Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice.

Wnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5 dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5 dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5 dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages.

Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone.

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.

An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions.

BACKGROUND: Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development. METHODOLOGY/PRINCIPAL FINDINGS: Mice were mutagenized with N-ethyl-N-nitrosourea (ENU) and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila]) and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib. CONCLUSIONS/SIGNIFICANCE: Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.

An ENU-induced point mutation in the mouse Btaf1 gene causes post-gastrulation embryonic lethality and protein instability.

The mouse Btaf1 gene, an ortholog of yeast MOT1, encodes a highly conserved general transcription factor. The function of this SNF2-like ATPase has been studied mainly in yeast and human cells, which has revealed that it binds directly to TBP, forming the B-TFIID complex. This complex binds to core promoters of RNA polymerase II-transcribed genes and, of crucial importance, BTAF1-TBP interactions have been shown to affect the kinetics of TBP-promoter interactions. Here we report the isolation of a mouse line carrying a Btaf1 allele containing an ENU-induced point mutation that causes a substitution mutation in the BTAF1 ATPase domain. Embryos homozygous for this loss-of-function mutation appear to be morphologically normal until early somite stages, but die between embryonic days 9 and 10.5 displaying growth arrest and edema. Analyses in vitro suggest that the altered protein is less stable and, independent from this, functionally impaired in releasing of TBP from chromatin, but not in binding to TBP.

Genetic analysis of Hedgehog signaling in ventral body wall development and the onset of omphalocele formation.

BACKGROUND: An omphalocele is one of the major ventral body wall malformations and is characterized by abnormally herniated viscera from the body trunk. It has been frequently found to be associated with other structural malformations, such as genitourinary malformations and digit abnormalities. In spite of its clinical importance, the etiology of omphalocele formation is still controversial. Hedgehog (Hh) signaling is one of the essential growth factor signaling pathways involved in the formation of the limbs and urogenital system. However, the relationship between Hh signaling and ventral body wall formation remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: To gain insight into the roles of Hh signaling in ventral body wall formation and its malformation, we analyzed phenotypes of mouse mutants of Sonic hedgehog (Shh), GLI-Kruppel family member 3 (Gli3) and Aristaless-like homeobox 4 (Alx4). Introduction of additional Alx4(Lst) mutations into the Gli3(Xt/Xt) background resulted in various degrees of severe omphalocele and pubic diastasis. In addition, loss of a single Shh allele restored the omphalocele and pubic symphysis of Gli3(Xt/+); Alx4(Lst/Lst) embryos. We also observed ectopic Hh activity in the ventral body wall region of Gli3(Xt/Xt) embryos. Moreover, tamoxifen-inducible gain-of-function experiments to induce ectopic Hh signaling revealed Hh signal dose-dependent formation of omphaloceles. CONCLUSIONS/SIGNIFICANCE: We suggest that one of the possible causes of omphalocele and pubic diastasis is ectopically-induced Hh signaling. To our knowledge, this would be the first demonstration of the involvement of Hh signaling in ventral body wall malformation and the genetic rescue of omphalocele phenotypes.

The planar cell polarity pathway in vertebrate development.

Directing the orientation of cells in three dimensions is a fundamental aspect of many of the processes underlying the generation of the appropriate shape and function of tissues and organs during embryonic development. In an epithelium, this requires not only the establishment of apicobasal polarity, but also cell arrangement in a specific direction in the plane of the cell sheet. The molecular pathway central to regulating this planar cell polarity (PCP) was originally discovered in the fruit fly Drosophila melanogaster and has more recently been shown to act in a highly analogous way in vertebrates, involving a strongly overlapping set of genes. Mutant studies and molecular analyses have led to insights into the role of ordered planar cell polarity in the development of a wide variety of organs and tissues. In this review, we give an overview of recent developments in the study of planar polarity signaling in vertebrates.

ß-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis.

Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ß-catenin genes underlies colorectal carcinogenesis. As a result, ß-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ß-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ß-catenin at tyrosine residues, which is thought to increase ß-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ß-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ß-catenin gene was introduced. Results This study provided in vivo evidence that ß-catenin(E654) is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ß-catenin(E654) targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ßcatenin. Surprisingly, the expression of ß-catenin(E654) did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ß-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ß-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.

Planar cell polarity defects and defective Vangl2 trafficking in mutants for the COPII gene Sec24b.

Among the cellular properties that are essential for the organization of tissues during animal development, the importance of cell polarity in the plane of epithelial sheets has become increasingly clear in the past decades. Planar cell polarity (PCP) signaling in vertebrates has indispensable roles in many aspects of their development, in particular, controlling alignment of various types of epithelial cells. Disrupted PCP has been linked to developmental defects in animals and to human pathology. Neural tube closure defects (NTD) and disorganization of the mechanosensory cells of the organ of Corti are commonly known consequences of disturbed PCP signaling in mammals. We report here a typical PCP phenotype in a mouse mutant for the Sec24b gene, including the severe NTD craniorachischisis, abnormal arrangement of outflow tract vessels and disturbed development of the cochlea. In addition, we observed genetic interaction between Sec24b and the known PCP gene, scribble. Sec24b is a component of the COPII coat protein complex that is part of the endoplasmic reticulum (ER)-derived transport vesicles. Sec24 isoforms are thought to be directly involved in cargo selection, and we present evidence that Sec24b deficiency specifically affects transport of the PCP core protein Vangl2, based on experiments in embryos and in cultured primary cells.

A novel mutant allele of Ncx1: a single amino acid substitution leads to cardiac dysfunction.

The biological role and structure-function relationship of the Na(+)Ca(2+) exchanger NCX1 have been the subject of much investigation. Subtle mutagenesis to study the function of a protein seems only feasible in in vitro systems, but genetic forward screens have the potential to provide in vivo models to study single amino acid substitutions. In a genetic screen in mouse, we have isolated a mutant line carrying a novel mutant allele of the mouse Ncx1 gene. In this allele, a point mutation causes the substitution of a highly conserved asparagine residue (N874) with lysine. Accepted models for NCX1 structure propose that the affected amino acid is located in one of the reentrant membrane loops and experiments in vitro have identified N874 as critical for the ion transport function of NCX1. We found severe circulation defects and defective placentation in homozygous Ncx1(N87K4) mutant embryos, making the phenotype essentially indistinguishable from those of previously described null mutants. By ex vivo analysis, we demonstrated intrinsic functional abnormalities of cardiomyocytes. Western blot analysis and immunohistochemistry demonstrated normal levels and subcellular localization of the altered protein, ruling out the possibility that the abnormalities are a mere consequence of a major disturbance of protein structure. This study confirms and extends studies in vitro indicating the significance of amino acid N874 for the function of the NCX1 protein. It provides an in vivo model for this mutation and demonstrates the potential of forward genetic screens in a mammalian system.

Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2 in sinoatrial and pacemaking development.

BACKGROUND: Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves. METHODS AND RESULTS: To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2-/- embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos. CONCLUSIONS: From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh.

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.

Genetics of shoulder girdle formation: roles of Tbx15 and aristaless-like genes.

The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. We have studied the impact of Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the mouse shoulder and pelvic girdles. We observed severe reduction of the scapula in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle.

Expression of retinaldehyde dehydrogenase II and sequential activation of 5' Hoxb genes in the mouse caudal hindbrain.

The precise anterior boundaries of Hox expression domains are critical for correct antero-posterior (A-P) patterning of the vertebrate longitudinal axis. Retinoic acid (RA) signalling has been shown to play an important role in the specification of pre-otic rhombomere boundaries, and in the regulation of 3' Hox expression within this territory. In addition, we recently showed that RA signalling controls 5'Hoxb gene expression in the caudal hindbrain, which had not been discovered before. We show here that the expression domain of these 5'Hoxb genes undergoes a sequential, colinear rostral expansion between E9.5 and E11.5 in the caudal hindbrain, and that this differential expansion occurs just rostrally to the localisation of the transcripts for the RA biosynthetic enzyme Raldh2 in the cervical mesenchyme.

Dorsoventral patterning of the mouse coat by Tbx15.

Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (de(H)), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (a(t)) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the de(H) mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in de(H) that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in de(H)/de(H) animals. Construction of a targeted allele of Tbx15 confirmed that the de(H) phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.

The direct context of a hox retinoic acid response element is crucial for its activity.

During embryogenesis, target genes of retinoid signaling are able to respond differently to identical concentrations of retinoids. Small differences in the retinoic acid response elements (RARE) may be essential for these distinct responses. Recently, we identified a RARE in a Hox enhancer (dubbed distal element) that is active relatively late during mouse development. We now show that the RARE motif in the distal element is necessary and sufficient for the induction of gene expression by retinoic acid (RA) in P19 embryonic carcinoma cells. Furthermore, the significance of these results was established by RA treatment of transgenic mouse lines carrying the distal element containing the wild-type or a mutated RARE. We compared the in vitro activity of the distal element-RARE with that of the direct repeat with 5-bp spacer RARE of the RARbeta2 gene, which is active during early during mouse development. We found that these RAREs, despite their similarity, responded differently to RA. By making single point mutations we show that the specificity resides in their retinoid X receptor-binding sites and is determined by base pairs located just outside the RARE consensus sequence. We suggest that the context of RARE motifs is important for the distinct transcriptional activities of genes under control of retinoid signaling.

The OAR/aristaless domain of the homeodomain protein Cart1 has an attenuating role in vivo.

Aristaless-related genes encode a structurally defined group of homeoproteins that share a C-terminal stretch of amino acids known as the OAR- or aristaless domain. Many aristaless-related genes have been linked to major developmental functions, but the function of the aristaless domain itself is poorly understood. Expression and functional studies have shown that a subgroup of these genes, including Prx1, Prx2, Alx3, Alx4 and Cart1, is essential for correct morphogenesis of the limbs and cranium. We now demonstrate the function of the aristaless domain in vivo by ectopically expressing normal and mutated forms of Cart1 and Alx3. Ectopic expression of Cart1 in transgenic mice does not disturb development, whereas expression of a Cart1 form from which the aristaless domain has been deleted results in severe cranial and vertebral malformations. The Alx3 protein contains a divergent aristaless domain that appears not to be functional, as ectopic expression of Alx3 results in an altered phenotype irrespective of the presence of this aristaless domain. Linking the Cart1 aristaless domain to Alx3 extinguishes teratogenicity. We show that, at the molecular level, the most important consequence of deleting the aristaless domain is increased DNA binding to its palindromic target sequence. This demonstrates that the aristaless domain functions as an intra-molecular switch to contain the activity of the transcription factor that it is part of.

Retinoids regulate the anterior expression boundaries of 5' Hoxb genes in posterior hindbrain.

We describe the regulatory interactions that cause anterior extension of the mouse 5' Hoxb expression domains from spinal cord levels to their definitive boundaries in the posterior hindbrain between embryonic day E10 and E11.5. This anterior expansion is retinoid dependent since it does not occur in mouse embryos deficient for the retinoic acid-synthesizing enzyme retinaldehyde dehydrogenase 2. A retinoic acid response element (RARE) was identified downstream of Hoxb5 and shown to be essential for expression of Hoxb5 and Hoxb8 reporter transgenes in the anterior neural tube. The spatio-temporal activity of this element overlaps with rostral extension of the expression domain of endogenous Hoxb5, Hoxb6 and Hoxb8 into the posterior hindbrain. The RARE and surrounding sequences are found at homologous positions in the human, mouse and zebrafish genome, which supports an evolutionarily conserved regulatory function.

Progression of vertebrate limb development through SHH-mediated counteraction of GLI3.

Distal limb development and specification of digit identities in tetrapods are under the control of a mesenchymal organizer called the polarizing region. Sonic Hedgehog (SHH) is the morphogenetic signal produced by the polarizing region in the posterior limb bud. Ectopic anterior SHH signaling induces digit duplications and has been suspected as a major cause underlying congenital malformations that result in digit polydactyly. Here, we report that the polydactyly of Gli3-deficient mice arises independently of SHH signaling. Disruption of one or both Gli3 alleles in mouse embryos lacking Shh progressively restores limb distal development and digit formation. Our genetic analysis indicates that SHH signaling counteracts GLI3-mediated repression of key regulator genes, cell survival, and distal progression of limb bud development.