Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of functional Arp2/3 complex expression as well as abolished lamellipodia formation and membrane ruffling, as expected. Interestingly, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in the rates of random cell migration. However, both exploration of new space by individual cells and collective migration were clearly compromised by the incapability to efficiently maintain directionality of migration, while the principal ability to chemotax was only moderately affected. Examination of actin remodeling at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation.

The Arp2/3 complex is crucial for colonisation of the mouse skin by melanoblasts.

The Arp2/3 complex is essential for the assembly of branched filamentous actin, but its role in physiology and development is surprisingly little understood. Melanoblasts deriving from the neural crest migrate along the developing embryo and traverse the dermis to reach the epidermis, colonising the skin and eventually homing within the hair follicles. We have previously established that Rac1 and Cdc42 direct melanoblast migration in vivo We hypothesised that the Arp2/3 complex might be the main downstream effector of these small GTPases. Arp3 depletion in the melanocyte lineage results in severe pigmentation defects in dorsal and ventral regions of the mouse skin. Arp3 null melanoblasts demonstrate proliferation and migration defects and fail to elongate as their wild-type counterparts. Conditional deletion of Arp3 in primary melanocytes causes improper proliferation, spreading, migration and adhesion to extracellular matrix. Collectively, our results suggest that the Arp2/3 complex is absolutely indispensable in the melanocyte lineage in mouse development, and indicate a significant role in developmental processes that require tight regulation of actin-mediated motility.

IL-22-Independent Protection from Colitis in the Absence of Nkx2.3 Transcription Factor in Mice.

The transcription factor Nkx2.3 regulates the vascular specification of Peyer patches in mice through determining endothelial addressin preference and may function as a susceptibility factor in inflammatory bowel diseases in humans. We wished to analyze the role of Nkx2.3 in colonic solitary intestinal lymphoid tissue composition and in colitis pathogenesis. We studied the colonic solitary intestinal lymphoid tissue of Nkx2.3-deficient mice with immunofluorescence and flow cytometry. Colitis was induced in mice using 2.5% dextran sodium sulfate, and severity was assessed with histology, flow cytometry, and quantitative PCR. We found that the lack of Nkx2.3 impairs maturation of isolated lymphoid follicles and attenuates dextran sodium sulfate-induced colitis independent of endothelial absence of mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which was also coupled with enhanced colonic epithelial regeneration. Although we observed increased numbers of group 3 innate lymphoid cells and Th17 cells and enhanced transcription of IL-22, Ab-mediated neutralization of IL-22 did not abolish the protection from colitis in Nkx2.3-deficient mice. Nkx2.3-/- hematopoietic cells could not rescue wild-type mice from colitis. Using LacZ-Nkx2.3 reporter mice, we found that Nkx2.3 expression was restricted to VAP-1+ myofibroblast-like pericryptal cells. These results hint at a previously unknown stromal role of Nkx2.3 as driver of colitis and indicate that Nkx2.3+ stromal cells play a role in epithelial cell homeostasis.

ARP3 Controls the Podocyte Architecture at the Kidney Filtration Barrier.

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion.

Niche Cadherins Control the Quiescence-to-Activation Transition in Muscle Stem Cells.

Many adult stem cells display prolonged quiescence, promoted by cues from their niche. Upon tissue damage, a coordinated transition to the activated state is required because non-physiological breaks in quiescence often lead to stem cell depletion and impaired regeneration. Here, we identify cadherin-mediated adhesion and signaling between muscle stem cells (satellite cells [SCs]) and their myofiber niche as a mechanism that orchestrates the quiescence-to-activation transition. Conditional removal of N-cadherin and M-cadherin in mice leads to a break in SC quiescence, with long-term expansion of a regeneration-proficient SC pool. These SCs have an incomplete disruption of the myofiber-SC adhesive junction and maintain niche residence and cell polarity, yet show properties of SCs in a state of transition from quiescence toward full activation. Among these is nuclear localization of ß-catenin, which is necessary for this phenotype. Injury-induced perturbation of niche adhesive junctions is therefore a likely first step in the quiescence-to-activation transition.

Single-Stranded DNA-Binding Transcriptional Regulator FUBP1 Is Essential for Fetal and Adult Hematopoietic Stem Cell Self-Renewal.

The ability of hematopoietic stem cells (HSCs) to self-renew is a prerequisite for the establishment of definitive hematopoiesis and life-long blood regeneration. Here, we report the single-stranded DNA-binding transcriptional regulator far upstream element (FUSE)-binding protein 1 (FUBP1) as an essential factor of HSC self-renewal. Functional inactivation of FUBP1 in two different mouse models resulted in embryonic lethal anemia at around E15.5 caused by severely diminished HSCs. Fetal and adult HSCs lacking FUBP1 revealed an HSC-intrinsic defect in their maintenance, expansion, and long-term blood reconstitution, but could differentiate into all hematopoietic lineages. FUBP1-deficient adult HSCs exhibit significant transcriptional changes, including upregulation of the cell-cycle inhibitor p21 and the pro-apoptotic Noxa molecule. These changes caused an increase in generation time and death of HSCs as determined by video-microscopy-based tracking. Our data establish FUBP1 and its recognition of single-stranded genomic DNA as an important element in the transcriptional regulation of HSC self-renewal.

Nkx2.2 and Nkx2.9 are the key regulators to determine cell fate of branchial and visceral motor neurons in caudal hindbrain.

Cranial motor nerves in vertebrates are comprised of the three principal subtypes of branchial, visceral, and somatic motor neurons, which develop in typical patterns along the anteroposterior and dorsoventral axes of hindbrain. Here we demonstrate that the formation of branchial and visceral motor neurons critically depends on the transcription factors Nkx2.2 and Nkx2.9, which together determine the cell fate of neuronal progenitor cells. Disruption of both genes in mouse embryos results in complete loss of the vagal and spinal accessory motor nerves, and partial loss of the facial and glossopharyngeal motor nerves, while the purely somatic hypoglossal and abducens motor nerves are not diminished. Cell lineage analysis in a genetically marked mouse line reveals that alterations of cranial nerves in Nkx2.2; Nkx2.9 double-deficient mouse embryos result from changes of cell fate in neuronal progenitor cells. As a consequence progenitors of branchiovisceral motor neurons in the ventral p3 domain of hindbrain are transformed to somatic motor neurons, which use ventral exit points to send axon trajectories to their targets. Cell fate transformation is limited to the caudal hindbrain, as the trigeminal nerve is not affected in double-mutant embryos suggesting that Nkx2.2 and Nkx2.9 proteins play no role in the development of branchiovisceral motor neurons in hindbrain rostral to rhombomere 4.

Generation of a Nkx2.2(Cre) knock-in mouse line: Analysis of cell lineages in the central nervous system.

A Nkx2.2(cre) knock-in mutant mouse line was generated that on the appropriate reporter strain enables cell fate analysis of the Nkx2.2 cell lineage in the central nervous system and elsewhere. We here demonstrate that Nkx2.2 lineage-marked cells reside in the ventral p3 region along the entire length of the CNS and also in pancreas of mouse embryos. Nkx2.2(+) progenitor cells develop into V3 interneurons in spinal cord and generate the branchio-visceral motor nuclei of cranial nerves in hindbrain. Nkx2.2(+) cells in hindbrain also form serotonergic neurons and oligodendrocytes during later developmental stages. In mouse mutants lacking Nkx2.2 protein the neuronal progenitor cells in spinal cord are transformed to the distinct fate of somatic motor neurons including their axonal projections that exit the CNS ventrally and no longer cross the midline at the commissure. These data identify Nkx2.2 as key regulator to determine neuronal subtypes in the p3 domain of the central nervous system.

Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development.

BACKGROUND: SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517-595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. RESULTS: By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516-584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1-639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. CONCLUSIONS: Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination.

Absence of Nkx2-3 homeodomain transcription factor reprograms the endothelial addressin preference for lymphocyte homing in Peyer's patches.

Although the homing of lymphocytes to GALT has been extensively studied, little is known about how high endothelial venules (HEVs) within Peyer's patches (PPs) are patterned to display dominantly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In this study, we report that Nkx2-3-deficient mice show gradual loss of MAdCAM-1 in PPs postnatally and increased levels of mRNA for peripheral lymph node addressin (PNAd) backbone proteins as well as enhanced expression of MECA79 sulfated glycoepitope at the luminal aspect of HEVs, thus replacing MAdCAM-1 with PNAd. Induction of PNAd in mutant PPs requires lymphotoxin ß receptor activity, and its upregulation needs the presence of mature T and B cells. Furthermore, treatment with MECA-79 anti-PNAd mAb in vivo effectively blocks lymphocyte homing to mutant PPs. Despite the replacement of MAdCAM-1 by PNAd in HEV endothelia, lymphocytes could efficiently home to PPs in mutant mice. We conclude that although Nkx2-3 activity controls the addressin balance of HEVs in GALT, the general HEV functionality is preserved independently from Nkx2-3, indicating a substantial plasticity in the specification of GALT HEV endothelium.

Generation and characterization of a tamoxifen-inducible Eomes(CreER) mouse line.

Transgenic mouse lines expressing inducible forms of Cre-recombinase in a tissue-specific manner are powerful genetic tools for studying aspects of development and various processes in the adult. The T-box transcription factor eomesodermin (Eomes) plays critical roles for maintenance and differentiation of different pools of stem and progenitor cells from early embryonic stages to adulthood. These include trophoblast stem cells, epiblast cells during the generation of the primary germ layers, neurogenic intermediate progenitor cells in embryonic and adult cortical neurogenesis, and maturing natural killer and T cells. Here, we report on the generation and analysis of an Eomes(CreER) -targeted allele by placing the tamoxifen-activatable Cre-recombinase (CreER) under the control of the Eomes genomic locus. We demonstrate that CreER expression recapitulates endogenous Eomes transcription within different progenitor cell populations. Tamoxifen administration specifically labels Eomes-expressing cells and their progeny as demonstrated by crossing Eomes(CreER) animals to different Cre-inducible reporter strains. In summary, this novel Eomes(CreER) allele can be used as elegant genetic tool that allows to follow the fate of Eomes-positive cells and to genetically manipulate them in a temporal specific manner.

Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice.

Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.

Absence of Nkx2-3 homeodomain transcription factor induces the formation of LYVE-1-positive endothelial cysts without lymphatic commitment in the spleen.

In contrast to peripheral lymph nodes possessing lymphatic and blood vasculature, the spleen in both humans and rodents is largely devoid of functioning lymphatic capillaries. Here it is reported that in mice lacking homeodomain transcription factor Nkx2-3, the spleen contains an extensive network of lymphocyte-filled sacs lined by cells expressing LYVE-1 antigen, a marker associated with lymphatic endothelium cells (LECs). Real-time quantitative PCR analyses of Nkx2-3 mutant spleen revealed a substantial increase of LYVE-1 and podoplanin mRNA levels, without the parallel increase of mRNA for VEGFR-3 (vascular endothelial growth factor receptor Type 3) and Prox1 (Prospero homeobobox protein 1), two markers specific for LECs. Although these structures express VEGFR-2/flk-1, they lack Prox1 protein, indicating their non-LEC endothelial origin. The LYVE-1(+) structures are bordered with ER-TR7(+) fibroblastic reticular cells with small clusters of macrophages expressing MARCO and sialoadhesin. Short-term cell-tracing studies using labeled lymphocytes indicate that these LYVE-1(+) cysts are largely excluded from the systemic circulation. Cells expressing LYVE-1 glycoprotein as putative precursors for such structures are detectable in the spleen of late-stage embryos, and the formation of LYVE-1(+) structures is independent from the activity of lymphotoxin ß-receptor. Thus the splenic vascular defects in Nkx2-3 deficiency include the generation of LYVE-1(+) cysts, comprised of endothelial cells without being committed along the LEC lineage.

N-terminal ataxin-3 causes neurological symptoms with inclusions, endoplasmic reticulum stress and ribosomal dislocation.

Mutant ataxin-3 is aberrantly folded and proteolytically cleaved in spinocerebellar ataxia type 3. The C-terminal region of the protein includes a polyglutamine stretch that is expanded in spinocerebellar ataxia type 3. Here, we report on the analysis of an ataxin-3 mutant mouse that has been obtained by gene trap integration. The ataxin-3 fusion protein encompasses 259 N-terminal amino acids including the Josephin domain and an ubiquitin-interacting motif but lacks the C-terminus with the polyglutamine stretch, the valosin-containing protein binding region and part of the ubiquitin-interacting motif 2. Homozygous ataxin-3 mutant mice were viable and showed no apparent anatomical defects at birth. However, at the age of 9 months, homozygous and heterozygous mutant mice revealed significantly altered behaviour and progressing deficits of motor coordination followed by premature death at ∼12 months. At this time, prominent extranuclear protein aggregates and neuronal cell death was found in mutant mice. This was associated with disturbances of the endoplasmic reticulum-mediated unfolded protein response, consistent with the normal role of ataxin-3 in endoplasmic reticulum homeostasis. Thus, the ataxin-3 gene trap model provides evidence for a contribution of the non-polyglutamine containing ataxin-3 N-terminus, which mimics a calpain fragment that has been observed in spinocerebellar ataxia type 3. Consistent with the disease in humans, gene trap mice develop cytoplasmic inclusion bodies and implicate impaired unfolded protein response in the pathogenesis of spinocerebellar ataxia type 3.

Transcription factor Nkx2-3 controls the vascular identity and lymphocyte homing in the spleen.

The vasculature in the spleen and peripheral lymph nodes (pLNs) is considerably different, which affects both homing of lymphocytes and antigenic access to these peripheral lymphoid organs. In this paper, we demonstrate that in mice lacking the homeodomain transcription factor Nkx2-3, the spleen develops a pLN-like mRNA expression signature, coupled with the appearance of high endothelial venules (HEVs) that mediate L-selectin-dependent homing of lymphocytes into the mutant spleen. These ectopic HEV-like vessels undergo postnatal maturation and progressively replace MAdCAM-1 by pLN addressin together with the display of CCL21 arrest chemokine in a process that is reminiscent of HEV formation in pLNs. Similarly to pLNs, development of HEV-like vessels in the Nkx2-3-deficient spleen depends on lymphotoxin-ß receptor-mediated signaling. The replacement of splenic vessels with a pLN-patterned vasculature impairs the recirculation of adoptively transferred lymphocytes and reduces the uptake of blood-borne pathogens. The Nkx2-3 mutation in BALB/c background causes a particularly disturbed splenic architecture, characterized by the near complete lack of the red pulp, without affecting lymph nodes. Thus, our observations reveal that the organ-specific patterning of splenic vasculature is critically regulated by Nkx2-3, thereby profoundly affecting the lymphocyte homing mechanism and blood filtering capacity of the spleen in a tissue-specific manner.

The transcription factors Nkx2.2 and Nkx2.9 play a novel role in floor plate development and commissural axon guidance.

The transcription factors Nkx2.2 and Nkx2.9 have been proposed to execute partially overlapping functions in neuronal patterning of the ventral spinal cord in response to graded sonic hedgehog signaling. The present report shows that in mice lacking both Nkx2 proteins, the presumptive progenitor cells in the p3 domain of the neural tube convert to motor neurons (MN) and never acquire the fate of V3 interneurons. This result supports the concept that Nkx2 transcription factors are required to establish V3 progenitor cells by repressing the early MN lineage-specific program, including genes like Olig2. Nkx2.2 and Nkx2.9 proteins also perform an additional, hitherto unknown, function in the development of non-neuronal floor plate cells. Here, we demonstrate that loss of both Nkx2 genes results in an anatomically smaller and functionally impaired floor plate causing severe defects in axonal pathfinding of commissural neurons. Defective floor plates were also seen in Nkx2.2(+/-);Nkx2.9(-/-) compound mutants and even in single Nkx2.9(-/-) mutants, suggesting that floor plate development is sensitive to dose and/or timing of Nkx2 expression. Interestingly, adult Nkx2.2(+/-);Nkx2.9(-/-) compound-mutant mice exhibit abnormal locomotion, including a permanent or intermittent hopping gait. Drug-induced locomotor-like activity in spinal cords of mutant neonates is also affected, demonstrating increased variability of left-right and flexor-extensor coordination. Our data argue that the Nkx2.2 and Nkx2.9 transcription factors contribute crucially to the formation of neuronal networks that function as central pattern generators for locomotor activity in the spinal cord. As both factors affect floor plate development, control of commissural axon trajectories might be the underlying mechanism.

A homeodomain feedback circuit underlies step-function interpretation of a Shh morphogen gradient during ventral neural patterning.

The deployment of morphogen gradients is a core strategy to establish cell diversity in developing tissues, but little is known about how small differences in the concentration of extracellular signals are translated into robust patterning output in responding cells. We have examined the activity of homeodomain proteins, which are presumed to operate downstream of graded Shh signaling in neural patterning, and describe a feedback circuit between the Shh pathway and homeodomain transcription factors that establishes non-graded regulation of Shh signaling activity. Nkx2 proteins intrinsically strengthen Shh responses in a feed-forward amplification and are required for ventral floor plate and p3 progenitor fates. Conversely, Pax6 has an opposing function to antagonize Shh signaling, which provides intrinsic resistance to Shh responses and is important to constrain the inductive capacity of the Shh gradient over time. Our data further suggest that patterning of floor plate cells and p3 progenitors is gated by a temporal switch in neuronal potential, rather than by different Shh concentrations. These data establish that dynamic, non-graded changes in responding cells are essential for Shh morphogen interpretation, and provide a rationale to explain mechanistically the phenomenon of cellular memory of morphogen exposure.

Transcriptional regulator BPTF/FAC1 is essential for trophoblast differentiation during early mouse development.

The putative transcriptional regulator BPTF/FAC1 is expressed in embryonic and extraembryonic tissues of the early mouse conceptus. The extraembryonic trophoblast lineage in mammals is essential to form the fetal part of the placenta and hence for the growth and viability of the embryo in utero. Here, we describe a loss-of-function allele of the BPTF/FAC1 gene that causes embryonic lethality in the mouse. BPTF/FAC1-deficient embryos form apparently normal blastocysts that implant and develop epiblast, visceral endoderm, and extraembryonic ectoderm including trophoblast stem cells. Subsequent development of mutants, however, is arrested at the early gastrula stage (embryonic day 6.5), and virtually all null embryos die before midgestation. Most notably, the ectoplacental cone is drastically reduced or absent in mutants, which may cause the embryonic lethality. Development of the mutant epiblast is also affected, as the anterior visceral endoderm and the primitive streak do not form correctly, while brachyury-expressing mesodermal cells arise but are delayed. The mutant phenotype suggests that gastrulation is initiated, but no complete anteroposterior axis of the epiblast appears. We conclude that BPTF/FAC1 is essential in the extraembryonic lineage for correct development of the ectoplacental cone and fetomaternal interactions. In addition, BPTF/FAC1 may also play a role either directly or indirectly in anterior-posterior patterning of the epiblast.

The hnRNP and cytoskeletal protein raver1 contributes to synaptic plasticity.

Raver1 is an hnRNP protein that interacts with the ubiquitous splicing regulator PTB and binds to cytoskeletal components like alpha-actinin and vinculin/metavinculin. Cell culture experiments suggested that raver1 functions as corepressor in PTB-regulated splicing reactions and may thereby increase proteome complexity. To determine the role of raver1 in vivo, we inactivated the gene by targeted disruption in the mouse. Here we report that raver1-deficient mice develop regularly to adulthood and show no obvious anatomical or behavioral defects. In keeping with this notion, cells from raver1-null mice were indistinguishable from wild type cells and displayed normal growth, motility, and cytoskeletal architecture in culture. Moreover, alternative splicing of exons, including the model exon 3 of alpha-tropomyosin, was not markedly changed in mutant mice, suggesting that the role of raver1 for PTB-mediated exon repression is not absolutely required to generate splice variants during mouse development. Interestingly however, loss of raver1 caused significantly reduced plasticity of synapses on acute hippocampal slices, as elicited by electrophysiological measurements of markedly lower LTP and LTD in mutant neurons. Our results provide evidence that raver1 may play an important role for the regulation of neuronal synaptic plasticity, possibly by controlling especially the late LTP via posttranscriptional mechanisms.

Arp3 is required during preimplantation development of the mouse embryo.

The role of Arp3 in mouse development was investigated utilizing a gene trap mutation in the Arp3 gene. Heterozygous Arp3(WT/GT) mice are normal, however, homozygous Arp3(GT/GT) embryos die at blastocyst stage. Earlier embryonic stages appear unaffected by the mutation, probably due to maternal Arp3 protein. Mutant blastocysts isolated at E3.5 fail to continue development in vitro, lack outgrowth of trophoblast-like cells in culture and express reduced levels of the trophoblast marker Cdx2, while markers for inner cell mass continue to be present. The recessive embryonic lethal phenotype indicates that Arp3 plays a vital role for early mouse development, possibly when trophoblast cells become critical for implantation.