SEK1 deficiency reveals mitogen-activated protein kinase cascade crossregulation and leads to abnormal hepatogenesis

SEK1 (MKK4/JNKK) is a mitogen-activated protein kinase activator that has been shown to participate in vitro in two stress-activated cascades terminating with the SAPK and p38 kinases. To define the role of SEK1 in vivo, we studied stress-induced signaling in SEK1^−/− embryonic stem and fibroblast cells and evaluated the phenotype of SEK1^−/− mouse embryos during development. Studies of SEK1^−/− embryonic stem cells demonstrated defects in stimulated SAPK phosphorylation but not in the phosphorylation of p38 kinase. In contrast, SEK1^−/− fibroblasts exhibited defects in both SAPK and p38 phosphorylation, demonstrating that crosstalk exists between the stress-activated cascades. Tumor necrosis factor α and interleukin 1 stimulation of both stress-activated cascades are severely affected in the SEK1^−/− fibroblast cells. SEK1 deficiency leads to embryonic lethality after embryonic day 12.5 and is associated with abnormal liver development. This phenotype is similar to c-jun null mouse embryos and suggests that SEK1 is required for phosphorylation and activation of c-jun during the organo-genesis of the liver.     

Endothelial expression of the Notch ligand Jagged1 is required for vascular smooth muscle development

The Notch ligand Jagged1 (Jag1) is essential for vascular remodeling and has been linked to congenital heart disease in humans, but its precise role in various cell types of the cardiovascular system has not been extensively investigated. We show that endothelial-specific deletion of Jag1 results in embryonic lethality and cardiovascular defects, recapitulating the Jag1 null phenotype. These embryos show striking deficits in vascular smooth muscle, whereas endothelial Notch activation and arterial-venous differentiation appear normal. Endothelial Jag1 mutant embryos are phenotypically distinct from embryos in which Notch signaling is inhibited in endothelium. Together, these results imply that the primary role of endothelial Jag1 is to potentiate the development of neighboring vascular smooth muscle.     

A distinct role for secreted fibroblast growth factor-binding proteins in development

FGFs modulate diverse biological processes including embryonic development. Secreted FGF-binding proteins (BPs) can release FGFs from their local extracellular matrix storage, chaperone them to their cognate receptors, and thus modulate FGF signaling. Here we describe 2 chicken BP homologs (chBP) that show distinct expression peaks at embryonic days E7.5 (chBP2) and E11.5 (chBP1), although their tissue distribution is similar (skin = intestine>lung>heart, liver). Embryos were grown ex ovo to monitor the phenotypic impact of a timed in vivo knockdown of expression peaks by microinjection of specific siRNAs targeted to either of the chBPs. Knockdown of peak expression of chBP2 caused embryonic lethality within <5 days. Surviving embryos showed defective ventral wall closure indicative of altered dorsoventral patterning. This defect coincided with reduced expression of HoxB7 but not HoxB8 that are involved in the control of thoracic/abdominal segment morphology. Also, MAPK phosphatase 3, a negative regulator of FGF signaling, and sonic hedgehog that can participate in feedback control of the FGF pathway were reduced, reflecting altered FGF signaling. Knockdown of the chBP1 expression peak caused embryonic lethality within <3 days although no distinct morphologic phenotype or pathways alterations were apparent. We conclude that BPs play a significant role in fine-tuning the complex FGF signaling network during distinct phases of embryonic development.     

GTF2IRD2 is located in the Williams-Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix-loop-helix repeats

Williams-Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix-loop-helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix-loop-helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams-Beuren syndrome critical region.     

Regulatory network analysis of hypertension and hypotension microarray data from mouse model

We aimed to identify the potential genes related to blood pressure regulation and screen target genes for high blood pressure (BPH) and low blood pressure (BPL) treatment. The GSE19817 microarray dataset, which included the aorta, liver, heart, and kidney samples from BPH, BPL, and normotensive mice, was downloaded from the Gene Expression Omnibus. Principal component analysis (PCA) was performed based on the entire expression profile. Differentially expressed genes (DEGs) were screened, followed by pathway enrichment analysis. Finally, gene regulatory networks were constructed based on BPH-related and BPL-related DEGs in the aorta, liver, heart, and kidney samples. As a result, DEGs were screened within their respective tissues due to high heterogeneity of different tissues. Totally, 2,726 BPH-related DEGs and 2,472 BPL-related DEGs were screened, which were mainly enriched in pathways such as immune response. The topology data of gene regulatory networks constructed by DEGs in the heart, kidney, and liver were similar than that in aorta. Finally, among BPH-related DEGs, Sept6 and Pigx were found in the top 10 differentially regulated DEGs by comparing the BPH-related DEGs of the aorta with the DEGs of the other 3 tissues in the regulatory network. Although among the top 10 differentially regulated BPL-related DEGs, no common differentially regulated DEGs were found, Wif1, Urb2, and Gtf2ird1 were found among the top ten DEGs in the three tissues other than the kidney tissue. Sept6 and Pigx might participate in the pathogenesis of BPH, whereas Gtf2ird1, Urb2, and Wif1 might be critical target genes for BPL treatment.     

Spontaneous Irs1 passenger mutation linked to a gene-targeted SerpinB2 allele

In characterizing mice with targeted disruption of the SerpinB2 gene, we observed animals that were small at birth with delayed growth and decreased life expectancy. Although this phenotype cosegregated with homozygosity for the inactive SerpinB2 allele, analysis of homozygous SerpinB2-deficient mice derived from two additional independent embryonic stem (ES) cell clones exhibited no growth abnormalities. Examination of additional progeny from the original SerpinB2-deficient line revealed recombination between the small phenotype (smla) and the SerpinB2 locus. The locus responsible for smla was mapped to a 2.78-Mb interval approximately 30 Mb proximal to SerpinB2, bounded by markers D1Mit382 and D1Mit216. Sequencing of Irs1 identified a nonsense mutation at serine 57 (S57X), resulting in complete loss of IRS1 protein expression. Analysis of ES cell DNA suggests that the S57X Irs1 mutation arose spontaneously in an ES cell subclone during cell culture. Although the smla phenotype is similar to previously reported Irs1 alleles, mice exhibited decreased survival, in contrast to the enhanced longevity reported for IRS1 deficiency generated by gene targeting. This discrepancy could result from differences in strain background, unintended indirect effects of the gene targeting, or the minimal genetic interference of the S57X mutation compared with the conventionally targeted Irs1-KO allele. Spontaneous mutations arising during ES cell culture may be a frequent but underappreciated occurrence. When linked to a targeted allele, such mutations could lead to incorrect assignment of phenotype and may account for a subset of markedly discordant results from experiments independently targeting the same gene.     

Noonan syndrome is associated with enhanced pERK activity,the repression of which can prevent craniofacial malformations

A gain of function mutation in SHP2, a protein phosphatase encoded by PTPN11, causes Noonan syndrome (NS), which is characterized in part by developmental deficits in both the cardiac and skull fields. Previously, we found that expression of the mutated protein SHP2 Q79R in the heart led to a phenotypic presentation that mimicked some aspects of NS and that this was dependent upon activation of the ERK1/2 pathway. To understand the role that ERK1/2 signaling plays in skull development through signaling in the neural crest, we explored the consequences of Q79R expression in neural crest cells, which contribute to a subset of the bony and cartilaginous structures of the skull. Hyperactivation of ERK1/2 led to craniofacial defects that included smaller skull lengths, greater inner canthal distances, and taller frontal bone heights. In proportion to the smaller skull length, mandibular bone length was also reduced. Inhibition of ERK1/2 hyperactivity as a result of Q79R expression was achieved by injection of the MAPK/ERK kinase inhibitor U0126 during pregnancy. The drug effectively decreased the severity of the craniofacial defects and restored normal skull shape and fontanelle closure. X-ray computer-assisted microtomography analysis of the head confirmed that decreasing ERK1/2 activity led to an abrogation of the craniofacial deficits and brain shape changes that presented in the mice. These data show that normal ERK1/2 signaling in the neural crest is imperative for normal craniofacial development and offer insight into how the heart and craniofacial developmental fields might be affected in some congenital syndromic presentations.     

VEGF-B-Neuropilin-1 signaling is spatiotemporally indispensable for vascular and neuronal development in zebrafish

Physiological functions of vascular endothelial growth factor (VEGF)-B remain an enigma, and deletion of the Vegfb gene in mice lacks an overt phenotype. Here we show that knockdown of Vegfba, but not Vegfbb, in zebrafish embryos by specific morpholinos produced a lethal phenotype owing to vascular and neuronal defects in the brain. Vegfba morpholinos also markedly prevented development of hyaloid vasculatures in the retina, but had little effects on peripheral vascular development. Consistent with phenotypic defects, Vegfba, but not Vegfaa, mRNA was primarily expressed in the brain of developing zebrafish embryos. Interestingly, in situ detection of Neuropilin1 (Nrp1) mRNA showed an overlapping expression pattern with Vegfba, and knockdown of Nrp1 produced a nearly identically lethal phenotype as Vegfba knockdown. Furthermore, zebrafish VEGF-Ba protein directly bound to NRP1. Importantly, gain-of-function by exogenous delivery of mRNAs coding for NRP1-binding ligands VEGF-B or VEGF-A to the zebrafish embryos rescued the lethal phenotype by normalizing vascular development. Similarly, exposure of zebrafish embryos to hypoxia also rescued the Vegfba morpholino-induced vascular defects in the brain by increasing VEGF-A expression. Independent evidence of VEGF-A gain-of-function was provided by using a functionally defective Vhl-mutant zebrafish strain, which again rescued the Vegfba morpholino-induced vascular defects. These findings show that VEGF-B is spatiotemporally required for vascular development in zebrafish embryos and that NRP1, but not VEGFR1, mediates the essential signaling.Angiogenesis is essential for embryonic development and contributes to the onset and development of many diseases (1). The angiogenic process is tightly regulated by angiogenic factors and inhibitors and involves cooperative and synchronized interactions between vascular endothelial cells and perivascular cells including pericytes and vascular smooth muscle cells. Among all known angiogenic factors, vascular endothelial growth factor (VEGF; also called VEGFA) is probably the best-characterized proangiogenic factor under physiological and pathological conditions (2, 3). There are five structurally and functionally related members in the VEGF family, which includes VEGF-A, -B, -C, and -D and placental growth factor (PlGF) (4). These factors bind primarily to three membrane tyrosine kinase receptors (TKRs), i.e., VEGFR1, VEGFR2, and VEGFR3, to display their biological functions (4). According to their receptor-binding patterns and biological functions, members of the VEGF family are divided into three subgroups: (i) VEGFA as the VEGFR1- and VEGFR2-binding ligand (5); (ii) VEGF-B and PlGF that exclusively bind to VEGFR1 (4, 6–8); and (iii) VEGF-C and VEGF-D as VEGFR3- and VEGFR2-binding ligands (9). Whereas VEGF-A potently stimulates angiogenesis, vascular permeability, and lymphangiogenesis, VEGF-C and VEGF-D primarily induce lymphangiogenesis although they also induce angiogenesis (9). VEGFR2 has been reported as the key receptor that transduces angiogenic and vascular permeability signals, and VEGFR3 is responsible mainly for lymphangiogenesis (10). In addition to TKRs, various heparin-binding isoforms of each member in the VEGF family have been reported to bind to neuropilins (NRPs), which is also crucial for angiogenesis, lymphangiogenesis, axon guidance, cell survival, migration, and invasion (11–14).VEGF-A is required for embryonic development in mammals, and deletion of only one allele of the Vegfa gene (haploinsufficiency) in mice results in a lethal embryonic phenotype, owing to inappropriate development of the vascular and hematopoietic systems (15, 16). Paradoxically, modest overexpression of VEGF-A in mice also causes embryonic lethality due to cardiovascular deficiency (17). These findings demonstrate that an optimal level of VEGF-A expression is needed for embryonic development. Unlike VEGF-A, deletion of the Vegfb gene in mice does not produce an overt phenotype, except slight cardiovascular impairments (18, 19). Recently, it has been found that VEGF-B–deficient animals exhibit defective lipid uptake in endothelial cells (20, 21). However, these findings could not be reproduced in another study (22). Based on these findings, VEGF-B is probably the least-characterized member in the VEGF-A family, and its physiological functions remain an enigmatic issue in mice (6). The key issue in VEGF-B research is what this factor does under physiological conditions. One of the main differences between developing mouse embryos and zebrafish embryos is the presence of tissue hypoxia during development. In mice and other mammals, embryonic tissues develop under a relatively hypoxic environment, and hypoxia is one of the key mechanisms behind up-regulation of VEGF-A expression (23). The increased VEGFA expression in various tissues would probably compensate the VEGF-B deletion-associated vascular and other defects. However, zebrafish embryos lack this hypoxia-related VEGF-A compensatory mechanism and allow us to study spatiotemporal functions of VEGF-B during embryonic development.To test this hypothesis, in the present study we have investigated the functions of VEGF-B in developing zebrafish embryos. Surprisingly, knockdown of the Vegfba gene in developing zebrafish embryos produced a lethal phenotype owing to vascular defects in the brain. The functional defects of VEGF-B–deficient zebrafish embryos impeccably correlate with the VEGF-B expression pattern in the developing brain in which VEGF-A expression is modestly low. Importantly, exposure of VEGF-B–defective zebrafish embryos to hypoxia rescues the VEGF-B deficiency-induced vascular defects by a VEGF-A–dependent mechanism. Our findings for the first time to our knowledge demonstrate the indispensable function of VEGF-B in vascular development in zebrafish embryos.     

Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts

Calcium entry into myocytes drives contraction of the embryonic heart. To prepare for the next contraction, myocytes must extrude calcium from intracellular space via the Na+/Ca2+ exchanger (NCX1) or sequester it into the sarcoplasmic reticulum, via the sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2). In mammals, defective calcium extrusion correlates with increased intracellular calcium levels and may be relevant to heart failure and sarcoplasmic dysfunction in adults. We report here that mutation of the cardiac-specific NCX1 (NCX1h) gene causes embryonic lethal cardiac arrhythmia in zebrafish tremblor (tre) embryos. The tre ventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias including fibrillation. Calcium extrusion defects in tre mutants correlate with severe disruptions in sarcomere assembly, whereas mutations in the L-type calcium channel that abort calcium entry do not produce this phenotype. Knockdown of SERCA2 activity by morpholino-mediated translational inhibition or pharmacological inhibition causes embryonic lethality due to defects in cardiac contractility and morphology but, in contrast to tre mutation, does not produce arrhythmia. Analysis of intracellular calcium levels indicates that homozygous tre embryos develop calcium overload, which may contribute to the degeneration of cardiac function in this mutant. Thus, the inhibition of NCX1h versus SERCA2 activity differentially affects the pathophysiology of rhythm in the developing heart and suggests that relative levels of NCX1 and SERCA2 function are essential for normal development.     

Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells

Loss-of-function mutations in the human ERG1 potassium channel (hERG1) frequently underlie the long QT2 (LQT2) syndrome. The role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous, loss-of-function LQT2 syndrome mutation. The hERG N629D mutation was introduced into the orthologous mouse gene, mERG, by homologous recombination in mouse embryonic stem cells. Intact homozygous embryos showed abrupt cessation of the heart beat. N629D/N629D embryos die in utero by embryonic day 11.5. Their developmental defects include altered looping architecture, poorly developed bulbus cordis, and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of I(Kr) function, depolarized resting potential, prolonged action potential duration (LQT), failure to repolarize, and propensity to oscillatory arrhythmias. N629D/N629D myocytes manifest calcium oscillations and increased sarcoplasmic reticulum Ca(+2) content. Although the N629D/N629D protein is synthesized, it is mainly located intracellularly, whereas +/+ mERG protein is mainly in plasmalemma. N629D/N629D embryos show robust apoptosis in craniofacial regions, particularly in the first branchial arch and, to a lesser extent, in the cardiac outflow tract. Because deletion of Hand2 produces apoptosis, in similar regions and with a similar final developmental phenotype, Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. In conclusion, loss of I(Kr) function in N629D/N629D cardiovascular system leads to defects in cardiac ontogeny in the first branchial arch, outflow tract, and the right ventricle.     

Essential roles for four cytoplasmic intermediate filament proteins in Caenorhabditis elegans development

The structural proteins of the cytoplasmic intermediate filaments (IFs) arise in the nematode Caenorhabditis elegans from eight reported genes and an additional three genes now identified in the complete genome. With the use of double-stranded RNA interference (RNAi) for all 11 C. elegans genes encoding cytoplasmic IF proteins, we observe phenotypes for the five genes A1, A2, A3, B1, and C2. These range from embryonic lethality (B1) and embryonic/larval lethality (A3) to larval lethality (A1 and A2) and a mild dumpy phenotype of adults (C2). Phenotypes A2 and A3 involve displaced body muscles and paralysis. They probably arise by reduction of hypodermal IFs that participate in the transmission of force from the muscle cells to the cuticle. The B1 phenotype has multiple morphogenetic defects, and the A1 phenotype is arrested at the L1 stage. Thus, at least four IF genes are essential for C. elegans development. Their RNAi phenotypes are lethal defects due to silencing of single IF genes. In contrast to C. elegans, no IF genes have been identified in the complete Drosophila genome, posing the question of how Drosophila can compensate for the lack of these proteins, which are essential in mammals and C. elegans. We speculate that the lack of IF proteins in Drosophila can be viewed as cytoskeletal alteration in which, for instance, stable microtubules, often arranged as bundles, substitute for cytoplasmic IFs.     

K-RasV14I recapitulates Noonan syndrome in mice

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-Ras^V14I, a recurrent KRAS mutation in NS patients. K-Ras^V14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-Ras^V14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome.Noonan syndrome (NS) (1) belongs to a group of clinically related developmental disorders known as “RASopathies” (2, 3). NS presents with an incidence of about 1/1,000–1/2,500 newborns, and patients display a broad spectrum of clinical symptoms including craniofacial dysmorphia, short stature, cardiovascular and skeletal defects, delayed puberty, and learning difficulties (4). About 10% of NS patients also exhibit myeloproliferative disorders (MPD), which usually are transient. Less frequently, these patients develop severe MPD, juvenile myelomonocytic leukemia (JMML), or other forms of leukemia (2, 5). NS is inherited in an autosomal dominant manner and results from germ-line mutations in at least 11 different genes including Protein Tyrosine Phosphatase Non-Receptor type 11 (PTPN11), Son of Sevenless homolog 1 (SOS1), KRAS, NRAS, RAF1, BRAF, MEK1, SHOC2, CBL, RIT1, and RRAS, most of which are involved in mediating RAS signaling (3, 6–8). Among these loci, PTPN11 is the most frequently mutated, in about 50% of NS patients. KRAS mutations have been identified in less than 5% of the patients (2).NS patients with KRAS mutations display more severe clinical and cognitive defects. However, the limited number of these patients makes it difficult to establish a clear genotype–phenotype correlation (3, 4). Thus, far, 18 different germ-line mutations have been reported in the KRAS locus of NS patients (NSEuroNet database: nseuronet.com). These mutations confer milder gain-of-function effects than somatically acquired cancer-associated mutations (9). Replacement of the valine residue located at position 14 by isoleucine is one of the most frequent KRAS mutations (10). Although this mutation is adjacent to amino acid residues typically altered in cancer, KRAS^V14I displays an intermediate intrinsic GTPase activity compared with wild-type and oncogenic isoforms (9). Moreover, the mutant KRAS^V14I protein shows an increase in nucleotide exchange activity that is likely to be responsible for its accumulation in the active guanosine triphosphate (GTP)-bound state (9).Here we describe the generation of a strain of mice carrying an endogenous K-Ras^V14I germ-line mutation. These mice displayed many of the phenotypic abnormalities observed in NS patients, including small size, craniofacial dysmorphism, and cardiac defects. Moreover, they develop fatal MPD, a disease reminiscent of the JMML characteristic of patients with NS. These mice offer a relevant experimental tool for studying the alterations underlying the clinical manifestations of NS and for testing new therapies aimed at preventing or ameliorating these deficits.     

Histone H3 lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodeling

HYPB is a human histone H3 lysine 36 (H3K36)–specific methyltransferase and acts as the ortholog of yeast Set2. This study explored the physiological function of mammalian HYPB using knockout mice. Homozygous disruption of Hypb impaired H3K36 trimethylation but not mono- or dimethylation, and resulted in embryonic lethality at E10.5-E11.5. Severe vascular defects were observed in the Hypb ^−/− embryo, yolk sac, and placenta. The abnormally dilated capillaries in mutant embryos and yolk sacs could not be remodeled into large blood vessels or intricate networks, and the aberrantly rounded mesodermal cells exhibited weakened interaction with endothelial cells. The embryonic vessels failed to invade the labyrinthine layer of placenta, which impaired the embryonic–maternal vascular connection. These defects could not be rescued by wild-type tetraploid blastocysts, excluding the possibility that they were caused by the extraembryonic tissues. Consistent with these phenotypes, gene expression profiling in wild-type and Hypb ^−/− yolk sacs revealed that the Hypb disruption altered the expression of some genes involved in vascular remodeling. At the cellular level, Hypb ^−/− embryonic stem cell–derived embryonic bodies, as well as in vitro–cultured human endothelial cells with siRNA-mediated suppression of HYPB, showed obvious defects in cell migration and invasion during vessel formation, suggesting an intrinsic role of Hypb in vascular development. Taken together, these results indicate that Hypb is required for embryonic vascular remodeling and provide a tool to study the function of H3K36 methylation in vasculogenesis/angiogenesis.     

Development of the mammalian axial skeleton requires signaling through the Gαi subfamily of heterotrimeric G proteins

129/SvEv mice with a loss-of-function mutation in the heterotrimeric G protein α-subunit gene Gnai3 have fusions of ribs and lumbar vertebrae, indicating a requirement for Gα_i (the “inhibitory” class of α-subunits) in somite derivatives. Mice with mutations of Gnai1 or Gnai2 have neither defect, but loss of both Gnai3 and one of the other two genes increases the number and severity of rib fusions without affecting the lumbar fusions. No myotome defects are observed in Gnai3/Gnai1 double-mutant embryos, and crosses with a conditional allele of Gnai2 indicate that Gα_i is specifically required in cartilage precursors. Penetrance and expressivity of the rib fusion phenotype is altered in mice with a mixed C57BL/6 × 129/SvEv genetic background. These phenotypes reveal a previously unknown role for G protein-coupled signaling pathways in development of the axial skeleton.     

The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis

The LIM-finger protein Lmo2, which is activated in T cell leukemias by chromosomal translocations, is required for yolk sac erythropoiesis. Because Lmo2 null mutant mice die at embryonic day 9–10, it prevents an assessment of a role in other stages of hematopoiesis. We have now studied the hematopoietic contribution of homozygous mutant Lmo2 −/− mouse embryonic stem cells and found that Lmo2 −/− cells do not contribute to any hematopoietic lineage in adult chimeric mice, but reintroduction of an Lmo2-expression vector rescues the ability of Lmo2 null embryonic stem cells to contribute to all lineages tested. This disruption of hematopoiesis probably occurs because interaction of Lmo2 protein with factors such as Tal1/Scl is precluded. Thus, Lmo2 is necessary for early stages of hematopoiesis, and the Lmo2 master gene encodes a protein that has a central and crucial role in the hematopoietic development.     

The mouse Pax21Neu mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney

We describe a new mouse frameshift mutation (Pax2^1Neu) with a 1-bp insertion in the Pax2 gene. This mutation is identical to a previously described mutation in a human family with renal-coloboma syndrome [Sanyanusin, P., McNoe, L. A., Sullivan, M. J., Weaver, R. G. & Eccles, M. R. (1995) Hum. Mol. Genet. 4, 2183–2184]. Heterozygous mutant mice exhibit defects in the kidney, the optic nerve, and retinal layer of the eye, and in homozygous mutant embryos, development of the optic nerve, metanephric kidney, and ventral regions of the inner ear is severely affected. In addition, we observe a deletion of the cerebellum and the posterior mesencephalon in homozygous mutant embryos demonstrating that, in contrast to mutations in Pax5, which is also expressed early in the mid-hindbrain region, loss of Pax2 gene function alone results in the early loss of the mid-hindbrain region. The mid-hindbrain phenotype is similar to Wnt1 and En1 mutant phenotypes, suggesting the conservation of gene regulatory networks between vertebrates and Drosophila.