Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates.

The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.

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 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.

Identification of FOXJ1 effectors during ciliogenesis in the foetal respiratory epithelium and embryonic left-right organiser of the mouse.

Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1+ vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto+ vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap - including 1700012B09Rik, 1700026L06Rik and Fam183b - we provide extended experimental evidence for a ciliary function.

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.

Differential regulation of node formation, nodal ciliogenesis and cilia positioning by Noto and Foxj1.

The mouse transcription factor Noto is expressed in the node and controls node morphogenesis, formation of nodal cilia and left-right asymmetry. Noto acts upstream of Foxj1, which regulates ciliogenesis in other mouse tissues. However, the significance of Foxj1 for the formation of cilia in the mouse node is unclear; in non-amniote species Foxj1 is required for ciliogenesis in the structures equivalent to the node. Here, we analyzed nodes, nodal cilia and nodal flow in mouse embryos in which we replaced the Noto-coding sequence with that of Foxj1, or in embryos that were deficient for Foxj1. We show that Foxj1 expressed from the Noto locus is functional and restores the formation of structurally normal motile cilia in the absence of Noto. However, Foxj1 is not sufficient for the correct positioning of cilia on the cell surface within the plane of the nodal epithelium, and cannot restore normal node morphology. We also show that Foxj1 is essential for ciliogenesis upstream of Rfx3 in the node. Thus, the function of Foxj1 in vertebrate organs of asymmetry is conserved, and Noto regulates node morphogenesis and the posterior localization of cilia on node cells independently of Foxj1.

Live imaging and genetic analysis of mouse notochord formation reveals regional morphogenetic mechanisms.

The node and notochord have been extensively studied as signaling centers in the vertebrate embryo. The morphogenesis of these tissues, particularly in mouse, is not well understood. Using time-lapse live imaging and cell lineage tracking, we show the notochord has distinct morphogenetic origins along the anterior-posterior axis. The anterior head process notochord arises independently of the node by condensation of dispersed cells. The trunk notochord is derived from the node and forms by convergent extension. The tail notochord forms by node-derived progenitors that actively migrate toward the posterior. We also reveal distinct genetic regulation within these different regions. We show that Foxa2 compensates for and genetically interacts with Noto in the trunk notochord, and that Noto has an evolutionarily conserved role in regulating axial versus paraxial cell fate. Therefore, we propose three distinct regions within the mouse notochord, each with unique morphogenetic origins.

The mouse homeobox gene Noto regulates node morphogenesis, notochordal ciliogenesis, and left right patterning.

The mouse homeobox gene Noto represents the homologue of zebrafish floating head (flh) and is expressed in the organizer node and in the nascent notochord. Previous analyses suggested that Noto is required exclusively for the formation of the caudal part of the notochord. Here, we show that Noto is also essential for node morphogenesis, controlling ciliogenesis in the posterior notochord, and the establishment of laterality, whereas organizer functions in anterior-posterior patterning are apparently not compromised. In mutant embryos, left-right asymmetry of internal organs and expression of laterality markers was randomized. Mutant posterior notochord regions were variable in size and shape, cilia were shortened with highly irregular axonemal microtubuli, and basal bodies were, in part, located abnormally deep in the cytoplasm. The transcription factor Foxj1, which regulates the dynein gene Dnahc11 and is required for the correct anchoring of basal bodies in lung epithelial cells, was down-regulated in mutant nodes. Likewise, the transcription factor Rfx3, which regulates cilia growth, was not expressed in Noto mutants, and various other genes important for cilia function or assembly such as Dnahc5 and Nphp3 were down-regulated. Our results establish Noto as an essential regulator of node morphogenesis and ciliogenesis in the posterior notochord, and suggest Noto acts upstream of Foxj1 and Rfx3.

Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6.

The vertebral column and skeletal muscles of vertebrates are derived from the paraxial mesoderm, which is laid down initially as two stripes of mesenchymal cells alongside the neural tube and subsequently segmented. Previous work has shown that the wingless-type MMTV integration site family (WNT), fibroblast growth factor- and Delta-Notch signalling pathways control presomitic mesoderm (psm) formation and segmentation. Here, we show that the expression of mesogenin 1, a basic helix-loop-helix transcription factor, which is essential for psm maturation, is regulated by synergism between WNT signalling and the T-box 6 transcription factor, involving a feed-forward control mechanism. These findings emphasize the crucial role of WNT signalling in the control of psm formation, maturation and segmentation.

The mouse homeobox gene Not is required for caudal notochord development and affected by the truncate mutation.

The floating head (flh) gene in zebrafish encodes a homeodomain protein, which is essential for notochord formation along the entire body axis. flh orthologs, termed Not genes, have been isolated from chick and Xenopus, but no mammalian ortholog has yet been identified. Truncate (tc) is an autosomal recessive mutation in mouse that specifically disrupts the development of the caudal notochord. Here, we demonstrate that truncate arose by a mutation in the mouse Not gene. The truncate allele (Nottc) contains a point mutation in the homeobox of Not that changes a conserved Phenylalanine residue in helix 1 to a Cysteine (F20C), and significantly destabilizes the homeodomain. Reversion of F20C in one allele of homozygous tc embryonic stem (ES) cells is sufficient to restore normal notochord formation in completely ES cell-derived embryos. We have generated a targeted mutation of Not by replacing most of the Not coding sequence, including the homeobox with the eGFP gene. The phenotype of NoteGFP/eGFP, NoteGFP/tc, and Nottc/tc embryos is very similar but slightly more severe in NoteGFP/eGFP than in Nottc/tc embryos. This confirms allelism of truncate and Not, and indicates that tc is not a complete null allele. Not expression is abolished in Foxa2 and T mutant embryos, suggesting that Not acts downstream of both genes during notochord development. This is in contrast to zebrafish embryos, in which flh interacts with ntl (zebrafish T) in a regulatory loop and is essential for development of the entire notochord, and suggests that different genetic control circuits act in different vertebrate species during notochord formation.