Cochleovestibular nerve development is integrated with migratory neural crest cells

The cochleovestibular (CV) nerve, which connects the inner ear to the brain, is the nerve that enables the senses of hearing and balance. The aim of this study was to document the morphological development of the mouse CV nerve with respect to the two embryonic cells types that produce it, specifically, the otic vesicle-derived progenitors that give rise to neurons, and the neural crest cell (NCC) progenitors that give rise to glia. Otic tissues of mouse embryos carrying NCC lineage reporter transgenes were whole mount immunostained to identify neurons and NCC. Serial optical sections were collected by confocal microscopy and were compiled to render the three dimensional (3D) structure of the developing CV nerve. Spatial organization of the NCC and developing neurons suggest that neuronal and glial populations of the CV nerve develop in tandem from early stages of nerve formation. NCC form a sheath surrounding the CV ganglia and central axons. NCC are also closely associated with neurites projecting peripherally during formation of the vestibular and cochlear nerves. Physical ablation of NCC in chick embryos demonstrates that survival or regeneration of even a few individual NCC from ectopic positions in the hindbrain results in central projection of axons precisely following ectopic pathways made by regenerating NCC.     

FGF signalling pathways in development of the midbrain and anterior hindbrain

Development of the central nervous system is coordinated by intercellular signalling centres established within the neural tube. The isthmic organizer (IsO), located between the midbrain and anterior hindbrain, is one such centre. Important signal molecules secreted by the IsO include members of the fibroblast growth factor and Wnt families. These signals are integrated with dorsally and ventrally derived signals to regulate development of the midbrain and rhombomere 1 of the hindbrain. The IsO is operational for a remarkably long period of time. Depending on the developmental stage, it controls a variety of processes such as cell survival, cell identity, neural precursor proliferation, neuronal differentiation and axon guidance. This review focuses on the fibroblast growth factor signalling, its novel molecular regulatory mechanisms and how this pathway regulates multiple aspects of cell behaviour in the developing midbrain and anterior hindbrain.     

The Zebrafish trilobite gene is essential for tangential migration of branchiomotor neurons

Newborn neurons migrate extensively in the radial and tangential directions to organize the developing vertebrate nervous system. We show here that mutations in zebrafish trilobite (tri) that affect gastrulation-associated cell movements also eliminate tangential migration of motor neurons in the hindbrain. In the wild-type hindbrain, facial (nVII) and glossopharyngeal (nIX) motor neurons are induced in rhombomeres 4 and 6, respectively, and migrate tangentially into r6 and r7 (nVII) and r7 (nIX). In all three tri alleles examined, although normal numbers of motor neurons are induced, nVII motor neurons are found exclusively in r4, and nIX-like motor neurons are found exclusively in r6. The migration of other neuronal and nonneuronal cell types is unaffected in tri mutants. Rhombomere formation and the development of other hindbrain neurons are also unaffected in tri mutants. Furthermore, tangential neuronal migration occurs normally in the gastrulation mutant knypek, indicating that the trilobite neuron phenotype does not arise nonspecifically from aberrant gastrulation-associated movements. We conclude that trilobite function is specifically required for two types of cell migration that occur at different stages of zebrafish development.     

Apoptosis of premigratory neural crest cells in rhombomeres 3 and 5: consequences for patterning of the branchial region

In the avian hindbrain, premigratory neural crest cells undergo programmed cell death (apoptosis) in rhombomeres 3 and 5 (r3, r5). Here, we have attempted to analyze the significance of the loss of neural crest cells from these odd-numbered rhombomeres. When apoptosis is prevented in r3 and r5, r3 crest migrate into the first arch and r5 into the third arch. Interestingly, these extra neural crest cells contributed to the formation of ectopic muscle attachment sites that are also found in those species in which r3 and r5 neural crest cells do not undergo apoptosis. Thus, apoptosis in the odd-numbered rhombomeres appears to be an evolutionarily derived mechanism that is required to eliminate r3 and r5 crest migration into first and third arches and thereby remove these muscle attachment sites.     

A unique expression pattern of Tbx10 in the hindbrain as revealed by Tbx10LacZ allele

To study the expression/function of Tbx10, a T‐box gene, Tbx10LacZ/+ mice were established by replacing the T‐box coding region with a LacZ gene. X‐gal staining showed that LacZ+ cells were localized to two‐cell populations in rhombomere 4 and rhombomere 6. No significant differences in the locations of LacZ+ cells were found between Tbx10LacZ/+ and Tbx10LacZ/LacZ mice, and the Tbx10LacZ/LacZ mice were viable and fertile. We found that the LacZ+ cells are present in both embryonic and adult mice. Histological studies suggest that the rhombomere 4‐derived LacZ+ cells are a subpopulation of the ventral interneurons in the pons.     

Utility of HoxB2 enhancer‐mediated Cre activity for functional studies in the developing inner ear

The rhombomere 4(r4)‐restricted expression of the mouse Hoxb2 gene is regulated by a 1.4‐kb enhancer‐containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2‐r4‐Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5–E12.5, in the cochleovestibular and facio‐acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2‐r4‐Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected. genesis 47:361–365, 2009. © 2009 Wiley‐Liss, Inc.     

Zebrafish Tshz3b negatively regulates Hox function in the developing hindbrain

In flies, the zinc-finger protein Teashirt promotes trunk segmental identities, in part, by repressing the expression and function of anterior hox paralog group (PG) 1-4 genes that specify head fates. Anterior-posterior patterning of the vertebrate hindbrain also requires Hox PG 1-4 function, but the role of vertebrate teashirt-related genes in this process has not been investigated. In this work, we use overexpression and structure-function analyses to show that zebrafish tshz3b antagonizes Hox-dependent hindbrain segmentation. Ectopic Tshz3b perturbs the specification of rhombomere identities and leads to the caudal expansion of r1, the only rhombomere whose identity is specified independently of Hox function. This overexpression phenotype does not require the homeodomain and C-terminal zinc fingers that are unique to vertebrate Teashirt-related proteins, but does require that Tshz3b function as a repressor. Together, these results argue that the negative regulation of Hox PG 1-4 function is a conserved characteristic of Teashirt-related proteins.     

Hox gene colinear expression in the avian medulla oblongata is correlated with pseudorhombomeric domains

The medulla oblongata (or caudal hindbrain) is not overtly segmented, since it lacks observable interrhombomeric boundaries. However, quail-chick fate maps showed that it is formed by 5 pseudorhombomeres (r7-r11) which were empirically found to be delimited consistently at planes crossing through adjacent somites (Cambronero and Puelles, 2000). We aimed to reexamine the possible segmentation or rostrocaudal regionalisation of this brain region attending to molecular criteria. To this end, we studied the expression of Hox genes from groups 3 to 7 correlative to the differentiating nuclei of the medulla oblongata. Our results show that these genes are differentially expressed in the mature medulla oblongata, displaying instances of typical antero-posterior (3′ to 5′) Hox colinearity. The different sensory and motor columns, as well as the reticular formation, appear rostrocaudally regionalised according to spaced steps in their Hox expression pattern. The anterior limits of the respective expression domains largely fit boundaries defined between the experimental pseudorhombomeres. Therefore the medulla oblongata shows a Hox-related rostrocaudal molecular regionalisation comparable to that found among rhombomeres, and numerically consistent with the pseudorhombomere list. This suggests that medullary pseudorhombomeres share some AP patterning mechanisms with the rhombomeres present in the rostral, overtly-segmented hindbrain, irrespective of variant boundary properties.     

Generation of a transgenic mouse line expressing GFP-Cre protein from a Hoxb4 neural enhancer

Here, we describe a transgenic mouse line, in which expression of green fluorescent protein fused to Cre recombinase (GFP-Cre) is directed by the early neuronal enhancer (ENE) of Hoxb4. In E9.0-13.5 transgenic embryos, Cre activity coincided with endogenous Hoxb4 throughout the neural tube up to the r6/r7 boundary in the hindbrain, the dorsal root ganglia, and the Xth cranial ganglia. Unexpectedly, Cre activity was also consistently detected in the trigeminal (Vth) cranial nerve, which is devoid of endogenous Hoxb4 expression. Strong GFP dependent fluorescence appeared slightly later in E9.5-E11.5 embryos, and reflected the later expression pattern expected for Hoxb4-ENE directed expression in the neural tube up to the r7/r8 not r6/r7 boundary. Thus, with the exception of the trigeminal nerve, this reporter faithfully reproduces endogenous embryonic neural Hoxb4 expression, and provides an excellent reagent for in vivo gene manipulations in neuronal Hoxb4 positive cells as well as the developing trigeminal nerve. This transgenic mouse line should prove especially useful for determining the fate map of neuronal populations arising in rhombomeres 7 and 8 on its own and in combination with the small set of other existing rhombomere-specific Cre recombinase expressing lines.