Sensory Placodes Research Articles

Development of the nervus terminalis: Origin and migration

The origin of the nervus terminalis is one of the least well understood developmental events involved in generating the cranial ganglia of the forebrain in vertebrate animals. This cranial nerve forms at the formidable interface of the anteriormost limits of migrating cranial neural crest cells, the terminal end of the neural tube and the differentiating olfactory and adenohypophyseal placodes. The complex cellular interactions that give rise to the various structures associated with the sensory placode (olfactory) and endocrine placode (adenohypophysis) surround and engulf this enigmatic cranial nerve. The tortured history of nervus terminalis development (see von Bartheld, this issue, pages 13-24) reflects the lack of consensus on the origin (or origins), as well as the experimental difficulties in uncovering the origin, of the nervus terminalis. Recent technical advances have allowed us to make headway in understanding the origin(s) of this nerve. The emergence of the externally fertilized zebrafish embryo as a model system for developmental biology and genetics has shed new light on this century-old problem. Coupled with new developmental models are techniques that allow us to trace lineage, visualize gene expression, and genetically ablate cells, adding to our experimental tools with which to follow up on studies provided by our scientific predecessors. Through these techniques, a picture is emerging in which the origin of at least a subset of the nervus terminalis cells lies in the cranial neural crest. In this review, the data surrounding this finding will be discussed in light of recent findings on neural crest and placode origins.

Expression of the polycomb group gene bmi-1 in the early chick embryo

Bmi-1 is a protooncogene of the polycomb group that has important functions in self-renewal of adult stem cells and maintenance of anterior identity along the embryonic body axis. Despite these important functions, surprisingly little is known about its distribution pattern in the embryo. Here, we have isolated the chick homologue of bmi-1 and examined its expression pattern from gastrulation through neurulation and establishment of organ rudiments. During gastrulation, bmi-1 transcripts were expressed in the epiblast cells adjacent to the primitive streak. During early nervous system development, robust expression was observed in the open neural plate and later in the dorsal neural tube and much of the brain. Bmi-1 expression was also present in the developing heart primordia and the sensory placodes. The data show that bmi-1 is present in, but not restricted to, tissue containing multipotent precursor cells.

DLX5 positions the neural crest and preplacode region at the border of the neural plate

The neural crest and sensory placodes arise from a region of the embryonic ectoderm that lies between the neural plate and future epidermis. While some of the signalling pathways that are involved in cell fate determination at the border of the neural plate have been characterised, it is still unclear how different signals are integrated. Transcription factors of the DLX gene family that may mediate such cell fate decisions are expressed at the border of the neural plate. Here, we demonstrate that DLX5 is involved in positioning this border by repressing neural properties and simultaneously by promoting the formation of border-like cells that express the neural fold markers MSX1 and BMP4 and the preplacodal region marker SIX4. However, DLX5 is not sufficient to impart epidermal character or to specify cell fates that arise at the border of the neural plate, like neural crest or fully formed sensory placodes, in a cell-autonomous manner. Additional signals are generated when mature neural plate and epidermis interact and these are required for neural crest formation. We propose that patterning of the embryonic ectoderm is a multistep process that sequentially subdivides the ectoderm into regions with defined cell fates.

Concerted action of two dlx paralogs in sensory placode formation.

Sensory placodes are ectodermal thickenings that give rise to elements of the vertebrate cranial sensory nervous system, including the inner ear and nose. Although mutations have been described in humans, mice and zebrafish that perturb ear and nose development, no mutation is known to prevent sensory placode formation. Thus, it has been postulated that a functional redundancy exists in the genetic mechanisms that govern sensory placode development. We describe a zebrafish deletion mutation, b380, which results in a lack of both otic and olfactory placodes. The b380 deletion removes several known genes and expressed sequence tags, including dlx3 and dlx7, two transcription factors that share a homoeobox domain similar in sequence to the Drosophila Distal-less gene. dlx3 and dlx7 are expressed in an overlapping pattern in the regions that produce the otic and olfactory placodes in zebrafish. We present evidence suggesting that it is specifically the removal of these two genes that leads to the otic and olfactory phenotype of b380 mutants. Using morpholinos, antisense oligonucleotides that effectively block translation of target genes, we find that functional reduction of both dlx genes contributes to placode loss. Expression patterns of the otic marker pax2.1, olfactory marker anxV and eya1, a marker of both placodes, in morpholino-injected embryos recapitulate the reduced expression of these genes seen in b380 mutants. We also examine expression of dlx3 and dlx7 in the morpholino-injected embryos and present evidence for existence of auto- and cross-regulatory control of expression among these genes. We demonstrate that dlx3 is necessary and sufficient for proper otic and olfactory placode development. However, our results indicate that dlx3 and dlx7 act in concert and their importance in placode formation is only revealed by inactivating both paralogs.

Cloning and expression of Ventrhoid, a novel vertebrate homologue of the Drosophila EGF pathway gene rhomboid

In Drosophila melanogaster, the seven-pass transmembrane protein Rhomboid (Rho) is a crucial positive modulator of EGF signaling playing a substantial role in patterning of the ventral neuroectoderm and fate specification of neuroblasts. Here, we describe the cloning and expression pattern of Ventrhoid ( Vrho), the novel evolutionarily conserved vertebrate cDNA related to fruit fly rho. Most importantly, like rho in Drosophila, Vrho is also expressed in a spatially restricted manner. Vrho expression is most prominent along the developing ventral neural tube, and is also detectable in the ventral forebrain, prospective diencephalon, otic vesicles, mandibular arches, cranial sensory placodes, last formed pair of somites and hindgut in midgestational mouse embryos.

The transcriptional regulator Id3 is expressed in cranial sensory placodes during early avian embryonic development.

The chick homologue of the helix-loop-helix gene Id3 was isolated, and its expression pattern was analyzed during early stages of chick development. Chick Id3 is dynamically expressed in the olfactory, lens, and otic placodes. It is also observed in the epiphysis, nephric primordium, stomodeum, dermomyotome, distal branchial arches, dorsolateral hindbrain, foregut endoderm, dorsal spinal cord, and somites.

Expression and phylogeny of claudins in vertebrate primordia.

Claudins, the major transmembrane proteins of tight junctions, are members of the tetraspanin superfamily of proteins that mediate cellular adhesion and migration. Their functional importance is demonstrated by mutations in claudin genes that eliminate tight junctions in myelin and the testis, abolish Mg(2+) resorption in the kidney, and cause autosomal recessive deafness. Here we report that two paralogs among 15 claudin genes in the zebrafish, Danio rerio, are expressed in the otic and lateral-line placodes at their earliest stages of development. Related claudins in amphibians and mammals are expressed in a similar manner in vertebrate primordia such as sensory placodes, branchial arches, and limb buds. We also show that the claudin gene family may have expanded along the chordate stem lineage from urochordates to gnathostomes, in parallel with the elaboration of vertebrate characters. We propose that tight junctions not only form barriers in mature epithelia, but also participate in vertebrate morphogenesis.

Origin of the vertebrate inner ear: evolution and induction of the otic placode.

The vertebrate inner ear forms a highly complex sensory structure responsible for the detection of sound and balance. Some new aspects on the evolutionary and developmental origin of the inner ear are summarised here. Recent molecular data have challenged the longstanding view that special sense organs such as the inner ear have evolved with the appearance of vertebrates. In addition, it has remained unclear whether the ear originally arose through a modification of the amphibian mechanosensory lateral line system or whether both evolved independently. A comparison of the developmental mechanisms giving rise to both sensory systems in different species should help to clarify some of these controversies. During embryonic development, the inner ear arises from a simple epithelium adjacent to the hindbrain, the otic placode, that is specified through inductive interactions with surrounding tissues. This review summarises the embryological evidence showing that the induction of the otic placode is a multistep process which requires sequential interaction of different tissues with the future otic ectoderm and the recent progress that has been made to identify some of the molecular players involved. Finally, the hypothesis is discussed that induction of all sensory placodes initially shares a common molecular pathway, which may have been responsible to generate an 'ancestral placode' during evolution.

Lamprey Dlx genes and early vertebrate evolution.

Gnathostome vertebrates have multiple members of the Dlx family of transcription factors that are expressed during the development of several tissues considered to be vertebrate synapomorphies, including the forebrain, cranial neural crest, placodes, and pharyngeal arches. The Dlx gene family thus presents an ideal system in which to examine the relationship between gene duplication and morphological innovation during vertebrate evolution. Toward this end, we have cloned Dlx genes from the lamprey Petromyzon marinus, an agnathan vertebrate that occupies a critical phylogenetic position between cephalochordates and gnathostomes. We have identified four Dlx genes in P. marinus, whose orthology with gnathostome Dlx genes provides a model for how this gene family evolved in the vertebrate lineage. Differential expression of these lamprey Dlx genes in the forebrain, cranial neural crest, pharyngeal arches, and sensory placodes of lamprey embryos provides insight into the developmental evolution of these structures as well as a model of regulatory evolution after Dlx gene duplication events.

Expression of three zebrafish Six4 genes in the cranial sensory placodes and the developing somites

The homeobox gene Six4/AREC3 is a member of the vertebrate Six family of transcription factor genes. In this study we describe the cloning and expression of three zebrafish homologues of Six4/AREC3, six4.1, six4.2 and six4.3. These zebrafish Six4 proteins show high homology to the mammalian Six4/AREC3 proteins in the most C-terminal region in addition to the Six domain and homeodomain. Whole-mount in situ hybridization showed that six4.1 is expressed in the presumptive cranial placodal precursor cells, and later in the olfactory, otic and lateral line placodes. six4.2 is expressed in the presomitic mesoderm, somites and pectoral fin bud. six4.3 appears to be a unique member of the Six4 proteins and is expressed ubiquitously at gastrulation and later in the tectum.

Ectopic Sox3 activity elicits sensory placode formation

The induction of sensory organ placodes, in particular the lens placode, represents the paradigm for induction. We show that medaka Sox3 is expressed in the neuroectoderm and in the placodes of all sensory organs prior to placode formation and subsequently in placode-derived tissues. Ectopic Sox3 expression leads to ectopic expression of Pax6 and Eya1 in embryonic ectoderm and causes ectopic lens and otic vesicle formation. The descendants of cells ectopically expressing Sox3-mRNA contribute to ectopic lens tissue. This suggests a permissive role for Sox3 in establishing a placodal competence. In addition, ectopic Sox3 expression leads to the dysgenesis of the endogenous sensory organs. Both effects of ectopic Sox3 expression can be separated by ectopic expression of a truncated Sox3 variant depending on its expression level. Our data suggests that Sox3 is a permissive factor for sensory placode formation and plays an important role in sensory organ development.

Induction of inner ear fate by FGF3.

Loss-of-function experiments in avians and mammals have provided conflicting results on the capacity of fibroblast growth factor 3 (FGF3) to act as a secreted growth factor responsible for induction and morphogenesis of the vertebrate inner ear. Using a novel technique for gene transfer into chicken embryos, we have readdressed the role of FGF3 during inner ear development in avians. We find that ectopic expression of FGF3 results in the formation of ectopic placodes which express otic marker genes. The ectopically induced placodes form vesicles which show the characteristic gene expression pattern of a developing inner ear. Ectopic expression of FGF3 also influences the formation of the normal orthotopic inner ear, whereas another member of the FGF family, FGF2, shows no effects on inner ear induction. These results demonstrate that a single gene can induce inner ear fate and reveal an unexpectedly widespread competence of the surface ectoderm to form sensory placodes in higher vertebrates.

Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages

Whole mount in situ hybridisation was used to study the embryonic expression of the mouse HMG box-containing genes Sox1, Sox2 and Sox3 between 6.5 and 9.0 days post coitum (dpc). Sox2 and Sox3 are expressed in the epiblast and extraembryonic ectoderm of the egg cylinder, becoming restricted to the prospective neural plate and chorion at the onset of gastrulation. Sox3 is upregulated in the posterior ectoderm during late streak and neural plate stages and is concomitantly downregulated in the chorion. Sox1 transcripts are first detected in the neural fold ectoderm at the headfold stage.During early somitogenesis, all three genes are expressed in the neuroectoderm, and Sox2 and Sox3 are also expressed in the primitive streak ectoderm, gut endoderm and prospective sensory placodes.

Placodal origin of Brn-3-expressing cranial sensory neurons.

The Brn-3 class of POU-domain transcription factors includes three genes in mammals which have key roles in the development of specific groups of sensory neurons. Here, we have identified three avian genes which correspond to the murine genes Brn-3.0, Brn-3.1, and Brn-3.2. Using an in situ hybridization probe generic for this gene class, the earliest detectable expression of Brn-3 in the chick is at stage 15, in placodal and migrating precursors of the trigeminal ganglion. By stage 19, Brn-3.0 protein is detectable in the trigeminal and vestibulocochlear ganglia with Brn-3.0-specific antisera, and Brn-3 message expression has extended to the dorsal root ganglia. At later stages, when condensation of the trigeminal ganglion is complete, Brn-3.0-immunoreactive neurons are concentrated in the portion of the ganglion distal to the brain stem. To examine the developmental origin of the Brn-3 expressing cells, we combined lipophilic dye (DiI) labeling with in situ hybridization. DiI labeling of the placodal surface ectoderm and of premigratory neural crest cells in the neural tube reveals that all, or nearly all, of the Brn3-expressing neurons in the trigeminal ganglia are derived from the sensory placodes and not from the neural crest, and thus, that Brn-3 is an early marker of the placode-derived sensory neural lineage.

Identification of a mechanism to localize generation of retinoic acid in rat embryos

Vitamin A (retinol) is essential for normal mammalian development. However, its biological activity depends upon its conversion to retinoic acid (RA), a local mediator of cellular proliferation and differentiation. Previous studies have shown that embryonic RA is found specifically in tissues known to depend upon vitamin A for normal development and that its production follows uptake of maternal retinol. The aim of this study was to identify the mechanism for tissue-specific generation of RA in developing rat embryos. Here we show immunohistochemical localization of the retinol binding protein receptor, cellular retinol binding protein, retinol dehydrogenase and retinal dehydrogenase in rat embryos (presomitic to the 25–30 somite pair stage). These proteins are proposed to be responsible for cellular uptake of retinol, its intracellular transport and its conversion to RA. Thus, they potentially constitute the entire metabolic pathway from vitamin A to RA. All four proteins were detected specifically in tissues that are known to depend upon vitamin A for normal development including the yolk sac, heart, gut, notochord, somites, sensory placodes and the limb. Furthermore, our previous studies have demonstrated that uptake of retinol into the yolk sac depends upon a retinol binding protein receptor. Here we provide evidence that this mechanism functions also in the heart. Colocalization of cellular retinol binding protein, retinol and retinal dehydrogenase with the retinol binding protein receptor in tissues dependent upon vitamin A for normal development suggests that coordinate functioning of these proteins is responsible for cellular uptake of circulating retinol and its metabolism to RA. This is the first evidence of a tissue-specific mechanism for generation of RA from its precursor retinol in the developing embryo.

Integrin alpha 6 expression is required for early nervous system development in Xenopus laevis.

The integrin alpha 6 subunit pairs with both the beta 1 and beta 4 subunits to form a subfamily of laminin receptors. Here we report the cDNA cloning and primary sequence for the Xenopus homologue of the mammalian integrin alpha 6 subunit. We present data demonstrating the spatial and temporal expression of alpha 6 mRNA and protein during early development. Initially, alpha 6 transcripts are expressed in the dorsal ectoderm and future neural plate at the end of gastrulation. Later in development, alpha 6 mRNAs are expressed in a variety of neural derivatives, including the developing sensory placodes (otic and olfactory) and commissural neurons within the neural tube. Integrin alpha 6 is also expressed in the elongating pronephric duct as well as a subset of the rhombencephalic neural crest, which will form the Schwann cells lining several cranial nerves (VII, VIII and X). In vivo expression of an alpha 6 antisense transcript in the animal hemisphere leads to a reduction in alpha 6 protein expression, a loss of adhesion to laminin, and severe defects in normal development. In 35% of cases, reduced levels of alpha 6 expression result in embryos that complete gastrulation normally but arrest at neurulation prior to the formation of the neural plate. In an additional 22% of cases, embryos develop with severe axial defects, including complete loss of head or tail structures. In contrast, overexpression of the alpha 6 subunit by injection of full-length mRNA has no apparent effect on embryonic development. Co-injection of antisense and sense plasmid constructs results in a partial rescue of the antisense-generated phenotypes. These data indicate that the integrin alpha 6 subunit is critical for the early development of the nervous system in amphibians.

Activin and Its Receptors during Gastrulation and the Later Phases of Mesoderm Development in the Chick Embryo

We have cloned chick homologues of the type-II activin receptor, which we have designated cActR-IIA and -IIB. Binding assays show that the two receptors are indistinguishable in their ability to bind activin-A, with comparablekds. Injection of mRNAs encoding these receptors intoXenopusembryos causes axial duplications. Expression of both receptors can first be detected in the primitive streak byin situhybridization. This suggests that these genes may be activated in response to mesoderm induction. In agreement with this, we find that treatment of preprimitive streak chick embryos with activin-A leads to rapid induction of the expression of cActR-IIB. At later stages, cActR-IIA transcripts become localized mainly in the notochord and myotome and cActR-IIB in the dorsal neural tube, proximal-anterior part of the limb bud, sensory placodes, and specific regions of the fore- and midbrain. To test the response of early chick embryonic tissues to activin, we designed a newin vitroassay for differentiation. We find that explants of area opaca epiblast or posterior primitive streak from various stages can respond to activin treatment by differentiating into a variety of mesodermal cell types in a dose-dependent manner. These results suggest that the importance of activin-related signaling pathways is not confined to pregastrulation stages and that these receptors may be involved in mediating the effects of inducing signals during later stages of development of the mesoderm, limbs, and nervous system.

Developmental expression of c-kit, a proto-oncogene encoded by the W locus

Developmental expression of the c-kit proto-oncogene, a receptor tyrosine kinase encoded by the W locus, was investigated by in situ hybridization in normal mouse embryos. Early after implantation transcripts were detectable only in the maternal placenta (6 1/2-7 1/2 days p.c.). Subsequently (8 1/2 days p.c.) numerous ectodermal (neural tube, sensory placodes) and endodermal (embryonic gut) derivatives expressed c-kit. Later transcripts were detected also in the blood islands of the yolk sac and in the embryonic liver, the main sites of embryonic hemopoiesis. Around midgestation, transcripts accumulated in the branchial pouches and also in primordial germ cells of the genital ridges. This complex pattern of expression remained characteristic also later in gestation, when c-kit was expressed in highly differentiated structures of the craniofacial area, in presumptive melanoblasts and in the CNS. In the adult ovary, maternal c-kit transcripts were detected. They were present in the oocytes of both immature and mature ovarian follicles, but not in the male germ line, where c-kit expression may be down regulated. Thus, c-kit activity is complex and appears in multiple tissues including those that also display defects in mutations at the W locus where c-kit is encoded. Correlation between W phenotypes and c-kit expression, as well as the regulation of the complex and multiple expression of polypeptide growth factors and receptors, is discussed.

Embryotoxic effects of sodium arsenite and sodium arsenate on mouse embryos in culture

Embryotoxic effects of two inorganic arsenic compounds, sodium arsenite (Asi) and sodium arsenate (Asa), on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with three to five somites exposed to 1-40 microM Asi or to 10-400 microM Asa were cultured for 48 hours and their development was compared with that of control embryos. Asi proved to be teratogenic between 3 and 4 microM and embryolethal at higher concentrations; Asa had similar activity but at concentrations ten times higher than for Asi. Both compounds produced a growth retardation and a similar pattern of defects. Growth retardation was indicated by a statistically significant reduction in crown-rump length, head length, and yolk sac diameter. Abnormal embryos were characterized by hypoplasia of the prosencephalon with open neural tube, hydropericardium, somite abnormalities, and failure of development of limb buds and sensory placodes. These results confirm that both Asa and Asi are embryotoxic compounds and that the Asi activity occurs at concentrations ten times lower than for Asa. Our results suggest that in humans both of these compounds may be involved in part of "unaccountable" early abortions and malformations claimed to be due to the toxicity of heavy metals.

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Peripheral sensory organs and ganglia found in the vertebrate head arise during embryonic development from distinct ectodermal thickenings, called cranial sensory placodes (adenohypophyseal, olfactory, lens, trigeminal, epibranchial, and otic). A series of patterning events leads to the establishment of these placodes. Subsequently, these placodes undergo specific morphogenetic movements and cell-type specification in order to shape the final placodal derivatives and to produce differentiated cell types necessary for their function. In this chapter, we will focus on recent studies in the zebrafish that have advanced our understanding of cranial sensory placode development. We will summarize the signaling events and their molecular effectors guiding the formation of the so-called preplacodal region, and the subsequent subdivision of this region along the anteroposterior axis that gives rise to specific placode identities as well as those controlling morphogenesis and neurogenesis. Finally, we will highlight the approaches used in zebrafish that have been established to precisely label cell populations, to follow their development, and/or to characterize cell fates within a specific placode.