Posterior Primitive Streak Research Articles

Control of Modular Tissue Flows Shaping the Embryo in Avian Gastrulation.

Avian gastrulation requires coordinated flows of thousands of cells to form the body plan. We quantified these flows using their fundamental kinematic units: one attractor and two repellers constituting its Dynamic Morphoskeleton (DM). We have also elucidated the mechanistic origin of the attractor, marking the primitive streak (PS), and controlled its shape, inducing gastrulation flows in the chick embryo that are typical of other vertebrates. However, the origins of repellers and dynamic embryo shape remain unclear. Here, we address these questions using active matter physics and experiments. Repeller 1, separating the embryo proper (EP) from extraembryonic (EE) tissues, arises from the tug-of-war between EE epiboly and EP isotropic myosin-induced active stress. Repeller 2, bisecting the anterior and posterior PS and associated with embryo shape change, arises from anisotropic myosin-induced active intercalation in the mesendoderm. Combining mechanical confinement with inhibition of mesendoderm induction, we eliminated either one or both repellers, as predicted by our model. Our results reveal a remarkable modularity of avian gastrulation flows delineated by the DM, uncovering the mechanistic roles of EE epiboly, EP active constriction, mesendoderm intercalation and ingression. These findings offer a new perspective for deconstructing morphogenetic flows, uncovering their modular origin, and aiding synthetic morphogenesis.

Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation invivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells invitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

Dissecting signaling hierarchies in the patterning of the mouse primitive streak using micropatterned EpiLC colonies

SummaryEmbryo studies have established that the patterning of the mouse gastrula depends on a regulatory network in which the WNT, BMP, and NODAL signaling pathways cooperate, but aspects of their respective contributions remain unclear. Studying their impact on the spatial organization and developmental trajectories of micropatterned epiblast-like cell (EpiLC) colonies, we show that NODAL is required prior to BMP action to establish the mesoderm and endoderm lineages. The presence of BMP then forces NODAL and WNT to support the formation of posterior primitive streak (PS) derivatives, while its absence allows them to promote that of anterior PS derivatives. Also, a Nodal mutation elicits more severe patterning defects in vitro than in the embryo, suggesting that ligands of extra-embryonic origin can rescue them. These results support the implication of a combinatorial process in PS patterning and illustrate how the study of micropatterned EpiLC colonies can complement that of embryos.

Residual pluripotency is required for inductive germ cell segregation

Fine-tuned dissolution of pluripotency is critical for proper cell differentiation. Here we show that the mesodermal transcription factor, T, globally affects the properties of pluripotency through binding to Oct4 and to the loci of other pluripotency regulators. Strikingly, lower T levels coordinately affect naïve pluripotency, thereby directly activating the germ cell differentiation program, in contrast to the induction of germ cell fate of primed models. Contrary to the effect of lower T levels, higher T levels more severely affect the pluripotency state, concomitantly enhancing the somatic differentiation program and repressing the germ cell differentiation program. Consistent with such in vitro findings, nascent germ cells in vivo are detected in the region of lower T levels at the posterior primitive streak. Furthermore, T and core pluripotency regulators co-localize at the loci of multiple germ cell determinants responsible for germ cell development. In conclusion, our findings indicate that residual pluripotency establishes the earliest and fundamental regulatory mechanism for inductive germline segregation from somatic lineages.

Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids.

Our understanding in the inherent properties of human pluripotent stem cells (hPSCs)have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids.Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensionsto generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We alsoprovidea concise description on howto assess renal commitmentduring the time course of kidney organoid generation. This includes the use offlow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.

Biophysical phenotypes and determinants of anterior vs. posterior primitive streak cells derived from human pluripotent stem cells

Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, our understanding of cellular mechanism(s) underlying cell fate diversification along the anterior-posterior axis of the PS remains incomplete. Furthermore, differences in biophysical phenotypes between anterior and posterior PS cells, which could affect their functions and regulate their fate decisions, remain uncharacterized. Herein, anterior and posterior PS cells were derived using human pluripotent stem cell (hPSC)-based in vitro culture systems. We observed that anterior and posterior PS cells displayed significantly different biophysical phenotypes, including cell morphology, migration, and traction force generation, which was further regulated by different levels of Activin A- and BMP4-mediated developmental signaling. Our data further suggested that intracellular cytoskeletal contraction could mediate anterior and posterior PS differentiation and phenotypic bifurcation through its effect on Activin A- and BMP4-mediated intracellular signaling events. Together, our data provide new information about biophysical phenotypes of anterior and posterior PS cells and reveal an important role of intracellular cytoskeletal contractility in regulating anterior and posterior PS differentiation of hPSCs. Statement of SignificanceFormation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, molecular and cellular mechanism(s) underlying functional diversification of embryonic cells along the anterior-posterior axis of the PS remains incompletely understood. This work describes the first study to characterize the biophysical properties of anterior and posterior PS cells derived from human pluripotent stem cells (hPSCs). Importantly, our data showing the important role of cytoskeleton contraction in controlling anterior vs. posterior PS cell phenotypic switch (through its effect on intracellular Smad signaling activities downstream of Activin A and BMP4) should shed new light on biomechanical regulations of the development and anterior-posterior patterning of the PS. Our work will contribute significantly to uncovering new biophysical principles and cellular mechanisms driving cell lineage diversification and patterning during the PS formation.

Specifying the Anterior Primitive Streak by Modulating YAP1 Levels in Human Pluripotent Stem Cells

SummarySpecifying the primitive streak (PS) guides stem cell differentiation in vitro; however, much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1−/− human embryonic stem cells (hESCs) in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1−/− hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and induced pluripotent stem cell differentiation into distinct mesendodermal lineages.

Identification of Novel Hemangioblast Genes in the Early Chick Embryo.

During early vertebrate embryogenesis, both hematopoietic and endothelial lineages derive from a common progenitor known as the hemangioblast. Hemangioblasts derive from mesodermal cells that migrate from the posterior primitive streak into the extraembryonic yolk sac. In addition to primitive hematopoietic cells, recent evidence revealed that yolk sac hemangioblasts also give rise to tissue-resident macrophages and to definitive hematopoietic stem/progenitor cells. In our previous work, we used a novel hemangioblast-specific reporter to isolate the population of chick yolk sac hemangioblasts and characterize its gene expression profile using microarrays. Here we report the microarray profile analysis and the identification of upregulated genes not yet described in hemangioblasts. These include the solute carrier transporters SLC15A1 and SCL32A1, the cytoskeletal protein RhoGap6, the serine protease CTSG, the transmembrane receptor MRC1, the transcription factors LHX8, CITED4 and PITX1, and the previously uncharacterized gene DIA1R. Expression analysis by in situ hybridization showed that chick DIA1R is expressed not only in yolk sac hemangioblasts but also in particular intraembryonic populations of hemogenic endothelial cells, suggesting a potential role in the hemangioblast-derived hemogenic lineage. Future research into the function of these newly identified genes may reveal novel important regulators of hemangioblast development.

A pluripotent stem cell-based model for post-implantation human amniotic sac development

Development of the asymmetric amniotic sac—with the embryonic disc and amniotic ectoderm occupying opposite poles—is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.

Making a Kidney Organoid Using the Directed Differentiation of Human Pluripotent Stem Cells.

An organoid can be defined as a three-dimensional organ-like structure formed from organ-specific progenitor cells. Organ progenitor cells were empirically found to self-organize three-dimensional tissues when they were aggregated and cultivated in vitro. While this nature power of progenitor cells has an amazing potential to recreate artificial organs in vitro, there had been difficulty to apply this technology to human organs due to the inaccessibility to human progenitor cells until human-induced pluripotent stem cell (hiPSC) was invented by Takahashi and Yamanaka in 2007. As embryonic stem cells do, hiPSCs also have pluripotency to give rise to any organs/tissues cell types, including the kidney, via directed differentiation. Here, we provide a detailed protocol for generating kidney organoids using human pluripotent stem cells. The protocol differentiates human pluripotent stem cells into the posterior primitive streak. This is followed by the simultaneous induction of posterior and anterior intermediate mesoderm that are subsequently aggregated and undergo self-organization into the kidney organoid. Such kidney organoids are comprised of all anticipated kidney cell types including nephrons segmented into the glomerulus, proximal tubule, loop of Henle, and distal tubule as well as the collecting duct, endothelial network, and renal interstitium.

Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis.

The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however,can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived invitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from hemogenic endothelium? Is the hemangioblast an invitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.

Generating a self-organizing kidney from pluripotent cells.

Recent studies on the directed differentiation of human pluripotent stem cells report tissue self-organization in vitro such that multiple component cell types arise in concert and arrange with respect to each, thereby recapitulating the morphogenetic events typical for that organ. Such self-organization has generated pituitary, optic cup, liver, brain, intestine, stomach and now kidney. Here, we will describe the cell types present within the self-organizing kidney, how these signal to each other to form a kidney organoid and the potential applications of kidney organoids. Protocols for the directed differentiation of human pluripotent cells focus on recapitulating the developmental steps required during embryogenesis. In the case of the kidney, this has involved mesodermal differentiation through posterior primitive streak and intermediate mesoderm. Recent studies have observed the simultaneous formation of both ureteric epithelium and nephron progenitors in vitro. These component cell types signal to each other to initiate nephron formation as would occur during development. The generation of kidney organoids is a major advance in nephrology. Such organoids may be useful for disease modelling and drug screening. Ultimately, our capacity to generate organoids may extend to the development of tissues for transplantation.

FOXF1 inhibits hematopoietic lineage commitment during early mesoderm specification.

The molecular mechanisms orchestrating early mesoderm specification are still poorly understood. In particular, how alternate cell fate decisions are regulated in nascent mesoderm remains mostly unknown. In the present study, we investigated both in vitro in differentiating embryonic stem cells, and in vivo in gastrulating embryos, the lineage specification of early mesodermal precursors expressing or not the Forkhead transcription factor FOXF1. Our data revealed that FOXF1-expressing mesoderm is derived from FLK1(+) progenitors and that in vitro this transcription factor is expressed in smooth muscle and transiently in endothelial lineages, but not in hematopoietic cells. In gastrulating embryos, FOXF1 marks most extra-embryonic mesoderm derivatives including the chorion, the allantois, the amnion and a subset of endothelial cells. Similarly to the in vitro situation, FOXF1 expression is excluded from the blood islands and blood cells. Further analysis revealed an inverse correlation between hematopoietic potential and FOXF1 expression in vivo with increased commitment toward primitive erythropoiesis in Foxf1-deficient embryos, whereas FOXF1-enforced expression in vitro was shown to repress hematopoiesis. Altogether, our data establish that during gastrulation, FOXF1 marks all posterior primitive streak extra-embryonic mesoderm derivatives with the remarkable exception of the blood lineage. Our study further suggests that this transcription factor is implicated in actively restraining the specification of mesodermal progenitors to hematopoiesis.

Mixl1 localizes to putative axial stem cell reservoirs and their posterior descendants in the mouse embryo

Mixl1 is thought to play important roles in formation of mesoderm and endoderm. Previously, Mixl1 expression was reported in the posterior primitive streak and allantois, but the precise spatiotemporal whereabouts of Mixl1 protein throughout gastrulation have not been elucidated. To localize Mixl1 protein, immunohistochemistry was carried out at 2–4h intervals on mouse gastrulae between primitive streak and 16-somite pair (s) stages (∼E6.5–9.5). Mixl1 localized to the entire primitive streak early in gastrulation. However, by headfold stages (∼E7.75–8.0), Mixl1 diminished within the mid-streak but remained concentrated at either end of the streak, and localized throughout midline posterior visceral endoderm. At the streak’s anterior end, Mixl1 was confined to the posterior crown cells of Hensen’s node, which contribute to dorsal hindgut endoderm, and the posterior notochord. In the posterior streak, Mixl1 localized to the Allantoic Core Domain (ACD), which is the source of most of the allantois and contributes to the posterior embryonic–extraembryonic interface. In addition, Mix1 co-localized with the early hematopoietic marker, Runx1, in the allantois and visceral yolk sac blood islands. During hindgut invagination (4–16s, ∼E8.5–9.5), Mixl1 localized to the hindgut lip, becoming concentrated within the midline anastomosis of the splanchnopleure, which appears to create the ventral component of the hindgut and omphalomesenteric artery. Surrounding the distal hindgut, Mixl1 identified midline cells within tailbud mesoderm. Mixl1 was also found in the posterior notochord. These findings provide a critical systematic, and tissue-level understanding of embryonic Mixl1 localization, and support its role in regulation of crucial posterior axial mesendodermal stem cell niches during embryogenesis.

Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney

With the prevalence of end-stage renal disease rising 8% per annum globally, there is an urgent need for renal regenerative strategies. The kidney is a mesodermal organ that differentiates from the intermediate mesoderm (IM) through the formation of a ureteric bud (UB) and the interaction between this bud and the adjacent IM-derived metanephric mesenchyme (MM). The nephrons arise from a nephron progenitor population derived from the MM (ref.). The IM itself is derived from the posterior primitive streak. Although the developmental origin of the kidney is well understood, nephron formation in the human kidney is completed before birth. Hence, there is no postnatal stem cell able to replace lost nephrons. In this study, we have successfully directed the differentiation of human embryonic stem cells (hESCs) through posterior primitive streak and IM under fully chemically defined monolayer culture conditions using growth factors used during normal embryogenesis. This differentiation protocol results in the synchronous induction of UB and MM that forms a self-organizing structure, including nephron formation, in vitro. Such hESC-derived components show broad renal potential ex vivo, illustrating the potential for pluripotent-stem-cell-based renal regeneration.

Widespread but tissue-specific patterns of interferon-induced transmembrane protein 3 (IFITM3, FRAGILIS, MIL-1) in the mouse gastrula

Interferon-induced transmembrane protein 3 (IFITM3; FRAGILIS; MIL-1) is part of a larger family of important small interferon-induced transmembrane genes and proteins involved in early development, cell adhesion, and cell proliferation, and which also play a major role in response to bacterial and viral infections and, more recently, in pronounced malignancies. IFITM3, together with tissue-nonspecific alkaline phosphatase (TNAP), PRDM1, and STELLA, has been claimed to be a hallmark of segregated primordial germ cells (PGCs) (Saitou et al., 2002). However, whether IFITM3, like STELLA, is part of a broader stem/progenitor pool that builds the posterior region of the mouse conceptus (Mikedis and Downs, 2012) is obscure. To discover the whereabouts of IFITM3 during mouse gastrulation (∼E6.5-9.0), systematic immunohistochemical analysis was carried out at closely spaced 2-4-h intervals. Results revealed diverse, yet consistent, profiles of IFITM3 localization throughout the gastrula. Within the putative PGC trajectory and surrounding posterior tissues, IFITM3 localized as a large cytoplasmic spot with or without staining in the plasma membrane. IFITM3, like STELLA, was also found in the ventral ectodermal ridge (VER), a posterior progenitor pool that builds the tailbud. The large cytoplasmic spot with plasma membrane staining was exclusive to the posterior region; the visceral yolk sac, non-posterior tissues, and epithelial tissues exhibited spots of IFITM3 without cell surface staining. Colocalization of the intracellular IFITM3 spot with the endoplasmic reticulum, Golgi apparatus, or endolysosomes was not observed. That relatively high levels of IFITM3 were found throughout the posterior primitive streak and its derivatives is consistent with evidence that IFITM3, like STELLA, is part of a larger stem/progenitor cell pool at the posterior end of the primitive streak that forms the base of the allantois and builds the fetal-umbilical connection, thus further obfuscating practical phenotypic distinctions between so-called PGCs and surrounding soma.

Retention of Stem Cell Plasticity in Avian Primitive Streak Cells and the Effects of Local Microenvironment

Primitive streak (PS) is the first structure occurring in embryonic gastrulation, in which the epiblast cells undergo the epithelial-mesenchymal transition to become the loose mesoderm cells subsequently. Because the mesoderm cells departing from different portions of PS are blessed with disparate migration trajectory and differentiation fate, one question is when the cell fate is determinated. To understand whether the cell fate and cell migration pattern will be alternated along with the microenvironment transformation, the traditional transplantation technology was used to replace the anterior PS cells in HH4 host embryo using posterior PS tissue labeled by green fluorescent protein (GFP) in the same stage donor embryo, and then, we tracked the migration trajectory of the GFP-positive cells with fluorescence stereomicroscope after incubation, and eventually verified the cell contribution from the transplants with in situ hybridization and immunocytochemistry. The same experimental strategy applied for posterior PS site replacement in host embryo. We found that the transplanted posterior PS cells to anterior part of streak followed the anterior PS cell migration pattern rather than kept its posterior streak cell migration trajectory, and so did vice versa. In addition, the transplants were involved in the contribution to the subsequent organogenesis as the local PS tissues affirmed by specific expression of myocardial or hematopoietic markers. Therefore, our data strongly suggest that the PS cells still keep stem cell plasticity during gastrulation and the eventual cell fate will depend on the spatial gene expression within local microenvironment along with development.

STELLA-positive subregions of the primitive streak contribute to posterior tissues of the mouse gastrula

The developmental relationship between the posterior embryonic and extraembryonic regions of the mammalian gastrula is poorly understood. Although many different cell types are deployed within this region, only the primordial germ cells (PGCs) have been closely studied. Recent evidence has suggested that the allantois, within which the PGCs temporarily take up residence, contains a pool of cells, called the Allantoic Core Domain (ACD), critical for allantoic elongation to the chorion. Here, we have asked whether the STELLA-positive cells found within this region, thought to be specified PGCs, are actually part of the ACD and to what extent they, and other ACD cells, contribute to the allantois and fetal tissues. To address these hypotheses, STELLA was immunolocalized to the mouse gastrula between Early Streak (ES) and 12-somite pair (-s) stages (~6.75–9.0days post coitum, dpc) in histological sections. STELLA was found in both the nucleus and cytoplasm in a variety of cell types, both within and outside of the putative PGC trajectory. Fate-mapping the headfold-stage (~7.75–8.0dpc) posterior region, by which time PGCs are thought to be segregated into a distinct lineage, revealed that the STELLA-positive proximal ACD and intraembryonic posterior primitive streak (IPS) contributed to a wide range of somatic tissues that encompassed derivatives of the three primary germ layers. This contribution included STELLA-positive cells localizing to tissues both within and outside of the putative PGC trajectory. Thus, while STELLA may identify a subpopulation of cells destined for the PGC lineage, our findings reveal that it may be part of a broader niche that encompasses the ACD and through which the STELLA population may contribute cells to a wide variety of posterior tissues of the mouse gastrula.

Mutated in colorectal cancer (Mcc), a candidate tumor suppressor, is dynamically expressed during mouse embryogenesis

Mutated in Colorectal Cancer (MCC) encodes a multiple PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein implicated, as its name suggests, in the pathogenesis of human colon cancer. To date, however, what role, if any, MCC plays in normal tissue homeostasis and development remains unclear. In an effort to expand our understanding of MCC function and distribution, we examined the expression of the evolutionarily conserved mouse Mcc homolog between embryonic days (E) 6.5 and 12.5 using conventional whole-mount in situ hybridization and two independent Mcc reporter alleles. Mcc is expressed in the posterior primitive streak during gastrulation and in diverse tissues of both mesodermal and endodermal origin. In addition, Mcc transcripts localize to the posterior neural tube and identify discrete neuronal subtypes and ganglia within the developing central nervous system. Genetically, however, Mcc is entirely dispensable, as mice homozygous for the Mcc(Gt(D062B07)) gene trap allele, which generates a loss-of-function mutation, are viable and fertile, with no ostensible phenotype.