Cell cycle-related kinase regulates mammalian eye development through positive and negative regulation of the Hedgehog pathway.

Cell cycle-related kinase (CCRK) is a conserved regulator of ciliogenesis whose loss in mice leads to a wide range of developmental defects, including exencephaly, preaxial polydactyly, skeletal abnormalities, and microphthalmia. Here, we investigate the role of CCRK in mouse eye development. Ccrk mutants show dramatic patterning defects, with an expansion of the optic stalk domain into the optic cup, as well as an expansion of the retinal pigment epithelium (RPE) into neural retina (NR) territory. In addition, Ccrk mutants display a shortened optic stalk. These defects are associated with bimodal changes in Hedgehog (Hh) pathway activity within the eye, including the loss of proximal, high level responses but a gain in distal, low level responses. We simultaneously removed the Hh activator GLI2 in Ccrk mutants (Ccrk-/-;Gli2-/-), which resulted in rescue of optic cup patterning and exacerbation of optic stalk length defects. Next, we disrupted the Hh pathway antagonist GLI3 in mutants lacking CCRK (Ccrk-/-;Gli3-/-), which lead to even greater expansion of the RPE markers into the NR domain and a complete loss of NR specification within the optic cup. These results indicate that CCRK functions in eye development by both positively and negatively regulating the Hh pathway, and they reveal distinct requirements for Hh signaling in patterning and morphogenesis of the eyes.

Proper ciliary assembly is critical for restricting Hedgehog signaling during early eye development in mice.

Patterning of the vertebrate eye into optic stalk, retinal pigment epithelium (RPE) and neural retina (NR) territories relies on a number of signaling pathways, but how these signals are interpreted by optic progenitors is not well understood. The primary cilium is a microtubule-based organelle that is essential for Hedgehog (Hh) signaling, but it has also been implicated in the regulation of other signaling pathways. Here, we show that the optic primordium is ciliated during early eye development and that ciliogenesis is essential for proper patterning and morphogenesis of the mouse eye. Ift172 mutants fail to generate primary cilia and exhibit patterning defects that resemble those of Gli3 mutants, suggesting that cilia are required to restrict Hh activity during eye formation. Ift122 mutants, which produce cilia with abnormal morphology, generate optic vesicles that fail to invaginate to produce the optic cup. These mutants also lack formation of the lens, RPE and NR. Such phenotypic features are accompanied by strong, ectopic Hh pathway activity, evidenced by altered gene expression patterns. Removal of GLI2 from Ift122 mutants rescued several aspects of optic cup and lens morphogenesis as well as RPE and NR specification. Collectively, our data suggest that proper assembly of primary cilia is critical for restricting the Hedgehog pathway during eye formation in the mouse.

Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components.

Primary cilia are required for proper Sonic Hedgehog (Shh) signaling in mammals. However, their role in the signal transduction process remains unclear. We have identified sister of open brain (sopb), a null allele of mouse Intraflagellar transport protein 122 (Ift122). IFT122 negatively regulates the Shh pathway in the cilium at a step downstream of the Shh ligand and the transmembrane protein Smoothened, but upstream of the Gli2 transcription factor. Ift122(sopb) mutants generate primary cilia, but they show features of defective retrograde intraflagellar transport. IFT122 controls the ciliary localization of Shh pathway regulators in different ways. Disruption of IFT122 leads to accumulation of Gli2 and Gli3 at cilia tips while blocking the ciliary localization of the antagonist TULP3. Suppressor of Fused and Smoothened localize to the cilium through an IFT122-independent mechanism. We propose that the balance between positive and negative regulators of the Shh pathway at the cilium tip controls the output of the pathway and that Shh signaling regulates this balance through intraflagellar transport.

Evolution of a chordate-specific mechanism for myoblast fusion.

Vertebrate myoblast fusion allows for multinucleated muscle fibers to compound the size and strength of mononucleated cells, but the evolution of this important process is unknown. We investigated the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here, we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed a complex evolutionary history of myoblast fusion. A prevertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Evolutionary comparisons between vertebrate and nonvertebrate Myomaker revealed key structural and mechanistic insights into myoblast fusion. Thus, our findings suggest an evolutionary model of chordate fusogens and illustrate how new genes shape the emergence of novel morphogenetic traits and mechanisms.

Tubby-like protein 3 (TULP3) regulates patterning in the mouse embryo through inhibition of Hedgehog signaling.

Tubby-like protein 3 (TULP3) is required for proper embryonic development in mice. Disruption of mouse Tulp3 results in morphological defects in the embryonic craniofacial regions, the spinal neural tube and the limbs. Here, we show that TULP3 functions as a novel negative regulator of Sonic hedgehog (Shh) signaling in the mouse. In Tulp3 mutants, ventral cell types in the lumbar neural tube, which acquire their identities in response to Shh signaling, are ectopically specified at the expense of dorsal cell types. Genetic epistasis experiments show that this ventralized phenotype occurs independently of Shh and the transmembrane protein Smoothened, but it is dependent on the transcription factor Gli2. The ventralized phenotype is also dependent on the kinesin II subunit Kif3A, which is required for intraflagellar transport and ciliogenesis. In addition, TULP3 is required for proper Shh-dependent limb patterning and for maintaining the correct balance between differentiation and proliferation in the neural tube. Finally, the localization of TULP3 to the tips of primary cilia raises the possibility that it regulates the Hedgehog pathway within this structure.

FKBP8 cell-autonomously controls neural tube patterning through a Gli2- and Kif3a-dependent mechanism.

Signaling by Sonic hedgehog (Shh) represents an important process by which many types of neural progenitor cells become properly organized along the dorsal-ventral axis of the vertebrate neural tube in a concentration-dependent manner. However, the mechanism by which Shh signals are transduced with high fidelity and the relationship between the Shh signaling pathway and other patterning systems remain unclear. Here we focus on the role of FK506-binding protein 8 (FKBP8) in controlling neural cell identity through its antagonism of the Shh pathway. Our data indicate that disruption of FKBP8 function activates the Shh signaling pathway cell-autonomously at a step that is independent of the transmembrane protein Smoothened but dependent on the Gli2 transcription factor. This activation is also dependent on the kinesin-2 subunit Kif3a, a component of the intraflagellar transport (IFT) machinery used to generate cilia. Our data also indicate that non-cell-autonomous effects of the Fkbp8 mutation further contribute to the neural patterning phenotype and suggest that FKBP8 plays an indirect role in promoting Bone morphogenetic protein (BMP) signaling through antagonism of the Shh pathway.

Mouse Dispatched homolog1 is required for long-range, but not juxtacrine, Hh signaling.

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, we have identified recessive ENU-induced mutations in six genes that prevent normal specification of ventral cell types in the spinal cord. We positionally cloned the genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. Our data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh.

Complex interactions between genes controlling trafficking in primary cilia.

Cilia-associated human genetic disorders are striking in the diversity of their abnormalities and their complex inheritance. Inactivation of the retrograde ciliary motor by mutations in DYNC2H1 causes skeletal dysplasias that have strongly variable expressivity. Here we define previously unknown genetic relationships between Dync2h1 and other genes required for ciliary trafficking. Mutations in mouse Dync2h1 disrupt cilia structure, block Sonic hedgehog signaling and cause midgestation lethality. Heterozygosity for Ift172, a gene required for anterograde ciliary trafficking, suppresses cilia phenotypes, Sonic hedgehog signaling defects and early lethality of Dync2h1 homozygotes. Ift122, like Dync2h1, is required for retrograde ciliary trafficking, but reduction of Ift122 gene dosage also suppresses the Dync2h1 phenotype. These genetic interactions illustrate the cell biology underlying ciliopathies and argue that mutations in intraflagellar transport genes cause their phenotypes because of their roles in cilia architecture rather than direct roles in signaling.

Broad-minded links cell cycle-related kinase to cilia assembly and hedgehog signal transduction.

Recent findings indicate that mammalian Sonic hedgehog (Shh) signal transduction occurs within primary cilia, although the cell biological mechanisms underlying both Shh signaling and ciliogenesis have not been fully elucidated. We show that an uncharacterized TBC domain-containing protein, Broad-minded (Bromi), is required for high-level Shh responses in the mouse neural tube. We find that Bromi controls ciliary morphology and proper Gli2 localization within the cilium. By use of a zebrafish model, we further show that Bromi is required for proper association between the ciliary membrane and axoneme. Bromi physically interacts with cell cycle-related kinase (CCRK), whose Chlamydomonas homolog regulates flagellar length. Biochemical and genetic interaction data indicate that Bromi promotes CCRK stability and function. We propose that Bromi and CCRK control the structure of the primary cilium by coordinating assembly of the axoneme and ciliary membrane, allowing Gli proteins to be properly activated in response to Shh signaling.

Analysis of hedgehog signaling in mouse intraflagellar transport mutants.

Intraflagellar transport (IFT) has been studied for decades in model systems such as Chlamydomonas and Caenorhabditis elegans. More recently, IFT has been investigated using genetic approaches in mammals using the mouse as a model system. Through such studies, a new appreciation of the importance of IFT and cilia in mammalian signal transduction has emerged. Specifically, IFT has been shown to play a key role in controlling signaling by Sonic and Indian Hedgehog (Hh) ligands. The effects of mutations in IFT components on Sonic Hh signaling in the embryo are complex and differ depending on the nature of the components, alleles, and tissues examined. For this reason, we provide a basis for analyzing the phenotype as a guide for those investigators who study IFT in cell culture or use invertebrate systems and wish to extend their studies to include development of the mouse embryo. We provide an overview of Sonic Hh-dependent tissue patterning in the developing neural tube and limb buds, the two systems in which it has been studied most extensively, and we show examples of how this patterning is disrupted by mutations in mouse IFT components.

Cilia and developmental signaling.

Recent studies have revealed unexpected connections between the mammalian Hedgehog (Hh) signal transduction pathway and the primary cilium, a microtubule-based organelle that protrudes from the surface of most vertebrate cells. Intraflagellar transport proteins, which are required for the construction of cilia, are essential for all responses to mammalian Hh proteins, and proteins required for Hh signal transduction are enriched in primary cilia. The phenotypes of different mouse mutants that affect ciliary proteins suggest that cilia may act as processive machines that organize sequential steps in the Hh signal transduction pathway. Cilia on vertebrate cells are likely to be important in additional developmental signaling pathways and are required for PDGF receptor alpha signaling in cultured fibroblasts. Cilia are not essential for either canonical or noncanonical Wnt signaling, although cell-type-specific modulation of cilia components may link cilia and Wnt signaling in some tissues. Because ciliogenesis in invertebrates is limited to a very small number of specialized cell types, the role of cilia in developmental signaling pathways is likely a uniquely vertebrate phenomenon.

Mouse Rab23 regulates hedgehog signaling from smoothened to Gli proteins.

Sonic hedgehog (Shh) signaling is required for the growth and patterning of many tissues in vertebrate embryos, but important aspects of the Shh signal transduction pathway are poorly understood. For example, the vesicle transport protein Rab23 is a cell autonomous negative regulator of Shh signaling, but the process affected by Rab23 has not been defined. Here, we demonstrate that Rab23 acts upstream of Gli transcription factors in patterning neural cell types in the spinal cord. Double mutant analysis indicates that the primary target of Rab23 is the Gli2 activator and that Rab23 and Gli3 repressor have additive effects on patterning. Analysis of Gli3 protein suggests that Rab23 also has a role in promoting the production of Gli3 repressor. Although the membrane proteins Patched and Smoothened change subcellular localization in response to Shh, double mutant analysis demonstrates that Rab23 does not work through either Patched or Smoothened. Instead, Rab23 appears to regulate subcellular localization of essential components of the Hedgehog pathway that act downstream of Smoothened and upstream of Gli proteins.

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.

FKBP8 is a negative regulator of mouse sonic hedgehog signaling in neural tissues.

Sonic hedgehog (SHH) is a secreted morphogen that regulates the patterning and growth of many tissues in the developing mouse embryo, including the central nervous system (CNS). We show that a member of the FK506-binding protein family, FKBP8, is an essential antagonist of SHH signaling in CNS development. Loss of FKBP8 causes ectopic and ligand-independent activation of the Shh pathway, leading to expansion of ventral cell fates in the posterior neural tube and suppression of eye development. Although it is expressed broadly, FKBP8 is required to antagonize SHH signaling primarily in neural tissues, suggesting that hedgehog signal transduction is subject to cell-type specific modulation during mammalian development.