Cortical development depends upon tightly controlled cell fate and cell survival decisions that generate a functional neuronal population, but the coordination of these two processes is poorly understood. Here we show that conditional removal of a key apical complex protein, Pals1, causes premature withdrawal from the cell cycle, inducing excessive generation of early-born postmitotic neurons followed by surprisingly massive and rapid cell death, leading to the abrogation of virtually the entire cortical structure. Pals1 loss shows exquisite dosage sensitivity, so that heterozygote mutants show an intermediate phenotype on cell fate and cell death. Loss of Pals1 blocks essential cell survival signals, including the mammalian target of rapamycin (mTOR) pathway, while mTORC1 activation partially rescues Pals1 deficiency. These data highlight unexpected roles of the apical complex protein Pals1 in cell survival through interactions with mTOR signaling.

Tubulin post-translational modifications generate microtubule heterogeneity and modulate microtubule function, and are catalyzed by tubulin tyrosine ligase-like (TTLL) proteins. Using antibodies specific to monoglycylated, polyglycylated, and glutamylated tubulin in whole mount immunostaining of zebrafish embryos, we observed distinct, tissue-specific patterns of tubulin modifications. Tubulin modification patterns in cilia correlated with the expression of ttll3 and ttll6 in ciliated cells. Expression screening of all zebrafish tubulin tyrosine ligase-like genes revealed additional tissue-specific expression of ttll1 in brain neurons, ttll4 in muscle, and ttll7 in otic placodes. Knockdown of ttll3 eliminated cilia tubulin glycylation but had surprisingly mild effects on cilia structure and motility. Similarly, knockdown of ttll6 strongly reduced cilia tubulin glutamylation but only partially affected cilia structure and motility. Combined loss of function of ttll3 and ttll6 caused near complete loss of cilia motility and induced a variety of axonemal ultrastructural defects similar to defects previously observed in zebrafish fleer mutants, which were shown to lack tubulin glutamylation. Consistently, we find that fleer mutants also lack tubulin glycylation. These results indicate that tubulin glycylation and glutamylation have overlapping functions in maintaining cilia structure and motility and that the fleer/ dyf-1 TPR protein is required for both types of tubulin posttranslational modification.Glycylation and glutamylation are post-translational modifications of tubulin subunits within stable microtubules of cilia, centrioles, neuronal axons, and mitotic spindles (1-3). Variable lengths of added glycyl or glutamyl side chains generate diverse tubulin subtypes and contribute to heterogeneity of tubulin function by modulating microtubule-protein interactions (2, 4, 5). For instance, glutamyl side chain length has been shown to differentially affect recruitment of the microtubule-associated proteins MAP1A and MAP2 (6), and to selectively enhance KIF1A-dependent vesicle transport in hippocampal neurons (7). In vertebrates, glutamylated tubulin is associated with cilia and neuronal axons, whereas glycylated tubulin is primarily localized to cilia (8 -11). Both modifications occur on an overlapping set of glutamates near the tubulin C terminus (3, 12, 13). Deletion of the ␤-tubulin C terminus is lethal in Tetrahymena and generates non-motile, short cilia lacking a portion of the microtubule doublet B-subfiber (14 -16). Drosophila mutants lacking the ␤-tubulin C terminus fail to assemble sperm axonemes, whereas cytoplasmic microtubules are not affected (17, 18). Axonemal B-subfiber defects and reduced cilia beat amplitude are also observed in the zebrafish fleer mutant, which lacks a TPR protein required for cilia polyglutamylation (19), as well as in Tetrahymena overexpressing the tubulin glutamylase, TTLL6Ap (20,21). Evidence for a role for tubulin glycylation in cilia elongation in zebrafish has recently been re...

Cilia are essential for normal organ function and developmental patterning, but their role in injury and regeneration responses is unknown. To probe the role of cilia in injury, we analyzed the function of foxj1, a transcriptional regulator of cilia genes, in response to tissue damage and renal cyst formation. Zebrafish foxj1a , but not foxj1b , was rapidly induced in response to epithelial distension and stretch, kidney cyst formation, acute kidney injury by gentamicin, and crush injury in spinal cord cells. Obstruction-induced up-regulation of foxj1a was not inhibited by cycloheximide, identifying foxj1a as a primary response gene to epithelial injury. Foxj1 was also dramatically up-regulated in murine cystic kidney disease epithelia [ jck/jck (nek8) and Ift88Tg737Rpw −/− ] as well as in response to kidney ischemia-reperfusion injury. Obstruction of the zebrafish pronephric tubule caused a rapid increase in cilia beat rate that correlated tightly with expanded tubule diameter and epithelial stretch. Zebrafish foxj1a was specifically required for cilia motility. Enhanced foxj1a expression in obstructed tubules induced cilia motility target genes efhc1 , tektin-1 , and dnahc9. foxj1a -deficient embryos failed to up-regulate efhc1 , tektin-1 , and dnahc9 and could not maintain enhanced cilia beat rates after obstruction, identifying an essential role for foxj1 in modulating cilia function after injury. These studies reveal that activation of a Foxj1 transcriptional network of ciliogenic genes is an evolutionarily conserved response to multiple forms of tissue damage and highlight enhanced cilia function as a previously uncharacterized component of organ homeostasis.

The molecular processes underlying regeneration remain largely unknown. Several potential factors have been elucidated by focusing on the regenerative function of genes originally identified in a developmental context. A complementary approach is to consider the roles of factors involved in wound healing. Here we focus on the Thrombospondins, a family of secreted extracellular matrix proteins that have been implicated in skin wound healing in mammals. We show that a subset of Thrombospondins are expressed at distinct times and in particular cell types during axolotl limb regeneration. Our studies have revealed the axolotl orthologs of thrombospondin-1 (tsp-1) and thrombospondin-4 (tsp-4) are highly upregulated during limb regeneration in patterns both distinct and similar to larval limb development. Our data suggest that thrombospondins may be key regulators of limb regeneration in axolotl, while their activation appears to be relegated solely to wound healing in vertebrates that have lost the ability to regenerate limbs.