Reverse and Forward Genetics Approaches Reveal the Gene Networks That Regulate Development of Inner Ear Neurons

Stato-Acoustic Ganglion (SAG) neurons originate from the floor of the otic vesicle during a brief developmental window. They subsequently leave the otic vesicle and undergo a phase of migration and proliferation (transit-amplification). Neuroblasts finally differentiate into mature SAG neurons and extend processes to connect sensory cells of the inner ear to the information processing centers in the brain. The goal of this dissertation has been to elucidate mechanisms controlling these diverse events, which have heretofore been only poorly understood. First we showed that a threshold level of Fgf signaling initially sets the neurogenic domain in the otic epithelium. However, the level of Fgf signaling increases during development and becomes inhibitory to otic neurogenesis. Specfically, fgf5 is expressed by accumulating SAG neurons, which serves to terminate specification of new neuroblasts and delay differentiation of transit-amplifying cells. Second, we tested the role of transcription factor tfap2a, which we found is expressed in the neurogenic domain in both zebrafish and chick. Gain and loss-of-function studies revealed that Tfap2a activates expression of bmp7a, which in turn partially inhibits Fgf and Notch signaling. By modulating the inhibitory functions of Fgf and Notch, Tfap2a regulates the duration, amount and speed of SAG development. Third, we investigated the mechanism by which SAG neuroblasts leave the otic epithelium. We showed that Goosecoid (Gsc) regulates epithelial-mesenchymal transition of the otic neuroblasts. Fgf signaling regulates expression of gsc in a region iii partially overlapping with the neurogenic otic domain. The medial marker Pax2a acts in opposition to Gsc and stabilizes otic epithelia in non-neurogenic parts of the otic vesicle. Lastly, we conducted a mutagenesis screen in zebrafish to identify ENU-induced mutations that affect SAG development. We recovered a SAG deficient mutation, termed sagd1 that strongly reduces a subset of SAG neurons required for vestibular (balance) functions. Whole genome sequencing revealed that sagd1 affects the glycolytic enzyme, Phosphoglycerate kinase-1 (Pgk1). Further analysis revealed that Pgk1 acts nonautonomously to augment Fgf signaling during early stages of otic neurogenesis. Together, these studies have uncovered a number of previously unknown mechanisms for dynamic regulation of Fgf to control specification, delamination, and maturation of SAG neurons

Spemann organizer gene Goosecoid promotes delamination of neuroblasts from the otic vesicle

Neurons of the Statoacoustic Ganglion (SAG), which innervate the inner ear, originate as neuroblasts in the floor of the otic vesicle and subsequently delaminate and migrate toward the hindbrain before completing differentiation. In all vertebrates, locally expressed Fgf initiates SAG development by inducing expression of Neurogenin1 (Ngn1) in the floor of the otic vesicle. However, not all Ngn1-positive cells undergo delamination, nor has the mechanism controlling SAG delamination been elucidated. Here we report that Goosecoid (Gsc), best known for regulating cellular dynamics in the Spemann organizer, regulates delamination of neuroblasts in the otic vesicle. In zebrafish, Fgf coregulates expression of Gsc and Ngn1 in partially overlapping domains, with delamination occurring primarily in the zone of overlap. Loss of Gsc severely inhibits delamination, whereas overexpression of Gsc greatly increases delamination. Comisexpression of Ngn1 and Gsc induces ectopic delamination of some cells from the medial wall of the otic vesicle but with a low incidence, suggesting the action of a local inhibitor. The medial marker Pax2a is required to restrict the domain of gsc expression, and misexpression of Pax2a is sufficient to block delamination and fully suppress the effects of Gsc. The opposing activities of Gsc and Pax2a correlate with repression or up-regulation, respectively, of E-cadherin (cdh1). These data resolve a genetic mechanism controlling delamination of otic neuroblasts. The data also elucidate a developmental role for Gsc consistent with a general function in promoting epithelial-to-mesenchymal transition (EMT)

A Spatial and Temporal Gradient of Fgf Differentially Regulates Distinct Stages of Neural Development in the Zebrafish Inner Ear

Neuroblasts of the statoacoustic ganglion (SAG) initially form in the floor of the otic vesicle during a relatively brief developmental window. They soon delaminate and undergo a protracted phase of proliferation and migration (transit-amplification). Neuroblasts eventually differentiate and extend processes bi-directionally to synapse with hair cells in the inner ear and various targets in the hindbrain. Our studies in zebrafish have shown that Fgf signaling controls multiple phases of this complex developmental process. Moderate levels of Fgf in a gradient emanating from the nascent utricular macula specify SAG neuroblasts in laterally adjacent otic epithelium. At a later stage, differentiating SAG neurons express Fgf5, which serves two functions: First, as SAG neurons accumulate, increasing levels of Fgf exceed an upper threshold that terminates the initial phase of neuroblast specification. Second, elevated Fgf delays differentiation of transit-amplifying cells, balancing the rate of progenitor renewal with neuronal differentiation. Laser-ablation of mature SAG neurons abolishes feedback-inhibition and causes precocious neuronal differentiation. Similar effects are obtained by Fgf5-knockdown or global impairment of Fgf signaling, whereas Fgf misexpression has the opposite effect. Thus Fgf signaling renders SAG development self-regulating, ensuring steady production of an appropriate number of neurons as the larva grows

CNS myelination requires VAMP2/3-mediated membrane expansion in oligodendrocytes

Myelin is required for rapid nerve signaling and is emerging as a key driver of CNS plasticity and disease. How myelin is built and remodeled remains a fundamental question of neurobiology. Central to myelination is the ability of oligodendrocytes to add vast amounts of new cell membrane, expanding their surface areas by many thousand-fold. However, how oligodendrocytes add new membrane to build or remodel myelin is not fully understood. Here, we show that CNS myelin membrane addition requires exocytosis mediated by the vesicular SNARE proteins VAMP2/3. Genetic inactivation of VAMP2/3 in myelinating oligodendrocytes caused severe hypomyelination and premature death without overt loss of oligodendrocytes. Through live imaging, we discovered that VAMP2/3-mediated exocytosis drives membrane expansion within myelin sheaths to initiate wrapping and power sheath elongation. In conjunction with membrane expansion, mass spectrometry of oligodendrocyte surface proteins revealed that VAMP2/3 incorporates axon-myelin adhesion proteins that are collectively required to form nodes of Ranvier. Together, our results demonstrate that VAMP2/3-mediated membrane expansion in oligodendrocytes is indispensable for myelin formation, uncovering a cellular pathway that could sculpt myelination patterns in response to activity-dependent signals or be therapeutically targeted to promote regeneration in disease

Tfap2a Promotes Specification and Maturation of Neurons in the Inner Ear through Modulation of Bmp, Fgf and Notch Signaling

<div><p>Neurons of the statoacoustic ganglion (SAG) transmit auditory and vestibular information from the inner ear to the hindbrain. SAG neuroblasts originate in the floor of the otic vesicle. New neuroblasts soon delaminate and migrate towards the hindbrain while continuing to proliferate, a phase known as transit amplification. SAG cells eventually come to rest between the ear and hindbrain before terminally differentiating. Regulation of these events is only partially understood. Fgf initiates neuroblast specification within the ear. Subsequently, Fgf secreted by mature SAG neurons exceeds a maximum threshold, serving to terminate specification and delay maturation of transit-amplifying cells. Notch signaling also limits SAG development, but how it is coordinated with Fgf is unknown. Here we show that transcription factor Tfap2a coordinates multiple signaling pathways to promote neurogenesis in the zebrafish inner ear. In both zebrafish and chick, Tfap2a is expressed in a ventrolateral domain of the otic vesicle that includes neurogenic precursors. Functional studies were conducted in zebrafish. Loss of Tfap2a elevated Fgf and Notch signaling, thereby inhibiting SAG specification and slowing maturation of transit-amplifying cells. Conversely, overexpression of Tfap2a inhibited Fgf and Notch signaling, leading to excess and accelerated SAG production. However, most SAG neurons produced by Tfap2a overexpression died soon after maturation. Directly blocking either Fgf or Notch caused less dramatic acceleration of SAG development without neuronal death, whereas blocking both pathways mimicked all observed effects of Tfap2a overexpression, including apoptosis of mature neurons. Analysis of genetic mosaics showed that Tfap2a acts non-autonomously to inhibit Fgf. This led to the discovery that Tfap2a activates expression of Bmp7a, which in turn inhibits both Fgf and Notch signaling. Blocking Bmp signaling reversed the effects of overexpressing Tfap2a. Together, these data support a model in which Tfap2a, acting through Bmp7a, modulates Fgf and Notch signaling to control the duration, amount and speed of SAG neural development.</p></div

<i>fgf5</i> from mature neurons terminates the phase of neuroblast specification.

<p>The icon at the top of the figure indicates that analysis focuses on initial formation of neuroblasts. Experimental manipulations in groups IV and V are briefly summarized at the tops of the corresponding data panels. (A–H) Expression of <i>neurog1</i> in control embryos (A–C), a <i>hs:fgf8</i> embryo (D), <i>fgf5</i> morphants (E–G), and a <i>hs:</i>fgf8 embryo injected with <i>fgf5</i>-MO (H) at the indicated stages. Transgenic embryos (D, H) were heat shocked for 30 minutes at 39°C beginning at 24 hpf. Vertical lines in (A, C–E, G, H) indicate the plane of transverse sections in (B, F, and insets in C, D, G and H). (I–L) Expression of <i>isl2b:Gfp</i> at 22 hpf (I, J) and <i>neurog1</i> at 30 hpf (K, L) in a specimen in which mature (<i>fgf5</i>-expressing) neurons were laser-ablated. The same specimen is shown in all panels. Mature SAG neurons expressing <i>isl2b:Gfp</i> were serially ablated on the left side at 22 hpf (J) and 25 hpf (not shown), and the embryo was fixed and sectioned at 30 hpf to examine <i>neurog1</i> expression (L). Images of the unablated right side (I, K) were inverted to facilitate comparison. The surface of the otic vesicle is outlined in all panels. Arrows in sections indicate the edges of <i>neurog1</i> domain in the otic floor. Note that the amount and duration of delamination of <i>neurog1⁺</i> neuroblasts is strongly enhanced by knockdown of <i>fgf5</i> (F, G) or ablation of mature neurons (L). Activation of <i>hs:fgf8</i> reverses the effects of <i>fgf5-</i>MO (H). Scale bar, 25 µm. Transverse sections are shown with lateral to the left and dorsal up. Wholemount images show dorsolateral views with anterior to the left.</p

Normal axial patterning in <i>fgf5</i> morphants.

<p>(A–H) Expression of regional patterning markers in control embryos (A–D) and <i>fgf5</i> morphants (E–H). Expression of <i>pax5</i> (A, E) and <i>pou3f3b</i> (B, F) labels anterior and posterior regions, respectively. Expression of <i>otx1a</i> (C, G) and <i>dlx3b</i> (D, H) labels ventromedial and dorsolateral regions, respectively. The otic vesicle is outlined. Images show dorsolateral views with anterior to the left. (I) The total number of hair cells in the utricular (ut) and saccular (sac) maculae of control embryos and <i>fgf5</i> morphants at 32 hpf. Data were obtained by counting GFP-positive hair cells (mean of total number ± standard deviation) in the sensory epithelia of <i>brn3c:Gfp</i> transgenic embryos. Data show means and standard deviations from 20 specimens each. Differences between control and experimental specimens were not statistically significant (p = 0.16 for the utricle, p = 0.67 for the saccule) based on Student's <i>t</i> tests.</p

Effects of altering Fgf on SAG maturation.

*<p>Rate of mature neuron production during the indicated interval.</p

Effects of altering Fgf on neuroblast specification.

<p>Effects of altering Fgf on neuroblast specification.</p

Tfap2a regulates the level of Fgf and Notch Signaling in the otic vesicle.

<p>(A-V) Whole-mount images (dorsal up, anterior left) showing dorsolateral views of the otic vesicle (outlined). (A-R) Expression of the indicated genes in wild-type embryos, <i>hs</i>:<i>tfap2a</i> embryos and <i>tfap2a</i>^-/- mutants (A-I) or <i>tfap2a</i> morphants (J-R) at 26 hpf (A-L, P-R) and 28 (M-O) hpf. (S, T) Cross-sections (dorsal up, medial left) passing through the utricular macula show <i>spry4</i> expression at 28 hpf in a control embryo and <i>tfap2a</i>^-/- mutant. (U-X) Whole-mounts showing expression of <i>etv5b</i> at 26 hpf. Activation of <i>hs</i>:<i>tfap2a</i> diminishes <i>etv5b</i> expression (U, V), activation of <i>hs</i>:<i>fgf8</i> leads to global upregulation of <i>etv5b</i> (W), and co-activation of <i>hs</i>:<i>fgf8</i> and <i>hs</i>:<i>tfap2a</i> restores <i>etv5b</i> to near normal (X). (Y) Cross-sections (dorsal up, medial left) passing just posterior to the utricular macula showing <i>ngn1</i> at 24 hpf following a 35°C heat shock at 23 hpf. Reduction in the <i>ngn1</i> domain caused by knockdown of <i>tfap2a</i> is rescued by weak activation of <i>hs</i>:<i>dnfgfr1</i>. (Z) Mean and standard deviation of the total number of <i>ngn1</i> positive cells in the otic epithelium at 24 hpf for the genotypes and knockdowns indicated in the color key (counted from serial sections, n = 3–6 ears per time point). Asterisks (*) indicate statistically significant differences between the groups indicated in brackets.</p