Background

Primary cilia are microtubule-based sensory organelles that play important roles in developmental signaling pathways. Recent work demonstrated that, in cell culture, the daughter cell that inherits the older mother centriole generates a primary cilium and responds to external stimuli prior to its sister cell. This asynchrony in timing of cilia formation could be especially critical during development as cell divisions are required for both differentiation and maintenance of progenitor cell niches.

Methods

Here we integrate several fluorescent markers and use ex vivo live imaging of a single cell division within the mouse E8.5 neuroepithelium to reveal both the formation of a primary cilium and the transcriptional response to Sonic hedgehog in the daughter cells.

Results

We show that, upon cell division, cilia formation and the Sonic hedgehog response are asynchronous between the daughter cells.

Conclusions

Our results demonstrate that we can directly observe single cell divisions within the developing neuroepithelium and concomitantly monitor cilium formation or Sonic hedgehog response. We expect this method to be especially powerful in examining whether cellular behavior can lead to both differentiation and maintenance of cells in a progenitor niche.

Primary cilia are present on almost all vertebrate cells, and they have diverse functions in distinct tissues. Cilia are important for sensation in multiple capacities in contexts as different as the retina, kidney, and inner ear. In addition to these roles, cilia play a critical part in various developmental processes. Of particular importance is the development of the neural tube, where cilia are essential for the transduction of the Sonic Hedgehog (Shh) signaling pathway that specifies neuronal cell fates. This relationship is well established and is the most recognizable function for cilia in the neural tube, but it may be part of a larger picture. Here, we discuss the links between cilia and Shh signaling, as well as suggesting additional roles for cilia, and mechanisms for their placement, in the neural tube.

In the embryonic neural tube, multiple signaling pathways work in concert to create functional neuronal circuits in the adult spinal cord. In the ventral neural tube, Sonic hedgehog (Shh) acts as a graded morphogen to specify neurons necessary for movement. In the dorsal neural tube, bone morphogenetic protein (BMP) and Wnt signals cooperate to specify neurons involved in sensation. Several signaling pathways, including Shh, rely on primary cilia in vertebrates. In this study, we used a mouse mutant with abnormal cilia, Arl13bhnn, to study the relationship between cilia, cell signaling, and neural tube patterning. Alr13bhnn mutants have abnormal ventral neural tube patterning due to disrupted Shh signaling; in addition, dorsal patterning defects occur, but the cause of these is unknown. Here we show that the Arl13bhnn dorsal patterning defects result from abnormal BMP signaling. In addition, we find that Wnt ligands are abnormally expressed in Arl13bhnn mutants; surprisingly, however, downstream Wnt signaling is normal. We demonstrate that Arl13b is required non-autonomously for BMP signaling and Wnt ligand expression, indicating that the abnormal Shh signaling environment in Arl13bhnn embryos indirectly causes dorsal defects.

Background

Primary cilia are microtubule-based sensory organelles that play important roles in developmental signaling pathways. Recent work demonstrated that, in cell culture, the daughter cell that inherits the older mother centriole generates a primary cilium and responds to external stimuli prior to its sister cell. This asynchrony in timing of cilia formation could be especially critical during development as cell divisions are required for both differentiation and maintenance of progenitor cell niches.

Methods

Here we integrate several fluorescent markers and use ex vivo live imaging of a single cell division within the mouse E8.5 neuroepithelium to reveal both the formation of a primary cilium and the transcriptional response to Sonic hedgehog in the daughter cells.

Results

We show that, upon cell division, cilia formation and the Sonic hedgehog response are asynchronous between the daughter cells.

Conclusions

Our results demonstrate that we can directly observe single cell divisions within the developing neuroepithelium and concomitantly monitor cilium formation or Sonic hedgehog response. We expect this method to be especially powerful in examining whether cellular behavior can lead to both differentiation and maintenance of cells in a progenitor niche.

We show Arl13b is localized to the ciliary membrane and regulates tubulin modifications and ciliary length in vitro. Significantly, we found that Smoothened is enriched in Arl13b null fibroblasts, even without Sonic hedgehog stimulation, but that Glis are not similarly enriched.

Arl13b, a ciliary protein within the ADP-ribosylation factor family and Ras superfamily of GTPases, is required for ciliary structure but has poorly defined ciliary functions. In this paper, we further characterize the role of Arl13b in cilia by examining mutant cilia in vitro and determining the localization and dynamics of Arl13b within the cilium. Previously, we showed that mice lacking Arl13b have abnormal Sonic hedgehog (Shh) signaling; in this study, we show the dynamics of Shh signaling component localization to the cilium are disrupted in the absence of Arl13b. Significantly, we found Smoothened (Smo) is enriched in Arl13b-null cilia regardless of Shh pathway stimulation, indicating Arl13b regulates the ciliary entry of Smo. Furthermore, our analysis defines a role for Arl13b in regulating the distribution of Smo within the cilium. These results suggest that abnormal Shh signaling in Arl13b mutant embryos may result from defects in protein localization and distribution within the cilium.

Forward genetic screens in Mus musculus have proved powerfully informative by revealing unsuspected mechanisms governing basic biological processes. This approach uses potent chemical mutagens, such as N-ethyl-N-nitrosourea (ENU), to randomly induce mutations in mice, which are then bred and phenotypically screened to identify lines that disrupt a specific biological process of interest. Although identifying a mutation using the rich resources of mouse genetics is straightforward, it is unfortunately neither fast nor cheap. Here we show that detecting newly induced causal variants in a forward genetic screen can be accelerated dramatically using a methodology that combines multiplex chromosome-specific exome capture, next-generation sequencing, rapid mapping, sequence annotation, and variation filtering. The key innovation of our method is multiplex capture and sequence that allows the simultaneous survey of both mutant, parental, and background strains in a single experiment. By comparing variants identified in mutant offspring with those found in dbSNP, the unmutagenized background strains, and parental lines, induced causative mutations can be distinguished immediately from preexisting variation or experimental artifact. Here we demonstrate this approach to find the causative mutations induced in four novel ENU lines identified from a recent ENU screen. In all four cases, after applying our method, we found six or fewer putative mutations (and sometimes only a single one). Determining the causative variant was then easily achieved through standard segregation approaches. We have developed this process into a community resource that will speed up individual labs’ ability to identify the genetic lesion in mutant mouse lines; all of our reagents and software tools are open source and available to the broader scientific community.

Apoptosis and necroptosis are complementary pathways controlled by common signaling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor (DR)-induced apoptosis. This caspase has also been implicated in nonapoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signaling and other less defined biological processes as diverse as innate immune signaling and myeloid or lymphoid differentiation patterns 1. Casp8 suppresses RIP3/RIP1 kinase complex-dependent 2–4 necroptosis 5 that follows DR-activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defense mechanism against murine cytomegalovirus (MCMV) 6. Disruption of Casp8 expression leads to embryonic lethality in mice between E10.5 and E11.5 7. Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to its role promoting apoptosis. We find that RIP3 is responsible for the midgestational death of Casp8-deficient embryos. Remarkably, Casp8−/−Rip3−/− double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice appear immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr). Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.

Methods of genomic selection that combine high-density oligonucleotide microarrays with next-generation DNA sequencing allow investigators to characterize genomic variation in selected portions of complex eukaryotic genomes. Yet choosing which specific oligonucleotides to be use can pose a major technical challenge. To address this issue, we have developed a software package called MOPeD (Microarray Oligonucleotide Probe Designer), which automates the process of designing genomic selection microarrays. This web-based software allows individual investigators to design custom genomic selection microarrays optimized for synthesis with Roche NimbleGen’s maskless photolithography. Design parameters include uniqueness of the probe sequences, melting temperature, hairpin formation, and the presence of single nucleotide polymorphisms. We generated probe databases for the human, mouse, and rhesus macaque genomes and conducted experimental validation of MOPeD-designed microarrays in human samples by sequencing the human X chromosome exome, where relevant sequence metrics indicated superior performance relative to a microarray designed by the Roche NimbleGen proprietary algorithm. We also performed validation in the mouse to identify known mutations contained within a 487-kb region from mouse chromosome 16, the mouse chromosome 16 exome (1.7 Mb), and the mouse chromosome 12 exome (3.3 Mb). Our results suggest that the open source MOPeD software package and website (http://moped.genetics.emory.edu/) will make a valuable resource for investigators in their sequence-based studies of complex eukaryotic genomes.

Humans with L1 cell adhesion molecule (L1CAM) mutations exhibit X-linked hydrocephalus, as well as other severe neurological disorders. L1-6D mutant mice, which are homozygous for a deletion that removes the sixth immunoglobulin-like domain of L1cam, seldom display hydrocephalus on the 129/Sv background. However, the same L1-6D mutation produces severe hydrocephalus on the C57BL/6J background. To begin to understand how L1cam deficiencies result in hydrocephalus and to identify modifier loci that contribute to X-linked hydrocephalus by genetically interacting with L1cam, we conducted a genome-wide scan on F2 L1-6D mice, bred from L1-6D 129S2/SvPasCrlf and C57BL/6J mice. Linkage studies, utilizing chi-square tests and quantitative trait loci mapping techniques, were performed. Candidate modifier loci were further investigated in an extension study. Linkage was confirmed for a locus on chromosome 5, which we named L1cam hydrocephalus modifier 1 (L1hydro1), p = 4.04 × 10−11.

Background

The enormous throughput and low cost of second-generation sequencing platforms now allow research and clinical geneticists to routinely perform single experiments that identify tens of thousands to millions of variant sites. Existing methods to annotate variant sites using information from publicly available databases via web browsers are too slow to be useful for the large sequencing datasets being routinely generated by geneticists. Because sequence annotation of variant sites is required before functional characterization can proceed, the lack of a high-throughput pipeline to efficiently annotate variant sites can act as a significant bottleneck in genetics research.

Results

SeqAnt (Sequence Annotator) is an open source web service and software package that rapidly annotates DNA sequence variants and identifies recessive or compound heterozygous loci in human, mouse, fly, and worm genome sequencing experiments. Variants are characterized with respect to their functional type, frequency, and evolutionary conservation. Annotated variants can be viewed on a web browser, downloaded in a tab-delimited text file, or directly uploaded in a BED format to the UCSC genome browser. To demonstrate the speed of SeqAnt, we annotated a series of publicly available datasets that ranged in size from 37 to 3,439,107 variant sites. The total time to completely annotate these data completely ranged from 0.17 seconds to 28 minutes 49.8 seconds.

Conclusion

SeqAnt is an open source web service and software package that overcomes a critical bottleneck facing research and clinical geneticists using second-generation sequencing platforms. SeqAnt will prove especially useful for those investigators who lack dedicated bioinformatics personnel or infrastructure in their laboratories.

Mammalian Argonaute proteins (EIF2C1−4) play an essential role in RNA-induced silencing. Here, we show that the loss of eIF2C2 (Argonaute2 or Ago2) results in gastrulation arrest, ectopic expression of Brachyury (T), and mesoderm expansion. We identify a genetic interaction between Ago2 and T, as Ago2 haploinsufficiency partially rescues the classic T/+ short-tail phenotype. Finally, we demonstrate that the ectopic T expression and concomitant mesoderm expansion result from disrupted fibroblast growth factor signaling, likely due to aberrant expression of Eomesodermin. Together, these data indicate that a factor best known as a key component of the RNA-induced silencing complex is required for proper fibroblast growth factor signaling during gastrulation, suggesting a possible micro-RNA function in the formation of a mammalian germ layer.

Author Summary

Gastrulation is a developmental phase that delineates the three embryonic germ layers: ectoderm, endoderm, and mesoderm. The gene Brachyury is essential for mesoderm development, and short-tail mice, which were later found to be carrying a Brachyury mutation, have been known since 1927. In this study, we found a genetic interaction between Brachyury and another gene in mouse, Argonaute2. We show that the loss of Argonauate2, a necessary component of a recently appreciated pathway of gene regulation called RNA interference, results in embryonic death during gastrulation, abnormal expression of Brachyury, and expansion of the mesoderm layer. This suggests that Argonaute2 is important in early development and in regulating Brachyury function. Consistent with this conclusion, we found that mice simultaneously carrying mutations in both Argonaute2 and Brachyury have significantly longer tails than mice with only a Brachyury mutation. A closer look at other genes involved in mesoderm development revealed that a disruption in fibroblast growth factor signaling may explain the mesoderm expansion in mice carrying the Argonaute2 mutation. Together this work demonstrates that a factor best known as a key component of RNA interference is required for the formation of a mammalian germ layer.

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57Kip2, as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57Kip2 during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57Kip2, and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57Kip2 have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57Kip2 is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).