Embryonic stem cells (ESCs) hold tremendous promise for cell based therapeutics and as a biological tool. As differentiation of ESCs mimics early development, ESCs also serve as an excellent tool to study the underlying mechanisms of many developmental processes and cancer. The transition from ESC to somatic cell requires proper signaling as well as many intricate layers of gene regulation. Clarifying the interplay between these different processes is important for understanding the many transitions that happen during development as well as those that occur upon cancer metastasis.

, To elucidate some of the molecular mechanisms of ESC differentiation, we explored different areas of regulation within ESCs including cell signaling as well as epigenetic and transcription gene regulation. To best investigate these areas, we developed a recombinant gene technology called the Floxin system, in which we specifically and efficiently tagged genes within their endogenous loci in ESCs. The gene trap and tagged ESC lines were used to identify novel binding partners and investigate disease alleles.

Through the Floxin system, we demonstrate that Ofd1, a protein required for ciliogenesis, restrains neural differentiation in embryoid bodies (EBs). We also show that Ofd1 is essential for proper Hh and Wnt signaling and that disease alleles of Ofd1 cannot rescue proper neural differentiation. Secondly, we investigate Polycomb-like 3 (Pcl3), a protein involved in histone modifications and gene repression. We found that Pcl3 incorporates into Polycomb repressive complex 2 (PRC2) and promotes complex function. Diminishment of Pcl3 decreased PRC2 binding as well as ESC self-renewal, indicating that Pcl3 enhances PRC2 targeting and ESC maintenance. Finally, we identify novel binding partners of Sall4, a transcription factor important for ESC pluripotency and reprogramming.

Notably, Ofd1, Pcl3, and Sall4 are all associated with human conditions and cancer. Thus besides understanding how these proteins function in ESC maintenance and differentiation, we have gained insight into the mechanisms by which disruption of these proteins contributes to human disease.

We describe here a resource of mouse embryonic stem (ES) cell lines that contain gene trap insertions capable of post-insertional modification. We demonstrate Floxin technology for efficient targeted modification of gene trap alleles. Loss-of-function gene trap mutations created with the pGTLxf and pGTLxr vectors are reverted and new DNA sequences inserted into the locus using Cre recombinase and a shuttle vector, pFloxin. As proof-of-principle, we used this strategy to create targeted modification of several gene trap alleles. pFloxin-contained DNA constructs are efficiently and precisely inserted, and are regulated by endogenous promoters. Possible applications include the generation of point mutations, humanized alleles, tagged alleles and insertion of non-homologous constructs such as fluorescent reporters. The resource contains ES cell lines with compatible gene traps in more than 4,500 genes, enabling the high-throughput modification of many genes in mouse ES cells.

Centrosomes and their component centrioles represent the principal microtubule organizing centers of animal cells. Here we show that the gene underlying Orofaciodigital Syndrome 1, Ofd1, is a component of the distal centriole that controls centriole length. In the absence of Ofd1, distal regions of centrioles, but not procentrioles, elongate abnormally. These long centrioles are structurally similar to normal centrioles, but contain destabilized microtubules with abnormal post-translational modifications. Ofd1 is also important for centriole distal appendage formation and centriolar recruitment of the intraflagellar transport protein Ift88. To model OFD1 Syndrome in embryonic stem cells, we replaced the Ofd1 gene with missense alleles from human OFD1 patients. Distinct disease-associated mutations cause different degrees of excessive or decreased centriole elongation, all of which are associated with diminished ciliogenesis. Our results indicate that Ofd1 acts at the distal centriole to build distal appendages, recruit Ift88, and stabilize centriolar microtubules at a defined length.

Ciliopathies, diseases that arise from defective ciliary function, afflict many tissues to various degrees. However the genetic basis underlying the range of phenotypes is unclear. Two protein complexes at the ciliary transition zone are linked to the ciliopathies Meckel syndrome (MKS) and nephronophthisis (NPHP); the BBSome is a third complex linked to Bardet-Biedl syndrome. To model the complex inheritance of ciliopathies, we made double mutants between MKS, NPHP, and BBS genes, focusing on tctn-1, the C. elegans ortholog of the Tectonic family of ciliopathy genes. Loss of TCTN-1 or TCTN-1 and an MKS complex component does not disrupt ciliary structure. In contrast, loss of TCTN-1 and an NPHP complex component disrupts ciliary structure, as does loss of an NPHP and a BBS complex component. Thus, the MKS, NPHP and BBS complexes have overlapping ciliogenic functions in C. elegans. Similar genetic interactions that modify ciliopathy phenotypes are conserved in mammals, as mice mutant for both an MKS family gene and an NPHP family gene display exacerbated phenotypes compared to single mutants. Tctn1 and Bbs1 also show a synthetic genetic interaction in mammals. Together, our data support the hypothesis that multiple mutations within an individual organism may explain the expressivity observed in ciliopathy patients.

In addition, we investigated the role of Odr4 in mammals, a gene that is required for the localization of GPCRs to cilia in C. elegans. Preliminary studies demonstrate that Odr4 is required for embryonic development and binds to the Ufm1-specific protease, UfSP2.

During development, the Hedgehog (Hh) signaling pathway controls growth, cell fate decisions and morphogenesis. Hh signaling has long been an intense area of research, as defects in this pathway are associated with a number of congenital defects and cancers. Within the past few years, studies have illuminated that vertebrate Hh signaling requires primary cilia, microtubule-based organelles that project from the surface of most cells. To elucidate how cilia regulate Hh signaling, we chose to focus on the two central activators of the pathway, the seven transmembrane protein, Smoothened, and its downstream effector, Gli2.

Vertebrate Smo (vSmo) translocation to the primary cilium is a necessary and regulated step in the Hh pathway. Cilia, however, are dispensable in Drosophila Hh signaling and dSmo requires other mechanisms for activation. To explore how Smo activation and the requirement for the primary cilium have evolutionarily diverged, we examined vSmo phosphorylation and generated a panel of dSmo-vSmo chimeric proteins. We demonstrate that vSmo may be phosphorylated by CKIä/å, proteins which localize to the base of the primary cilium. Although phosphorylation by Grk2 and interaction with âarrestin2 have been implicated in vSmo ciliary localization and activation, we show that neither are necessary. Through our chimeric analyses, we observe that the cytoplasmic tail, which contains a previously described ciliary localization motif, is necessary, but not sufficient, for ciliary translocation.

Gli2 is the primary transcriptional activator of the Hh pathway and has been shown to be enriched at the primary cilium, particularly upon pathway activation. However, the molecular mechanisms that regulate this translocation remain largely unexplored. To investigate Gli2 subcellular dynamics, we used recombinant gene technology to specifically modify the Gli2 locus in mouse embryonic stem cells. We generated a knock-in mouse model expressing Gli2-GFP and find that this allele functions as a hypomorph in vivo. We demonstrate how specific domains and post-translational modifications regulate Gli2 nuclear enrichment, binding partners and function. We identify a central region of Gli2 that is required, but not sufficient for Gli2 ciliary localization. Together, these studies provide insight into how Gli2 may be functioning at the subcellular level, particularly at the primary cilium.

Human accelerated regions (HARS), which are genome sequences that have evolved at an accelerated rate in the human lineage, have previously been shown to include developmental enhancers. I have used a massively parallel enhancer reporter assay (MPRA) to test a library of human and chimp HAR orthologs in a cell model of mammalian testes, in order to find HARS which have evolved in human and may underly unique human reproductive traits. Through luciferase assay, 4C-seq, transcriptional analysis and Crispr/Cas9 deletion I have validated a candidate and shown that it is an enhancer of the hedgehog signaling pathway effector Gli2 in the testis cell model. Furthermore, I have shown that the enhancer is required for normal Gli2 expression in Leydig cells, as well as normal testis vascular development and normal inter-male social behavior.

Primary cilia are signaling organelles that project from the apical surface of almost all vertebrate cells. The signaling capabilities of primary cilia stem from their unique membrane protein composition: even though the ciliary membrane is continuous with the plasma membrane, certain membrane proteins localize specifically to the ciliary membrane. This dissertation describes how the ciliary membrane protein composition is maintained, and how the complex responsible for ciliary membrane composition is localized. Using a combination of molecular biology, cell biology, and biochemistry, I have investigated the role of TMEM231 in the formation and function of the transition zone in cilia during development and in disease, and the role of INTU in localizing TMEM231 during ciliogenesis.

I show that TMEM231 is a component of the Meckel Syndrome (MKS) complex, and is responsible for organizing the MKS complex at the transition zone. In the absence of Tmem231, mouse embryos display phenotypes similar to those found in MKS, which is a genetic disorder characterized by polydactyly, cystic kidneys, and exencephaly. Using co-immunoprecipitation, I confirmed that TMEM231 physically interacts with other known MKS complex components, including B9D1 and MKS1. In addition, I show that TMEM231 functions with the MKS complex, which is required for ciliary membrane proteins to localize properly. In collaboration with human geneticists, we identified compound heterozygous mutations in TMEM231 in Oro-facio-digital syndrome type III (OFD3) patients, as well as homozygous mutations in TMEM231 in MKS patients. I discovered that these mutations are hypomorphic, as they are unable to restore full ciliary membrane protein localization in a rescue assay. These findings confirm that TMEM231 is a functional component of the MKS complex, and suggest that the mutations identified in OFD3 and MKS patients underlie the disease phenotypes.

In addition, I investigated the role of INTU in cilia function, because Intu mutant mice display phenotypes similar to MKS complex mutant mice. First, I confirm that INTU is necessary for cilia formation, and that this is not due to loss of IFT-B components. I observed that MKS complex components, including TMEM231 and TMEM67, are lost in the absence of Intu. Interestingly, another complex called the Nephronophthisis (NPHP) complex is also mislocalized. These results suggest that Intu is required for both NPHP and MKS complexes to localize properly. Finally, I looked at RPGRIP1L, the previously identified lynchpin of the NPHP and MKS complexes, and found that RPGRIP1L localizes normally in the absence of Intu. These data suggest that INTU functions downstream of RPGRIP1L to localize both the NPHP and MKS complexes. Cumulatively, I have shown that TMEM231 and INTU are required for cilia to form and function properly, and that cilia are important for regulating vertebrate development and adult tissue homeostasis.

Historically, the ribosome has been viewed as a ribozyme with constitutive rather than regulatory capacity in mRNA translation. However, emerging studies reveal that ribosome activity may be highly regulated to impart a new layer of specificity in control of gene expression and organismal biology. Heterogeneity in the composition of the ribosome may generate variations to the translation machinery that significantly impact on how the genomic template is translated into functional proteins.

Studies in our laboratory have shown that ribosomal protein RPL38 confers specificity to the ribosome and is responsible for specifically translating a subset of Homeobox (Hox) mRNAs to regulate the mammalian body plan. In Rpl38 mutant embryos, global protein synthesis is unchanged while the translation of 8 of the 39 Hox mRNAs is specifically affected. Unexpectedly, Rpl38 transcripts are highly enriched in a tissue specific manner, most notably in somites and within the neural tube. Together, these studies led us to propose that ribosome in distinct regions of the embryo may be highly specialized to exert transcript-specific translational control. However how transcripts are recognized and selected by RPL38 remains unknown.

Here I uncover a unique set of RNA regulons embedded in the 5'UTRs of Hox genes which are critical for ribosome-directed control of their spatiotemporal expression patterns. These structured RNA elements, resembling Viral Internal Ribosome Entry Sites (IRES), are positioned close to the AUG of subsets of Hox mRNA to interact with ribosomal proteins and directly recruit the ribosome. Using Hoxa9 as a model, I find that the IRES element folds into a stable structure in vitro and can recruit ribosomes directly to the mRNA. By generating the first targeted knock-out of an IRES element in the mouse, I show that the Hoxa9 transcript, despite numerous layers of transcriptional control, requires an IRES-element for accurate spatiotemporal expression and axial skeletal patterning. The unexpected requirement for specialized translation control of Hox transcripts is further explained by the activity of a novel Translational Inhibitory Element (TIE), which inhibits general cap-dependent translation, thereby converting a generic mode of mRNA translation into a highly specialized one. Together, these studies uncover a new paradigm of specialized translational regulation that is achieved through a combination of unique RNA regulons to facilitate ribosome-mediate control of gene expression.

Primary cilia are membrane-bound microtubule-based structures present on most mammalian cells and are important for intercellular signaling. Cilia are present on a subset of endothelial cells, which form the innermost layer of blood vessels, where they project into the vessel lumen and are implicated as mechanical sensors of blood flow. To test the in vivo role of endothelial cilia, I conditionally deleted Ift88, a gene required for ciliogenesis, in endothelial cells of mice. I found that endothelial primary cilia are dispensable for vascular development. Removing endothelial cilia increased atherosclerosis in Apoe-/- mice fed a western-type diet, indicating that cilia protect against atherosclerosis. Consistent with this result, Apoe-/- mice lacking endothelial cilia had increased inflammatory gene expression.