Distinct roles and requirements for Ras pathway signaling in visceral versus somatic muscle founder specification.

Pleiotropic signaling pathways must somehow engender specific cellular responses. In the Drosophila mesoderm, Ras pathway signaling specifies muscle founder cells from among the broader population of myoblasts. For somatic muscles, this is an inductive process mediated by the ETS-domain downstream Ras effectors Pointed and Aop (Yan). We demonstrate here that for the circular visceral muscles, despite superficial similarities, a significantly different specification mechanism is at work. Not only is visceral founder cell specification not dependent on Pointed or Aop, but Ras pathway signaling in its entirety can be bypassed. Our results show that de-repression, not activation, is the predominant role of Ras signaling in the visceral mesoderm and that, accordingly, Ras signaling is not required in the absence of repression. The key repressor acts downstream of the transcription factor Lame duck and is likely a member of the ETS transcription factor family. Our findings fit with a growing body of data that point to a complex interplay between the Ras pathway, ETS transcription factors, and enhancer binding as a crucial mechanism for determining unique responses to Ras signaling.

ROSA26 reporter mouse lines and image analyses reveal distinct region-specific cell behaviors in the visceral endoderm.

The early post-implantation mouse embryo changes dramatically in both size and shape. These morphological changes are based on characteristic cellular behaviors, including cell growth and allocation. To perform clonal analysis, we established a Cre/loxP-based reporter mouse line, R26R-ManGeKyou, that enables clonal labeling with multiple colors. We also developed a novel ImageJ plugin, LP-Clonal, for quantitative measurement of the tilt angle of clonal cluster shape, enabling identification of the direction of cluster expansion. We carried out long-term and short-term lineage tracking. We also performed time-lapse imaging to characterize cellular behaviors using R26-PHA7-EGFP and R26R-EGFP These images were subjected to quantitative image analyses. We found that the proximal visceral endoderm overlying the extra-embryonic ectoderm shows coherent cell growth in a proximal-anterior to distal-posterior direction. We also observed that directional cell migration is coupled with cell elongation in the anterior region. Our observations suggest that the behaviors of visceral endoderm cells vary between regions during peri-implantation stages.

Computed tomography study of the fetal development of the dairy cow stomach complex.

In the fetal development of animals, critical physiological and anatomical events influence the long-term health and performance of the offspring. To identify the critical growth phases of the fetal bovine stomach, we used computed tomography imaging on 30 German Holstein fetuses to examine the fetal bovine stomach in situ. Computed tomography allows the study of diverse parameters such as the volume of the stomach chambers in situ without the need for sophisticated filling preparation techniques. The absolute volume, relative volume, and monthly volume increase of each stomach chamber were determined. Computed tomography was a reliable method for in situ examination of the fetal bovine stomach complex from the third month of gestation onward. It was able to detect an abnormal position of the abomasum in 2 fetuses. The crown-rump length of the fetuses studied ranged from 9.5 to 89 cm (from 2.2 to 8.3 mo of gestation). Over this timeline, the changes in the relative volumes of the ruminoreticulum and abomasum were inversely related. Until mo 5 of gestation, the relative volume of the ruminoreticulum increased steadily, whereas that of the abomasum decreased. Thereafter, the relative volume of the ruminoreticulum became gradually smaller, and that of the abomasum became larger; by mo 8, the abomasum was larger than the ruminoreticulum. All stomach chambers had large increases in volume over the gestation period and we observed differences in development patterns and volume changes of the individual stomach chambers over this period. The largest monthly volume increase of the stomach complex was between mo 4 and 5 of gestation. In this period, the volume of the ruminoreticulum increased 43.8 times, that of the omasum 38.9 times, and that of the abomasum 30.03 times. Between mo 5 and 6 of gestation, the abomasum had another growth spurt, with a monthly volume increase of 10.4 times. These 2 time points in the gestation period may be critical phases of fetal development that should be considered in the management of pregnant cattle.

Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm.

The behavior of visceral endoderm cells was examined as the anterior visceral endoderm (AVE) formed from the distal visceral endoderm (DVE) using the mouse lines R26-H2B-EGFP and R26-PHA7-EGFP to visualize cell nuclei and adherens junction, respectively. The analysis using R26-H2B-EGFP demonstrated global cell rearrangement that was not specific to the DVE cells in the monolayer embryonic visceral endoderm sheet; each population of the endoderm cells moved collectively in a swirling movement as a whole. Most of the AVE cells at E6.5 were not E5.5 DVE cells but were E5.5 cells that were located caudally behind them, as previously reported (Hoshino et al., 2015; Takaoka et al., 2011). In the rearrangement, the posterior embryonic visceral endoderm cells did not move, as extraembryonic visceral endoderm cells did not, and they constituted a distinct population during the process of anterior-posterior axis formation. The analysis using R26-PHA7-EGFP suggested that constriction of the apical surfaces of the cells in prospective anterior portion of the DVE initiated the global cellular movement of the embryonic visceral endoderm to drive AVE formation.

Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs.

Pitx2 is a conserved homeodomain transcription factor that has multiple functions during embryonic development. Mutations in human PITX2 cause autosomal dominant Axenfeld-Rieger syndrome (ARS), characterized by congenital eye and tooth malformations. Pitx2(-/-) knockout mouse models recapitulate aspects of ARS, but are embryonic lethal. To date, ARS treatments remain limited to managing individual symptoms due to an incomplete understanding of PITX2 function. In addition to regulating eye and tooth development, Pitx2 is a target of a conserved Nodal (TGFß) signaling pathway that mediates left-right (LR) asymmetry of visceral organs. Based on its highly conserved asymmetric expression domain, the Nodal-Pitx2 axis has long been considered a common denominator of LR development in vertebrate embryos. However, functions of Pitx2 during asymmetric organ morphogenesis are not well understood. To gain new insight into Pitx2 function we used genome editing to create mutations in the zebrafish pitx2 gene. Mutations in the pitx2 homeodomain caused phenotypes reminiscent of ARS, including aberrant development of the cornea and anterior chamber of the eye and reduced or absent teeth. Intriguingly, LR asymmetric looping of the heart and gut was normal in pitx2 mutants. These results suggest conserved roles for Pitx2 in eye and tooth development and indicate Pitx2 is not required for asymmetric looping of zebrafish visceral organs. This work establishes zebrafish pitx2 mutants as a new animal model for investigating mechanisms underlying congenital malformations in ARS and high-throughput drug screening for ARS therapeutics. Additionally, pitx2 mutants present a unique opportunity to identify new genes involved in vertebrate LR patterning. We show Nodal signaling-independent of Pitx2-controls asymmetric expression of the fatty acid elongase elovl6 in zebrafish, pointing to a potential novel pathway during LR organogenesis.

Establishment of the Visceral Embryonic Midline Is a Dynamic Process that Requires Bilaterally Symmetric BMP Signaling.

The vertebrate body plan contains both dorsal and ventral midline structures. While dorsal midline structures have been extensively studied, formation of ventral midline structures, and how they become aligned with the dorsal midline, is a fundamental aspect of vertebrate development that is poorly understood. This study uses the chick dorsal mesentery (DM) as a model for investigating the formation of ventral midline structures. We document formation of the DM by epithelial-to-mesenchymal transition (EMT) and medial ingression of the lateral plate coelomic lining and show that DM positioning is a fundamentally dynamic process regulated by relative levels of bone morphogenetic protein (BMP) signaling in the two sides of the ingressing lateral plate. Disruption of this process causes misalignment of the DM and disturbances during initial stages of lung morphogenesis. Since the dorsal midline is a source of BMP antagonists, these results suggest a mechanism for aligning the dorsal and ventral embryonic midlines.

A novel approach to assessing fetal tissue stiffness using virtual touch tissue quantification.

AIMS: Virtual touch tissue quantification (VTTQ) has been widely used in adults, but its application in fetuses has not been reported. The purposes of this study were to describe the normal shear wave velocity (SWV) values of fetal brain, lung, and liver by VTTQ and to examine the clinical usefulness of this procedure in THE evaluation of gestational age-related SWV changes in the fetal brain, lung and liver. MATERIAL AND METHODS: One hundred fetuses were enrolled in this study and were evaluated at 18 weeks (second trimester) and 35 weeks (third trimester) during pregnancy. The SWV was measured at the cerebral parenchyma, thalamus, cerebellum, choroid plexus, left lung, right lung, right and left lobe of the liver in each participant. RESULTS: The SWV at the cerebral parenchyma were all significantly greater in the third trimesters than in the second trimesters (3.29+/-1.05 vs. 2.22+/-0.97 m/s, p<0.001), while the velocities at thalamus, cerebellum, choroid plexus, left lung, right lung, right lobe of the liver and left lobe of the liver did not differ between the second and the third trimesters (p>0.05). Moreover, the SWV at the cerebral parenchyma correlated significantly with gestational age (r=0.47, p<0.001). CONCLUSIONS: VTTQ can provide numerical measurements of fetal brain, lung and liver stiffness and can effectively and objectively indicate gestational age-related changes in cerebral parenchyma stiffness by measuring SWV values.

Coelomic epithelium-derived cells in visceral morphogenesis.

Coelomic cavities of vertebrates are lined by a mesothelium which develops from the lateral plate mesoderm. During development, the coelomic epithelium is a highly active cell layer, which locally is able to supply mesenchymal cells that contribute to the mesodermal elements of many organs and provide signals which are necessary for their development. The relevance of this process of mesenchymal cell supply to the developing organs is becoming clearer because genetic lineage tracing techniques have been developed in recent years. Body wall, heart, liver, lungs, gonads, and gastrointestinal tract are populated by cells derived from the coelomic epithelium which contribute to their connective and vascular tissues, and sometimes to specialized cell types such as the stellate cells of the liver, the Cajal interstitial cells of the gut or the Sertoli cells of the testicle. In this review we collect information about the contribution of coelomic epithelium derived cells to visceral development, their developmental fates and signaling functions. The common features displayed by all these processes suggest that the epithelial-mesenchymal transition of the embryonic coelomic epithelium is an underestimated but key event of vertebrate development, and probably it is shared by all the coelomate metazoans.

[Artificial Inversion of the Left-Right Visceral Asymmetry in Vertebrates: Conceptual Approaches and Experimental Solutions].

Externally, vertebrates are bilaterally symmetrical; however, left-right asymmetry is observed in the structure of their internal organs and systems of organs (circulatory, digestive, and respiratory). In addition to the asymmetry of internal organs (visceral), there is also functional (i.e., asymmetrical functioning of organs on the left and right sides of the body) and behavioral asymmetry. The question of a possible association between different types of asymmetry is still open. The study of the mechanisms of such association, in addition to the fundamental interest, has important applications for biomedicine, primarily for the understanding of the brain functioning in health and disease and for the development of methods of treatment of certain mental diseases, such as schizophrenia and autism, for which the disturbance of left-right asymmetry of the brain was shown. To study the deep association between different types of asymmetry, it is necessary to obtain adequate animal models (primarily animals with inverted visceral organs, situs inversus totalis). There are two main possible approaches to obtaining such model organisms: mutagenesis followed by selection of mutant strains with mutations in the genes that affect the formation of the left-right visceral asymmetry and experimental obtaining of animals with inverted internal organs. This review focuses on the second approach. We describe the theoretical models for establishing left-right asymmetry and possible experimental approaches to obtaining animals with inverted internal organs.

Environmental concentration of nonylphenol alters the development of urogenital and visceral organs in avian model.

Nonylphenol (NP) is an endocrine disruptor with harmful effects including feminization and carcinogenesis on various organisms. This substance is a degradation product of nonylphenol ethoxylates (NPEO) that is used in several industrial and agricultural processes. In this paper, we examined the assessment of NP exposure on chick embryo development, using a concentration consistent with the environmental concentrations of NP. With this aim, NP (between 0.1 and 50 µg/egg) was injected into the yolk of egg through a small needle hole in the shell. We report the effect of NP on chick reproductive system development although the effect we observed is lower than those observed by exposition to other endocrine disruptors. However, histological analysis highlighted a decrease of intraluminal seminiferous surface area in 64.12% of case (P=0.0086) and an heterogeneous organization of the renal tubules when 10 µg/egg were injected. Moreover, an impairment of liver development with an abnormal bile spillage was observed when higher concentration of NP was injected (50 µg/egg).

Study for collecting background data on Wistar Hannover [Crl:WI(Han)] rats in embryo-fetal development studies--comparative data to Sprague Dawley rats.

The purpose of the present study was to collect the background data on Wistar Hannover [Crl:WI(Han)] (hereafter Wistar Han) rats in embryo-fetal development studies from the 6 safety research facilities of pharmaceutical companies and contract research organizations. In each facility, 20 or 22 female rats were dosed with vehicle solution during the organogenesis period. As a result, no abnormalities in clinical signs and necropsy findings in dams were found. Body weights and food consumption in dams were lower than those in Sprague Dawley (SD) rats. The number of corpora lutea (13.3 vs. 16.0 in SD) and implantations (11.8 vs. 14.7) were fewer, and fetal body weights (3.66 vs. 3.70) and placental weights (0.42 vs. 0.45) tended to be lower than those in SD rats. Regarding the fetal abnormalities, the incidence of several findings such as the persistent left umbilical artery (10.4% vs. 1.1%) and cervical (5.2% vs. 0.4%), full (7.4% vs. 0.9%) or short supernumerary (64.5% vs. 9.9%) and wavy ribs (6.6% vs. 0.3%) was higher than that in SD rats. Our present study showed that they maintained a sufficient number of live fetuses and the difference in the fetal sex ratio was not observed. In conclusion, Wistar Han rats were considered to be a suitable strain for embryo-fetal development toxicity study. Since the incidence of several abnormalities was higher than that in SD rats, it may be said that to accumulate background control data is important to evaluate the embryo-fetal development toxicity study using Wistar Han rats.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine.

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

Microautophagy in the visceral endoderm is essential for mouse early development.

During early embryogenesis, before the conceptus forms the placenta, maternal nutrients as well as signaling molecules must reach the embryo proper through a tightly sealed epithelial tissue, the visceral endoderm (VE). The VE serves as a signaling center for embryogenesis, where exocytic and endocytic processes integrate signal production, perception and termination. However, the endocytic process in this important tissue has not been well characterized. We show that endocytic delivery to the lysosomes occurs via RAB7-dependent microautophagy. This process is essential for early mammalian development.

Activation of the PTHRP/adenylate cyclase pathway promotes differentiation of rat XEN cells into parietal endoderm, whereas Wnt/ß-catenin signaling promotes differentiation into visceral endoderm.

During early mammalian development, primitive endoderm (PrE) is specified and segregated away from the pluripotent epiblast. At a later developmental stage, PrE forms motile parietal endoderm (PE) lying proximal to the trophectoderm, and visceral endoderm (VE) that contacts the developing epiblast and extraembryonic ectoderm. Mouse extraembryonic endoderm (XEN) cells were isolated and became widely used to study signals governing lineage specification. Rat XEN cell lines have also been derived, but were distinguished from mouse by expression of SSEA1 and Oct4. We showed here that rat XEN cells grown in the presence of a GSK3 inhibitor or overexpressing ß-catenin exhibited enhanced formation of cell contacts and decreased motility. Rat XEN cells treated with BMP4 revealed similar morphological changes. Furthermore, we observed that rat XEN cells cultured with GSK3 inhibitor formed adhesion and tight junctions, and acquired bottom-top polarity, indicating the formation of VE cells. In contrast, forskolin, an activator of the cAMP pathway, induced the disruption of cell contacts in rat XEN cells. Treatment with forskolin induced PE formation and epithelial-mesenchymal transition (EMT) in rat XEN cells. Using microarray and real-time PCR assays, we found that VE versus PE formation of rat XEN cells was correlated with change in expression levels of VE or PE marker genes. Similar to forskolin, EMT was prompted upon treatment of rat XEN cells with recombinant parathyroid hormone related peptide (PTHRP), an activator of the cAMP pathway in vivo. Taken together, our data suggest that rat XEN cells are PrE-like cells. The activation of Wnt or BMP4 pathways in rat XEN cells leads to the acquisition of VE characteristics, whereas the activation of the PTHRP/cAMP pathway leads to EMT and the formation of PE.

Study on the developmental toxicity of combined artesunate and mefloquine antimalarial drugs on rats.

Antimalarial drug combinations containing artemisinins (ACTs) have become first choice therapies for Plasmodium falciparum malaria. Data on safety of ACTs in pregnancy are limited and no previous study has been conducted on the developmental toxicity of artesunate-mefloquine combinations on the first trimester of gestation. To evaluate the developmental toxicity of an artesunate/mefloquine combination, pregnant rats were treated orally with artesunate (15 and 40 mg/kg bwt/day), mefloquine (30 and 80 mg/kg bwt/day) and artesunate/mefloquine (15/30 and 40/80 mg/kg bwt/day) on gestation days 9-11. Dams were C-sectioned on day 20, and their uteri and fetuses removed and examined for soft tissue and skeleton abnormalities. Artesunate increased embryolethality and the incidence of limb long bone malformations on the absence of overt maternal toxicity. Mefloquine (80 mg/kg bwt/day) was maternally toxic and enhanced fetal variations. Combination of artesunate and mefloquine did not enhance their toxicity compared to the toxicity observed after its separate administration. Embryotoxicity of artesunate was apparently attenuated when it is co-administered with mefloquine.

The FGF8-related signals Pyramus and Thisbe promote pathfinding, substrate adhesion, and survival of migrating longitudinal gut muscle founder cells.

Fibroblast growth factors (FGFs) frequently fulfill prominent roles in the regulation of cell migration in various contexts. In Drosophila, the FGF8-like ligands Pyramus (Pyr) and Thisbe (Ths), which signal through their receptor Heartless (Htl), are known to regulate early mesodermal cell migration after gastrulation as well as glial cell migration during eye development. Herein, we show that Pyr and Ths also exert key roles during the long-distance migration of a specific sub-population of mesodermal cells that migrate from the caudal visceral mesoderm within stereotypic bilateral paths along the trunk visceral mesoderm toward the anterior. These cells constitute the founder myoblasts of the longitudinal midgut muscles. In a forward genetic screen for regulators of this morphogenetic process we identified loss of function alleles for pyr. We show that pyr and ths are expressed along the paths of migration in the trunk visceral mesoderm and endoderm and act largely redundantly to help guide the founder myoblasts reliably onto and along their substrate of migration. Ectopically-provided Pyr and Ths signals can efficiently re-rout the migrating cells, both in the presence and absence of endogenous signals. Our data indicate that the guidance functions of these FGFs must act in concert with other important attractive or adhesive activities of the trunk visceral mesoderm. Apart from their guidance functions, the Pyr and Ths signals play an obligatory role for the survival of the migrating cells. Without these signals, essentially all of these cells enter cell death and detach from the migration substrate during early migration. We present experiments that allowed us to dissect the roles of these FGFs as guidance cues versus trophic activities during the migration of the longitudinal visceral muscle founders.

Sensitive windows of skeletal development in rabbits determined by hydroxyurea exposure at different times throughout gestation.

The critical periods of axial skeletal development in rats and mice have been well characterized, however the timing of skeletal development in rabbits is not as well known. It is important to have a more precise understanding of this timing of axial skeletal development in rabbits due to the common use of this species in standard nonclinical studies to assess embryo-fetal developmental toxicity. Hydroxyurea, a teratogen known to induce a variety of fetal skeletal malformations, was administered to New Zealand White rabbits as a single dose (500 mg/kg) on individual days during gestation (gestation day, GD 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 19) and fetal external, visceral, and skeletal morphology was examined following cesarean sections on GD 29. A wide range of fetal skeletal effects was observed following hydroxyurea treatment, with a progression of malformations from anterior to posterior structures over time, as well as from proximal to distal structures over time. The sensitive window of axial skeletal development was determined to be GD 8 to 13, while disruption of appendicular and cranio-facial skeletal development occurred primarily from GD 11 to 16 and GD 11 to 12, respectively. The results of this study provide a better understanding of the critical developmental window for different segments of the rabbit skeleton, which will aid in the design of window studies to investigate teratogenicity in rabbits.

Developmental toxicity of lersivirine in rabbits when administered throughout organogenesis and when limited to sensitive windows of axial skeletal development.

BACKGROUND: Lersivirine is a second-generation nonnucleoside reverse transcriptase inhibitor undergoing clinical development for the treatment of human immunodeficiency virus-1. An embryo-fetal development study was performed to evaluate the potential for maternal and developmental toxicity of lersivirine. METHODS: Pregnant New Zealand White rabbits were administered 0, 100, 250, and 500 mg/kg lersivirine by oral gavage once daily on gestation days (GDs) 7 to 19, followed by cesarean section on GD 29 and fetal evaluation. RESULTS: Maternal toxicity was noted at all dose levels (decreased food consumption and body weight gain), with fetal toxicity at 500 mg/kg (decreased fetal weights, increased postimplantation loss). Equivocal findings for axial skeletal malformations were observed in three fetuses at 500 mg/kg. To better understand if these malformations were related to treatment with lersivirine, a follow-up rabbit embryo-fetal development study was performed with 1000 mg/kg/day lersivirine (500 mg/kg BID, 12-hr interdose interval) for two different 3-day windows, GDs 8 to 10 or GDs 11 to 13, which represent the sensitive windows of axial skeletal development in rabbits. Control rabbits were administered vehicle following the same dosing regimen from GDs 8 to 13. Cesarean sections were performed on GD 29, and fetal skeletons were examined for the potential of lersivirine to cause skeletal malformations in rabbits. At maternal exposure levels higher than the initial study, lersivirine did not induce fetal skeletal malformations when administered in the sensitive windows of axial skeletal development. CONCLUSION: The results of these studies indicate that lersivirine did not exhibit any evidence of teratogenicity in rabbits.

Foetal volumetry using magnetic resonance imaging in intrauterine growth restriction.

OBJECTIVES: We used magnetic resonance imaging (MRI) to perform volumetry of foetuses with and without growth restriction, and identify deviations in organ growth. STUDY DESIGN: 20 growth restricted and 19 normal foetuses were scanned once during pregnancy at gestational age 20.53-36.57 weeks. MRI scans were performed on a 1.5T system using ssFSE sequences. Manual segmentation of whole body, brain, heart, lung, liver, thymus and kidney volume was performed. Data on the severity of foetal growth restriction and pregnancy outcome was collected. RESULTS: There was a significant reduction in foetal whole body volume and volume of all internal organs except the brain in growth restricted foetuses. A brain:liver ratio above 3.0 was associated with a 3.3 fold increase in risk of perinatal mortality (95% CI=1.68-6.47). CONCLUSION: MRI provides an accurate assessment of foetal organ growth. It may have a role to play in monitoring disease severity and the effect of future interventions.

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity.

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.