadily through pseudotime with precise and consistent dynamics.

SUMMARY Hematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells sitting at the very top1,2. Multiple models have been proposed as to what the earliest lineage choices are in these primitive hematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them3–10. Given that the bulk of studies addressing lineage outcomes have been performed in the context of hematopoietic transplantation, current lineage branching models are more likely to represent roadmaps of lineage potential rather than native fate. Here, we utilize transposon (Tn) tagging to clonally trace the fates of progenitors and stem cells in unperturbed hematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte (Mk) lineage arises largely independently of other hematopoietic fates. Our data, combined with single cell RNAseq, identify a functional hierarchy of uni- and oligolineage producing clones within the MPP population. Finally, our results demonstrate that traditionally defined long-term HSCs (LT-HSCs) are a significant source of Mk-restricted progenitors, suggesting that the Mk-lineage is the predominant native fate of LT-HSCs. Our study provides evidence for a substantially revised roadmap for unperturbed hematopoiesis, and highlights unique properties of MPPs and HSCs in situ.