A quantitative model of cellular decision making in direct neuronal reprogramming.

The direct reprogramming of adult skin fibroblasts to neurons is thought to be controlled by a small set of interacting gene regulators. Here, we investigate how the interaction dynamics between these regulating factors coordinate cellular decision making in direct neuronal reprogramming. We put forward a quantitative model of the governing gene regulatory system, supported by measurements of mRNA expression. We found that nPTB needs to feed back into the direct neural conversion network most likely via PTB in order to accurately capture quantitative gene interaction dynamics and correctly predict the outcome of various overexpression and knockdown experiments. This was experimentally validated by nPTB knockdown leading to successful neural conversion. We also proposed a novel analytical technique to dissect system behaviour and reveal the influence of individual factors on resulting gene expression. Overall, we demonstrate that computational analysis is a powerful tool for understanding the mechanisms of direct (neuronal) reprogramming, paving the way for future models that can help improve cell conversion strategies.

Roles of PTBP1 in alternative splicing, glycolysis, and oncogensis.

Polypyrimidine tract-binding protein 1 (PTBP1) plays an essential role in splicing and is expressed in almost all cell types in humans, unlike the other proteins of the PTBP family. PTBP1 mediates several cellular processes in certain types of cells, including the growth and differentiation of neuronal cells and activation of immune cells. Its function is regulated by various molecules, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and RNA-binding proteins. PTBP1 plays roles in various diseases, particularly in some cancers, including colorectal cancer, renal cell cancer, breast cancer, and glioma. In cancers, it acts mainly as a regulator of glycolysis, apoptosis, proliferation, tumorigenesis, invasion, and migration. The role of PTBP1 in cancer has become a popular research topic in recent years, and this research has contributed greatly to the formulation of a useful therapeutic strategy for cancer. In this review, we summarize recent findings related to PTBP1 and discuss how it regulates the development of cancer cells.

Polypyrimidine tract-binding proteins are essential for B cell development.

Polypyrimidine tract-binding protein 1 (PTBP1) is a RNA-binding protein (RBP) expressed throughout B cell development. Deletion of Ptbp1 in mouse pro-B cells results in upregulation of PTBP2 and normal B cell development. We show that PTBP2 compensates for PTBP1 in B cell ontogeny as deletion of both Ptbp1 and Ptbp2 results in a complete block at the pro-B cell stage and a lack of mature B cells. In pro-B cells PTBP1 ensures precise synchronisation of the activity of cyclin dependent kinases at distinct stages of the cell cycle, suppresses S-phase entry and promotes progression into mitosis. PTBP1 controls mRNA abundance and alternative splicing of important cell cycle regulators including CYCLIN-D2, c-MYC, p107 and CDC25B. Our results reveal a previously unrecognised mechanism mediated by a RBP that is essential for B cell ontogeny and integrates transcriptional and post-translational determinants of progression through the cell cycle.

Roles of PTBP1 in alternative splicing, glycolysis, and oncogensis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Polypyrimidine tract-binding protein 1 (PTBP1) plays an essential role in splicing and is expressed in almost all cell types in humans, unlike the other proteins of the PTBP family. PTBP1 mediates several cellular processes in certain types of cells, including the growth and differentiation of neuronal cells and activation of immune cells. Its function is regulated by various molecules, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and RNA-binding proteins. PTBP1 plays roles in various diseases, particularly in some cancers, including colorectal cancer, renal cell cancer, breast cancer, and glioma. In cancers, it acts mainly as a regulator of glycolysis, apoptosis, proliferation, tumorigenesis, invasion, and migration. The role of PTBP1 in cancer has become a popular research topic in recent years, and this research has contributed greatly to the formulation of a useful therapeutic strategy for cancer. In this review, we summarize recent findings related to PTBP1 and discuss how it regulates the development of cancer cells.

Role of polypyrimidine tract-binding protein 1/yin yang 2 signaling in regulating vascular smooth muscle cell proliferation and neointima hyperplasia.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular restenosis. We investigated whether polypyrimidine tract-binding protein 1 (PTBP1), a novel regulator of cell proliferation and differentiation, is implicated in VSMC proliferation and neointima hyperplasia responding to injury. C57BL/6 J mice of 10-12 weeks old were randomly divided into sham and carotid artery injury group. Primary VSMCs obtained from thoracic aortas of 10- to 12-week-old mice were treated with physiological saline and platelet derived growth factor-BB (PDGF-BB). Adenovirus expressing shCon, shPTBP1 or shYY2 were transfected into the injured common carotid artery or VSMCs. qRT-PCR and immunoblotting were used to determine the mRNA and protein expression levels, respectively. Immunohistochemical staining of H&E and Ki-67 were used to evaluate restenosis of vessels. Cell counting kit-8 assay and Ki-67 immunofluorescent staining were utilized to evaluate the rate of VSMC proliferation. The expression of PTBP1 were upregulated both in injured arteries and in PDGF-BB-treated VSMCs. PTBP1 inhibition significantly attenuated neointima hyperplasia and Ki-67 positive area induced by injury. Knockdown of PTBP1 in vitro also suppressed VSMC proliferation after PDGF-BB treatment. The effects of PTBP1 inhibition mentioned above were all abolished by knockdown of YY2. Finally, we identified four cell cycle regulators (p53, p21, Cdkn1c, Cdkn2b) that were regulated by PTBP1/YY2 axis both in vitro and in vivo. These findings demonstrated that PTBP1 is a critical regulator of VSMC proliferation and neointima hyperplasia via modulating the expression of YY2.

PTBP1 contributes to spermatogenesis through regulation of proliferation in spermatogonia.

Polypyrimidine tract-binding protein 1 (PTBP1) is a highly conserved RNA-binding protein that is a well-known regulator of alternative splicing. Testicular tissue is one of the richest tissues with respect to the number of alternative splicing mRNA isoforms, but the molecular role(s) of PTBP1 in the regulation of these isoforms during spermatogenesis is still unclear. Here, we developed a germ cell-specific Ptbp1 conditional knockout (cKO) mouse model by using the Cre-loxP system to investigate the role of PTBP1 in spermatogenesis. Testis weight in Ptbp1 cKO mice was comparable to that in age-matched controls until 3 weeks of age; at ≥ 2 months old, testis weight was significantly lighter in cKO mice than in age-matched controls. Sperm count in Ptbp1 cKO mice at 2 months old was comparable to that in controls, whereas sperm count significantly decreased at 6 months old. Seminiferous tubules that exhibited degeneration in spermatogenic function were more evident in the 2-month-old Ptbp1 cKO mice than in controls. In addition, the early neonatal proliferation of spermatogonia, during postnatal days 1-5, was significantly retarded in Ptbp1 cKO mice compared with that in controls. An in vitro spermatogonia culture model (germline stem cells) revealed that hydroxytamoxifen-induced deletion of PTBP1 from germline stem cells caused severe proliferation arrest accompanied by an increase of apoptotic cell death. These data suggest that PTBP1 contributes to spermatogenesis through regulation of spermatogonia proliferation.

PTBP1 promotes tumorigenesis by regulating apoptosis and cell cycle in colon cancer.

Increased expression of polypyrimidine tract-binding protein 1 (PTBP1) has been observed in human ovarian tumors, glioblastomas, and breast cancer, but its biological roles in tumorigenesis is not fully clear. In the present research, we investigated the biological role of PTBP1 in colon cancer. We found that PTBP1 was overexpressed both in colon cancer cell lines and tissues. Tissue microarray analysis (TMA) indicated that low PTBP1 expression predicted a favorable overall survival for colon cancer patients. Using small interfering RNA technology, we found that down-regulation of PTBP1 significantly inhibited colon cancer cell growth/proliferation, and induced cell cycle arrest as well as apoptosis in vitro. Western blot analysis showed that siRNA PTBP1 could up-regulate the expression of cytoC, p53 and Bax as well as down-regulated p85, p-AKT, cyclinD1, CDK4 and Bcl2 compared to the control. Furthermore, Caspase-3 and PARP1 were activated when PTBP1 is knockdown. This study implies that PTBP1 plays an important role in tumorigenesis of colon cancer.

A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival.

Functions of many long noncoding RNAs (lncRNAs) depend on their ability to interact with multiple copies of specific RNA-binding proteins (RBPs). Here, we devised a workflow combining bioinformatics and experimental validation steps to systematically identify RNAs capable of multivalent RBP recruitment. This uncovered a number of previously unknown transcripts encoding high-density RBP recognition arrays within genetically normal short tandem repeats. We show that a top-scoring hit in this screen, lncRNA PNCTR, contains hundreds of pyrimidine tract-binding protein (PTBP1)-specific motifs allowing it to sequester a substantial fraction of PTBP1 in a nuclear body called perinucleolar compartment. Importantly, PNCTR is markedly overexpressed in a variety of cancer cells and its downregulation is sufficient to induce programmed cell death at least in part by stimulating PTBP1 splicing regulation activity. This work expands our understanding of the repeat-containing fraction of the human genome and illuminates a novel mechanism driving malignant transformation of cancer cells.

cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation.

Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.

Ptbp2 Controls an Alternative Splicing Network Required for Cell Communication during Spermatogenesis.

Alternative splicing has essential roles in development. Remarkably, spermatogenic cells express more alternatively spliced RNAs compared to most whole tissues; however, regulation of these RNAs remains unclear. Here, we characterize the alternative splicing landscape during spermatogenesis and reveal an essential function for the RNA-binding protein Ptbp2 in this highly regulated developmental program. We found that Ptbp2 controls a network of genes involved in cell adhesion, migration, and polarity, suggesting that splicing regulation by Ptbp2 is critical for germ cell communication with Sertoli cells (multifunctional somatic cells necessary for spermatogenesis). Indeed, Ptbp2 ablation in germ cells resulted in disorganization of the filamentous actin (F-actin) cytoskeleton in Sertoli cells, indicating that alternative splicing regulation is necessary for cellular crosstalk during germ cell development. Collectively, the data delineate an alternative splicing regulatory network essential for spermatogenesis, the splicing factor that controls it, and its biological importance in germ-Sertoli communication.

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis.

Regulation of alternative splicing is an important process for cell differentiation and development. Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exon-centric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed. We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.

A mechanism underlying position-specific regulation of alternative splicing.

Many RNA-binding proteins including a master regulator of splicing in developing brain and muscle, polypyrimidine tract-binding protein 1 (PTBP1), can either activate or repress alternative exons depending on the pre-mRNA recruitment position. When bound upstream or within regulated exons PTBP1 tends to promote their skipping, whereas binding to downstream sites often stimulates inclusion. How this switch is orchestrated at the molecular level is poorly understood. Using bioinformatics and biochemical approaches we show that interaction of PTBP1 with downstream intronic sequences can activate natural cassette exons by promoting productive docking of the spliceosomal U1 snRNP to a suboptimal 5' splice site. Strikingly, introducing upstream PTBP1 sites to this circuitry leads to a potent splicing repression accompanied by the assembly of an exonic ribonucleoprotein complex with a tightly bound U1 but not U2 snRNP. Our data suggest a molecular mechanism underlying the transition between a better-known repressive function of PTBP1 and its role as a bona fide splicing activator. More generally, we argue that the functional outcome of individual RNA contacts made by an RNA-binding protein is subject to extensive context-specific modulation.

Linc-ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR-124/PTBP1/PKM2 axis.

PURPOSE: In this study, we investigated the regulation of linc-ROR on autophagy and gemcitabine resistance of pancreatic cancer cells and further studied the underlying involvement of the miR-124/PTBP1/PKM2 axis in this regulation. METHODS: Pancreatic cancer cell lines PANC-1 and MIAPaCa-2 cells were used as in vitro model. Autophagy was assessed by western blot of LC3 I/II and observation GFP-LC3 puncta. Cell viability was examined using CCK-8 assay. Cell apoptosis was examined by flow cytometric analysis of Annexin V/PI staining. QRT-PCR, RNA fluorescence in situ hybridization and dual luciferase assay were used to study the expression and the binding between linc-ROR and miR-124. RESULTS: Linc-ROR siRNA significantly sensitized PANC-1 and MIAPaCa-2 cells to gemcitabine, while linc-ROR overexpression significantly reduced the sensitivity. Linc-ROR knockdown reduced basal autophagy, while linc-ROR overexpression markedly increased basal autophagy in the cells. Linc-ROR siRNA showed similar effect as 3-MA on enhancing gemcitabine-induced cell apoptosis and also reduced PKM2 expression. MiR-124 overexpression restored PKM1 and reduced PKM2 levels in the cells. In addition, miR-124 mimics also alleviated autophagy in pancreatic cancer cells. Both miR-124 mimics and PKM2 siRNA enhanced gemcitabine-induced cell apoptosis. In both pancreatic cell lines and PADC tissues, linc-ROR is negatively correlated with miR-124 expression. In addition, dual luciferase assay verified two 8mer binding sites between miR-124 and linc-ROR. CONCLUSION: Linc-ROR confers gemcitabine resistance to pancreatic cancer cells at least partly via inducing autophagy. There is a linc-ROR/miR-124/PTBP1/PKM2 axis involved in regulation of gemcitabine resistance in pancreatic cancer cells.

Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and incurable disease. Poor prognosis is due to multiple reasons, including acquisition of resistance to gemcitabine, the first-line chemotherapeutic approach. Thus, there is a strong need for novel therapies, targeting more directly the molecular aberrations of this disease. We found that chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that are drug-resistant (DR-PDAC cells). Importantly, alternative splicing (AS) of the pyruvate kinase gene (PKM) was differentially modulated in DR-PDAC cells, resulting in promotion of the cancer-related PKM2 isoform, whose high expression also correlated with shorter recurrence-free survival in PDAC patients. Switching PKM splicing by antisense oligonucleotides to favor the alternative PKM1 variant rescued sensitivity of DR-PDAC cells to gemcitabine and cisplatin, suggesting that PKM2 expression is required to withstand drug-induced genotoxic stress. Mechanistically, upregulation of the polypyrimidine-tract binding protein (PTBP1), a key modulator of PKM splicing, correlated with PKM2 expression in DR-PDAC cell lines. PTBP1 was recruited more efficiently to PKM pre-mRNA in DR- than in parental PDAC cells. Accordingly, knockdown of PTBP1 in DR-PDAC cells reduced its recruitment to the PKM pre-mRNA, promoted splicing of the PKM1 variant and abolished drug resistance. Thus, chronic exposure to gemcitabine leads to upregulation of PTBP1 and modulation of PKM AS in PDAC cells, conferring resistance to the drug. These findings point to PKM2 and PTBP1 as new potential therapeutic targets to improve response of PDAC to chemotherapy.

[RNA-binding protein PTB in spermatogenesis: Progress in studies].

RNA binding proteins (RBPs) regulate the function of cells by interacting with nascent transcripts and therefore are receiving increasing attention from researchers for their roles in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. Further investigations on the post-transcriptional regulation mechanisms and isoforms of PTB proteins in the spermatogenesis show that PTB protein 1 (Ptbp1) is a predominant isoform in mitotic cells (spermatogonia), while Ptbp2 predominates in meiotic spermatocytes and postmeiotic spermatids and binds to the specific 3' untranslated region (3' UTR) of the phosphoglycerate kinase 2 (Pgk-2) mRNA, which helps to stabilize Pgk-2 mRNA in male mouse germ cells. In case of Ptbp2 inactivation in the testis, the differentiation of germ cells arrests in the stage of round spermatids, with proliferation of multinucleated cells in the seminiferous tubule, increased apoptosis of spermatocytes, atrophy of seminiferous tubules, and lack of elongating spermatids, which consequently affects male fertility. This article presents an overview on the structure of the PTB protein and its role in regulating mammalian spermatogenesis.

In silico to in vivo splicing analysis using splicing code models.

With the growing appreciation of RNA splicing's role in gene regulation, development, and disease, researchers from diverse fields find themselves investigating exons of interest. Commonly, researchers are interested in knowing if an exon is alternatively spliced, if it is differentially included in specific tissues or in developmental stages, and what regulatory elements control its inclusion. An important step towards the ability to perform such analysis in silico was made with the development of computational splicing code models. Aimed as a practical how-to guide, we demonstrate how researchers can now use these code models to analyze a gene of interest, focusing on Bin1 as a case study. Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein known to be functionally regulated through alternative splicing in a tissue-specific manner. Specific Bin1 isoforms have been associated with muscular diseases and cancers, making the study of its splicing regulation of wide interest. Using AVISPA, a recently released web tool based on splicing code models, we show that many Bin1 tissue-dependent isoforms are correctly predicted, along with many of its known regulators. We review the best practices and constraints of using the tool, demonstrate how AVISPA is used to generate high confidence novel regulatory hypotheses, and experimentally validate predicted regulators of Bin1 alternative splicing.

PTB deficiency causes the loss of adherens junctions in the dorsal telencephalon and leads to lethal hydrocephalus.

Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cre-mediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.

Polymorphism rs11085226 in the gene encoding polypyrimidine tract-binding protein 1 negatively affects glucose-stimulated insulin secretion.

OBJECTIVE: Polypyrimidine tract-binding protein 1 (PTBP1) promotes stability and translation of mRNAs coding for insulin secretion granule proteins and thereby plays a role in ß-cells function. We studied whether common genetic variations within the PTBP1 locus influence insulin secretion, and/or proinsulin conversion. METHODS: We genotyped 1,502 healthy German subjects for four tagging single nucleotide polymorphisms (SNPs) within the PTBP1 locus (rs351974, rs11085226, rs736926, and rs123698) covering 100% of genetic variation with an r(2)≥0.8. The subjects were metabolically characterized by an oral glucose tolerance test with insulin, proinsulin, and C-peptide measurements. A subgroup of 320 subjects also underwent an IVGTT. RESULTS: PTBP1 SNP rs11085226 was nominally associated with lower insulinogenic index and lower cleared insulin response in the OGTT (p≤0.04). The other tested SNPs did not show any association with the analyzed OGTT-derived secretion parameters. In the IVGTT subgroup, SNP rs11085226 was accordingly associated with lower insulin levels within the first ten minutes following glucose injection (p = 0.0103). Furthermore, SNP rs351974 was associated with insulin levels in the IVGTT (p = 0.0108). Upon interrogation of MAGIC HOMA-B data, our rs11085226 result was replicated (MAGIC p = 0.018), but the rs351974 was not. CONCLUSIONS: We conclude that common genetic variation in PTBP1 influences glucose-stimulated insulin secretion. This underlines the importance of PTBP1 for beta cell function in vivo.

Polypyrimidine tract-binding protein regulates the cell cycle through IRES-dependent translation of CDK11(p58) in mouse embryonic stem cells.

Polypyrimidine tract-binding protein (PTB/PTBP1/hnRNP I) is a member of the heterogeneous nuclear ribonucleoprotein family that binds specifically to pyrimidine-rich sequences of RNAs. Although PTB is a multifunctional protein involved in RNA processing and internal ribosome entry site (IRES)-dependent translation, the role of PTB in early mouse development is unclear. Ptb knockout mice exhibit embryonic lethality shortly after implantation and Ptb-/- embryonic stem (ES) cells have a severe proliferation defect that includes a prolonged G2/M phase. The present study shows that PTB promotes M phase progression by the direct repression of CDK11(p58) IRES activity in ES cells. The protein expression and IRES activity of CDK11(p58) in Ptb-/- ES cells is higher than that of wild-type ES cells, indicating that PTB is involved in the repression of CDK11(p58) expression through IRES-dependent translation in ES cells. Interestingly, CDK11(p58) IRES activity is activated by upstream of N-Ras (UNR) in 293T and NIH3T3 cells, whereas UNR is not present in the Cdk11 mRNA-protein complex in ES cells. In addition, PTB interacts directly with the IRES region of CDK11(p58) in ES cells. These results suggest that PTB regulates the precise expression of CDK11(p58) through direct interaction with CDK11(p58) IRES and promotes M phase progression in ES cells.

PTBP1 is required for embryonic development before gastrulation.

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.