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Convergent extension (CE) is an evolutionarily conserved developmental process whereby polarized collective cell movements drive the elongation of tissues or organs, and defects in CE are associated with multiple human birth defects. Here, we combined embryology with cell biology and newly developed biomechanical tools to interrogate the link between cell behavior and tissue-scale CE. We found that unique patterns of polarized cortex tension are required to resolve individual cell intercalation events, and this in turn maintains normal cell packing configurations that favor the planar propagation of the cellular forces across the tissue, and thus normal CE. Our data suggest that planar polarized force propagation plays a critical role in the propagation of cell behaviors via cellular mechanoreciprocity, underpinning the propagation of tissue-scale CE.

Cell-type specific RNA-associated proteins are essential for development and homeostasis in animals. Despite a massive recent effort to systematically identify RNA-associated proteins, we currently have few comprehensive rosters of cell-type specific RNA-associated proteins in vertebrate tissues. Here, we demonstrate the feasibility of determining the RNA-interacting proteome of a defined vertebrate embryonic tissue using DIF-FRAC, a systematic and universal (i.e., label-free) method. Application of DIF-FRAC to cultured tissue explants of Xenopus mucociliary epithelium identified dozens of known RNA-associated proteins as expected, but also several novel RNA-associated proteins, including proteins related to assembly of the mitotic spindle and regulation of ciliary beating. In particular, we show that the inner dynein arm tether Cfap44 is an RNA-associated protein that localizes not only to axonemes, but also to liquid-like organelles in the cytoplasm called DynAPs. This result led us to discover that DynAPs are generally enriched for RNA. Together, these data provide a useful resource for a deeper understanding of mucociliary epithelia and demonstrate that DIF-FRAC will be broadly applicable for systematic identification of RNA-associated proteins from embryonic tissues.

SummaryConvergent extension (CE) is a collective cell movement required for embryonic axis elongation, neural tube closure, and kidney tubule elongation. During CE, iterative cell intercalations drive the elongation of a tissue 1. In order to move, cells reorganize their actin cytoskeleton; the implication of actin regulators during CE, then, is expected. Interestingly, two other groups of proteins have been shown to be required for this cell behavior: Planar Cell Polarity proteins (PCP)2-4 and Septins5, both of which interact with the actin cytoskeleton 6-14. Disruption of any of these systems have substantial overlap in terms of embryonic phenotypes, but how they interact to govern CE is largely unknown15-17. Here, we find that PCP proteins and Septins sequester actomyosin activity, create flows of actin across the anterior side of cells, and bundle actin into a node and cable system that displays a planar polarized gradient of stiffness.Key findings-PCP- and Septin-dependent actin node and cable structures form and become anteriorized over time-Actin cytoskeletal reorganization correlates with PCP protein polarization.-Actomyosin flows are directed to anterior PCP puncta and PCP movement relies on actin turnover.-Septins localize to anterior nodes and bundle actin filaments into anterior actin cable.-PCP- and Septin-dependent anterior actin cables increase local cellular stiffness and create a planar polarized stiffness gradient across the cell.

The dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells and in vitro systems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type-specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin-severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1 (Twf1) in Xenopus. We find that Twf1 is essential for convergent extension, and loss of Twf1 results in a disruption of lamellipodial dynamics and polarity. Moreover, Twf1 loss results in a failure to assemble polarized cytoplasmic actin cables, which are essential for convergent extension. These data provide an in vivo complement to our more-extensive understanding of Twf1 action in vitro and provide new links between the core machinery of actin regulation and the specialized cell behaviors of embryonic morphogenesis.

The dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells andin vitrosystems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1. We confirm previous results on the role of Twf1 in lamellipodia and extend those findings by linking Twf1, actin turnover, and cell polarization required for convergent extension during vertebrate gastrulation.

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we usein vivoimaging to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity purification mass-spectrometry ofin vivointeraction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

Cell-type specific RNA-associated proteins (RAPs) are essential for development and homeostasis in animals. Despite a massive recent effort to systematically identify RAPs, we currently have few comprehensive rosters of cell-type specific RAPs in vertebrate tissues. Here, we demonstrate the feasibility of determining the RNA-interacting proteome of a defined vertebrate embryonic tissue using DIF-FRAC, a systematic and universal (i.e., label-free) method. Application of DIF-FRAC to cultured tissue explants of Xenopus mucociliary epithelium identified dozens of known RAPs as expected, but also several novel RAPs, including proteins related to assembly of the mitotic spindle and regulation of ciliary beating. In particular, we show that the inner dynein arm tether Cfap44 is an RNA-associated protein that localizes not only to axonemes, but also to liquid-like organelles in the cytoplasm called DynAPs. This result led us to discover that DynAPs are generally enriched for RNA. Together, these data provide a useful resource for a deeper understanding of mucociliary epithelia and demonstrate that DIF-FRAC will be broadly applicable for systematic identification of RAPs from embryonic tissues.

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins – p16(lnk4a), which blocks cyclin-dependent kinase activity, and p19(Arf), which is best known as a positive regulator of the p53 tumor suppressor – and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts (MEFs) generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFβ-2-dependent induction of the Arf reporter in the eye - a process essential for normal eye development - does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.

Arf encodes an important tumor suppressor, p19Arf, which also plays a critical role to control hyperplasia in the primary vitreous during mouse eye development. In the absence of Arf, mice are born blind and display a phenotype closely mimicking severe forms of the human eye disease, persistent hyperplastic primary vitreous (PHPV). In this report, we characterize p19Arf expression in perivascular cells that normally populate the primary vitreous and express the Arf promoter. Using a new ex vivo model, we show that these cells respond to exogenous Tgfβ, despite being isolated at a time when Tgfβ has already turned on the Arf promoter. Treatment of the cells with PDGF-B ligand doubles the population of cells in S-phase and ectopic expression of Arf blunts that effect. We show this effect is mediated through Pdgfrβ as expression of Arf represses expression of Pdgfrβ mRNA and protein to approximately 60%. p53 is not required for Arf-dependent blockade of PDGF-B driven proliferation and repression of Pdgfrβ protein as ectopic expression of Arf is still able to inhibit the 2-fold increase in the S-phase fraction of cells upon treatment with PDGF-B. Finally, induction of mature miR-34a, a microRNA previously identified to be regulated by p19Arf does not depend on p53 while the expression of the primary transcript does require p53. These data corroborate that, as in vivo, p19Arf functions to inhibit PDGF-B driven proliferation ex vivo.

Although many researchers have successfully uncovered novel functions of the tumor suppressor p19Arf utilizing various types of cultured cancer cells and immortalized fibroblasts, these systems do not accurately reflect the endogenous environment in which Arf is developmentally expressed. We addressed this by isolating perivascular cells (PVCs) from the primary vitreous of the mouse eye. This rare cell type normally expresses the p19Arf tumor suppressor in a non-pathological, developmental context. We utilized fluorescence activated cell sorting (FACS) to purify the cells by virtue of a GFP reporter driven by the native Arf promoter and then characterized their morphology and gene expression pattern. We further examined the effects of reintroduction of Arf expression in the Arf GFP/GFP PVCs to verify expected downstream effectors of p19Arf as well as uncover novel functions of Arf as a regulator of vasculogenesis. This methodology and cell culture model should serve as a useful tool to examine p19Arf biology.

Recent studies show that Arf, a bona fide tumor suppressor, also plays an essential role during mouse eye development. Tgfβ is required for Arf promoter activation in developing mouse eyes, and its capacity to induce Arf depends on Smads 2/3 as well as p38 Mapk. Substantial delay between activation of these pathways and increased Arf transcription imply that changes in the binding of additional transcription factors help orchestrate changes in Arf expression. Focusing on proteins with putative DNA binding elements near the mouse Arf transcription start, we now show that Tgfβ induction of this gene correlated with decreased expression and DNA binding of C/ebpβ to the proximal Arf promoter. Ectopic expression of C/ebpβ in mouse embryo fibroblasts (MEFs) blocked Arf induction by Tgfβ. Although basal levels of Arf mRNA were increased by C/ebpβ loss in MEFs and in the developing eye, Tgfβ was still able to increase Arf, indicating that derepression was not the sole factor. Chromatin immunoprecipitation (ChIP) assay showed increased Sp1 binding to the Arf promotor at 24 and 48 hours after Tgfβ treatment, at which time points Arf expression was significantly induced by Tgfβ. Chemical inhibition of Sp1 and its knockdown by RNA interference blocked Arf induction by Tgfβ in MEFs. In summary, our results indicate that C/ebpβ and Sp1 are negative and positive Arf regulators that are influenced by Tgfβ.

The Arf tumor suppressor represents one of several genes encoded at the Cdkn 2 a and Cdkn 2 b loci in the mouse. Beyond its role blunting the growth of incipient cancer cells, the Arf gene also plays an essential role in development: its gene product, p19 Arf , is induced by Tgfβ2 in the developing eye to dampen proliferative signals from Pdgfrβ, which effect ultimately fosters the vascular remodeling required for normal vision in the mouse. Mechanisms underlying Arf induction by Tgfβ2 are not fully understood. Using the chr 4 Δ70 kb/Δ70 kb mouse, we now show that deletion of the coronary artery disease (CAD) risk interval lying upstream of the Cdkn 2 a / b locus represses developmentally-timed induction of Arf resulting in eye disease mimicking the persistent hyperplastic primary vitreous (PHPV) found in Arf -null mice and in children. Using mouse embryo fibroblasts, we demonstrate that Arf induction by Tgfβ is blocked in cis to the 70 kb deletion, but Arf induction by activated RAS and cell culture “shock” is not. Finally, we show that Arf induction by Tgfβ is derailed by preventing RNA polymerase II recruitment following Smad 2/3 binding to the promoter. These findings provide the first evidence that the CAD risk interval, located at a distance from Arf , acts as a cis enhancer of Tgfβ2-driven induction of Arf during development.