Heterozygous Embryos Research Articles

A Screen for Genes Regulating the Wingless Gradient in Drosophila Embryos

During the development of the Drosophila embryonic epidermis, the secreted Wingless protein initially spreads symmetrically from its source. At later stages, Wingless becomes asymmetrically distributed in a Hedgehog-dependent manner, to control the patterning of the embryonic epidermis. When Wingless is misexpressed in engrailed cells in hedgehog heterozygous mutant embryos, larvae show a dominant phenotype consisting of patches of naked cuticle in denticle belts. This dose-sensitive phenotype is a direct consequence of a change in Wg protein distribution. We used this phenotype to carry out a screen for identifying genes regulating Wingless distribution or transport in the embryonic epidermis. Using a third chromosome deficiency collection, we found several genomic regions that showed a dominant interaction. After using a secondary screen to test for mutants and smaller deficiencies, we identified three interacting genes: dally, notum, and brahma. We confirmed that dally, as well as its homolog dally-like, and notum affect Wingless distribution in the embryonic epidermis, directly or indirectly. Thus, our assay can be used effectively to screen for genes regulating Wingless distribution or transport.

Reduced proportion of Purkinje cells expressing paternally derived mutant Mecp2308 allele in female mouse cerebellum is not due to a skewed primary pattern of X-chromosome inactivation

Rett syndrome (RTT) is an X-linked disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. The pattern of X-chromosome inactivation (XCI) is thought to play a role in phenotypic severity. In the present study, patterns of XCI were assessed by lacZ staining of embryos and adult brains of mice heterozygous for a X-linked Hmgcr-nls-lacZ transgene on a mutant mouse model of RTT. We found that there was no difference between the lacZ staining patterns in the brain of wild-type and heterozygous mutant embryos at embryonic day 9.5 (E9.5) suggesting that Mecp2 has no effect on the primary pattern of XCI. At 20 weeks of age, there was no significant difference between XCI patterns in the Purkinje cells in the cerebellum of heterozygous mutant and wild-type mice when the mutant allele was inherited from the mother. However, when the mutant allele was paternally inherited, a significant difference was detected. Thus, parental origin of the mutation may have a bearing on phenotype through XCI patterns. An estimation of the Purkinje cell precursor number based on XCI mosaicism revealed that, when the mutation was paternally inherited, the precursor number was less than that in the wild-type mice. Therefore, it is likely that the number of precursor cells allocated to the Purkinje cell lineage is affected by a paternally inherited mutation in Mecp2. We also observed that the pattern of XCI in cultured fibroblasts was significantly correlated with patterns in the Purkinje cells in mutant animals but not in wild-type mice.

Dosage-dependent requirement for mouse Vezf1 in vascular system development

Vezf1 is an early development gene that encodes a zinc finger transcription factor. In the developing embryo, Vezf1 is expressed in the yolk sac mesoderm and the endothelium of the developing vasculature and, in addition, in mesodermal and neuronal tissues. Targeted inactivation of Vezf1 in mice reveals that it acts in a closely regulated, dose-dependent fashion on the development of the blood vascular and lymphatic system. Homozygous mutant embryos display vascular remodeling defects and loss of vascular integrity leading to localized hemorrhaging. Ultrastructural analysis shows defective endothelial cell adhesion and tight junction formation in the mutant vessels. Moreover, in heterozygous embryos, haploinsufficiency is observed that is characterized by lymphatic hypervascularization associated with hemorrhaging and edema in the jugular region; a phenotype reminiscent of the human congenital lymphatic malformation syndrome cystic hygroma.

N-Myristoyltransferase 1 Is Essential in Early Mouse Development

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a beta-galactosidase marker gene. beta-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by approximately 95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.

Genetic Deletion of the Repressor of Estrogen Receptor Activity (REA) Enhances the Response to Estrogen in Target Tissues In Vivo

We previously identified a coregulator, repressor of estrogen receptor activity (REA), that directly interacts with estrogen receptor (ER) and represses ER transcriptional activity. Decreasing the intracellular level of REA by using small interfering RNA knockdown or antisense RNA approaches in cells in culture resulted in a significant increase in the level of up-regulation of estrogen-stimulated genes. To elucidate the functional activities of REA in vivo, we have used targeted disruption to delete the REA gene in mice. The targeting vector eliminated, by homologous recombination, the REA exon sequences encoding amino acids 12 to 201, which are required for REA repressive activity and for interaction with ER. The viability of heterozygous animals was similar to that of the wild type, whereas homozygous animals did not develop, suggesting a crucial role for REA in early development. Female, but not male, heterozygous animals had an increased body weight relative to age-matched wild-type animals beginning after puberty. REA mRNA and protein levels in uteri of heterozygous animals were half that of the wild type, and studies with heterozygous animals revealed a greater uterine weight gain and epithelial hyperproliferation in response to estradiol (E2) and a substantially greater stimulation by E2 of a number of estrogen up-regulated genes in the uterus. Even more dramatic in REA heterozygous animals was the loss of down regulation by E2 of genes in the uterus that are normally repressed by estrogen in wild-type animals. Mouse embryo fibroblasts derived from heterozygous embryos also displayed a greater transcriptional response to E2. These studies demonstrate that REA is a significant modulator of estrogen responsiveness in vivo: it normally restrains estrogen actions, moderating ER stimulation and enhancing ER repression of E2-regulated genes.

The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse

Blimp1, a zinc-finger containing DNA-binding transcriptional repressor, functions as a master regulator of B cell terminal differentiation. Considerable evidence suggests that Blimp1 is required for the establishment of anteroposterior axis formation and the formation of head structures during early vertebrate development. In mouse embryos, Blimp1 is strongly expressed in axial mesendoderm, the tissue known to provide anterior patterning signals during gastrulation. Here, we describe for the first time the defects caused by loss of Blimp1 function in the mouse. Blimp1 deficient embryos die at mid-gestation, but surprisingly early axis formation, anterior patterning and neural crest formation proceed normally. Rather, loss of Blimp1 expression disrupts morphogenesis of the caudal branchial arches and leads to a failure to correctly elaborate the labyrinthine layer of the placenta. Blimp1 mutant embryos also show widespread blood leakage and tissue apoptosis, and, strikingly, Blimp1 homozygous mutants entirely lack PGCs. At the time of PGC allocation around 7.25 days post coitum, Blimp1 heterozygous embryos exhibit decreased numbers of PCGs. Thus Blimp1 probably acts to turn off the default pathway that allows epiblast cells to adopt a somatic cell fate, and shifts the transcriptional program so that they become exclusively allocated into the germ cell lineage.

JAM-A expression during embryonic development

Cell adhesion molecules of the immunoglobulin superfamily play an important role in embryonic development. We have shown recently that JAM-A, a member of this family expressed at endothelial and epithelial tight junctions, is involved in platelet activation, leukocyte transmigration, and angiogenesis. Here, we determine the expression pattern of the JAM-A gene during embryogenesis using transgenic mice expressing lacZ under the control of the endogenous JAM-A promoter. Histochemical staining for beta-galactosidase in heterozygous mouse embryos was first seen in the inner cell mass and trophectoderm of the blastocyst. By 8.5 days post coitum (dpc), JAM-A gene activity was detected in the endoderm and part of the surface ectoderm. At 9.5 dpc, JAM-A expression began to localize to certain organ systems, most notably the developing inner ear and early vasculature. Localization of JAM-A to embryonic vasculature was confirmed by double-staining with antibodies against JAM-A and platelet endothelial cell adhesion molecule-1, a known endothelial cell marker. As organogenesis progressed, high levels of JAM-A expression continued in the epithelial component of the inner ear as well as the epithelium of the developing skin, olfactory system, lungs, and kidneys. In addition, JAM-A gene activity was found in the developing liver, choroid plexuses, and gut tubes. Immunofluorescent staining with a JAM-A antibody was performed to confirm that expression of the JAM-A-beta-galactosidase fusion protein accurately represented endogenous JAM-A protein. Thus, JAM-A is prominently expressed in embryonic vasculature and the epithelial components of several organ systems and may have an important role in their development.

Versican expression during skeletal/joint morphogenesis and patterning of muscle and nerve in the embryonic mouse limb

Versican, an extracellular matrix proteoglycan, has been implicated in limb development and is expressed in precartilage mesenchymal condensations. However, studies have lacked precise spatial and temporal investigation of versican localization during skeletogenesis and its relationship to patterning of muscle and nerve during mammalian limb development. The transgenic mouse line hdf (heart defect), which bears a lacZ reporter construct disrupting Cspg2 encoding versican, allowed ready detection of hdf transgene expression through histochemical analysis. Hdf transgene expression in whole mount heterozygous embryos and localization of versican relative to cartilage, muscle, and nerve tissues in paraffin-embedded limb sections of wild-type embryos from 10.5-14 days postcoitum were evaluated by lacZ histochemistry, immunohistochemistry, and in situ hybridization. Versican was localized within precartilage condensations and nascent cartilages with expression diminishing during maturation of the cartilage model at later time points. Interestingly, versican remained highly expressed in developing synovial joint interzones, suggesting potential function for versican in joint morphogenesis. Isolated myoblasts, incipient skeletal muscle masses, and neurites were not present in areas of strong versican expression within the developing limb. Versican-expressing tissues may reserve space for the future limb skeleton and developing joints and may aid in patterning of muscle and nerve by deterring muscle migration and innervation into these regions.

Allelic loss underlies type 2 segmental Hailey-Hailey disease, providing molecular confirmation of a novel genetic concept

Hailey-Hailey disease (HHD) is an autosomal dominant trait characterized by erythematous and oozing skin lesions preponderantly involving the body folds. In the present unusual case, however, unilateral segmental areas along the lines of Blaschko showing a rather severe involvement were superimposed on the ordinary symmetrical phenotype. Based on this observation and similar forms of mosaicism as reported in other autosomal dominant skin disorders, we postulated that in such cases, 2 different types of segmental involvement can be distinguished. Accordingly, the linear lesions as noted in the present case would exemplify type 2 segmental HHD. In the heterozygous embryo, loss of heterozygosity occurring at an early developmental stage would have given rise to pronounced linear lesions reflecting homozygosity or hemizygosity for the mutation. By analyzing DNA and RNA derived from blood and skin samples as well as keratinocytes of the index patient with various molecular techniques including RT-PCR, real-time PCR, and microsatellite analysis, we found a consistent loss of the paternal wild-type allele in more severely affected segmental skin regions, confirming this hypothesis for the first time, to our knowledge, at the molecular and cellular level.

Allelic loss underlies type 2 segmental Hailey-Hailey disease, providing molecular confirmation of a novel genetic concept

Hailey-Hailey disease (HHD) is an autosomal dominant trait characterized by erythematous and oozing skin lesions preponderantly involving the body folds. In the present unusual case, however, unilateral segmental areas along the lines of Blaschko showing a rather severe involvement were superimposed on the ordinary symmetrical phenotype. Based on this observation and similar forms of mosaicism as reported in other autosomal dominant skin disorders, we postulated that in such cases, 2 different types of segmental involvement can be distinguished. Accordingly, the linear lesions as noted in the present case would exemplify type 2 segmental HHD. In the heterozygous embryo, loss of heterozygosity occurring at an early developmental stage would have given rise to pronounced linear lesions reflecting homozygosity or hemizygosity for the mutation. By analyzing DNA and RNA derived from blood and skin samples as well as keratinocytes of the index patient with various molecular techniques including RT-PCR, real-time PCR, and microsatellite analysis, we found a consistent loss of the paternal wild-type allele in more severely affected segmental skin regions, confirming this hypothesis for the first time, to our knowledge, at the molecular and cellular level.

The tyrosine phosphatase, OST‐PTP, is expressed in mesenchymal progenitor cells early during skeletogenesis in the mouse

Osteotesticular protein tyrosine phosphatase (OST-PTP; OST), is a signaling molecule which catalyzes the removal of phosphates from tyrosine residues. It is known to be highly regulated in bone cells and has been shown to be important for the in vitro progression from a preosteoblast to a mature, mineralizing cell. However, the in vivo expression of this phosphatase during skeletogenesis has not been examined. Using Northern analysis and in situ hybridization (ISH), we have observed that this gene is strongly expressed early during the formation of the mouse skeleton. By 12.5 days postcoitum (dpc), expression of OST mRNA transcripts increases and is localized within the mesenchyme of craniofacial bones, ribs, limbs, and Meckel's cartilage. Following initiation of chondrogenesis, OST mRNA becomes restricted to the perichondrium of all endochondral elements. With ossification, this gene is also expressed by cells, presumably osteoblasts, at the chondro-osseous border and along cortical and trabecular bone surfaces. Unlike other bone markers examined such as Osterix and type II collagen, OST transcripts do not appear to be expressed by chondrocytes of epiphyseal cartilage or by non-hypertrophic or hypertrophic chondrocytes. Because the temporal expression patterns of OST and Runx2 were similar suggesting a potential interrelationship in bone regulation and function, OST expression was examined in transgenic mice lacking a functional Runx2/Cbfal protein (Runx2/Cbfal delta C (deltaC)) and possessing a cartilaginous skeleton. Interestingly, the OST gene was expressed with localization similar in wild-type, homozygous, and heterozygous embryos. These studies suggest that the expression of the OST gene may be important during skeletogenesis, potentially from commitment of mesenchymal cells to the ossification of new bones. Early in embryogenesis, regulation of OST expression may be independent of Runx2/Cbfa1.

Abnormalities of Interstitial Cells of Cajal in Dominant Megacolon Mice

Interstitial cells of Cajal (ICCs) in the gastrointestinal tract are a group of cells interacting with enteric neurons and smooth muscle cells. They serve as mediators of neurotransmission and pacemakers of the peristaltic movement of the gut. Previous investigations on patients with Hirschsprung’s disease and mice with aganglionic megacolon showed contrasting results on the relationship between the abnormalities of ICCs and enteric neurons. In order to ascertain whether the development of ICCs is also perturbed in the aganglionic segment of the gut, we used Dominant megacolon (Dom) mice as an animal model and an antibody specific to c-kit (a tyrosine kinase receptor expressed in ICCs) to examine the spatial distribution of ICCs in the developing and adult colon. Our results showed that in the wild-type adult colon, ICCs were bipolar in shape in the smooth muscle layer and dense networks of ICCs were observed in the myenteric and deep muscular plexuses, whereas in the aganglionic colon of Dom heterozygous mice, no ICCs could be found in the smooth muscle layer and disrupted patterns of ICC networks were observed in the myenteric and deep muscular plexuses. On 14.5 days of development, the levels of expression of c-kit and its ligand (stem cell factor) were similar in the wild-type, heterozygous and homozygous Dom embryos. However, by 18.5 days, the numbers of ICCs were greatly reduced in the myenteric and submucosal plexuses of the terminal colon of the heterozygous embryos comparing with the wild-type counterparts. Based on our results, we postulate that the abnormal development of ICCs may disrupt the neurotransmission and pacemaker activity of the gut, leading to interrupted peristalsis.

Deletion of the mouse P450c17 gene causes early embryonic lethality.

Dehydroepiandrosterone (DHEA), a 19-carbon precursor of sex steroids, is abundantly produced in the human but not the mouse adrenal. However, mice produce DHEA and DHEA-sulfate (DHEAS) in the fetal brain. DHEA stimulates axonal growth from specific populations of mouse neocortical neurons in vitro, while DHEAS stimulates dendritic growth from those cells. The synthesis of DHEA and sex steroids, but not mouse glucocorticoids and mineralocorticoids, requires P450c17, which catalyzes both 17 alpha-hydroxylase and 17,20-lyase activities. We hypothesized that P450c17-knockout mice would have disordered sex steroid synthesis and disordered brain DHEA production and thus provide phenotypic clues about the functions of DHEA in mouse brain development. We deleted the mouse P450c17 gene in 127/SvJ mice and obtained several lines of mice from two lines of targeted embryonic stem cells. Heterozygotes were phenotypically and reproductively normal, but in all mouse lines, P450c17(-/-) zygotes died by embryonic day 7, prior to gastrulation. The cause of this early lethality is unknown, as there is no known function of fetal steroids at embryonic day 7. Immunocytochemistry identified P450c17 in embryonic endoderm in E7 wild-type and heterozygous embryos, but its function in these cells is unknown. Enzyme assays of wild-type embryos showed a rapid rise in 17-hydroxylase activity between E6 and E7 and the presence of C(17,20)-lyase activity at E7. Treatment of pregnant females with subcutaneous pellets releasing DHEA or 17-OH pregnenolone at a constant rate failed to rescue P450c17(-/-) fetuses. Treatment of normal pregnant females with pellets releasing pregnenolone or progesterone did not cause fetal demise. These data suggest that steroid products of P450c17 have heretofore-unknown essential functions in early embryonic mouse development.

Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells.

At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1-2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.

Increased neuromuscular activity causes axonal defects and muscular degeneration.

Before establishing terminal synapses with their final muscle targets, migrating motor axons form en passant synaptic contacts with myotomal muscle. Whereas signaling through terminal synapses has been shown to play important roles in pre- and postsynaptic development, little is known about the function of these early en passant synaptic contacts. Here, we show that increased neuromuscular activity through en passant synaptic contacts affects pre- and postsynaptic development. We demonstrate that in zebrafish twister mutants, prolonged neuromuscular transmission causes motor axonal extension and muscular degeneration in a dose-dependent manner. Cloning of twister reveals a novel, dominant gain-of-function mutation in the muscle-specific nicotinic acetylcholine receptor alpha-subunit, CHRNA1. Moreover, electrophysiological analysis demonstrates that the mutant subunit increases synaptic decay times, thereby prolonging postsynaptic activity. We show that as the first en passant synaptic contacts form, excessive postsynaptic activity in homozygous embryos severely impedes pre- and postsynaptic development, leading to degenerative defects characteristic of the human slow-channel congenital myasthenic syndrome. By contrast, in heterozygous embryos, transient and mild increase in postsynaptic activity does not overtly affect postsynaptic morphology but causes transient axonal defects, suggesting bi-directional communication between motor axons and myotomal muscle. Together, our results provide compelling evidence that during pathfinding, myotomal muscle cells communicate extensively with extending motor axons through en passant synaptic contacts.

Binding sites of gamma-secretase inhibitors in rodent brain: distribution, postnatal development, and effect of deafferentation.

gamma-Secretase is a multimeric complex consisted of presenilins (PSs) and three other proteins. PSs appear to be key contributors for the enzymatic center, the potential target of a number of recently developed gamma-secretase inhibitors. Using radiolabeled and unlabeled inhibitors as ligands, this study was aimed to determine the in situ distribution of gamma-secretase in the brain. Characterization using PS-1 knock-out mouse embryos revealed 50 and 80% reductions of gamma-secretase inhibitor binding density in the heterozygous (PS-1(+/-)) and homozygous (PS-1-/-) embryos, respectively, relative to the wild type (PS-1(+/+)). The pharmacological profile from competition binding assays suggests that the ligands may target at the N- and C-terminal fragments of PS essential for gamma-secretase activity. In the adult rat brain, the binding sites existed mostly in the forebrain, the cerebellum, and discrete brainstem areas and were particularly abundant in areas rich in neuronal terminals, e.g., olfactory glomeruli, CA3-hilus area, cerebellar molecular layer, and pars reticulata of the substantia nigra. In the developing rat brain, diffuse and elevated expression of binding sites occurred at the early postnatal stage relative to the adult. The possible association of binding sites with neuronal terminals in the adult brain was further investigated after olfactory deafferentation. A significant decrease with subsequent recovery of binding sites was noted in the olfactory glomeruli after chemical damage of the olfactory epithelium. The findings in this study support a physiological role of PS or gamma-secretase complex in neuronal and synaptic development and plasticity.

A Twist to Bone Development

The Runx2 transcription factor controls bone development during early mammalian embryogenesis. However, a delay between Runx2 expression and osteoblast differentiation has suggested that this key transcription factor is held in check by another regulatory protein. Bialek et al. have identified two transcription factors of the Twist family, Twist-1 and -2, that inhibit osteoblast differentiation by blocking Runx2 function, which indicates that initiation of skeletogenesis occurs when an inhibition is relieved. Mouse osteoblasts were found to express Twist proteins before they expressed Runx2. After a decrease in Twist expression, expression of Runx2 gene targets commenced. Premature osteoblast differentiation was observed in mice heterozygous for Twist-1. Runx2 expression was not affected in the heterozygous embryos nor in an osteosarcoma cell line overexpressing Twist proteins. Expression of Twist proteins also decreased Runx2 activity in a transactivation-reporter gene assay. Purified recombinant Twist and Runx proteins interacted directly in vitro, and the authors further mapped the sites of interaction in both proteins. The study also points to premature activation of Runx2 as the molecular defect in Saethre-Chotzen syndrome, a skeletal disorder caused by a haplo-insufficiency in the TWIST locus. P. Bialek, B. Kern, X. Yang, M. Schrock, D. Sosic, N. Hong, H. Wu, K. Yu, D. M. Ornitz, E. N. Olson, M. J. Justice, G. Karsenty, A Twist code determines the onset of osteoblast differentiation. Dev. Cell 6 , 423-435 (2004). [Online Journal]

An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos

The present study describes a simple, rapid protocol for culture for limb tissue from individual 10.5-day post coitum mouse embryos that supports cartilage differentiation over a six-day period. This technique differs from other commonly used methods utilizing pooled limb tissue in that: 1) forelimbs from individual embryos were used as donor tissue; 2) limb tissue was dissociated by very gentle enzymatic digest (0.1% trypsin, 5 min); and, 3) cell suspensions were plated at a lower density (1 x 10(7) vs. 2 x 10(7) cells/ml) in a reduced volume of 3-5 microl. Under these modified conditions to increase limb cell yield from each embryo, histochemical and immunohistochemical analyses demonstrated reproducible formation of precartilage aggregates and subsequent overt chondrogenesis over a predictable time course. Using this culture protocol, analysis of limb mesenchyme from heterozygous hdf embryos, which bear an insertional mutation of the Cspg2 gene encoding the core protein of the chondroitin sulfate proteoglycan, versican, revealed an overall similar chondrogenic potential to that observed for wild-type littermates. This technique readily enables in vitro culture of limb bud mesenchyme from individual mouse embryos at this developmental stage and may be utilized by investigators to study the effects of the hdf and other transgenic mutations on mammalian limb development in vitro.

Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse.

Fibrillarin, a protein component of C/D box small nucleolar ribonucleoproteins (snoRNPs), directs 2'-O-methylation of rRNA and is also involved in other aspects of rRNA processing. A gene trap screen in embryonic stem (ES) cells resulted in an insertion mutation in the fibrillarin gene. This insertion generated a fusion protein that contained the N-terminal 132 amino acids of fibrillarin fused to a beta-galactosidase-neomycin phosphotransferase reporter. As a result, the N-terminal GAR domain was present in the fusion protein but the methyltransferase-like domain was missing. The ES cell line with the targeted fibrillarin allele was transmitted through the mouse germ line, creating heterozygous animals. Western blot analyses showed a reduction in fibrillarin protein levels in the heterozygous knockout animals. Animals homozygous for the mutation were inviable, and massive apoptosis was observed in early Fibrillarin(-/-) embryos, showing that fibrillarin is essential for development. Fibrillarin(+/-) live-born mice displayed no obvious growth defect, but heterozygous intercrosses revealed a reduced ratio of +/- to +/+ mice, showing that some of the Fibrillarin heterozygous embryos die in utero. Analyses of tissue samples and cultured embryonic fibroblasts showed no discernible alteration in pre-rRNA processing or the level of the U3 snoRNA. However, the level of the intron-encoded box C/D snoRNA U76 was clearly reduced. This suggests a high requirement for snoRNA synthesis during an early stage in development.

Six1 is required for the early organogenesis of mammalian kidney.

The murine Six gene family, homologous to Drosophila sine oculis (so) which encodes a homeodomain transcription factor, is composed of six members (Six1-6). Among the six members, only the Six2 gene has been previously shown to be expressed early in kidney development, but its function is unknown. We have recently found that the Six1 gene is also expressed in the kidney. In the developing kidney, Six1 is expressed in the uninduced metanephric mesenchyme at E10.5 and in the induced mesenchyme around the ureteric bud at E11.5. At E17.5 to P0, Six1 expression became restricted to a subpopulation of collecting tubule epithelial cells. To study its in vivo function, we have recently generated Six1 mutant mice. Loss of Six1 leads to a failure of ureteric bud invasion into the mesenchyme and subsequent apoptosis of the mesenchyme. These results indicate that Six1 plays an essential role in early kidney development. In Six1(-/-) kidney development, we have found that Pax2, Six2 and Sall1 expression was markedly reduced in the metanephric mesenchyme at E10.5, indicating that Six1 is required for the expression of these genes in the metanephric mesenchyme. In contrast, Eya1 expression was unaffected in Six1(-/-) metanephric mesenchyme at E10.5, indicating that Eya1 may function upstream of Six1. Moreover, our results show that both Eya1 and Six1 expression in the metanephric mesenchyme is preserved in Pax2(-/-) embryos at E10.5, further indicating that Pax2 functions downstream of Eya1 and Six1 in the metanephric mesenchyme. Thus, the epistatic relationship between Pax, Eya and Six genes in the metanephric mesenchyme during early kidney development is distinct from a genetic pathway elucidated in the Drosophila eye imaginal disc. Finally, our results show that Eya1 and Six1 genetically interact during mammalian kidney development, because most compound heterozygous embryos show hypoplastic kidneys. These analyses establish a role for Six1 in the initial inductive step for metanephric development.