Tubb4b is required for multi-ciliogenesis in the mouse.

Cilia are microtubule (MT)-based organelles present on the surface of nearly all vertebrate cells. MTs are polymers of α- and ß-tubulins that are each encoded by multiple, individual isotype genes. Tubulin isotype composition is thought to influence MT behaviors. Ciliary MTs differ from other MTs in the cell in terms of organization, stability and post-translational modifications. However, little is known about the tubulin isotypes that build ciliary MTs and the functional requirements for tubulin isotypes in cilia have not been examined in vertebrates. Here, we have tested the role of the ß-tubulin isotype genes in the mouse that harbor a conserved amino acid motif associated with ciliated organisms. We found that Tubb4b localizes to cilia in multi-ciliated cells (MCCs) specifically. In respiratory and oviduct MCCs, Tubb4b is asymmetrically localized within multi-cilia, indicating that the tubulin isotype composition changes along the length of the ciliary axonemal MTs. Deletion of Tubb4b resulted in striking structural defects within the axonemes of multi-cilia, without affecting primary cilia. These studies show that Tubb4b is essential for the formation of a specific MT-based subcellular organelle and sheds light on the requirements of tubulin isotypes in cilia.

Interactions between TULP3 tubby domain and ARL13B amphipathic helix promote lipidated protein transport to cilia.

The primary cilium is a nexus for cell signaling and relies on specific protein trafficking for function. The tubby family protein TULP3 transports integral membrane proteins into cilia through interactions with the intraflagellar transport complex-A (IFT-A) and phosphoinositides. It was previously shown that short motifs called ciliary localization sequences (CLSs) are necessary and sufficient for TULP3-dependent ciliary trafficking of transmembrane cargoes. However, the mechanisms by which TULP3 regulates ciliary compartmentalization of nonintegral, membrane-associated proteins and whether such trafficking requires TULP3-dependent CLSs is unknown. Here we show that TULP3 is required for ciliary transport of the Joubert syndrome-linked palmitoylated GTPase ARL13B through a CLS. An N-terminal amphipathic helix, preceding the GTPase domain of ARL13B, couples with the TULP3 tubby domain for ciliary trafficking, irrespective of palmitoylation. ARL13B transport requires TULP3 binding to IFT-A but not to phosphoinositides, indicating strong membrane-proximate interactions, unlike transmembrane cargo transport requiring both properties of TULP3. TULP3-mediated trafficking of ARL13B also regulates ciliary enrichment of farnesylated and myristoylated downstream effectors of ARL13B. The lipidated cargoes show distinctive depletion kinetics from kidney epithelial cilia with relation to Tulp3 deletion-induced renal cystogenesis. Overall, these findings indicate an expanded role of the tubby domain in capturing analogous helical secondary structural motifs from diverse cargoes.

A dominant tubulin mutation causes cerebellar neurodegeneration in a genetic model of tubulinopathy.

Mutations in tubulins cause distinct neurodevelopmental and degenerative diseases termed "tubulinopathies"; however, little is known about the functional requirements of tubulins or how mutations cause cell-specific pathologies. Here, we identify a mutation in the gene Tubb4a that causes degeneration of cerebellar granule neurons and myelination defects. We show that the neural phenotypes result from a cell type-specific enrichment of a dominant mutant form of Tubb4a relative to the expression other ß-tubulin isotypes. Loss of Tubb4a function does not underlie cellular pathology but is compensated by the transcriptional up-regulation of related tubulin genes in a cell type-specific manner. This work establishes that the expression of a primary tubulin mutation in mature neurons is sufficient to promote cell-autonomous cell death, consistent with a causative association of microtubule dysfunction with neurodegenerative diseases. These studies provide evidence that mutations in tubulins cause specific phenotypes based on expression ratios of tubulin isotype genes.

Mutations in Ciliary Trafficking Genes affect Sonic Hedgehog-dependent Neural Tube Patterning Differentially along the Anterior-Posterior Axis.

Cell specification in the ventral spinal cord is a well-studied model system to understand how tissue pattern develops in response to a morphogen gradient. Ventral cell types including motor neurons (MNs) are induced in the neural tube in response to graded Sonic Hedgehog (Shh) signaling. We performed a forward genetic screen in the mouse that incorporated a GFP-expressing transgene to visualize MNs to identify genes regulating ventral patterning. Here we contrast the neural patterning phenotypes of two mouse lines carrying induced mutations in ciliary trafficking genes. We show that a hypomorphic mutation in the gene Tubby-like protein 3 (Tulp3) resulted in a dorsal expansion of MNs consistent with an up-regulation of Shh signaling. Interestingly, patterning defects in Tulp3 mutants were restricted to posterior regions of the spinal cord as patterning was similar to WT in the anterior spinal cord. In contrast, a mutation in the ciliary trafficking gene cytoplasmic dynein 2 heavy chain 1 (Dync2h1), led to a complete loss of MNs in anterior regions of the spinal cord, indicating a strong down-regulation of Shh signaling. However, this severe phenotype was restricted to the cervical region as MNs developed posteriorly. Mutations in cilia trafficking genes affect Shh-dependent signaling in the neural tube differentially along the anterior-posterior (A-P) axis in a process that is not understood.

The Ciliary Protein Arl13b Functions Outside of the Primary Cilium in Shh-Mediated Axon Guidance.

The small GTPase Arl13b is enriched in primary cilia and regulates Sonic hedgehog (Shh) signaling. During neural development, Shh controls patterning and proliferation through a canonical, transcription-dependent pathway that requires the primary cilium. Additionally, Shh controls axon guidance through a non-canonical, transcription-independent pathway whose connection to the primary cilium is unknown. Here we show that inactivation of Arl13b results in defective commissural axon guidance in vivo. In vitro, we demonstrate that Arl13b functions autonomously in neurons for their Shh-dependent guidance response. We detect Arl13b protein in axons and growth cones, far from its well-established ciliary enrichment. To test whether Arl13b plays a non-ciliary function, we used an engineered, cilia-localization-deficient Arl13b variant and found that it was sufficient to mediate Shh axon guidance in vitro and in vivo. Together, these results indicate that, in addition to its ciliary role in canonical Shh signaling, Arl13b plays a cilia-independent role in Shh-mediated axon guidance.

Cell-Autonomous Hedgehog Signaling Is Not Required for Cyst Formation in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: PKD1 or PKD2, the two main causal genes for autosomal dominant polycystic kidney disease (ADPKD), encode the multipass transmembrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Polycystins localize to the primary cilium, an organelle essential for cell signaling, including signal transduction of the Hedgehog pathway. Mutations in ciliary genes that build and maintain the cilium also cause renal cystic disease through unknown pathways. Although recent studies have found alterations in Hedgehog signaling in ADPKD-related models and tissues, the relationship between Hedgehog and polycystic kidney disease is not known. METHODS: To examine the potential role of cell-autonomous Hedgehog signaling in regulating kidney cyst formation in vivo in both early- and adult-onset mouse models of ADPKD, we used conditional inactivation of Pkd1 combined with conditional modulation of Hedgehog signaling components in renal epithelial cells, where mutations in Pkd1 initiate cyst formation. After increasing or decreasing levels of Hedgehog signaling in cells that underwent inactivation of Pkd1, we evaluated the effects of these genetic manipulations on quantitative parameters of polycystic kidney disease severity. RESULTS: We found that in Pkd1 conditional mutant mouse kidneys, neither downregulation nor activation of the Hedgehog pathway in epithelial cells along the nephron significantly influenced the severity of the polycystic kidney phenotype in mouse models of developmental or adult-onset of ADPKD. CONCLUSIONS: These data suggest that loss of Pkd1 function results in kidney cysts through pathways that are not affected by the activity of the Hedgehog pathway.

Tulp3 Is a Ciliary Trafficking Gene that Regulates Polycystic Kidney Disease.

The primary cilium is an organelle essential for cell signaling pathways. One of the most common human genetic diseases is autosomal dominant polycystic kidney disease (ADPKD), which is caused by mutations in the PKD1 or PKD2 genes that encode Polycystin 1 and 2 (PC1/2), transmembrane proteins that translocate to the cilium. Mutations in genes that disrupt ciliogenesis also cause kidney cysts as part of a "ciliopathic" disease spectrum. The molecular mechanisms that link cilia function with renal cystic diseases are not well understood, and the mechanistic relationship between ADPKD and ciliopathic PKD is not known. Here we identify the gene Tubby-like protein-3 (Tulp3) as a key regulator of renal cystic disease from a forward genetic screen in the mouse. Mice homozygous for a hypomorphic missense mutation within the conserved Tubby domain of Tulp3 develop cysts at late embryonic stages, leading to severe postnatal loss of kidney function. In contrast to other ciliopathic disease models, Tulp3 mutations do not affect ciliogenesis. Instead, we demonstrate that Tulp3 is essential for the trafficking of the Joubert syndrome-associated small GTPase Arl13b into kidney cilia. We show that reduction of Pkd1 dosage promotes cystogenesis in the Tulp3 conditional ciliopathic PKD model. However, in an adult model of ADPKD utilizing inducible conditional Pkd1 deletion, concomitant removal of Tulp3 surprisingly ameliorates cystic disease. Therefore, Tulp3 controls distinct ciliary pathways that positively or negatively regulate cystogenesis depending on the cellular context.

Differential timing of granule cell production during cerebellum development underlies generation of the foliation pattern.

BACKGROUND: The mouse cerebellum (Cb) has a remarkably complex foliated three-dimensional (3D) structure, but a stereotypical cytoarchitecture and local circuitry. Little is known of the cellular behaviors and genes that function during development to determine the foliation pattern. In the anteroposterior axis the mammalian cerebellum is divided by lobules with distinct sizes, and the foliation pattern differs along the mediolateral axis defining a medial vermis and two lateral hemispheres. In the vermis, lobules are further grouped into four anteroposterior zones (anterior, central, posterior and nodular zones) based on genetic criteria, and each has distinct lobules. Since each cerebellar afferent group projects to particular lobules and zones, it is critical to understand how the 3D structure of the Cb is acquired. During cerebellar development, the production of granule cells (gcs), the most numerous cell type in the brain, is required for foliation. We hypothesized that the timing of gc accumulation is different in the four vermal zones during development and contributes to the distinct lobule morphologies. METHODS AND RESULTS: In order to test this idea, we used genetic inducible fate mapping to quantify accumulation of gcs in each lobule during the first two postnatal weeks in mice. The timing of gc production was found to be particular to each lobule, and delayed in the central zone lobules relative to the other zones. Quantification of gc proliferation and differentiation at three time-points in lobules representing different zones, revealed the delay involves a later onset of maximum differentiation and prolonged proliferation of gc progenitors in the central zone. Similar experiments in Engrailed mutants (En1 (-/+) ;En2 (-/-) ), which have a smaller Cb and altered foliation pattern preferentially outside the central zone, showed that gc production, proliferation and differentiation are altered such that the differences between zones are attenuated compared to wild-type mice. CONCLUSIONS: Our results reveal that gc production is differentially regulated in each zone of the cerebellar vermis, and our mutant analysis indicates that the dynamics of gc production plays a role in determining the 3D structure of the Cb.

A Mathematical Model of Granule Cell Generation During Mouse Cerebellum Development.

Determining the cellular basis of brain growth is an important problem in developmental neurobiology. In the mammalian brain, the cerebellum is particularly amenable to studies of growth because it contains only a few cell types, including the granule cells, which are the most numerous neuronal subtype. Furthermore, in the mouse cerebellum granule cells are generated from granule cell precursors (gcps) in the external granule layer (EGL), from 1 day before birth until about 2 weeks of age. The complexity of the underlying cellular processes (multiple cell behaviors, three spatial dimensions, time-dependent changes) requires a quantitative framework to be fully understood. In this paper, a differential equation-based model is presented, which can be used to estimate temporal changes in granule cell numbers in the EGL. The model includes the proliferation of gcps and their differentiation into granule cells, as well as the process by which granule cells leave the EGL. Parameters describing these biological processes were derived from fitting the model to histological data. This mathematical model should be useful for understanding altered gcp and granule cell behaviors in mouse mutants with abnormal cerebellar development and cerebellar cancers.

Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum.

The mammalian cerebellum consists of folds of different sizes and shapes that house distinct neural circuits. A crucial factor underlying foliation is the generation of granule cells (gcs), the most numerous neuron type in the brain. We used clonal analysis to uncover global as well as folium size-specific cellular behaviors that underlie cerebellar morphogenesis. Unlike most neural precursors, gc precursors divide symmetrically, accounting for their massive expansion. We found that oriented cell divisions underlie an overall anteroposteriorly polarized growth of the cerebellum and gc clone geometry. Clone geometry is further refined by mediolateral oriented migration and passive dispersion of differentiating gcs. Most strikingly, the base of each fissure acts as a boundary for gc precursor dispersion, which we propose allows each folium to be regulated as a developmental unit. Indeed, the geometry and size of clones in long and short folia are distinct. Moreover, in engrailed 1/2 mutants with shorter folia, clone cell number and geometry are most similar to clones in short folia of wild-type mice. Thus, the cerebellum has a modular mode of development that allows the plane of cell division and number of divisions to be differentially regulated to ensure that the appropriate number of cells are partitioned into each folium.

Microdissection and visualization of individual hair follicles for lineage tracing studies.

In vivo lineage tracing is a valuable technique to study cellular behavior. Our lab developed a lineage tracing method, based on the Cre/lox system, to genetically induce clonal labelling of cells and follow their progeny. Here we describe a protocol for temporally controlled clonal labelling and for microdissection of individual mouse hair follicles. We further present staining and visualization techniques used in our lab to analyze clones issued from genetically induced labelling.

Genetic fate mapping using site-specific recombinases.

Understanding how cells are assembled in three dimensions to generate an organ, or a whole organism, is a pivotal question in developmental biology. Similarly, it is critical to understand how adult stem cells integrate into an existing organ during regeneration or in response to injury. Key to discovering the answers to these questions is being able to study the various behaviors of distinct cell types during development or regeneration. Fate mapping techniques are fundamental to studying cell behaviors such as proliferation, movement, and lineage segregation, as the techniques allow precursor cells to be marked and their descendants followed and characterized over time. The generation of transgenic mice, combined with the use of site-specific recombinases (SSR) in the mouse genome, has provided a means to develop powerful genetic fate mapping approaches. A key advantage of genetic fate mapping is that it allows cells to be genetically marked, and therefore the mark is transmitted to all the descendants of the initially marked cells. By making modifications to the SSRs that render their enzymatic activity inducible, and the development of an assortment of reporter alleles for marking cells, increasingly sophisticated genetic fate mapping studies can be performed. In this chapter, we review the four main genetic fate mapping methods that utilize intrachromosomal recombination to mark cells (cumulative, inducible, clonal, and intersectional) and one interchromosomal method, the tools required to carry out each approach, and the practical considerations that have to be taken into account before embarking on each type of genetic fate mapping study.

Hair follicle renewal: authentic morphogenesis that depends on a complex progression of stem cell lineages.

The hair follicle (HF) grows during the anagen phase from precursors in the matrix that give rise to each differentiated HF layer. Little is known about the lineal relationship between these layer-restricted precursors and HF stem cells. To understand how the HF stem cells regenerate the typical anagen organization, we conducted in vivo clonal analysis of key stages of the HF cycle in mice. Unexpectedly, we found that the pool of HF stem cells contains precursors with both multipotent and restricted contributions. This implies that the lineal relationships between HF stem cells (persisting during telogen) and layer-restricted precursors (in the germinative layer), responsible for HF elongation during anagen, are not stereotyped. Formation of the matrix at each cycle is accompanied by the transient expansion of an intermediary pool of precursors at the origin of the germinative layer and by the progressive restriction of cell dispersion. The regionalization of clonal patterns within the outer HF structure (the outer root sheath) suggests that the position of the precursors might be a crucial factor in determining their fate. The presence of HF stem cells with multipotent contribution and the progressive segregation of HF lineages upon anagen activation indicate that each HF renewal cycle constitutes an authentic morphogenetic process. A comprehensive model was constructed based on the different clonal patterns observed. In this model, the positions of the precursors relative to the dermal papilla together with the progressive restriction of cell dispersion are part of the mechanism that restricts their contribution to the different HF lineages.

Defective planar cell polarity in polycystic kidney disease.

Morphogenesis involves coordinated proliferation, differentiation and spatial distribution of cells. We show that lengthening of renal tubules is associated with mitotic orientation of cells along the tubule axis, demonstrating intrinsic planar cell polarization, and we demonstrate that mitotic orientations are significantly distorted in rodent polycystic kidney models. These results suggest that oriented cell division dictates the maintenance of constant tubule diameter during tubular lengthening and that defects in this process trigger renal tubular enlargement and cyst formation.

Hair follicle renewal: organization of stem cells in the matrix and the role of stereotyped lineages and behaviors.

Hair follicles (HFs) are renewed via multipotent stem cells located in a reservoir (the bulge); however, little is known about how they generate multi-tissue HFs from a proliferative zone (the matrix). To address this issue, we temporally induced clonal labeling during HF growth. Challenging the prevailing hypothesis, we found that the matrix contains restricted self-renewing stem cells for each inner structure. These cells are located around the dermal papilla forming a germinative layer. They occupy different proximodistal sectors and produce differentiated cells along the matrix radial axis via stereotyped lineages and cell behavior. By contrast, the outer layer of HFs displays a mode of growth involving apoptosis that coordinates the development of outer and inner structures. HF morphology is therefore determined by the organization of cell fates along the proximodistal axis and by cell behavior along the radial (lateral) axis in the matrix. Thus, our studies suggest that fate and behavior are organized by two systems (uncoupled), and this uncoupling may represent a fundamental way to simplify morphogenesis.

Methods in clonal analysis and applications.

During development, embryonic cells display a large variety of behaviors that lead to the formation of embryonic structures that are frequently transient. Simultaneously, cells progress towards a specific fate. The current challenge for embryologists is to resolve how these two distinct aspects of development co-exist. As cell behaviors (including elementary cellular operations such as motility, adhesiveness, polarization, change in shape, division and death) and their control are much less well understood than the genetic aspects of cell fate determination, there is currently much interest in the study of cell behaviors. This mainly consists of labeling groups of cells or, less frequently, single cells and observing their descendants. In this review, we describe a few techniques for labeling groups of cells and we discuss prospective and retrospective clonal analysis, in particular the LaacZ system, in detail. We examine the information generated by these approaches.