The role of Wnt/planar cell polarity signaling in mouse facial branchiomotor neuron migration

Title from PDF of title page (University of Missouri--Columbia, viewed on May 21, 2012).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Anand ChandrasekharVita."July 2011"Neuronal migration is essential for the formation of distinct neural layers and functional neural networks in the developing central nervous system. As a model, we study the caudal migration of facial branchiomotor neurons (FBMNs) from rhombomere 4 (r4) to r6 within the developing mouse hindbrain. Since Wnt/planar cell polarity (PCP) signaling components had been implicated in zebrafish FBMN migration, we tested whether they also were required in mice. FBMNs failed to migrate caudally in Vangl2 (Looptail) mutants, Vangl2 knockout embryos, and Ptk7 mutants, indicating a specific role for Vangl2 and Wnt/PCP signaling in FBMN migration. However, FBMNs migrated normally in Dishevelled 1/2 double mutants and in zebrafish embryos with disrupted dishevelled signaling. These results suggest strongly that the caudal migration of FBMNs is controlled by multiple components of the Wnt/PCP pathway, yet may not require the central signaling molecule Dishevelled. Interestingly, in Celsr1 (Crash) mutants, many FBMNs migrated rostrally instead of caudally, indicating a specific role for Celsr1 in the directionality of FBMN migration. To better understand how Celsr1 functions, we inactivated Celsr1 in specific hindbrain tissues and found that it functions within the ventricular zone of rhombomeres 3 through 5 to regulate FBMN directionality. Using anterograde labeling with lipophilic dyes, we also found that the starting positions of individual FBMNs within r4 correlated with the direction of migration in Celsr1Crsh/+ mutants. Together, these results indicate that Celsr1 is required in the ventricular zone of multiple rhombomeres to regulate the direction of FBMN migration, and provides insight as to how only a subset of FBMNs is affected in Celsr1 mutants.Includes bibliographical reference

The mouse Wnt/PCP protein Vangl2 is necessary for migration of facial branchiomotor neurons, and functions independently of Dishevelled

AbstractDuring development, facial branchiomotor (FBM) neurons, which innervate muscles in the vertebrate head, migrate caudally and radially within the brainstem to form a motor nucleus at the pial surface. Several components of the Wnt/planar cell polarity (PCP) pathway, including the transmembrane protein Vangl2, regulate caudal migration of FBM neurons in zebrafish, but their roles in neuronal migration in mouse have not been investigated in detail. Therefore, we analyzed FBM neuron migration in mouse looptail (Lp) mutants, in which Vangl2 is inactivated. In Vangl2Lp/+ and Vangl2 Lp/Lp embryos, FBM neurons failed to migrate caudally from rhombomere (r) 4 into r6. Although caudal migration was largely blocked, many FBM neurons underwent normal radial migration to the pial surface of the neural tube. In addition, hindbrain patterning and FBM progenitor specification were intact, and FBM neurons did not transfate into other non-migratory neuron types, indicating a specific effect on caudal migration.Since loss-of-function in some zebrafish Wnt/PCP genes does not affect caudal migration of FBM neurons, we tested whether this was also the case in mouse. Embryos null for Ptk7, a regulator of PCP signaling, had severe defects in caudal migration of FBM neurons. However, FBM neurons migrated normally in Dishevelled (Dvl) 1/2 double mutants, and in zebrafish embryos with disrupted Dvl signaling, suggesting that Dvl function is essentially dispensable for FBM neuron caudal migration. Consistent with this, loss of Dvl2 function in Vangl2Lp/+ embryos did not exacerbate the Vangl2Lp/+ neuronal migration phenotype. These data indicate that caudal migration of FBM neurons is regulated by multiple components of the Wnt/PCP pathway, but, importantly, may not require Dishevelled function. Interestingly, genetic-interaction experiments suggest that rostral FBM neuron migration, which is normally suppressed, depends upon Dvl function

Brainstem Respiratory Oscillators Develop Independently of Neuronal Migration Defects in the Wnt/PCP Mouse Mutant looptail

The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone) to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp) mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP) protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF) settled at the ventral surface of the medulla in Vangl2Lp/+ and Vangl2Lp/Lp embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function

Acetaminophen Disrupts the Development of Pharyngeal Arch-Derived Cartilage and Muscle in Zebrafish

Acetaminophen is a common analgesic, but its potential effects on early embryonic development are not well understood. Previous studies using zebrafish (Danio rerio) have described the effects of acetaminophen on liver development and physiology, and a few have described gross physiological and morphological defects. Using a high but non-embryonic lethal dose of acetaminophen, we probed for defects in zebrafish craniofacial cartilage development. Strikingly, acetaminophen treatment caused severe craniofacial cartilage defects, primarily affecting both the presence and morphology of pharyngeal arch-derived cartilages of the viscerocranium. Delaying acetaminophen treatment restored developing cartilages in an order correlated with their corresponding pharyngeal arches, suggesting that acetaminophen may target pharyngeal arch development. Craniofacial cartilages are derived from cranial neural crest cells; however, many neural crest cells were still seen along their expected migration paths, and most remaining cartilage precursors expressed the neural crest markers sox9a and sox10, then eventually col2a1 (type II collagen). Therefore, the defects are not primarily due to an early breakdown of neural crest or cartilage differentiation. Instead, apoptosis is increased around the developing pharyngeal arches prior to chondrogenesis, further suggesting that acetaminophen may target pharyngeal arch development. Many craniofacial muscles, which develop in close proximity to the affected cartilages, were also absent in treated larvae. Taken together, these results suggest that high amounts of acetaminophen can disrupt multiple aspects of craniofacial development in zebrafish

The atypical cadherin Celsr1 functions non-cell autonomously to block rostral migration of facial branchiomotor neurons in mice.

The caudal migration of facial branchiomotor (FBM) neurons from rhombomere (r) 4 to r6 in the hindbrain is an excellent model to study neuronal migration mechanisms. Although several Wnt/Planar Cell Polarity (PCP) components are required for FBM neuron migration, only Celsr1, an atypical cadherin, regulates the direction of migration in mice. In Celsr1 mutants, a subset of FBM neurons migrates rostrally instead of caudally. Interestingly, Celsr1 is not expressed in the migrating FBM neurons, but rather in the adjacent floor plate and adjoining ventricular zone. To evaluate the contribution of different expression domains to neuronal migration, we conditionally inactivated Celsr1 in specific cell types. Intriguingly, inactivation of Celsr1 in the ventricular zone of r3-r5, but not in the floor plate, leads to rostral migration of FBM neurons, greatly resembling the migration defect of Celsr1 mutants. Dye fill experiments indicate that the rostrally-migrated FBM neurons in Celsr1 mutants originate from the anterior margin of r4. These data suggest strongly that Celsr1 ensures that FBM neurons migrate caudally by suppressing molecular cues in the rostral hindbrain that can attract FBM neurons

Atypical Cadherins Celsr1-3 Differentially Regulate Migration of Facial Branchiomotor Neurons in Mice

During hindbrain development, facial branchiomotor neurons (FBM neurons) migrate from medial rhombomere (r) 4 to lateral r6. In zebrafish, mutations in planar cell polarity genes celsr2 and frizzled3a block caudal migration of FBM neurons. Here, we investigated the role of cadherins Celsr1-3, and Fzd3 in FBM neuron migration in mice. In Celsr1 mutants (knock-out and Crash alleles), caudal migration was compromised and neurons often migrated rostrally into r2 and r3, as well as laterally. These phenotypes were not caused by defects in hindbrain patterning or neuronal specification. Celsr1 is expressed in FBM neuron precursors and the floor plate, but not in FBM neurons. Consistent with this, conditional inactivation showed that the function of Celsr1 in FBM neuron migration was non-cell autonomous. In Celsr2 mutants, FBM neurons initiated caudal migration but moved prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3 in FBM neurons and mimicked by inactivation of Fzd3. Furthermore, Celsr2 was epistatic to Celsr1. These data indicate that Celsr1-3 differentially regulate FBM neuron migration. Celsr1 helps to specify the direction of FBM neuron migration, whereas Celsr2 and 3 control its ability to migrate

Atypical Cadherins Celsr1-3 Differentially Regulate Migration of Facial Branchiomotor Neurons in Mice.

During hindbrain development, facial branchiomotor neurons (FBM neurons) migrate from medial rhombomere (r) 4 to lateral r6. In zebrafish, mutations in planar cell polarity genes celsr2 and frizzled3a block caudal migration of FBM neurons. Here, we investigated the role of cadherins Celsr1-3, and Fzd3 in FBM neuron migration in mice. In Celsr1 mutants (knock-out and Crash alleles), caudal migration was compromised and neurons often migrated rostrally into r2 and r3, as well as laterally. These phenotypes were not caused by defects in hindbrain patterning or neuronal specification. Celsr1 is expressed in FBM neuron precursors and the floor plate, but not in FBM neurons. Consistent with this, conditional inactivation showed that the function of Celsr1 in FBM neuron migration was non-cell autonomous. In Celsr2 mutants, FBM neurons initiated caudal migration but moved prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3 in FBM neurons and mimicked by inactivation of Fzd3. Furthermore, Celsr2 was epistatic to Celsr1. These data indicate that Celsr1-3 differentially regulate FBM neuron migration. Celsr1 helps to specify the direction of FBM neuron migration, whereas Celsr2 and 3 control its ability to migrate

Functional analysis of the preBötC oscillator in the <i>Lp</i> mutant.

<p>A: Photomicrograph of a transverse medullary slice through the preBötC obtained from an E16.5 <i>+/+</i> embryo loaded with calcium green 1-AM observed in direct fluorescence (left panel) and as relative changes in fluorescence (ΔF/F, right panel). The red circles indicate the position of the bilaterally distributed preBötC oscillators. Traces below (ΔF/F preBötC) indicate calcium transients measured in the preBötC region in control conditions (top trace), 10⁻⁷ M Substance P (SP, second trace), 10 µM Riluzole (third trace) and 10 µM CNQX (fourth trace). B and C: Same legend as in A for a <i>Lp/+</i> embryo (B) and a <i>Lp/Lp</i> embryo (C). D: Graph representing the mean frequency of calcium transients measured in the preBötC region in different experimental conditions (indicated below and color coded) for <i>+/+</i> (left part), <i>Lp/+</i> (middle part) and <i>Lp/Lp</i> (right part). Numbers in brackets indicate the number of preparations tested. Asterisks indicate statistically different means. Frequency is increased in the presence of SP, unchanged in the presence of riluzole and blocked in the presence of CNQX for all genotypes. Hence, the preBötC oscillator is functionally preserved in <i>Lp/Lp</i> mutants. D: dorsal, L: lateral.</p

Active cells form a functional e-pF oscillator in <i>Lp</i> mutant embryos.

<p>A: Ventral view of the hindbrain over the area encompassing the FMN and the active region. Preparations were obtained from an E15.5 <i>+/+</i> embryo, loaded with calcium Green 1-AM and observed in direct fluorescence (left panel) or as relative changes in fluorescence (ΔF/F, right panel). The yellow oval indicates the position of the FMN, the e-pF network is encircled in red. Traces below indicate calcium transients (ΔF/F) measured in the e-pF region in control conditions (top trace), pH 7.2 (second trace), 10 µM CNQX (third trace), 10 µM Riluzole (fourth trace) and 10⁻⁷ M Substance P (SP, fifth trace). B and C: Same legend as in A for a <i>Lp/+</i> embryo (B) and a <i>Lp/Lp</i> embryo (C). D: Graphs representing the mean frequency of calcium transients measured in the e-pF region in different experimental conditions (indicated below and color coded) for <i>+/+</i> (left part), <i>Lp/+</i> (middle part) and <i>Lp/Lp</i> (right part). Numbers in brackets indicate the number of preparations tested. Asterisks indicate statistically different means. Frequency is increased in the presence of CNQX, SP and in pH 7.2 for all genotypes, while riluzole blocks the rhythmic activity in the active region. The active network found in the mutant shares pharmacological characteristics of the e-pF recorded in the wild type littermates. L: lateral, R: rostral.</p

Altered distribution of active cells at the pial surface of <i>Lp</i> mutant hindbrain preparations.

<p>A–C: Ventral view of whole hindbrain preparations from E15.5 <i>+/+</i> (A), <i>Lp/+</i> (B) and <i>Lp/Lp</i> (C) embryos loaded with Calcium Green 1-AM and observed in direct fluorescence. Yellow ovals indicate the position of the facial motor nucleus (FMN) that is clearly visible in direct light (see right side of preparations in A and B). D–F: Maps for rhythmic active cells (red circles) detected at a high magnification for corresponding genotypes in the region delimited by the white rectangles in A–C. Active cells located at the ventral surface of the preparations are found for all genotypes. G–I: Histograms of the rostro-caudal distribution of active cells relative to the constant preBötC position for 2 wild-type (G), 3 <i>Lp/+</i> (H), and 3 <i>Lp/Lp</i> (I) embryos. The red arrows indicate the rostral and the caudal extremity of the FMN. The distribution of active cells shows a significant rostral displacement in <i>Lp/Lp</i> embryos. J–L: Calcium transients illustrated as relative fluorescent changes (ΔF/F) recorded in the region encompassing the active cells (delimited by the blue rectangles in A–C). Relative fluorescent changes intensity are color-coded, white corresponding to the strongest activity (see the color scale at the bottom). The traces below show the spontaneous calcium changes recorded over time in the entire active region for each genotype. R: rostral.</p