Loss of zebrafish pkd1l1 causes biliary defects that have implications for biliary atresia splenic malformation.

Biliary atresia is a fibroinflammatory neonatal disease with no effective therapies. A subset of cases (10-20%) is associated with laterality defects - labeled biliary atresia splenic malformation (BASM) syndrome. Recently, whole-exome sequencing of patients with BASM identified deleterious variants in PKD1L1. PKD1L1 is involved in left-right axis determination; however, its role in cholangiocytes is unknown. We generated the pkd1l1hsc117 allele using CRISPR/Cas9 mutagenesis in zebrafish to determine the role of Pkd1l1 in biliary development and function. Wild-type and mutant larvae were assessed for laterality defects, biliary function and biliary tree architecture at 5 days post fertilization. pkd1l1hsc117 mutant larvae exhibited early left-right patterning defects. The gallbladder was positioned on the left in 47% of mutants compared to 4% of wild-type larvae. Accumulation of PED6 in the gallbladder, an indicator of hepatobiliary function, was significantly reduced in pkd1l1hsc117 mutants (46%) compared to wild-type larvae (4%). pkd1l1hsc117 larvae exhibited fewer biliary epithelial cells and reduced density of the intrahepatic biliary network compared to those in wild-type larvae. These data highlight the essential role of pkd1l1 in normal development and function of the zebrafish biliary system, supporting a role for this gene as a cause of BASM.

Resolving primary pathomechanisms driving idiopathic-like spinal curvature using a new katnb1 scoliosis model.

Idiopathic scoliosis (IS) refers to abnormal spinal curvatures that occur in the absence of vertebral or neuromuscular defects. IS accounts for 80% of human spinal deformity, afflicts ∼3% of children worldwide, yet pathogenic mechanisms are poorly understood. A key role for cerebrospinal fluid (CSF) homeostasis in zebrafish spine development has been identified. Specifically, defects in cilia motility of brain ependymal cells (EC), CSF flow, and/or Reissner fiber (RF) assembly are observed to induce neuroinflammation, oxidative stress, abnormal CSF-contacting neuron activity, and urotensin peptide expression, all associating with scoliosis. However, the functional relevance of these observations to IS remains unclear. Here we characterize zebrafish katnb1 mutants as a new IS model. We define essential roles for Katnb1 in motile ciliated lineages, uncouple EC cilia and RF formation defects from spinal curvature, and identify abnormal CSF flow and cell stress responses as shared pathogenic signatures associated with scoliosis across diverse zebrafish models.

SCO-Spondin Defects and Neuroinflammation Are Conserved Mechanisms Driving Spinal Deformity across Genetic Models of Idiopathic Scoliosis.

Adolescent idiopathic scoliosis (AIS) affects 3% to 4% of children between the ages of 11 and 18 [1, 2]. This disorder, characterized by abnormal three-dimensional spinal curvatures that typically develop during periods of rapid growth, occurs in the absence of congenital vertebral malformations or neuromuscular defects [1]. Genetic heterogeneity [3] and a historical lack of appropriate animal models [4] have confounded basic understanding of AIS biology; thus, treatment options remain limited [5, 6]. Recently, genetic studies using zebrafish have linked idiopathic-like scoliosis to irregularities in motile cilia-mediated cerebrospinal fluid flow [7-9]. However, because loss of cilia motility in human primary ciliary dyskinesia patients is not fully associated with scoliosis [10, 11], other pathogenic mechanisms remain to be determined. Here, we demonstrate that zebrafish scospondin (sspo) mutants develop late-onset idiopathic-like spinal curvatures in the absence of obvious cilia motility defects. Sspo is a large secreted glycoprotein functionally associated with the subcommissural organ and Reissner's fiber [12]-ancient and enigmatic organs of the brain ventricular system reported to govern cerebrospinal fluid homeostasis [13, 14], neurogenesis [12, 15-18], and embryonic morphogenesis [19]. We demonstrate that irregular deposition of Sspo within brain ventricles is associated with idiopathic-like scoliosis across diverse genetic models. Furthermore, Sspo defects are sufficient to induce oxidative stress and neuroinflammatory responses implicated in AIS pathogenesis [9]. Through screening for chemical suppressors of sspo mutant phenotypes, we also identify potent agents capable of blocking severe juvenile spine deformity. Our work thus defines a new preclinical model of AIS and provides tools to realize novel therapeutic strategies.