GNB5 Mutations Cause an Autosomal-Recessive Multisystem Syndrome with Sinus Bradycardia and Cognitive Disability.

GNB5 encodes the G protein ß subunit 5 and is involved in inhibitory G protein signaling. Here, we report mutations in GNB5 that are associated with heart-rate disturbance, eye disease, intellectual disability, gastric problems, hypotonia, and seizures in nine individuals from six families. We observed an association between the nature of the variants and clinical severity; individuals with loss-of-function alleles had more severe symptoms, including substantial developmental delay, speech defects, severe hypotonia, pathological gastro-esophageal reflux, retinal disease, and sinus-node dysfunction, whereas related heterozygotes harboring missense variants presented with a clinically milder phenotype. Zebrafish gnb5 knockouts recapitulated the phenotypic spectrum of affected individuals, including cardiac, neurological, and ophthalmological abnormalities, supporting a direct role of GNB5 in the control of heart rate, hypotonia, and vision.

Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina.

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

M13 phage grafted with peptide motifs as a tool to detect amyloid-ß oligomers in brain tissue.

Oligomeric clusters of amyloid-ß (Aß) are one of the major biomarkers for Alzheimer's disease (AD). However, proficient methods to detect Aß-oligomers in brain tissue are lacking. Here we show that synthetic M13 bacteriophages displaying Aß-derived peptides on their surface preferentially interact with Aß-oligomers. When exposed to brain tissue isolated from APP/PS1-transgenic mice, these bacteriophages detect small-sized Aß-aggregates in hippocampus at an early age, prior to the occurrence of Aß-plaques. Similarly, the bacteriophages reveal the presence of such small Aß-aggregates in post-mortem hippocampus tissue of AD-patients. These results advocate bacteriophages displaying Aß-peptides as a convenient and low-cost tool to identify Aß-oligomers in post-mortem brain tissue of AD-model mice and AD-patients.

Loss of Hoxb8 alters spinal dorsal laminae and sensory responses in mice.

Although Hox gene expression has been linked to motoneuron identity, a role of these genes in development of the spinal sensory system remained undocumented. Hoxb genes are expressed at high levels in the dorsal horn of the spinal cord. Hoxb8 null mutants manifest a striking phenotype of excessive grooming and hairless lesions on the lower back. Applying local anesthesia underneath the hairless skin suppressed excessive grooming, indicating that this behavior depends on peripheral nerve activity. Functional ablation of mouse Hoxb8 also leads to attenuated response to nociceptive and thermal stimuli. Although spinal ganglia were normal, a lower postmitotic neural count was found in the dorsalmost laminae at lumbar levels around birth, leading to a smaller dorsal horn and a correspondingly narrowed projection field of nociceptive and thermoceptive afferents. The distribution of the dorsal neuronal cell types that we assayed, including neurons expressing the itch-specific gastrin-releasing peptide receptor, was disorganized in the lumbar region of the mutant. BrdU labeling experiments and gene-expression studies at stages around the birth of these neurons suggest that loss of Hoxb8 starts impairing development of the upper laminae of the lumbar spinal cord at approximately embryonic day (E)15.5. Because none of the neuronal markers used was unexpressed in the adult dorsal horn, absence of Hoxb8 does not impair neuronal differentiation. The data therefore suggest that a lower number of neurons in the upper spinal laminae and neuronal disorganization in the dorsal horn underlie the sensory defects including the excessive grooming of the Hoxb8 mutant.

Pannexin 1 Is Critically Involved in Feedback from Horizontal Cells to Cones.

Retinal horizontal cells (HCs) feed back negatively to cone photoreceptors and in that way generate the center/surround organization of bipolar cell receptive fields. The mechanism by which HCs inhibit photoreceptors is a matter of debate. General consensus exists that horizontal cell activity leads to the modulation of the cone Ca-current. This modulation has two components, one fast and the other slow. Several mechanisms for this modulation have been proposed: a fast ephaptic mechanism, and a slow pH mediated mechanism. Here we test the hypothesis that the slow negative feedback signal from HCs to cones is mediated by Panx1 channels expressed at the tips of the dendrites of horizontal cell. We generated zebrafish lacking Panx1 and found that the slow component of the feedback signal was strongly reduced in the mutants showing that Panx1 channels are a fundamental part of the negative feedback pathway from HCs to cones.

Hox cluster polarity in early transcriptional availability: a high order regulatory level of clustered Hox genes in the mouse.

The molecular mechanism underlying the 3' to 5' polarity of induction of mouse Hox genes is still elusive. While relief from a cluster-encompassing repression was shown to lead to all Hoxd genes being expressed like the 3'most of them, Hoxd1 (Kondo and Duboule, 1999), the molecular basis of initial activation of this 3'most gene, is not understood yet. We show that, already before primitive streak formation, prior to initial expression of the first Hox gene, a dramatic transcriptional stimulation of the 3'most genes, Hoxb1 and Hoxb2, is observed upon a short pulse of exogenous retinoic acid (RA), whereas it is not in the case for more 5', cluster-internal, RA-responsive Hoxb genes. In contrast, the RA-responding Hoxb1lacZ transgene that faithfully mimics the endogenous gene (Marshall et al., 1994) did not exhibit the sensitivity of Hoxb1 to precocious activation. We conclude that polarity in initial activation of Hoxb genes reflects a greater availability of 3'Hox genes for transcription, suggesting a pre-existing (susceptibility to) opening of the chromatin structure at the 3' extremity of the cluster. We discuss the data in the context of prevailing models involving differential chromatin opening in the directionality of clustered Hox gene transcription, and regarding the importance of the cluster context for correct timing of initial Hox gene expression.Interestingly, Cdx1 manifested the same early transcriptional availability as Hoxb1.

Cdx and Hox genes differentially regulate posterior axial growth in mammalian embryos.

Hox and Cdx transcription factors regulate embryonic positional identities. Cdx mutant mice display posterior body truncations of the axial skeleton, neuraxis, and caudal urorectal structures. We show that trunk Hox genes stimulate axial extension, as they can largely rescue these Cdx mutant phenotypes. Conversely, posterior (paralog group 13) Hox genes can prematurely arrest posterior axial growth when precociously expressed. Our data suggest that the transition from trunk to tail Hox gene expression successively regulates the construction and termination of axial structures in the mouse embryo. Thus, Hox genes seem to differentially orchestrate posterior expansion of embryonic tissues during axial morphogenesis as an integral part of their function in specifying head-to-tail identity. In addition, we present evidence that Cdx and Hox transcription factors exert these effects by controlling Wnt signaling. Concomitant regulation of Cyp26a1 expression, restraining retinoic acid signaling away from the posterior growth zone, may likewise play a role in timing the trunk-tail transition.

The Cdx4 mutation affects axial development and reveals an essential role of Cdx genes in the ontogenesis of the placental labyrinth in mice.

Caudal related homeobox (Cdx) genes have so far been shown to be important for embryonic axial elongation and patterning in several vertebrate species. We have generated a targeted mutation of mouse Cdx4, the third member of this family of transcription factor encoding genes and the last one to be inactivated genetically. Cdx4-null embryos were born healthy and appeared morphologically normal. A subtle contribution of Cdx4 to anteroposterior (AP) vertebral patterning was revealed in Cdx1/Cdx4 and Cdx2/Cdx4 compound mutants. Neither Cdx4-null nor Cdx1/Cdx4 double mutants are impaired in their axial elongation, but a redundant contribution of Cdx4 in this function was unveiled when combined with a Cdx2 mutant allele. In addition, inactivation of Cdx4 combined with heterozygous loss of Cdx2 results in embryonic death around E10.5 and reveals a novel function of Cdx genes in placental ontogenesis. In a subset of Cdx2/Cdx4 compound mutants, the fully grown allantois failed to fuse with the chorion. The remaining majority of these mutants undergo successful chorio-allantois fusion but fail to properly extend their allantoic vascular network into the chorionic ectoderm and do not develop a functional placental labyrinth. We present evidence that Cdx4 plays a crucial role in the ontogenesis of the allantoic component of the placental labyrinth when one Cdx2 allele is inactivated. The axial patterning role of Cdx transcription factors thus extends posteriorly to the epiblast-derived extra-embryonic mesoderm and, consequent upon the evolution of placental mammals, is centrally involved in placental morphogenesis. The relative contribution of Cdx family members in the stepwise ontogenesis of a functional placenta is discussed, with Cdx2 playing an obligatory part, assisted by Cdx4. The possible participation of Cdx1 was not documented but cannot be ruled out until allelic combinations further decreasing Cdx dose have been analyzed. Cdx genes thus operate in a redundant way during placentogenesis, as they do during embryonic axial elongation and patterning, and independently from the previously reported early Cdx2-specific role in the trophectoderm at implantation.

Cdx2 is essential for axial elongation in mouse development.

Inactivation of Cdx2 leads to preimplantation embryonic lethality. Rescue of the implantation defect by tetraploid fusion established that Cdx2 is necessary for trophoblastic development, vasculogenesis in the yolk sac mesoderm, allantoic growth, and chorioallantoic fusion. "Rescued" Cdx2 mutants die at late gastrulation stages because of failure of placental development. Cdx2 is also needed for the completion of the normal process of gastrulation and tail bud elongation. Presegmental paraxial mesoderm is severely restricted in amount and somites posterior to somite 5 are abnormal. The Cdx2 mutation, like mutations impairing Wnt and Fgf signaling, causes posterior truncations and disturbs axial patterning of the embryonic structures, indicated by changes in the Hox expression domains. The gene appears to be important in the integration of the pathways controlling embryonic axial elongation, and anterior-posterior patterning.

Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation.

Mouse Cdx and Hox genes presumably evolved from genes on a common ancestor cluster involved in anteroposterior patterning. Drosophila caudal (cad) is involved in specifying the posterior end of the early embryo, and is essential for patterning tissues derived from the most caudal segment, the analia. Two of the three mouse Cdx paralogues, Cdx 1 and Cdx2, are expressed early in a Hox-like manner in the three germ layers. In the nascent paraxial mesoderm, both genes are expressed in cells contributing first to the most rostral, and then to progressively more caudal parts of the vertebral column. Later, expression regresses from the anterior sclerotomes, and is only maintained for Cdx1 in the dorsal part of the somites, and for both genes in the tail bud. Cdx1 null mutants show anterior homeosis of upper cervical and thoracic vertebrae. Cdx2-null embryos die before gastrulation, and Cdx2 heterozygotes display anterior transformations of lower cervical and thoracic vertebrae. We have analysed the genetic interactions between Cdx1 and Cdx2 in compound mutants. Combining mutant alleles for both genes gives rise to anterior homeotic transformations along a more extensive length of the vertebral column than do single mutations. The most severely affected Cdx1 null/Cdx2 heterozygous mice display a posterior shift of their cranio-cervical, cervico-thoracic, thoraco-lumbar, lumbo-sacral and sacro-caudal transitions. The effects of the mutations in Cdx1 and Cdx2 were co-operative in severity, and a more extensive posterior shift of the expression of three Hox genes was observed in double mutants. The alteration in Hox expression boundaries occurred early. We conclude that both Cdx genes cooperate at early stages in instructing the vertebral progenitors all along the axis, at least in part by setting the rostral expression boundaries of Hox genes. In addition, Cdx mutants transiently exhibit alterations in the extent of Hox expression domains in the spinal cord, reminding of the strong effects of overexpressing Cdx genes on Hox gene expression in the neurectoderm. Phenotypical alterations in the peripheral nervous system were observed at mid-gestation stages. Strikingly, the altered phenotype at caudal levels included a posterior truncation of the tail, mildly affecting Cdx2 heterozygotes, but more severely affecting Cdx1/Cdx2 double heterozygotes and Cdx1 null/Cdx2 heterozygotes. Mutations in Cdx1 and Cdx2 therefore also interfere with axis elongation in a cooperative way. The function of Cdx genes in morphogenetic processes during gastrulation and tail bud extension, and their relationship with the Hox genes are discussed in the light of available data in Amphioxus, C. elegans, Drosophila and mice.