Restriction of retinoic acid activity by Cyp26b1 is required for proper timing and patterning of osteogenesis during zebrafish development.

Skeletal syndromes are among the most common birth defects. Vertebrate skeletogenesis involves two major cell types: cartilage-forming chondrocytes and bone-forming osteoblasts. In vitro, both are under the control of retinoic acid (RA), but its exact in vivo effects remained elusive. Here, based on the positional cloning of the dolphin mutation, we have studied the role of the RA-oxidizing enzyme Cyp26b1 during cartilage and bone development in zebrafish. cyp26b1 is expressed in condensing chondrocytes as well as in osteoblasts and their precursors. cyp26b1 mutants and RA-treated wild-type fish display a reduction in midline cartilage and the hyperossification of facial and axial bones, leading to fusions of vertebral primordia, a defect not previously described in the context of RA signaling. Fusions of cervical vertebrae were also obtained by treating mouse fetuses with the specific Cyp26 inhibitor R115866. Together with data on the expression of osteoblast markers, our results indicate that temporal and spatial restriction of RA signaling by Cyp26 enzymes is required to attenuate osteoblast maturation and/or activity in vivo. cyp26b1 mutants may serve as a model to study the etiology of human vertebral disorders such as Klippel-Feil anomaly.

The multidomain protein Brpf1 binds histones and is required for Hox gene expression and segmental identity.

The Trithorax group (TrxG) is composed of diverse, evolutionary conserved proteins that form chromatin-associated complexes accounting for epigenetic transcriptional memory. However, the molecular mechanisms by which particular loci are marked for reactivation after mitosis are only partially understood. Here, based on genetic analyses in zebrafish, we identify the multidomain protein Brpf1 as a novel TrxG member with a central role during development. brpf1 mutants display anterior transformations of pharyngeal arches due to progressive loss of anterior Hox gene expression. Brpf1 functions in association with the histone acetyltransferase Moz (Myst3), an interaction mediated by the N-terminal domain of Brpf1, and promotes histone acetylation in vivo. Brpf1 recruits Moz to distinct sites of active chromatin and remains at chromosomes during mitosis, mediated by direct histone binding of its bromodomain, which has a preference for acetylated histones, and its PWWP domain, which binds histones independently of their acetylation status. This is the first demonstration of histone binding for PWWP domains. Mutant analyses further show that the PWWP domain is absolutely essential for Brpf1 function in vivo. We conclude that Brpf1, coordinated by its particular set of domains, acts by multiple mechanisms to mediate Moz-dependent histone acetylation and to mark Hox genes for maintained expression throughout vertebrate development.

REREa/Atrophin-2 interacts with histone deacetylase and Fgf8 signaling to regulate multiple processes of zebrafish development.

The transcriptional regulator RERE/Atrophin-2 (RERE) is required for the normal patterning of the early vertebrate embryo, including the central nervous system, pharyngeal arches, and limbs. Consistent with a role as a transcriptional corepressor, RERE binds histone deacetylase 1 and 2 (HDAC1/2), and orphan nuclear receptors such as Tlx. Here, we identify the zebrafish babyface (bab) as a mutant in rerea and show that it interacts genetically with fibroblast growth factor 8 (fgf8). We suggest that this finding is largely due to its interactions with HDAC, because genetic or pharmacological disruptions of HDAC phenocopy many features of the bab mutant. Furthermore, removing the functions of either REREa or HDAC synergizes with loss of Fgf8 function to disrupt posterior mesoderm formation during somitogenesis, midbrain-hindbrain boundary maintenance, and pharyngeal cartilage development. Together, these results reveal novel in vivo roles for REREa in HDAC-mediated regulation of Fgf signaling. We present a model for RERE-dependent patterning in which tissue-specific transcriptional repression, by means of an REREa-HDAC complex, modulates growth factor signaling during embryogenesis.

Defective potassium channel Kir2.1 trafficking underlies Andersen-Tawil syndrome.

Andersen-Tawil syndrome is a skeletal and cardiac muscle disease with developmental features caused by mutations in the inward rectifier K+ channel gene KCNJ2. Patients harboring these mutations exhibit extremely variable expressivities. To explore whether these mutations can be correlated with a specific patient phenotype, we expressed both wild-type (WT) and mutant genes cloned into a bi-cistronic vector. Functional expression in human embryonic kidney 293 cells showed that none of the mutant channels express current when present alone. When co-expressed with WT channels, only construct V302M-WT yields inward current. Confocal microscopy fluorescence revealed three patterns of channel expression in the cell: 1) mutations D71V, N216H, R218Q, and pore mutations co-assemble and co-localize to the membrane with the WT and exert a dominant-negative effect on the WT channels; 2) mutation V302M leads to channels that lose their ability to co-assemble with WT and traffic to the cell surface; 3) deletions Delta 95-98 and Delta 314-315 lead to channels that do not traffic to the membrane but retain their ability to co-assemble with WT channels. These data show that the Andersen-Tawil syndrome phenotype may occur through a dominant-negative effect as well as through haplo-insufficiency and reveal amino acids critical in trafficking and conductance of the inward rectifier K+ channels.

Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome).

Andersen syndrome (AS) is a rare, inherited disorder characterized by periodic paralysis, long QT (LQT) with ventricular arrhythmias, and skeletal developmental abnormalities. We recently established that AS is caused by mutations in KCNJ2, which encodes the inward rectifier K(+) channel Kir2.1. In this report, we characterized the functional consequences of three novel and seven previously described KCNJ2 mutations using a two-microelectrode voltage-clamp technique and correlated the findings with the clinical phenotype. All mutations resulted in loss of function and dominant-negative suppression of Kir2.1 channel function. In mutation carriers, the frequency of periodic paralysis was 64% and dysmorphic features 78%. LQT was the primary cardiac manifestation, present in 71% of KCNJ2 mutation carriers, with ventricular arrhythmias present in 64%. While arrhythmias were common, none of our subjects suffered sudden cardiac death. To gain insight into the mechanism of arrhythmia susceptibility, we simulated the effect of reduced Kir2.1 using a ventricular myocyte model. A reduction in Kir2.1 prolonged the terminal phase of the cardiac action potential, and in the setting of reduced extracellular K(+), induced Na(+)/Ca(2+) exchanger-dependent delayed afterdepolarizations and spontaneous arrhythmias. These findings suggest that the substrate for arrhythmia susceptibility in AS is distinct from the other forms of inherited LQT syndrome.