Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo.

OBJECTIVE: The mammalian Notch ligand DLL1 has essential functions during development. To visualise DLL1 in tissues, for sorting and enrichment of DLL1-expressing cells, and to efficiently purify DLL1 protein complexes we tagged DLL1 in mice with AcGFPHA or Strep/FLAG. RESULTS: We generated constructs to express DLL1 that carried C-terminal in-frame an AcGFPHA tag flanked by loxP sites followed by a Strep/FLAG (SF) tag out of frame. Cre-mediated recombination replaced AcGFP-HA by SF. The AcGFPHAstopSF cassette was added to DLL1 for tests in cultured cells and introduced into endogenous DLL1 in mice by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cells and embryo lysates. In CHO cells the AcGFP fluorophore fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the SF tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or SF-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.

The highly conserved FOXJ1 target CFAP161 is dispensable for motile ciliary function in mouse and Xenopus.

Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.

The FOXJ1 target Cfap206 is required for sperm motility, mucociliary clearance of the airways and brain development.

Cilia are complex cellular protrusions consisting of hundreds of proteins. Defects in ciliary structure and function, many of which have not been characterised molecularly, cause ciliopathies: a heterogeneous group of human syndromes. Here, we report on the FOXJ1 target gene Cfap206, orthologues of which so far have only been studied in Chlamydomonas and Tetrahymena In mouse and Xenopus, Cfap206 was co-expressed with and dependent on Foxj1 CFAP206 protein localised to the basal body and to the axoneme of motile cilia. In Xenopus crispant larvae, the ciliary beat frequency of skin multiciliated cells was enhanced and bead transport across the epidermal mucociliary epithelium was reduced. Likewise, Cfap206 knockout mice revealed ciliary phenotypes. Electron tomography of immotile knockout mouse sperm flagella indicated a role in radial spoke formation reminiscent of FAP206 function in Tetrahymena Male infertility, hydrocephalus and impaired mucociliary clearance of the airways in the absence of laterality defects in Cfap206 mutant mice suggests that Cfap206 may represent a candidate for the subgroup of human primary ciliary dyskinesias caused by radial spoke defects.

CFAP43 modulates ciliary beating in mouse and Xenopus.

Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development.

The ectodomains determine ligand function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro.

DLL1 and DLL4 are Notch ligands with high structural similarity but context-dependent functional differences. Here, we analyze their functional divergence using cellular co-culture assays, biochemical studies, and in vivo experiments. DLL1 and DLL4 activate NOTCH1 and NOTCH2 differently in cell-based assays and this discriminating potential lies in the region between the N-terminus and EGF repeat three. Mice expressing chimeric ligands indicate that the ectodomains dictate ligand function during somitogenesis, and that during myogenesis even regions C-terminal to EGF3 are interchangeable. Substitution of NOTCH1-interface residues in the MNNL and DSL domains of DLL1 with the corresponding amino acids of DLL4, however, does not disrupt DLL1 function in vivo. Collectively, our data show that DLL4 preferentially activates NOTCH1 over NOTCH2, whereas DLL1 is equally effective in activating NOTCH1 and NOTCH2, establishing that the ectodomains dictate selective ligand function in vivo, and that features outside the known binding interface contribute to their differences.

The evolutionary conserved FOXJ1 target gene Fam183b is essential for motile cilia in Xenopus but dispensable for ciliary function in mice.

The transcription factor FOXJ1 is essential for the formation of motile cilia throughout the animal kingdom. Target genes therefore likely constitute an important part of the motile cilia program. Here, we report on the analysis of one of these targets, Fam183b, in Xenopus and mice. Fam183b encodes a protein with unknown function which is conserved from the green algae Chlamydomonas to humans. Fam183b is expressed in tissues harbouring motile cilia in both mouse and frog embryos. FAM183b protein localises to basal bodies of cilia in mIMCD3 cells and of multiciliated cells of the frog larval epidermis. In addition, FAM183b interacts with NUP93, which also localises to basal bodies. During frog embryogenesis, Fam183b was dispensable for laterality specification and brain development, but required for ciliogenesis and motility of epidermal multiciliated cells and nephrostomes, i.e. the embryonic kidney. Surprisingly, mice homozygous for a null allele did not display any defects indicative of disrupted motile ciliary function. The lack of a cilia phenotype in mouse and the limited requirements in frog contrast with high sequence conservation and the correlation of gene expression with the presence of motile cilia. This finding may be explained through compensatory mechanisms at sites where no defects were observed in our FAM183b-loss-of-function studies.

Functional characterization of the mouse Serpina1 paralog DOM-7.

The generation of authentic mouse-models for human α1-antitrypsin (A1AT)-deficiency is difficult due to the high complexity of the mouse Serpina1 gene locus. Depending on the exact mouse strain, three to five paralogs are expressed, with different proteinase inhibitory properties. Nowadays with CRISPR-technology, genome editing of complex genomic loci is feasible and could be employed for the generation of A1AT-deficiency mouse models. In preparation of a CRISPR/Cas9-based genome-engineering approach we identified cDNA clones with a functional CDS for the Serpina1-paralog DOM-7. Here, we show that DOM-7 functionally inhibits neutrophil elastase (ELANE) and chymotrypsin, and therefore needs to be considered when aiming at the generation of A1AT-deficient models.

A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

1700012B09Rik, a FOXJ1 effector gene active in ciliated tissues of the mouse but not essential for motile ciliogenesis.

In humans and mice, motile cilia occur on the surface of the embryonic ventral node, on respiratory and ependymal epithelia and in reproductive organs where they ensure normal left-right asymmetry of the organism, mucociliary clearance of airways, homeostasis of the cerebrospinal fluid and fertility. The genetic programme for the formation of motile cilia, thus critical for normal development and health, is switched on by the key transcription factor FOXJ1. In previous microarray screens for murine FOXJ1 effectors, we identified candidates for novel factors involved in motile ciliogenesis, including both genes that are well conserved throughout metazoa and beyond, like FOXJ1 itself, and genes without overt homologues outside higher vertebrates. Here we examine one of the novel murine FOXJ1 effectors, the uncharacterised 1700012B09Rik whose homologues appear to be restricted to higher vertebrates. In mouse embryos and adults, 1700012B09Rik is predominantly expressed in motile ciliated tissues in a FOXJ1-dependent manner. 1700012B09RIK protein localises to basal bodies of cilia in cultured cells. Detailed analysis of 1700012B09RiklacZ knock-out mice reveals no impaired function of motile cilia or non-motile cilia. In conclusion, this novel FOXJ1 effector is associated mainly with motile cilia but - in contrast to other known FOXJ1 targets - its putative ciliary function is not essential for development or health in the mouse, consistent with a late emergence during evolution of motile ciliogenesis.

CFAP157 is a murine downstream effector of FOXJ1 that is specifically required for flagellum morphogenesis and sperm motility.

Motile cilia move extracellular fluids or mediate cellular motility. Their function is essential for embryonic development, adult tissue homeostasis and reproduction throughout vertebrates. FOXJ1 is a key transcription factor for the formation of motile cilia but its downstream genetic programme is only partially understood. Here, we characterise a novel FOXJ1 target, Cfap157, that is specifically expressed in motile ciliated tissues in mouse and Xenopus in a FOXJ1-dependent manner. CFAP157 protein localises to basal bodies and interacts with tubulin and the centrosomal protein CEP350. Cfap157 knockout mice appear normal but homozygous males are infertile. Spermatozoa display impaired motility and a novel phenotype: Cfap157-deficient sperm exhibit axonemal loops, supernumerary axonemal profiles with ectopic accessory structures, excess cytoplasm and clustered mitochondria in the midpiece regions, and defective axonemes along the flagella. Our study thus demonstrates an essential sperm-specific function for CFAP157 and suggests that this novel FOXJ1 effector is part of a mechanism that acts during spermiogenesis to suppress the formation of supernumerary axonemes and ensures a correct ultrastructure.

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1.

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.

Generation of an 870 kb deletion encompassing the Skt/Etl4 locus by combination of inter- and intra-chromosomal recombination.

BACKGROUND: Etl4(lacZ) (Enhancer trap locus 4) and Skt(Gt) (Sickle tail) are lacZ reporter gene integrations into the same locus on mouse chromosome 2 targeting a gene that is expressed in the notochord of early embryos and in multiple epithelia during later development. Both insertions caused recessive mutations that resulted exclusively in mild defects in the caudal vertebral column. Since notochord-derived signals are essential for formation of the vertebral column the phenotypes suggested that the lacZ insertions interfered with some notochord-dependent aspect of vertebral development. As both insertions occurred in introns it was unclear whether they represent hypomorphic alleles or abolish gene function. Here, we have generated a definitive null allele of the Skt/Etl4 gene and analysed homozygous mutants. RESULTS: We have introduced loxP sites into three positions of the gene based on additional upstream exons that we identified, and deleted approximately 870 kb of the locus by a combination of inter- and intra-chromosomal Cre-mediated recombinations in the female germ line of mice. This deletion removes about 90 % of the coding region and results in the loss of the SKT/ETL4 protein. Similar to the Etl4(lacZ) and Skt(Gt) alleles our deletion mutants are viable and fertile and show only mild defects in caudal vertebrae due to abnormal intervertebral disc development, although with higher penetrance. No other tissue with Skt/Etl4 expression that we analysed showed obvious defects. CONCLUSION: The complete loss of Skt/Etl4 function affects only development of caudal notochord derivatives and is compensated for in its other expression domains.

Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo.

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1Dll4ki/+ mice, the Dll1Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.

O-fucosylation of DLL3 is required for its function during somitogenesis.

Delta-like 3 (DLL3) is a member of the DSL family of Notch ligands in amniotes. In contrast to DLL1 and DLL4, the other Delta-like proteins in the mouse, DLL3 does not bind in trans to Notch and does not activate the receptor, but shows cis-interaction and cis-inhibitory properties on Notch signaling in vitro. Loss of the DSL protein DLL3 in the mouse results in severe somite patterning defects, which are virtually indistinguishable from the defects in mice that lack lunatic fringe (LFNG), a glycosyltransferase involved in modifying Notch signaling. Like LFNG, DLL3 is located within the trans-Golgi, however, its biochemical function is still unclear. Here, we show that i) both proteins interact, ii) epidermal growth factor like repeats 2 and 5 of DLL3 are O-fucosylated at consensus sites for POFUT1, and iii) further modified by FNG proteins in vitro. Embryos double homozygous for null mutations in Dll3 and Lfng are phenotypically indistinguishable from the single mutants supporting a potential common function. Mutation of the O-fucosylation sites in DLL3 does not disrupt the interaction of DLL3 with LFNG or full length Notch1or DLL1, and O-fucosylation-deficient DLL3 can still inhibit Notch in cis in vitro. However, in contrast to wild type DLL3, O-fucosylation-deficient DLL3 cannot compensate for the loss of endogenous DLL3 during somitogenesis in the embryo. Together our results suggest that the cis-inhibitory activity of DLL3 observed in cultured cells might not fully reflect its assumed essential physiological property, suggest that DLL3 and LFNG act together, and strongly supports that modification of DLL3 by O-linked fucose is essential for its function during somitogenesis.

Upk3b is dispensable for development and integrity of urothelium and mesothelium.

The mesothelium, the lining of the coelomic cavities, and the urothelium, the inner lining of the urinary drainage system, are highly specialized epithelia that protect the underlying tissues from mechanical stress and seal them from the overlying fluid space. The development of these epithelia from simple precursors and the molecular characteristics of the mature tissues are poorly analyzed. Here, we show that uroplakin 3B (Upk3b), which encodes an integral membrane protein of the tetraspanin superfamily, is specifically expressed both in development as well as under homeostatic conditions in adult mice in the mesothelia of the body cavities, i.e., the epicardium and pericardium, the pleura and the peritoneum, and in the urothelium of the urinary tract. To analyze Upk3b function, we generated a creERT2 knock-in allele by homologous recombination in embryonic stem cells. We show that Upk3bcreERT2 represents a null allele despite the lack of creERT2 expression from the mutated locus. Morphological, histological and molecular analyses of Upk3b-deficient mice did not detect changes in differentiation or integrity of the urothelium and the mesothelia that cover internal organs. Upk3b is coexpressed with the closely related Upk3a gene in the urothelium but not in the mesothelium, leaving the possibility of a functional redundancy between the two genes in the urothelium only.

Genetic deletion of SEPT7 reveals a cell type-specific role of septins in microtubule destabilization for the completion of cytokinesis.

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.

S/T phosphorylation of DLL1 is required for full ligand activity in vitro but dispensable for DLL1 function in vivo during embryonic patterning and marginal zone B cell development.

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.

Normal development in mice over-expressing the intracellular domain of DLL1 argues against reverse signaling by DLL1 in vivo.

The Notch signaling pathway mediates the direct communication between adjacent cells and regulates multiple developmental processes. Interaction of the Notch receptor with its ligands induces the liberation of the intracellular portion of Notch (NICD) referred to as regulated intramembraneous proteolysis (RIP). NICD translocates to the nucleus, and by complexing with the DNA binding protein RBPjκ and other cofactors activates transcription of bHLH genes. RIP-like processing of various mammalian Notch ligands (DLL1, JAG1 and JAG2) and the translocation of their intracellular domains (ICDs) to the nucleus has also been observed. These observations together with effects of over-expressed ligand ICDs in cultured cells on cell proliferation, differentiation, and Notch activity and target gene expression have led to the idea that the intracellular domains of Notch ligands have signaling functions. To test this hypothesis in vivo we have generated ES cells and transgenic mice that constitutively express various versions of the intracellular domain of mouse DLL1. In contrast to other cell lines, expression of DICDs in ES cells did not block proliferation or stimulate neuronal differentiation. Embryos with ubiquitous DICD expression developed to term without any apparent phenotype and grew up to viable and fertile adults. Early Notch-dependent processes or expression of selected Notch target genes were unaltered in transgenic embryos. In addition, we show that mouse DICD enters the nucleus inefficiently. Collectively, our results argue against a signaling activity of the intracellular domain of DLL1 in mouse embryos in vivo.

Tbx2 terminates shh/fgf signaling in the developing mouse limb bud by direct repression of gremlin1.

Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.

Tbx2 controls lung growth by direct repression of the cell cycle inhibitor genes Cdkn1a and Cdkn1b.

Vertebrate organ development relies on the precise spatiotemporal orchestration of proliferation rates and differentiation patterns in adjacent tissue compartments. The underlying integration of patterning and cell cycle control during organogenesis is insufficiently understood. Here, we have investigated the function of the patterning T-box transcription factor gene Tbx2 in lung development. We show that lungs of Tbx2-deficient mice are markedly hypoplastic and exhibit reduced branching morphogenesis. Mesenchymal proliferation was severely decreased, while mesenchymal differentiation into fibrocytes was prematurely induced. In the epithelial compartment, proliferation was reduced and differentiation of alveolar epithelial cells type 1 was compromised. Prior to the observed cellular changes, canonical Wnt signaling was downregulated, and Cdkn1a (p21) and Cdkn1b (p27) (two members of the Cip/Kip family of cell cycle inhibitors) were strongly induced in the Tbx2-deficient lung mesenchyme. Deletion of both Cdkn1a and Cdkn1b rescued, to a large degree, the growth deficits of Tbx2-deficient lungs. Prolongation of Tbx2 expression into adulthood led to hyperproliferation and maintenance of mesenchymal progenitor cells, with branching morphogenesis remaining unaffected. Expression of Cdkn1a and Cdkn1b was ablated from the lung mesenchyme in this gain-of-function setting. We further show by ChIP experiments that Tbx2 directly binds to Cdkn1a and Cdkn1b loci in vivo, defining these two genes as direct targets of Tbx2 repressive activity in the lung mesenchyme. We conclude that Tbx2-mediated regulation of Cdkn1a and Cdkn1b represents a crucial node in the network integrating patterning information and cell cycle regulation that underlies growth, differentiation, and branching morphogenesis of this organ.