Genetic analysis reveals a hierarchy of interactions between polycystin-encoding genes and genes controlling cilia function during left-right determination

During mammalian development, left-right (L-R) asymmetry is established by a cilia-driven leftward fluid flow within a midline embryonic cavity called the node. This ‘nodal flow’ is detected by peripherally-located crown cells that each assemble a primary cilium which contain the putative Ca2+ channel PKD2. The interaction of flow and crown cell cilia promotes left side-specific expression of Nodal in the lateral plate mesoderm (LPM). Whilst the PKD2-interacting protein PKD1L1 has also been implicated in L-R patterning, the underlying mechanism by which flow is detected and the genetic relationship between Polycystin function and asymmetric gene expression remains unknown. Here, we characterize a Pkd1l1 mutant line in which Nodal is activated bilaterally, suggesting that PKD1L1 is not required for LPM Nodal pathway activation per se, but rather to restrict Nodal to the left side downstream of nodal flow. Epistasis analysis shows that Pkd1l1 acts as an upstream genetic repressor of Pkd2. This study therefore provides a genetic pathway for the early stages of L-R determination. Moreover, using a system in which cultured cells are supplied artificial flow, we demonstrate that PKD1L1 is sufficient to mediate a Ca2+ signaling response after flow stimulation. Finally, we show that an extracellular PKD domain within PKD1L1 is crucial for PKD1L1 function; as such, destabilizing the domain causes L-R defects in the mouse. Our demonstration that PKD1L1 protein can mediate a response to flow coheres with a mechanosensation model of flow sensation in which the force of fluid flow drives asymmetric gene expression in the embryo

CRISIS AFAR: an international collaborative study of the impact of the COVID-19 pandemic on mental health and service access in youth with autism and neurodevelopmental conditions

BackgroundHeterogeneous mental health outcomes during the COVID-19 pandemic are documented in the general population. Such heterogeneity has not been systematically assessed in youth with autism spectrum disorder (ASD) and related neurodevelopmental disorders (NDD). To identify distinct patterns of the pandemic impact and their predictors in ASD/NDD youth, we focused on pandemic-related changes in symptoms and access to services.MethodsUsing a naturalistic observational design, we assessed parent responses on the Coronavirus Health and Impact Survey Initiative (CRISIS) Adapted For Autism and Related neurodevelopmental conditions (AFAR). Cross-sectional AFAR data were aggregated across 14 European and North American sites yielding a clinically well-characterized sample of N = 1275 individuals with ASD/NDD (age = 11.0 ± 3.6 years; n females = 277). To identify subgroups with differential outcomes, we applied hierarchical clustering across eleven variables measuring changes in symptoms and access to services. Then, random forest classification assessed the importance of socio-demographics, pre-pandemic service rates, clinical severity of ASD-associated symptoms, and COVID-19 pandemic experiences/environments in predicting the outcome subgroups.ResultsClustering revealed four subgroups. One subgroup-broad symptom worsening only (20%)-included youth with worsening across a range of symptoms but with service disruptions similar to the average of the aggregate sample. The other three subgroups were, relatively, clinically stable but differed in service access: primarily modified services (23%), primarily lost services (6%), and average services/symptom changes (53%). Distinct combinations of a set of pre-pandemic services, pandemic environment (e.g., COVID-19 new cases, restrictions), experiences (e.g., COVID-19 Worries), and age predicted each outcome subgroup.LimitationsNotable limitations of the study are its cross-sectional nature and focus on the first six months of the pandemic.ConclusionsConcomitantly assessing variation in changes of symptoms and service access during the first phase of the pandemic revealed differential outcome profiles in ASD/NDD youth. Subgroups were characterized by distinct prediction patterns across a set of pre- and pandemic-related experiences/contexts. Results may inform recovery efforts and preparedness in future crises; they also underscore the critical value of international data-sharing and collaborations to address the needs of those most vulnerable in times of crisis

Heavy and light roles: myosin in the morphogenesis of the heart

Myosin is an essential component of cardiac muscle, from the onset of cardiogenesis through to the adult heart. Although traditionally known for its role in energy transduction and force development, recent studies suggest that both myosin heavy-chain and myosin lightchain proteins are required for a correctly formed heart. Myosins are structural proteins that are not only expressed from early stages of heart development, but when mutated in humans they may give rise to congenital heart defects. This review will discuss the roles of myosin, specifically with regards to the developing heart. The expression of each myosin protein will be described, and the effects that altering expression has on the heart in embryogenesis in different animal models will be discussed. The human molecular genetics of the myosins will also be reviewed

Early doors (Edo) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping

Early doors ( Edo

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1–CLOCK complexes is suppressed by PER–CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping

Phenotyping of <i>Pkd1l1</i>^{<i>tm1/tm1</i>} Mutants.

<p>(A) Schematic diagram of PKD1L1 and PKD2 showing protein domains and the nature of the <i>Pkd1l1</i>^<i>rks</i> and <i>Pkd2</i>^<i>lrm4</i> point mutations. The double headed red arrow denotes the site of interaction between PKD1L1 and PKD2. PKD—Polycystic Kidney Disease; REJ—Receptor for Egg Jelly; GPS—G-protein Coupled Receptor Proteolytic Site; PLAT—Polycystin-1, Liopoxygenase, Alpha-Toxin. (B) <i>Pkd1l1</i>^{<i>tm1/tm1</i>} and sibling control showing reversed and normal situs, respectively. White arrows indicate stomach position. (C) Heart-stomach discordance (H-S Disc.) in <i>Pkd1l1</i>^{<i>tm1/tm1</i>}, <i>Dnah11</i>^<i>iv/iv</i> and <i>Pkd1l1</i>^{<i>rks/rks</i>} mutants scored at E13.5. Normally, the heart apex and stomach are positioned to the left. H-S Disc. is defined as the heart apex and stomach being on opposite sides. ns—not significant; *—p<0.05; **—p<0.001, Fisher’s Exact Test applied. (D-F) Lung situs assessed at E13.5 for embryos of the indicated genotypes with the ratio of lung lobes between left and right sides given. The percentage and total numbers of embryos showing each phenotype are indicated in <i>(F)</i>. (G-P) Expression patterns of <i>Nodal</i>, <i>Pitx2</i>, and <i>Lefty1/2</i> in embryos at E8.5 of the indicated genotypes, with the percentage number of embryos exhibiting each phenotype and the total number given. Embryos exhibiting bilateral marker expression are further categorized by whether they show equal or biased expression between the left and right sides. The inset in <i>(M)</i> shows a <i>Pkd1l1</i>^{<i>tm1/tm1</i>} embryo with bilateral <i>Pitx2</i> expression but with a right-sided bias. Arrowheads in <i>(N)</i> and <i>(O)</i> indicate midline <i>Lefty1</i> expression. <i>t</i> is shorthand for <i>Pkd1l1</i>^<i>tm1</i>. (Q-R) Sonic hedgehog (<i>Shh</i>) expression in the node (n) and notochord (nc) at E8.5.</p

Destabilization of a PKD Domain by the <i>Pkd1l1</i>^<i>rks</i> Mutation.

<p>(A-C) Structure of human PKD1 PKD domain 1 (<i>A</i>) and models of mouse PKD1L1 PKD domain 2; wild-type (<i>B</i>) or <i>rks</i>-mutated (<i>C</i>). Domains are largely composed of β-sheets (block arrows). The aspartic acid mutated in <i>Pkd1l1</i>^<i>rks</i>, or its equivalent in PKD1, is shown in space-fill. The asterisks denote loss of secondary structure in the <i>rks</i>-mutated domain. (D) SRCD spectroscopy of mouse PKD1L1 PKD domain 2 for wild-type and <i>rks</i>-mutated domains. Spectra are consistent with decreased stability (decreased secondary structure) in mutated domains. (E) Thermal denaturation analysis of PKD1L1 PKD domain 2: a reduced melting temperature (Tm) of 56.4°C is evident in the <i>rks</i>-mutated domain; in wild-type controls a Tm of 68.6°C is detected.</p

Multi-repression Model for L-R Asymmetry Determination in Crown Cells.

<p>(A) Schematic of a 3 ss flat-mounted mouse embryo showing somites (yellow), and node (blue). (B) Pictorial representation of the multi-repression model in which flow represses <i>Pkd1l1</i> on the left side, resulting in the derepression of <i>Pkd2</i>, inhibition of <i>Cerl2</i> and, as a result, higher NODAL activity on the left. (C-E) Predictions of the multi-repression model in various genetic mutants including the impact on the crown cell genetic pathway as well as the predicted LPM Nodal cascade activity.</p

Flow-Induced Ca²⁺ Signaling Depends on PKD1L1.

<p>(A-B) <i>Pkd1</i>^<i>+/+</i> <i>(A)</i> and <i>Pkd1</i>^{<i>–/–</i>}<i>(B)</i> cells were transfected with vector-GFP alone, PKD1L1-GFP or PKD1L1^rks-GFP. Successfully transfected cells had green fluorescence (GFP), and the entire cell population was observed by DIC. After baseline Ca²⁺ level was taken, fluid-shear stress was applied to cells (arrow). Numbers indicate time in seconds (s). Color bars indicate Ca²⁺ level (pseudocoloured), where black-purple and yellow-red represent low and high Ca²⁺ levels, respectively (C-D) Quantitation from independent experiments of <i>Pkd1</i>^<i>+/+</i> <i>(C)</i> and <i>Pkd1</i>^{<i>–/–</i>}<i>(D)</i> cells was averaged and plotted in line graphs. Within the same cell population, successfully transfected (GFP+) and non-transfected (GFP-) cells were analyzed separately. Arrows indicate the start of fluid-shear stress. Time is indicated in seconds (s). (E) Statistical analysis was done by analyzing the peak changes of intracellular Ca²⁺. While vector-GFP is used as a negative control, non-transfected cells (GFP-) were also used as an internal control. n = 150 cells for each group in three independent transfections. *—p<0.05.</p

Cilia and PKD2 Localization and Function.

<p>(A-E) PKD2 localization in nodal cilia of embryos of the indicated genotype. Staining was divided into categories and quantitation is given in (<i>A</i>). In <i>(A)</i>, all genotypes are statistically significantly different from each other (p<0.001) except for <i>Pkd2</i>^{<i>+/lrm4</i>} and <i>Pkd1l1</i>^<i>+/tm1</i> which are statistically not significantly different.</p