Role of ciliopathy protein TMEM107 in eye development: insights from a mouse model and retinal organoid.

Primary cilia are cellular surface projections enriched in receptors and signaling molecules, acting as signaling hubs that respond to stimuli. Malfunctions in primary cilia have been linked to human diseases, including retinopathies and ocular defects. Here, we focus on TMEM107, a protein localized to the transition zone of primary cilia. TMEM107 mutations were found in patients with Joubert and Meckel-Gruber syndromes. A mouse model lacking Tmem107 exhibited eye defects such as anophthalmia and microphthalmia, affecting retina differentiation. Tmem107 expression during prenatal mouse development correlated with phenotype occurrence, with enhanced expression in differentiating retina and optic stalk. TMEM107 deficiency in retinal organoids resulted in the loss of primary cilia, down-regulation of retina-specific genes, and cyst formation. Knocking out TMEM107 in human ARPE-19 cells prevented primary cilia formation and impaired response to Smoothened agonist treatment because of ectopic activation of the SHH pathway. Our data suggest TMEM107 plays a crucial role in early vertebrate eye development and ciliogenesis in the differentiating retina.

Morphological changes and two Nodal paralogs drive left-right asymmetry in the squamate veiled chameleon (C. calyptratus).

The ancestral mode of left-right (L-R) patterning involves cilia in the L-R organizer. However, the mechanisms regulating L-R patterning in non-avian reptiles remains an enigma, since most squamate embryos are undergoing organogenesis at oviposition. In contrast, veiled chameleon (Chamaeleo calyptratus) embryos are pre-gastrula at oviposition, making them an excellent organism for studying L-R patterning evolution. Here we show that veiled chameleon embryos lack motile cilia at the time of L-R asymmetry establishment. Thus, the loss of motile cilia in the L-R organizers is a synapomorphy of all reptiles. Furthermore, in contrast to avians, geckos and turtles, which have one Nodal gene, veiled chameleon exhibits expression of two paralogs of Nodal in the left lateral plate mesoderm, albeit in non-identical patterns. Using live imaging, we observed asymmetric morphological changes that precede, and likely trigger, asymmetric expression of the Nodal cascade. Thus, veiled chameleons are a new and unique model for studying the evolution of L-R patterning.

Nr6a1 controls Hox expression dynamics and is a master regulator of vertebrate trunk development.

The vertebrate main-body axis is laid down during embryonic stages in an anterior-to-posterior (head-to-tail) direction, driven and supplied by posteriorly located progenitors. Whilst posterior expansion and segmentation appears broadly uniform along the axis, there is developmental and evolutionary support for at least two discrete modules controlling processes within different axial regions: a trunk and a tail module. Here, we identify Nuclear receptor subfamily 6 group A member 1 (Nr6a1) as a master regulator of trunk development in the mouse. Specifically, Nr6a1 was found to control vertebral number and segmentation of the trunk region, autonomously from other axial regions. Moreover, Nr6a1 was essential for the timely progression of Hox signatures, and neural versus mesodermal cell fate choice, within axial progenitors. Collectively, Nr6a1 has an axially-restricted role in all major cellular and tissue-level events required for vertebral column formation, supporting the view that changes in Nr6a1 levels may underlie evolutionary changes in axial formulae.

Loss of ciliary transition zone protein TMEM107 leads to heterotaxy in mice.

Cilia in most vertebrate left-right organizers are involved in the original break in left-right (L-R) symmetry, however, less is known about their roles in subsequent steps of the cascade - relaying the signaling and maintaining the established asymmetry. Here we describe the L-R patterning cascades in two mutants of a ciliary transition zone protein TMEM107, revealing that near-complete loss of cilia in Tmem107null leads to left pulmonary isomerism due to the failure of the midline barrier. Contrary, partially retained cilia in the node and the midline of a hypomorphic Tmem107schlei mutant appear sufficient for the formation of the midline barrier and establishment and maintenance of the L-R asymmetry. Despite misregulation of Shh signaling in both mutants, the presence of normal Lefty1 expression and midline barrier formation in Tmem107schlei mutants, suggests a requirement for cilia, but not necessarily Shh signaling for Lefty1 expression and midline barrier formation.

Filling in the phylogenetic gaps: Induction, migration, and differentiation of neural crest cells in a squamate reptile, the veiled chameleon (Chamaeleo calyptratus).

Neural crest cells comprise a migratory progenitor cell population that differentiate into cell types such as neurons and glia of the peripheral nervous system, pigment cells, hormone secreting cells in glands, and skeletal and connective tissue in the head, thus making important contributions to most tissues and organs throughout the vertebrate body. The evolutionary appearance of neural crest cells is considered synonymous with the origin of vertebrates and their subsequent diversification and radiation. While the comparative biology of neural crest cells has been studied for a century and a half beginning with their discovery by Wilhelm His in 1868, most of our understanding of their development and function has come from a small number of species. Thus, critical gaps exist in our understanding of how neural crest cells mediate evolution and development. This is particularly true with respect to squamate reptiles (lizards, snakes, amphisbaenians), which account for approximately one-third of all living tetrapods. Here, we present veiled chameleons (Chamaeleo calyptratus) as a model system for studying neural crest cell development in squamates. Chameleons exhibit various morphological specializations associated with an arboreal lifestyle that may have been facilitated through neural crest cells acting as a conduit for evolutionary change.

The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28.

KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity.

Of Mice and Snakes: A Tail of Oct4.

The vertebrate axial skeleton comprises regions of specialized vertebrae, which vary in length between lineages. Aires et al. (2016) uncover a key role for Oct4 in determining trunk length in mice. Additionally, a heterochronic shift in Oct4 expression may underlie the extreme elongation of the trunk in snakes.

TMEM107 Is a Critical Regulator of Ciliary Protein Composition and Is Mutated in Orofaciodigital Syndrome.

The proximate causes of multiple human genetic syndromes (ciliopathies) are disruptions in the formation or function of the cilium, an organelle required for a multitude of developmental processes. We previously identified Tmem107 as a critical regulator of cilia formation and embryonic organ development in the mouse. Here, we describe a patient with a mutation in TMEM107 that developed atypical Orofaciodigital syndrome (OFD), and show that the OFD patient shares several morphological features with the Tmem107 mutant mouse including polydactyly and reduced numbers of ciliated cells. We show that TMEM107 appears to function within cilia to regulate protein content, as key ciliary proteins do not localize normally in cilia derived from the Tmem107 mouse mutant and the human patient. These data indicate that TMEM107 plays a key, conserved role in regulating ciliary protein composition, and is a novel candidate for ciliopathies of unknown etiology.

The membrane-anchored serine protease prostasin (CAP1/PRSS8) supports epidermal development and postnatal homeostasis independent of its enzymatic activity.

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8(-/-)) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8(Cat-/Cat-) mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8(-/-) and Prss8(Cat-/Cat-) mice born within the same litter. Furthermore, Prss8(Cat-/Cat-) mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease.

Pax7 is regulated by cMyb during early neural crest development through a novel enhancer.

The neural crest (NC) is a migratory population of cells unique to vertebrates that generates many diverse derivatives. NC cells arise during gastrulation at the neural plate border (NPB), which is later elevated as the neural folds (NFs) form and fuse in the dorsal region of the closed neural tube, from where NC cells emigrate. In chick embryos, Pax7 is an early marker, and necessary component of NC development. Unlike other early NPB markers, which are co-expressed in lateral ectoderm, medial neural plate or posterior-lateral mesoderm, Pax7 early expression seems more restricted to the NPB. However, the molecular mechanisms controlling early Pax7 expression remain poorly understood. Here, we identify a novel enhancer of Pax7 in avian embryos that replicates the expression of Pax7 associated with early NC development. Expression from this enhancer is found in early NPB, NFs and early emigrating NC, but unlike Pax7, which is also expressed in mesodermal derivatives, this enhancer is not active in somites. Further analysis demonstrates that cMyb is able to interact with this enhancer and modulates reporter and endogenous early Pax7 expression; thus, cMyb is identified as a novel regulator of Pax7 in early NC development.

Reduced prostasin (CAP1/PRSS8) activity eliminates HAI-1 and HAI-2 deficiency-associated developmental defects by preventing matriptase activation.

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.