Ann M. Lawler

Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11.

The bones that comprise the axial skeleton have distinct morphological features characteristic of their positions along the anterior/posterior axis. We previously described a novel TGF-β family member, myostatin (encoded by the gene Mstn, formerly Gdf8), that has an essential role in regulating skeletal muscle mass. We also identified a gene related to Mstn by low-stringency screening. While the work described here was being completed, the cloning of this gene, designated Gdf11 (also called Bmp11), was also reported by other groups. Here we show that Gdf11, a new transforming growth factor β (TGFβ) superfamily member, has an important role in establishing this skeletal pattern. During early mouse embryogenesis, Gdf11 is expressed in the primitive streak and tail bud regions, which are sites where new mesodermal cells are generated. Homozygous mutant mice carrying a targeted deletion of Gdf11 exhibit anteriorly directed homeotic transformations throughout the axial skeleton and posterior displacement of the hindlimbs. The effect of the mutation is dose dependent, as Gdf11+/– mice have a milder phenotype than Gdf11–/– mice. Mutant embryos show alterations in patterns of Hox gene expression, suggesting that Gdf11 acts upstream of the Hox genes. Our findings suggest that Gdf11 is a secreted signal that acts globally to specify positional identity along the anterior/posterior axis.

Regulation of left-right patterning in mice by growth/differentiation factor-1

The transforming growth factor-β (TGF-β) superfamily encompasses a large group of structurally related polypeptides that are capable of regulating cell growth and differentiation in a wide range of embryonic and adult tissues. Growth/differentiation factor-1 (Gdf-1, encoded by Gdf1 ) is a TGF-β family member of unknown function that was originally isolated from an early mouse embryo cDNA library and is expressed specifically in the nervous system in late-stage embryos and adult mice. Here we show that at early stages of mouse development, Gdf1 is expressed initially throughout the embryo proper and then most prominently in the primitive node, ventral neural tube, and intermediate and lateral plate mesoderm. To examine its biological function, we generated a mouse line carrying a targeted mutation in Gdf1. Gdf1−/− mice exhibited a spectrum of defects related to left-right axis formation, including visceral situs inversus, right pulmonary isomerism and a range of cardiac anomalies. In most Gdf1−/− embryos, the expression of Ebaf (formerly lefty-1) in the left side of the floor plate and Leftb (formerly lefty-2),nodal and Pitx2 in the left lateral plate mesoderm was absent, suggesting that Gdf1 acts upstream of these genes either directly or indirectly to activate their expression. Our findings suggest that Gdf1 acts early in the pathway of gene activation that leads to the establishment of left-right asymmetry.

Growth retardation and neonatal lethality in mice with a homozygous deletion in the C-terminal domain of RNA polymerase II.

Abstract The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandem repeats of the consensus heptapeptide YSPTSPS. Deletion studies in tissue culture cells have indicated that the CTD plays an essential role in transcription, although the nature of this essential function remains unclear. About half of the CTD can be deleted without affecting the viability of cells in tissue culture. Paradoxically, the dispensable CTD repeats are precisely conserved among all mammals whose CTD sequences are known. To determine whether the mammalian CTD is important in transcription during mouse development, we developed a gene targeting approach to introduce deletions into the CTD coding region of mouse embryonic stem (ES) cells. To maintain a functional Rpo2-1 gene, the neo marker in the targeting vector was positioned outside of the Rpo2-1 transcribed region, 1.2 kb from the site of the CTD deletion. G418-resistant clones were screened for co-integration of the CTD deletion, and the resulting ES lines were used to create germline chimeric mice. Stable heterozygous lines were established and mated to produce animals homozygous for the CTD deletion. We show here that mice homozygous for a deletion of thirteen of the 52 heptapeptide repeats are smaller than wild-type littermates and have a high rate of neonatal lethality. Surviving adults, although small, appear morphologically normal and are fertile. This result suggests that the CTD plays a role in regulating growth during mammalian development. The gene targeting approach described here should be useful for making further deletions in the CTD and may be of general applicability where it is desirable to engineer specific mutations in the germline of mice.

Perinatal loss of Ts65Dn Down syndrome mice.

Ts65Dn mice inherit a marker chromosome, T(1716)65Dn, producing segmental trisomy for orthologs of about half of the genes on human chromosome 21. These mice display a number of phenotypes that are directly comparable to those in humans with trisomy 21 and are the most widely used animal model of Down syndrome (DS). However, the husbandry of Ts65Dn mice is complicated. Males are sterile, and only 20–40% of the offspring of Ts65Dn mothers are trisomic at weaning. The lower-than-expected frequency of trisomic offspring has been attributed to losses at meiosis, during gestation and at postnatal stages, but no systematic studies support any of these suppositions. We show that the T(1716)65Dn marker chromosome is inherited at expected frequency and is fully compatible with development to midgestation. Disproportional loss of trisomic offspring occurs in late gestation and continues through birth to weaning. Different maternal H2 haplotypes are significantly associated with the frequency of trisomy at weaning in patterns different from those reported previously. The proportion of trisomic mice per litter decreases with age of the Ts65Dn mother. These results provide the first statistical and numerical evidence supporting the prenatal and perinatal pattern of loss in the Ts65Dn mouse model of DS.

Craniofacial abnormalities resulting from targeted disruption of the murine Sim2 gene.

Sim2 is a member of the basic helix‐loop‐helix PAS transcription factor gene family and is evolutionarily related to the Drosophila single‐minded gene, a key regulator of central nervous system midline development. In an effort to determine the biological roles of Sim2 in mammalian development, we disrupted the murine Sim2 gene through gene targeting. Mice homozygous for the disrupted allele (Sim2 ‐/‐) exhibit a cleft of the secondary palate and malformations of the tongue and pterygoid processes of the sphenoid bone. These craniofacial malformations are the most probable cause of aerophagia (air swallowing with subsequent accumulation of air in the gastrointestinal tract) and postnatal death exhibited by Sim2 ‐/‐ mice. The developing palates of the Sim2 ‐/‐ mice are hypocellular, and at embryonic day 14.5 contain excess extracellular matrix component hyaluronan (HA) compared with heterozygotes and homozygous wild‐type littermates. HA plays an important role in the regulation and mechanics of palate development. Its premature accumulation in Sim2 ‐/‐ animal palates suggests a regulatory role for Sim2 in HA synthesis and in the establishment of craniofacial architecture.

Increased male reproductive success in Ts65Dn “Down syndrome” mice

The Ts65Dn mouse is trisomic for orthologs of about half the genes on Hsa21. A number of phenotypes in these trisomic mice parallel those in humans with trisomy 21 (Down syndrome), including cognitive deficits due to hippocampal malfunction that are sufficiently similar to human that “therapies” developed in Ts65Dn mice are making their way to human clinical trials. However, the impact of the model is limited by availability. Ts65Dn cannot be completely inbred and males are generally considered to be sterile. Females have few, small litters and they exhibit poor care of offspring, frequently abandoning entire litters. Here we report identification and selective breeding of rare fertile males from two working colonies of Ts65Dn mice. Trisomic offspring can be propagated by natural matings or by in vitro fertilization (IVF) to produce large cohorts of closely related siblings. The use of a robust euploid strain as recipients of fertilized embryos in IVF or as the female in natural matings greatly improves husbandry. Extra zygotes cultured to the blastocyst stage were used to create trisomic and euploid embryonic stem (ES) cells from littermates. We developed parameters for cryopreserving sperm from Ts65Dn males and used it to produce trisomic offspring by IVF. Use of cryopreserved sperm provides additional flexibility in the choice of oocyte donors from different genetic backgrounds, facilitating rapid production of complex crosses. This approach greatly increases the power of this important trisomic model to interrogate modifying effects of trisomic or disomic genes that contribute to trisomic phenotypes.

Alterations of yeast artificial chromosome transgenic sequences in stretched embryonic stem-cell chromatin visualized by fluorescence in situ hybridization

Transgenic mice have been generated from embryonic stem (ES) cells carrying functional genes cloned within yeast artificial chromosomes (YACs). Information on the integrity and organization of the inserted sequences, including the number of copies and their orientation to each other, is still limited by current methods. We have applied fluorescence in situ hybridization to stretched chromatin preparations from YAC-transfected ES cells to analyze the organization and copy number of the integrated sequences.

Linkage of two pseudogenes from Vκ1 and Vκ9 murine immunoglobulin families

As an initial step towards the molecular analysis of the murine V kappa locus, a cosmid library from BALB/cJ mouse liver DNA was screened with probes representing 10 V kappa families. Of eight cosmids that were isolated from the initial screen, five contained a single restriction fragment that hybridized to the probes. Two cosmids contained two fragments that hybridized to the same probe, V kappa 4, indicating that some V kappa 4 gene segments are linked. One cosmid had two genes that belonged to different families, V kappa 1 and V kappa 9. The two gene segments were located within 12 kb of each other and lay in the same transcriptional orientation. Linkage of gene segments from the V kappa 1 and V kappa 9 families is consistent with a genetic map of the locus, and provides physical evidence for the first time that two genes from different families are closely linked in the murine kappa locus. Sequence analysis revealed that both genes are pseudogenes: the V kappa psi 1.7 gene segment has eight mutations, including termination codons, insertions, and deletions, and the V kappa psi 9B.8 gene segment has two mutations of an insertion and altered RNA splice site. Both genes have the potential to rearrange based on the sequence of their heptamer-nonamer motifs. The identification of pseudogenes raises the question of how many nonfunctional genes are present in the murine germline repertoire.

Generation of antibody diversity before and after immunization.

Immunoglobulin variable genes are a large, multigene family whose members rearrange at different times in development. The hallmark of the immune system is diversity, and it is intriguing to observe how B cells, which start out with unrearranged genes or zero diversity, rearrange germline genes to build a repertoire of greater than lo6 specificities. Which genes rearrange first? Is there a developmental program to rearrangement? How quickly does the repertoire diversify, and does it occur at a molecular level by randomly rearranging genes or at a cellular level after selection by antigen? Analysis of rearranged genes in B cells early in ontogeny has provided striking insights into the formation of the repertoire. Despite over 1,000 variable genes for the heavy chain (V,) in the germline, only a few, located near the joining (JH) genes, are rearranged.’” Although less is known about rearrangement of variable genes for the kappa light chain (VK), preliminary data indicate that genes from many different subfamilies are rearranged. Further analyses of rearranged VK genes may provide a molecular basis for the phenomenon of the programmed appearance of certain B cells during ontogeny.‘ Additional diversity is generated in immunoglobulin genes after antigen stimulation by somatic mutation. Much is known about the structural hallmarks of mutation in genes coding for antibodies from secondary responses. Nucleotide substitutions are clustered in a discrete region of DNA surrounding and including the rearranged V genesV6; they occur at a frequency of around 1%; and they are more common in IgG and IgA molecules than in I g h molecules.’ The elucidation of this unusual mechanism clearly requires studies of a more dynamic character. We have approached this problem by attempting to identify a population of B cells that is actively undergoing somatic mutation. The results indicate that mutation occurs early after B cells are stimulated with a primary injection of antigen. Mutated antibodies then undergo intense selection by antigen to produce those with high affinity. We propose that the major effect of somatic hypermutation is to increase affinity and not to create new specificities that would allow other gene products to participate in an immune response.”

Targeted Disruption of the 70kDa Peroxisomal Membrane Protein (PMP70) Gene Produces Dicarboxylic Aciduria and Peroxisome Proliferation |[bull]| 720

ABC transporters are members of a superfamily of membrane proteins involved in the transport of a variety of molecules across biological membranes. PMP70 is one of four ABC transporters in the mammalian peroxisome membrane; others are ALDP, ALDP-related (ALDR) and PMP70-related (PMP70R). Mutations in ALD result in accumulation of VLCFA due to impaired β-oxidation in peroxisomes and cause X-linked adrenoleukodystrophy. The function of PMP70 is unknown although PMP70 mutations have been observed in 2 patients with Zellweger syndrome. To understand its function and relationship to human disease, we targeted the murine PMP70 gene and produced PMP70 knockout mice. Products of heterozygous matings were born in expected Mendelian ratios. The knockout pups show a slower growth rate (70%) than that of wild type littermates for the first month of life but eventually catch up. PMP70 is absent by Northern and immunoblot analysis. Immunohistochemistry and electron microscopy show that peroxisomes are present and contain normal amounts of PTS1 and PTS2 targeted matrix proteins. β-oxidation of monocarboxylic fatty acid in PMP70-/- fibroblasts is normal. In liver, the peroxisome number appears to be increased approximately 2-fold. Metabolic studies in the PMP70-/- mice show a striking dicarboxylic aciduria. This result is particularly interesting because oxidation of dicarboxylic acids occurs mainly in peroxisomes. To explain our results, we suggest that PMP70 function involves transport of dicarboxylic acids into peroxisomes. In the absence of this transport, peroxisome β-oxidation of dicarboxylic acids is reduced; these compounds accumulate and cause peroxisome proliferation.