Engrailed 1 deficiency induces changes in ciliogenesis during human neuronal differentiation.

A key pathological feature of Parkinson's Disease (PD) is the progressive degeneration of dopaminergic neurons (DAns) in the substantia nigra pars compacta. Considering the major role of EN1 in the development and maintenance of these DAns and the implications from En1 mouse models, it is highly interesting to study the molecular and protective effect of EN1 also in a human cellular model. Therefore, we generated EN1 knock-out (ko) human induced pluripotent stem cell (hiPSCs) lines and analyzed these during neuronal differentiation. Although the EN1 ko didn't interfere with neuronal differentiation and generation of tyrosine hydroxylase positive (TH+) neurons per se, the neurons exhibited shorter neurites. Furthermore, mitochondrial respiration, as well as mitochondrial complex I abundance was significantly reduced in fully differentiated neurons. To understand the implications of an EN1 ko during differentiation, we performed a transcriptome analysis of human neuronal precursor cells (hNPCs) which unveiled alterations in cilia-associated pathways. Further analysis of ciliary morphology revealed an elongation of primary cilia in EN1-deficient hNPCs. Besides, also Wnt signaling pathways were severely affected. Upon stimulating hNPCs with Wnt which drastically increased EN1 expression in WT lines, the phenotypes concerning mitochondrial function and cilia were exacerbated in EN1 ko hNPCs. They failed to enhance the expression of the complex I subunits NDUFS1 and 3, and now displayed a reduced mitochondrial respiration. Furthermore, Wnt stimulation decreased ciliogenesis in EN1 ko hNPCs but increased ciliary length even further. This further highlights the relevance of primary cilia next to mitochondria for the functionality and correct maintenance of human DAns and provides new possibilities to establish neuroprotective therapies for PD.

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration.

BACKGROUND: The reactivation of genetic programs from early development is a common mechanism for injury-induced organ regeneration. T-box 3 (TBX3) is a member of the T-box family of transcription factors previously shown to regulate pluripotency and subsequent lineage commitment in a number of tissues, including limb and lung. TBX3 is also involved in lung and heart organogenesis. Here, we provide a comprehensive and thorough characterization of TBX3 and its role during pancreatic organogenesis and regeneration. RESULTS: We interrogated the level and cell specificity of TBX3 in the developing and adult pancreas at mRNA and protein levels at multiple developmental stages in mouse and human pancreas. We employed conditional mutagenesis to determine its role in murine pancreatic development and in regeneration after the induction of acute pancreatitis. We found that Tbx3 is dynamically expressed in the pancreatic mesenchyme and epithelium. While Tbx3 is expressed in the developing pancreas, its absence is likely compensated by other factors after ablation from either the mesenchymal or epithelial compartments. In an adult model of acute pancreatitis, we found that a lack of Tbx3 resulted in increased proliferation and fibrosis as well as an enhanced inflammatory gene programs, indicating that Tbx3 has a role in tissue homeostasis and regeneration. CONCLUSIONS: TBX3 demonstrates dynamic expression patterns in the pancreas. Although TBX3 is dispensable for proper pancreatic development, its absence leads to altered organ regeneration after induction of acute pancreatitis.

Comprehensive single cell mRNA profiling reveals a detailed roadmap for pancreatic endocrinogenesis.

Deciphering mechanisms of endocrine cell induction, specification and lineage allocation in vivo will provide valuable insights into how the islets of Langerhans are generated. Currently, it is ill defined how endocrine progenitors segregate into different endocrine subtypes during development. Here, we generated a novel neurogenin 3 (Ngn3)-Venus fusion (NVF) reporter mouse line, that closely mirrors the transient endogenous Ngn3 protein expression. To define an in vivo roadmap of endocrinogenesis, we performed single cell RNA sequencing of 36,351 pancreatic epithelial and NVF+ cells during secondary transition. This allowed Ngn3low endocrine progenitors, Ngn3high endocrine precursors, Fev+ endocrine lineage and hormone+ endocrine subtypes to be distinguished and time-resolved, and molecular programs during the step-wise lineage restriction steps to be delineated. Strikingly, we identified 58 novel signature genes that show the same transient expression dynamics as Ngn3 in the 7260 profiled Ngn3-expressing cells. The differential expression of these genes in endocrine precursors associated with their cell-fate allocation towards distinct endocrine cell types. Thus, the generation of an accurately regulated NVF reporter allowed us to temporally resolve endocrine lineage development to provide a fine-grained single cell molecular profile of endocrinogenesis in vivo.

Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios.

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

Anatomical and cellular heterogeneity in the mouse oviduct-its potential roles in reproduction and preimplantation development†.

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive organ that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are consistent coiling patterns and turning points in the coiled mouse oviduct that serve as reliable landmarks for luminal morphological regionalities. We also found previously unrecognized anatomical structures in the isthmus and uterotubal junction, which likely play roles in reproduction. Furthermore, we demarcated the ampulla-isthmus junction as a distinct region. Taken together, the oviduct mucosal epithelium has highly diverse structures with distinct epithelial cell populations, reflecting its complex functions in reproduction.

Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2.

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.

Generation of a Novel Nkx6-1 Venus Fusion Reporter Mouse Line.

Nkx6-1 is a member of the Nkx family of homeodomain transcription factors (TFs) that regulates motor neuron development, neuron specification and pancreatic endocrine and ß-cell differentiation. To facilitate the isolation and tracking of Nkx6-1-expressing cells, we have generated a novel Nkx6-1 Venus fusion (Nkx6-1-VF) reporter allele. The Nkx6-1-VF knock-in reporter is regulated by endogenous cis-regulatory elements of Nkx6-1 and the fluorescent protein fusion does not interfere with the TF function, as homozygous mice are viable and fertile. The nuclear localization of Nkx6-1-VF protein reflects the endogenous Nkx6-1 protein distribution. During embryonic pancreas development, the reporter protein marks the pancreatic ductal progenitors and the endocrine lineage, but is absent in the exocrine compartment. As expected, the levels of Nkx6-1-VF reporter are upregulated upon ß-cell differentiation during the major wave of endocrinogenesis. In the adult islets of Langerhans, the reporter protein is exclusively found in insulin-secreting ß-cells. Importantly, the Venus reporter activities allow successful tracking of ß-cells in live-cell imaging and their specific isolation by flow sorting. In summary, the generation of the Nkx6-1-VF reporter line reflects the expression pattern and dynamics of the endogenous protein and thus provides a unique tool to study the spatio-temporal expression pattern of this TF during organ development and enables isolation and tracking of Nkx6-1-expressing cells such as pancreatic ß-cells, but also neurons and motor neurons in health and disease.

A novel Cre-inducible knock-in ARL13B-tRFP fusion cilium reporter.

Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre-inducible cilium-specific reporter mouse line expressing an ARL13B-tRFP fusion protein driven by a CMV enhancer/chicken ß actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono- and multiciliated tissues and by time-lapse live-cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live-cell imaging. Thus, the ARL13B-tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue-specific manner to understand processes, such as ciliary protein trafficking or cilium-dependent signaling in vitro and in vivo.

miR-335 promotes mesendodermal lineage segregation and shapes a transcription factor gradient in the endoderm.

Transcription factors (TFs) pattern developing tissues and determine cell fates; however, how spatio-temporal TF gradients are generated is ill defined. Here we show that miR-335 fine-tunes TF gradients in the endoderm and promotes mesendodermal lineage segregation. Initially, we identified miR-335 as a regulated intronic miRNA in differentiating embryonic stem cells (ESCs). miR-335 is encoded in the mesoderm-specific transcript (Mest) and targets the 3'-UTRs of the endoderm-determining TFs Foxa2 and Sox17. Mest and miR-335 are co-expressed and highly accumulate in the mesoderm, but are transiently expressed in endoderm progenitors. Overexpression of miR-335 does not affect initial mesendoderm induction, but blocks Foxa2- and Sox17-mediated endoderm differentiation in ESCs and ESC-derived embryos. Conversely, inhibition of miR-335 activity leads to increased Foxa2 and Sox17 protein accumulation and endoderm formation. Mathematical modeling predicts that transient miR-335 expression in endoderm progenitors shapes a TF gradient in the endoderm, which we confirm by functional studies in vivo. Taken together, our results suggest that miR-335 targets endoderm TFs for spatio-temporal gradient formation in the endoderm and to stabilize lineage decisions during mesendoderm formation.

Wnt/ß-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse.

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse ß-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of ß-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for ß-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of ß-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/ß-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/ß-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.

Insulin regulates human pancreatic endocrine cell differentiation in vitro.

OBJECTIVE: The consequences of mutations in genes associated with monogenic forms of diabetes on human pancreas development cannot be studied in a time-resolved fashion in vivo. More specifically, if recessive mutations in the insulin gene influence human pancreatic endocrine lineage formation is still an unresolved question. METHODS: To model the extremely reduced insulin levels in patients with recessive insulin gene mutations, we generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSC) line expressing no insulin upon differentiation to stem cell-derived (SC-) ß cells in vitro. Differentiation of iPSCs into the pancreatic and endocrine lineage, combined with immunostaining, Western blotting and proteomics analysis phenotypically characterized the insulin gene deficiency in SC-islets. Furthermore, we leveraged FACS analysis and confocal microscopy to explore the impact of insulin shortage on human endocrine cell induction, composition, differentiation and proliferation. RESULTS: Interestingly, insulin-deficient SC-islets exhibited low insulin receptor (IR) signaling when stimulated with glucose but displayed increased IR sensitivity upon treatment with exogenous insulin. Furthermore, insulin shortage did not alter neurogenin-3 (NGN3)-mediated endocrine lineage induction. Nevertheless, lack of insulin skewed the SC-islet cell composition with an increased number in SC-ß cell formation at the expense of SC-α cells. Finally, insulin deficiency reduced the rate of SC-ß cell proliferation but had no impact on the expansion of SC-α cells. CONCLUSIONS: Using iPSC disease modelling, we provide first evidence of insulin function in human pancreatic endocrine lineage formation. These findings help to better understand the phenotypic impact of recessive insulin gene mutations during pancreas development and shed light on insulin gene function beside its physiological role in blood glucose regulation.

T-bet+ B cells are activated by and control endogenous retroviruses through TLR-dependent mechanisms.

Endogenous retroviruses (ERVs) are an integral part of the mammalian genome. The role of immune control of ERVs in general is poorly defined as is their function as anti-cancer immune targets or drivers of autoimmune disease. Here, we generate mouse-strains where Moloney-Murine Leukemia Virus tagged with GFP (ERV-GFP) infected the mouse germline. This enables us to analyze the role of genetic, epigenetic and cell intrinsic restriction factors in ERV activation and control. We identify an autoreactive B cell response against the neo-self/ERV antigen GFP as a key mechanism of ERV control. Hallmarks of this response are spontaneous ERV-GFP+ germinal center formation, elevated serum IFN-γ levels and a dependency on Age-associated B cells (ABCs) a subclass of T-bet+ memory B cells. Impairment of IgM B cell receptor-signal in nucleic-acid sensing TLR-deficient mice contributes to defective ERV control. Although ERVs are a part of the genome they break immune tolerance, induce immune surveillance against ERV-derived self-antigens and shape the host immune response.

Foxa2-venus fusion reporter mouse line allows live-cell analysis of endoderm-derived organ formation.

The Foxa2-winged helix/forkhead box transcription factor (TF) is absolutely required for endoderm formation and organogenesis. Foxa2 plays essential roles during lung, liver, pancreas, and gastrointestinal tract development and regulates cell-type specific programs in the adult organism. To specifically address Foxa2 function during organ development and homeostasis, we generated a Foxa2-Venus fusion (FVF) reporter protein by gene targeting in embryonic stem (ES) cells. The FVF knock-in reporter is expressed under endogenous Foxa2 control and the fluorescent protein fusion does not interfere with TF function, as homozygous mice are viable and fertile. Moreover, the FVF protein localizes to the nucleus, associates with chromatin during mitosis, and reflects the endogenous Foxa2 protein distribution pattern in several tissues in heterozygous animals. Importantly, live-cell imaging on single-cell level of the FVF and Sox17-Cherry fusion double knock-in reporter ES cell line reveals the dynamics of endoderm TF accumulation during ES cell differentiation. The FVF reporter also allowed us to identify the endoderm progenitors during gastrulation and to visualize the different branching morphogenesis modes of the lung and pancreas epithelium in ex vivo embryo and organ cultures. In summary, the generation of the FVF reporter line adds an important new tool to visualize and analyse endoderm-derived organ development and homeostasis on the cellular and molecular level.

The Sox17CreERT2 knock-in mouse line displays spatiotemporal activation of Cre recombinase in distinct Sox17 lineage progenitors.

The HMG-box transcription factor Sox17 is essential for endoderm formation, vascular development, and definitive hematopoiesis. To investigate the fate of distinct Sox17-expressing progenitor cells in a spatiotemporal manner, we generated a hormone-inducible CreERT2 knock-in mouse line. By homologous recombination we fused a codon improved, ligand-dependent estrogen receptor Cre recombinase by an intervening viral T2A sequence for co-translational cleavage to the 3' coding region of Sox17. Induction of Cre activity by administration of tamoxifen at defined time points of early mouse development and subsequent genetic lineage tracing confirmed the inducibility and tissue specificity of Cre recombination. Furthermore, Cre activity could be selectively induced in extra-embryonic and embryonic endoderm lineages, the primitive gut tube, and in endothelial cells of the vascular system as well as in the hemogenic endothelium of the dorsal aorta. The Sox17CreERT2 mouse line therefore represents a new tool for genetic lineage tracing in a tissue-specific manner and in addition enables lineage-restricted functional analysis.

Fltp(T2AiCre): a new knock-in mouse line for conditional gene targeting in distinct mono- and multiciliated tissues.

We recently identified Flattop (Fltp; 1700009p17Rik) in a screen for potential Foxa2 target and novel mouse organizer genes. Besides its expression in the embryonic node, we found that Fltp is active in other monociliated tissues such as the sensory organs of the inner ear, duct and islets of the pancreas as well as in testis. Additionally, Fltp mRNA is expressed in multiciliated epithelial cells of the lung and of the choroid plexi in the brain. To genetically lineage trace these cells during development and injury as well as to conditionally inactivate genes in these tissues, we generated a Cre recombinase knock-in mouse line using the Fltp gene locus. By homologous recombination we have fused the Fltp open-reading frame to a tandem affinity purification (TAP) tag followed by an intervening viral T2A sequence for co-translational cleavage and an improved Cre recombinase (iCre). This strategy allows both the analysis of the tagged Fltp-TAP-T2A protein and the usage of the iCre recombinase for conditional targeting approaches. Using the ROSA26 reporter mouse line we show that Fltp(T2AiCre) is first active in the monociliated cells of the node, notochord, floorplate and prechordal plate, consistent with the Fltp-TAP-T2A protein production in the node progenitor cells. Furthermore iCre recombinase activity is detected in multiciliated tissues such as choroid plexi of the brain and epithelial cells of the lung with the onset at E10.5 and E13.5, respectively. In the pancreas, ß-galactosidase activity is seen in the monociliated cells of the pancreatic duct and islet of Langerhans. Intercrossing Fltp(T2AiCre) mice with the CAG-CAT-EGFP reporter mouse line further confirms iCre activity in multiciliated cells of the lung and brain on a cellular level. Thus, the Fltp(T2AiCre) line is a powerful tool to conditionally inactivate genes in distinct mono- and multiciliated tissues and to analyze the tagged Fltp protein in vivo.

NEUROD2 function is dispensable for human pancreatic ß cell specification.

Introduction: The molecular programs regulating human pancreatic endocrine cell induction and fate allocation are not well deciphered. Here, we investigated the spatiotemporal expression pattern and the function of the neurogenic differentiation factor 2 (NEUROD2) during human endocrinogenesis. Methods: Using Crispr-Cas9 gene editing, we generated a reporter knock-in transcription factor (TF) knock-out human inducible pluripotent stem cell (iPSC) line in which the open reading frame of both NEUROD2 alleles are replaced by a nuclear histone 2B-Venus reporter (NEUROD2nVenus/nVenus). Results: We identified a transient expression of NEUROD2 mRNA and its nuclear Venus reporter activity at the stage of human endocrine progenitor formation in an iPSC differentiation model. This expression profile is similar to what was previously reported in mice, uncovering an evolutionarily conserved gene expression pattern of NEUROD2 during endocrinogenesis. In vitro differentiation of the generated homozygous NEUROD2nVenus/nVenus iPSC line towards human endocrine lineages uncovered no significant impact upon the loss of NEUROD2 on endocrine cell induction. Moreover, analysis of endocrine cell specification revealed no striking changes in the generation of insulin-producing b cells and glucagon-secreting a cells upon lack of NEUROD2. Discussion: Overall, our results suggest that NEUROD2 is expendable for human b cell formation in vitro.

The Sox17-mCherry fusion mouse line allows visualization of endoderm and vascular endothelial development.

Sox17 is a HMG-box transcription factor that has been shown to play important roles in both cardio-vascular development and endoderm formation. To analyze these processes in greater detail, we have generated a Sox17-mCherry fusion (SCF) protein by gene targeting in ES cells. SCF reporter mice are homozygous viable and faithfully reflect the endogenous Sox17 protein localization. We report that SCF positive cells constitute a subpopulation in the visceral endoderm before gastrulation and time-lapse imaging reveals that SCF monitors the nascent definitive endoderm during epithelialization. After gastrulation, SCF marks the mid- and hindgut endoderm and vascular endothelial network, which can be imaged during establishment in allantois explant cultures. The SCF reporter is downregulated in the endoderm epithelium and upregulated in endothelial cells of the intestine, lung, and pancreas during organogenesis. In summary, the generation of the Sox17-mCherry reporter mouse line allows direct visualization of endoderm and vascular development in culture and the mouse embryo.

Mitochondrial impairment and intracellular reactive oxygen species alter primary cilia morphology.

Primary cilia have recently emerged as cellular signaling organelles. Their homeostasis and function require a high amount of energy. However, how energy depletion and mitochondria impairment affect cilia have barely been addressed. We first studied the spatial relationship between a mitochondria subset in proximity to the cilium in vitro, finding similar mitochondrial activity measured as mitochondrial membrane potential compared with the cellular network. Next, using common primary cilia cell models and inhibitors of mitochondrial energy production, we found alterations in cilia number and/or length due to energy depletion and mitochondrial reactive oxygen species (ROS) overproduction. Finally, by using a mouse model of type 2 diabetes mellitus, we provided in vivo evidence that cilia morphology is impaired in diabetic nephropathy, which is characterized by ROS overproduction and impaired mitochondrial metabolism. In conclusion, we showed that energy imbalance and mitochondrial ROS affect cilia morphology and number, indicating that conditions characterized by mitochondria and radicals imbalances might lead to ciliary impairment.

Synaptotagmin-13 orchestrates pancreatic endocrine cell egression and islet morphogenesis.

During pancreas development endocrine cells leave the ductal epithelium to form the islets of Langerhans, but the morphogenetic mechanisms are incompletely understood. Here, we identify the Ca2+-independent atypical Synaptotagmin-13 (Syt13) as a key regulator of endocrine cell egression and islet formation. We detect specific upregulation of the Syt13 gene and encoded protein in endocrine precursors and the respective lineage during islet formation. The Syt13 protein is localized to the apical membrane of endocrine precursors and to the front domain of egressing endocrine cells, marking a previously unidentified apical-basal to front-rear repolarization during endocrine precursor cell egression. Knockout of Syt13 impairs endocrine cell egression and skews the α-to-ß-cell ratio. Mechanistically, Syt13 is a vesicle trafficking protein, transported via the microtubule cytoskeleton, and interacts with phosphatidylinositol phospholipids for polarized localization. By internalizing a subset of plasma membrane proteins at the front domain, including α6ß4 integrins, Syt13 modulates cell-matrix adhesion and allows efficient endocrine cell egression. Altogether, these findings uncover an unexpected role for Syt13 as a morphogenetic driver of endocrinogenesis and islet formation.