Numerous tissue transplantations have demonstrated that otocysts can develop into normal ears in any location in all vertebrates tested thus far, though the pattern of innervation of these transplanted ears has largely been understudied. Here, expanding on previous findings that transplanted ears demonstrate capability of local brainstem innervation and can also be innervated themselves by efferents, we show that inner ear afferents grow toward the spinal cord mostly along existing afferent and efferent fibers and preferentially enter the dorsal spinal cord. Once in the dorsal funiculus of the spinal cord, they can grow toward the hindbrain and can diverge into vestibular nuclei. Inner ear afferents can also project along lateral line afferents. Likewise, lateral line afferents can navigate along inner ear afferents to reach hair cells in the ear. In addition, transplanted ears near the heart show growth of inner ear afferents along epibranchial placode-derived vagus afferents. Our data indicate that inner ear afferents can navigate in foreign locations, likely devoid of any local ear-specific guidance cues, along existing nerves, possibly using the nerve-associated Schwann cells as substrate to grow along. However, within the spinal cord and hindbrain, inner ear afferents can navigate to vestibular targets, likely using gradients of diffusible factors that define the dorso-ventral axis to guide them. Finally, afferents of transplanted ears functionally connect to native hindbrain vestibular circuitry, indicated by eliciting a startle behavior response, and providing excitatory input to specific sets of extraocular motoneurons.
Abstract Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions, namely the dorsal morula, yolk syncytial layer, and distal hypoblast/anterior visceral endoderm (in amphibians, teleosts and mammals, respectively), require the localised stabilisation of nuclear Beta-catenin (Ctnnb1), implying that localised Wnt/Beta-catenin signalling activity is critical in their establishment. However, it is becoming increasingly apparent that the stabilisation of Beta-catenin in this context may be initiated independently of secreted Wnt growth factor activity. In Xenopus , dorsal Beta-catenin stabilisation is initiated by a requisite microtubule-mediated symmetry-breaking event in the fertilised egg: “cortical rotation”. Vegetally-localised wnt11b mRNA has been implicated upstream of Beta-catenin in this context, as has the dorsal enrichment of Wnt ligand-independent activators of Beta-catenin, but the extent that each of these processes contribute to axis formation in this paradigm remains unclear. Here we describe a maternal effect mutation in Xenopus laevis wnt11b . L , generated by CRISPR mutagenesis. We demonstrate a maternal requirement for timely and complete gastrulation morphogenesis and a zygotic requirement for proper left-right asymmetry. We also show that a subset of maternal wnt11b mutants have axis and dorsal gene expression defects, but that Wnt11b likely does not act through the Wnt coreceptor Lrp6 or through Dishevelled, which we additionally show (using exogenous constructs) do not exhibit patterns of activity consistent with roles in early Beta-catenin stabilisation. Instead, we find that microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs, leading to less organised and directed vegetal microtubule arrays. In conclusion, we propose that Wnt11b signals to the cytoskeleton in the egg or early zygote to enable robust cortical rotation, and thus acts in the distribution of putative dorsal determinants rather than as a component or effector of the determinants themselves.
The Wnt signaling network has major roles in developmental processes, human diseases and stem cell biology. A protein complex that includes Axin, APC and GSK3 tightly regulates this signaling. Axin, a key player in this complex, contains four critical domains: a β‐catenin binding domain, a GSK binding domain, an RGS domain and a DIX domain. Our study focuses on the role of the Axin‐RGS domain. Typically, RGS domains contain a conserved asparagine (Asn), critical for the ability to bind heterotrimeric G‐proteins. Interestingly, we find that the Axin‐RGS domain encodes a glutamine (Gln) at the equivalent position, suggesting a novel function for Axin‐RGS. We tested whether the Axin‐RGS domain functions in the context of Wnt signaling. We generated point mutations that converts the original Gln to Asn, predicted to increase affinity for Gα, and that converts Gln to Ala, predicted to abolish binding activity. We tested for the ability of the mutant forms to rescue Axin function in masterblind ( mbl ) zebrafish mutant embryos and in antisense morpholino knockdown of Axin in zebrafish and frog ( Xenopus laevis ). We found that the mutant constructs showed a differential ability to restore normal development in fish and frog embryos. These results suggest that the Axin‐RGS domain may be playing a role in regulating zygotic Wnt signaling. Grant Funding Source Roy J. Carver Charitable Trust
The evolutionary origin of novelties is a central problem in biology. At a cellular level this requires, for example, molecularly resolving how brainstem motor neurons change their innervation target from muscle fibers (branchial motor neurons) to neural crest-derived ganglia (visceral motor neurons) or ear-derived hair cells (inner ear and lateral line efferent neurons). Transplantation of various tissues into the path of motor neuron axons could determine the ability of any motor neuron to innervate a novel target. Several tissues that receive direct, indirect, or no motor innervation were transplanted into the path of different motor neuron populations in Xenopus laevis embryos. Ears, somites, hearts, and lungs were transplanted to the orbit, replacing the eye. Jaw and eye muscle were transplanted to the trunk, replacing a somite. Applications of lipophilic dyes and immunohistochemistry to reveal motor neuron axon terminals were used. The ear, but not somite-derived muscle, heart, or liver, received motor neuron axons via the oculomotor or trochlear nerves. Somite-derived muscle tissue was innervated, likely by the hypoglossal nerve, when replacing the ear. In contrast to our previous report on ear innervation by spinal motor neurons, none of the tissues (eye or jaw muscle) was innervated when transplanted to the trunk. Taken together, these results suggest that there is some plasticity inherent to motor innervation, but not every motor neuron can become an efferent to any target that normally receives motor input. The only tissue among our samples that can be innervated by all motor neurons tested is the ear. We suggest some possible, testable molecular suggestions for this apparent uniqueness.
Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover.
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