Mosmo Is Required for Zebrafish Craniofacial Formation

Hedgehog (Hh) signaling is a highly regulated molecular pathway implicated in many developmental and homeostatic events. Mutations in genes encoding primary components or regulators of the pathway cause an array of congenital malformations or postnatal pathologies, the extent of which is not yet fully defined. Mosmo (Modulator of Smoothened) is a modulator of the Hh pathway, which encodes a membrane tetraspan protein. Studies in cell lines have shown that Mosmo promotes the internalization and degradation of the Hh signaling transducer Smoothened (Smo), thereby down-modulating pathway activation. Whether this modulation is essential for vertebrate embryonic development remains poorly explored. Here, we have addressed this question and show that in zebrafish embryos, the two mosmo paralogs, mosmoa and mosmob, are expressed in the head mesenchyme and along the entire ventral neural tube. At the cellular level, Mosmoa localizes at the plasma membrane, cytoplasmic vesicles and primary cilium in both zebrafish and chick embryos. CRISPR/Cas9 mediated inactivation of both mosmoa and mosmob in zebrafish causes frontonasal hypoplasia and craniofacial skeleton defects, which become evident in the adult fish. We thus suggest that MOSMO is a candidate to explain uncharacterized forms of human congenital craniofacial malformations, such as those present in the 16p12.1 chromosomal deletion syndrome encompassing the MOSMO locus.

303 Identification of candidate genes related to temperament in Brahman cattle

The objective of this study was to identify genomic regions and genes associated with beef cattle temperament. Temperament, measured as exit velocity (EV; m/s), was recorded in 1,370 Brahman cattle from Texas A&M AgriLife Research at Overton, TX. We identified two groups of temperament-contrasting animals. Cows were calm if their EV of 0.16–3.41 m/s and bulls if their EV was 0.4–3.12 m/s (n-119). Cows were temperamental if their EV was 3.55–7.66 m/s and bulls if their EV was 3.13–10.83 m/s (n = 79). The 198 animals were genotyped using the GGP-HD-150K chip. 139,376 SNPs were evaluated for association with temperament. 13 SNP′s were associated with EV (P < 4.0E-05). The SNPs GABRG2-26484, NRXN3-26436 and TBX20-191081 are located in introns of the GABRG2, NRXN3 and TBX20 genes, respectively. The GABRG2 gene encodes a GABA receptor, the major inhibitory neurotransmitter in the mammalian brain. The NRXN3 gene encodes receptor proteins related to chemical transmission at synapses. TBX20 is a member of the T-box transcription factor family expressed in the developing stages of heart, limbs, eye and ventral neural tube. To test the effect of these 3 SNP′s on EV, Pen-Score and Temperament-Score, a general linear model was fitted including the fixed effects of sex of calf and year of birth, and the individual effect of the 3 SNPs. The marker TBX20-191081 was associated with the three traits evaluated (P < 0.01), where the GG genotype was associated with the calmest temperament. The GG genotype had a significant effect on EV (P < 0.0001) that was 1.35 and 1.95 m/s slower than AG and AA, respectively. For TS, the GG genotype had a TS that was 1.41 and 1.24 DS less than those of the AA and GA genotypes. Our study indicates that genetic control of cattle temperament has a wide network of genes with divergent functions and genetic background specificity.

Critical roles of ARHGAP36 as a signal transduction mediator of Shh pathway in lateral motor columnar specification

During spinal cord development, Sonic hedgehog (Shh), secreted from the floor plate, plays an important role in the production of motor neurons by patterning the ventral neural tube, which establishes MN progenitor identity. It remains unknown, however, if Shh signaling plays a role in generating columnar diversity of MNs that connect distinct target muscles. Here, we report that Shh, expressed in MNs, is essential for the formation of lateral motor column (LMC) neurons in vertebrate spinal cord. This novel activity of Shh is mediated by its downstream effector ARHGAP36, whose expression is directly induced by the MN-specific transcription factor complex Isl1-Lhx3. Furthermore, we found that AKT stimulates the Shh activity to induce LMC MNs through the stabilization of ARHGAP36 proteins. Taken together, our data reveal that Shh, secreted from MNs, plays a crucial role in generating MN diversity via a regulatory axis of Shh-AKT-ARHGAP36 in the developing mouse spinal cord.

Slit/Robo signals prevent spinal motor neuron emigration by organizing the spinal cord basement membrane

The developing spinal cord builds a boundary between the CNS and the periphery, in the form of a basement membrane. The spinal cord basement membrane is a barrier that retains CNS neuron cell bodies, while being selectively permeable to specific axon types. Spinal motor neuron cell bodies are located in the ventral neural tube next to the floor plate and project their axons out through the basement membrane to peripheral targets. However, little is known about how spinal motor neuron cell bodies are retained inside the ventral neural tube, while their axons can exit. In previous work, we found that disruption of Slit/Robo signals caused motor neuron emigration outside the spinal cord. In the current study, we investigate how Slit/Robo signals are necessary to keep spinal motor neurons within the neural tube. Our findings show that when Slit/Robo signals were removed from motor neurons, they migrated outside the spinal cord. Furthermore, this emigration was associated with abnormal basement membrane protein expression in the ventral spinal cord. Using Robo2 and Slit2 conditional mutants, we found that motor neuron-derived Slit/Robo signals were required to set up a normal basement membrane in the spinal cord. Together, our results suggest that motor neurons produce Slit signals that are required for the basement membrane assembly to retain motor neuron cell bodies within the spinal cord.

A Scube2-Shh feedback loop links morphogen release to morphogen signaling to enable scale invariant patterning of the ventral neural tube

To enable robust patterning, morphogen systems should be resistant to variations in gene expression and tissue size. Here we explore how a Shh morphogen gradient in the ventral neural tube enables proportional patterning in embryos of varying sizes. Using a surgical technique to reduce the size of zebrafish embryos and quantitative confocal microscopy, we find that patterning of neural progenitors remains proportional after size reduction. Intriguingly, a protein necessary for Shh release, Scube2, is expressed far from the source of sonic hedgehog production. scube2 expression levels control Shh signaling extent during ventral neural patterning and conversely Shh signaling represses the expression of scube2, thereby restricting its own signaling. scube2 is disproportionately downregulated in size-reduced embryos, providing a potential mechanism for size-dependent regulation of Shh. This regulatory feedback is necessary for pattern scaling, as demonstrated by a loss of scaling in scube2 overexpressing embryos. In a manner akin to the expander-repressor model of morphogen scaling, we conclude that feedback between Shh signaling and scube2 expression enables proportional patterning in the ventral neural tube by encoding a tissue size dependent morphogen signaling gradient.Summary StatementThe Shh morphogen gradient can scale to different size tissues by feedback between Scube2 mediated release of Shh and Shh based inhibition of Scube2 expressionAuthor ContributionsZ.M.C. conducted experiments and data analysis. Z.M.C and S.G.M. conceived the study, designed the experiments, and wrote the paper. K.I and Z.M.C. developed the size reduction technique. T.Y.C.T helped develop the image analysis technique and generated the tg(shha:memCherry) reporter line. S.G.M. supervised the overall study.

Gastric pouches and the mucociliary sole: setting the stage for nervous system evolution

Prerequisite for tracing nervous system evolution is understanding of the body plan, feeding behaviour and locomotion of the first animals in which neurons evolved. Here, a comprehensive scenario is presented for the diversification of cell types in early metazoans, which enhanced feeding efficiency and led to the emergence of larger animals that were able to move. Starting from cup-shaped, gastraea-like animals with outer and inner choanoflagellate-like cells, two major innovations are discussed that set the stage for nervous system evolution. First, the invention of a mucociliary sole entailed a switch from intra- to extracellular digestion and increased the concentration of nutrients flowing into the gastric cavity. In these animals, an initial nerve net may have evolved via division of labour from mechanosensory-contractile cells in the lateral body wall, enabling coordinated movement of the growing body that involved both mucociliary creeping and changes of body shape. Second, the inner surface of the animals folded into metameric series of gastric pouches, which optimized nutrient resorption and allowed larger body sizes. The concomitant acquisition of bilateral symmetry may have allowed more directed locomotion and, with more demanding coordinative tasks, triggered the evolution of specialized nervous subsystems. Animals of this organizational state would have resembled Ediacarian fossils such as Dickinsonia and may have been close to the cnidarian–bilaterian ancestor. In the bilaterian lineage, the mucociliary sole was used mostly for creeping, or frequently lost. One possible remnant is the enigmatic Reissner's fibre in the ventral neural tube of cephalochordates and vertebrates.