Bacterial c-di-GMP has a key role in establishing host-microbe symbiosis.

Most microbes evolve faster than their hosts and should therefore drive evolution of host-microbe interactions. However, relatively little is known about the characteristics that define the adaptive path of microbes to host association. Here we identified microbial traits that mediate adaptation to hosts by experimentally evolving the free-living bacterium Pseudomonas lurida with the nematode Caenorhabditis elegans as its host. After ten passages, we repeatedly observed the evolution of beneficial host-specialist bacteria, with improved persistence in the nematode being associated with increased biofilm formation. Whole-genome sequencing revealed mutations that uniformly upregulate the bacterial second messenger, cyclic diguanylate (c-di-GMP). We subsequently generated mutants with upregulated c-di-GMP in different Pseudomonas strains and species, which consistently increased host association. Comparison of pseudomonad genomes from various environments revealed that c-di-GMP underlies adaptation to a variety of hosts, from plants to humans. This study indicates that c-di-GMP is fundamental for establishing host association.

A History of Mouse Genetics: From Fancy Mice to Mutations in Every Gene.

The laboratory mouse has become the model organism of choice in numerous areas of biological and biomedical research, including the study of congenital birth defects. The appeal of mice for these experimental studies stems from the similarities between the physiology, anatomy, and reproduction of these small mammals with our own, but it is also based on a number of practical reasons: mice are easy to maintain in a laboratory environment, are incredibly prolific, and have a relatively short reproductive cycle. Another compelling reason for choosing mice as research subjects is the number of tools and resources that have been developed after more than a century of working with these small rodents in laboratory environments. As will become obvious from the reading of the different chapters in this book, research in mice has already helped uncover many of the genes and processes responsible for congenital birth malformations and human diseases. In this chapter, we will provide an overview of the methods, scientific advances, and serendipitous circumstances that have made these discoveries possible, with a special emphasis on how the use of genetics has propelled scientific progress in mouse research and paved the way for future discoveries.

Secretory pathway calcium ATPase 1 (SPCA1) controls mouse neural tube closure by regulating cytoskeletal dynamics.

Neural tube closure relies on the apical constriction of neuroepithelial cells. Research in frog and fly embryos has found links between the levels of intracellular calcium, actomyosin dynamics and apical constriction. However, genetic evidence for a role of calcium in apical constriction during mammalian neurulation is still lacking. Secretory pathway calcium ATPase (SPCA1) regulates calcium homeostasis by pumping cytosolic calcium into the Golgi apparatus. Loss of function in Spca1 causes cranial exencephaly and spinal cord defects in mice, phenotypes previously ascribed to apoptosis. However, our characterization of a novel allele of Spca1 revealed that neurulation defects in Spca1 mutants are not due to cell death, but rather to a failure of neuroepithelial cells to apically constrict. We show that SPCA1 influences cell contractility by regulating myosin II localization. Furthermore, we found that loss of Spca1 disrupts actin dynamics and the localization of the actin remodeling protein cofilin 1. Taken together, our results provide evidence that SPCA1 promotes neurulation by regulating the cytoskeletal dynamics that promote apical constriction and identify cofilin 1 as a downstream effector of SPCA1 function.

Pbx loss in cranial neural crest, unlike in epithelium, results in cleft palate only and a broader midface.

Orofacial clefting represents the most common craniofacial birth defect. Cleft lip with or without cleft palate (CL/P) is genetically distinct from cleft palate only (CPO). Numerous transcription factors (TFs) regulate normal development of the midface, comprising the premaxilla, maxilla and palatine bones, through control of basic cellular behaviors. Within the Pbx family of genes encoding Three Amino-acid Loop Extension (TALE) homeodomain-containing TFs, we previously established that in the mouse, Pbx1 plays a preeminent role in midfacial morphogenesis, and Pbx2 and Pbx3 execute collaborative functions in domains of coexpression. We also reported that Pbx1 loss from cephalic epithelial domains, on a Pbx2- or Pbx3-deficient background, results in CL/P via disruption of a regulatory network that controls apoptosis at the seam of frontonasal and maxillary process fusion. Conversely, Pbx1 loss in cranial neural crest cell (CNCC)-derived mesenchyme on a Pbx2-deficient background results in CPO, a phenotype not yet characterized. In this study, we provide in-depth analysis of PBX1 and PBX2 protein localization from early stages of midfacial morphogenesis throughout development of the secondary palate. We further establish CNCC-specific roles of PBX TFs and describe the developmental abnormalities resulting from their loss in the murine embryonic secondary palate. Additionally, we compare and contrast the phenotypes arising from PBX1 loss in CNCC with those caused by its loss in the epithelium and show that CNCC-specific Pbx1 deletion affects only later secondary palate morphogenesis. Moreover, CNCC mutants exhibit perturbed rostro-caudal organization and broadening of the midfacial complex. Proliferation defects are pronounced in CNCC mutants at gestational day (E)12.5, suggesting altered proliferation of mutant palatal progenitor cells, consistent with roles of PBX factors in maintaining progenitor cell state. Although the craniofacial skeletal abnormalities in CNCC mutants do not result from overt patterning defects, osteogenesis is delayed, underscoring a critical role of PBX factors in CNCC morphogenesis and differentiation. Overall, the characterization of tissue-specific Pbx loss-of-function mouse models with orofacial clefting establishes these strains as unique tools to further dissect the complexities of this congenital craniofacial malformation. This study closely links PBX TALE homeodomain proteins to the variation in maxillary shape and size that occurs in pathological settings and during evolution of midfacial morphology.

The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28.

KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity.

TRIM28 Controls Genomic Imprinting through Distinct Mechanisms during and after Early Genome-wide Reprogramming.

Genomic imprinting depends on the establishment and maintenance of DNA methylation at imprinting control regions. However, the mechanisms by which these heritable marks influence allele-specific expression are not fully understood. By analyzing maternal, zygotic, maternal-zygotic, and conditional Trim28 mutants, we found that the transcription factor TRIM28 controls genomic imprinting through distinct mechanisms at different developmental stages. During early genome-wide reprogramming, both maternal and zygotic TRIM28 are required for the maintenance of methylation at germline imprints. However, in conditional Trim28 mutants, Gtl2-imprinted gene expression was lost despite normal methylation levels at the germline IG-DMR. These results provide evidence that TRIM28 controls imprinting after early embryonic reprogramming through a mechanism other than the maintenance of germline imprints. Additionally, our finding that secondary imprints were hypomethylated in TRIM28 mutants uncovers a requirement of TRIM28 after genome-wide reprogramming for interpreting germline imprints and regulating DNA methylation at imprinted gene promoters.

The ENU-induced cetus mutation reveals an essential role of the DNA helicase DDX11 for mesoderm development during early mouse embryogenesis.

BACKGROUND: DDX11 is a DNA helicase of the conserved FANCJ/RAD3/XPD family involved in maintaining genome stability. Studies in yeast and humans have shown requirements for DDX11 in sister chromatid cohesion and DNA repair. In mouse, loss of Ddx11 results in embryonic lethality. However, the developmental defects of Ddx11 mutants are poorly understood. RESULTS: We describe the characterization and positional cloning of cetus, a mouse ENU-induced mutation in Ddx11. We demonstrate that cetus causes a nonconservative amino acid change in DDX11 motif V and that this mutation is a null allele of Ddx11. cetus mutant embryos failed to thrive beyond embryonic day 8.5 and displayed placental defects similar to those described in Ddx11 null embryos. Additionally, our characterization of Ddx11(cetus) mutants identified embryonic phenotypes that had not been previously reported in Ddx11(KO) embryos, including loss of somitic mesoderm, an open kinked neural tube and abnormal heart looping. We show that loss of Ddx11 causes widespread apoptosis from early embryonic stages and that loss of Ddx11 disrupts somitic mesoderm more dramatically than other embryonic cells. CONCLUSIONS: Our results identify novel roles of Ddx11 during embryo morphogenesis and demonstrate that the activity of its motif V is essential for DDX11 function.

Modulation of WNT signaling activity is key to the formation of the embryonic head.

The formation of the embryonic head begins with the assembly of the progenitor tissues of the brain, the head and face primordia and the foregut that are derived from the primary germ layers during gastrulation. Specification of the anterior-posterior polarity of major body parts and the morphogenesis of the head and brain specifically is driven by inductive signals including those mediated by BMP, Nodal, FGF and WNT. A critical role of ß-catenin dependent WNT signalling activity for head morphogenesis has been revealed through the analysis of the phenotypic impact of loss of function mutation of an antagonist: DKK1, a transcriptional repressor: GSC; and the outcome of interaction of Dkk1 with genes coding three components of the canonical signalling pathway: the ligand WNT3, the co-receptor LRP6 and the transcriptional co-factor, ß-catenin. The findings highlight the requirement of a stringent control of the timing, domain and level of canonical WNT signalling activity for the formation of the embryonic head.

TRIM28 is required by the mouse KRAB domain protein ZFP568 to control convergent extension and morphogenesis of extra-embryonic tissues.

TRIM28 is a transcriptional regulator that is essential for embryonic development and is implicated in a variety of human diseases. The roles of TRIM28 in distinct biological processes are thought to depend on its interaction with factors that determine its DNA target specificity. However, functional evidence linking TRIM28 to specific co-factors is scarce. chatwo, a hypomorphic allele of Trim28, causes embryonic lethality and defects in convergent extension and morphogenesis of extra-embryonic tissues. These phenotypes are remarkably similar to those of mutants in the Krüppel-associated box (KRAB) zinc finger protein ZFP568, providing strong genetic evidence that ZFP568 and TRIM28 control morphogenesis through a common molecular mechanism. We determined that chatwo mutations decrease TRIM28 protein stability and repressive activity, disrupting both ZFP568-dependent and ZFP568-independent roles of TRIM28. These results, together with the analysis of embryos bearing a conditional inactivation of Trim28 in embryonic-derived tissues, revealed that TRIM28 is differentially required by ZFP568 and other factors during the early stages of mouse embryogenesis. In addition to uncovering novel roles of TRIM28 in convergent extension and morphogenesis of extra-embryonic tissues, our characterization of chatwo mutants demonstrates that KRAB domain proteins are essential to determine some of the biological functions of TRIM28.

The mouse KRAB zinc-finger protein CHATO is required in embryonic-derived tissues to control yolk sac and placenta morphogenesis.

Yolk sac and placenta are required to sustain embryonic development in mammals, yet our understanding of the genes and processes that control morphogenesis of these extraembryonic tissues is still limited. The chato mutation disrupts ZFP568, a Krüppel-Associated-Box (KRAB) domain Zinc finger protein, and causes a unique set of extraembryonic malformations, including ruffling of the yolk sac membrane, defective extraembryonic mesoderm morphogenesis and vasculogenesis, failure to close the ectoplacental cavity, and incomplete placental development. Phenotypic analysis of chato embryos indicated that ZFP568 does not control proliferation or differentiation of extraembryonic lineages but rather regulates the morphogenetic events that shape extraembryonic tissues. Analysis of chimeric embryos showed that Zfp568 function is required in embryonic-derived lineages, including the extraembryonic mesoderm. Depleting Zfp568 affects the ability of extraembryonic mesoderm cells to migrate. However, explanted Zfp568 mutant cells could migrate properly when plated on appropriate extracellular matrix conditions. We show that expression of Fibronectin and Indian Hedgehog are reduced in chato mutant yolk sacs. These data suggest that ZFP568 controls the production of secreted factors required to promote morphogenesis of extraembryonic tissues. Our results support previously undescribed roles of the extraembryonic mesoderm in yolk sac morphogenesis and in the closure of the ectoplacental cavity and identify a novel role of ZFP568 in the development of extraembryonic tissues.

Chato, a KRAB zinc-finger protein, regulates convergent extension in the mouse embryo.

In Xenopus and zebrafish embryos, elongation of the anterior-posterior body axis depends on convergent extension, a process that involves polarized cell movements and is regulated by non-canonical Wnt signaling. The mechanisms that control axis elongation of the mouse embryo are much less well understood. Here, we characterize the ENU-induced mouse mutation chato, which causes arrest at midgestation and defects characteristic of convergent extension mutants, including a shortened body axis, mediolaterally extended somites and an open neural tube. The chato mutation disrupts Zfp568, a Krüppel-associated box (KRAB) domain zinc-finger protein. Morphometric analysis revealed that the definitive endoderm of mouse wild-type embryos undergoes cell rearrangements that lead to convergent extension during early somite stages, and that these cell rearrangements fail in chato embryos. Although non-canonical Wnt signaling is important for convergent extension in the mouse notochord and neural plate, the results indicate that chato regulates body axis elongation in all embryonic tissues through a process independent of non-canonical Wnt signaling.

Analysis of mouse embryonic patterning and morphogenesis by forward genetics.

Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.

Tcf3: a transcriptional regulator of axis induction in the early embryo.

The roles of Lef/Tcf proteins in determining cell fate characteristics have been described in many contexts during vertebrate embryogenesis, organ and tissue homeostasis, and cancer formation. Although much of the accumulated work on these proteins involves their ability to transactivate target genes when stimulated by beta-catenin, Lef/Tcf proteins can repress target genes in the absence of stabilized beta-catenin. By ablating Tcf3 function, we have uncovered an important requirement for a repressor function of Lef/Tcf proteins during early mouse development. Tcf3-/- embryos proceed through gastrulation to form mesoderm, but they develop expanded and often duplicated axial mesoderm structures, including nodes and notochords. These duplications are preceded by ectopic expression of Foxa2, an axial mesoderm gene involved in node specification, with a concomitant reduction in Lefty2, a marker for lateral mesoderm. By contrast, expression of a beta-catenin-dependent, Lef/Tcf reporter (TOPGal), is not ectopically activated but is faithfully maintained in the primitive streak. Taken together, these data reveal a unique requirement for Tcf3 repressor function in restricting induction of the anterior-posterior axis.

Essential role of glycosaminoglycans in Fgf signaling during mouse gastrulation.

In vitro studies have suggested that proteoglycans facilitate signaling by mammalian growth factors, but genetic evidence supporting this role has been lacking. Here, we characterize the ENU-induced mutation lazy mesoderm (lzme), which disrupts the single mouse gene encoding UDP-glucose dehydrogenase (Ugdh), an enzyme required for the synthesis of the glycosaminoglycan (GAG) side chains of proteoglycans. lzme mutants arrest during gastrulation with defects in migration of mesoderm and endoderm, a phenotype similar to that of mutants in the fibroblast growth factor (Fgf) pathway. Analysis of the expression of molecular markers indicates that Fgf signaling is blocked in lzme mutant embryos. In contrast, signaling by the growth factors Nodal and Wnt3, which are also essential during mouse gastrulation, appears to be normal in lzme embryos. The results demonstrate that proteoglycans are required during mouse gastrulation specifically to promote Fgf signaling.