Retinoids and nonvertebrate chordate development

Retinoic acid (RA) is required for the differentiation and morphogenesis of chordate-specific features, such as the antero-posterior regionalization of the dorsal hollow nerve cord and neural crest cells. RA receptors (RARs) have been reported exclusively in chordates, suggesting that the acquisition of the RAR gene was important for chordate evolution. A scenario is presented here for the establishment of an RAR-mediated developmental regulatory system during the course of chordate evolution. In the common chordate ancestor, RAR came to control the spatial expression pattern of Hox genes in the ectoderm and endoderm along the antero-posterior axis. In these germ layers, RA was required for the differentiation of epidermal sensory neurons and the morphogenesis of pharyngeal gill slits, respectively. As the diffuse epidermal nerve net in the chordate ancestor became centralized to form the dorsal nerve cord, the epidermal Hox expression pattern was carried into the central nervous system. Because the Hox code here came to specify neuronal identity along the antero-posterior axis, RA became inextricably linked to the antero-posterior patterning of the chordate central nervous system.     

Identification of a retinoic acid‐responsive neural enhancer in the Ciona intestinalis Hox1 gene

The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci‐Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci‐Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid‐response element (RARE). Mutation of the RARE in the Ci‐Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci‐Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA‐dependent and one RA‐independent.     

A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus

Retinoic acid (RA) mediates both anterior/posterior patterning and neuronal specification in the vertebrate central nervous system (CNS). However, the molecular mechanisms downstream of RA are not well understood. To investigate these mechanisms, we used the invertebrate chordate amphioxus, in which the CNS, although containing only about 20,000 neurons in adults, like the vertebrate CNS, has a forebrain, midbrain, hindbrain, and spinal cord and is regionalized by RA-signaling. Here we show, first, that domains of genes with expression normally limited to diencephalon and midbrain are generally not affected by altered RA-signaling, second, that contrary to previous reports, not only Hox1, 3, and 4, but also Hox2 and Hox6 are collinearly expressed in the amphioxus CNS, and third, that collinear expression of all these Hox genes is controlled by RA-signaling. Finally, we show that Hox1 is involved in mediating both the role of RA-signaling in regionalization of the hindbrain and in specification of hindbrain motor neurons. Thus, morpholino knock-down of the single amphioxus Hox1 mimics the effects of treatments with an RA-antagonist. This analysis establishes RA-dependent regulation of collinear Hox expression as a feature common to the chordate CNS and indicates that the RA-Hox hierarchy functions both in proper anterior/posterior patterning of the developing CNS and in specification of neuronal identity.     

The first steps towards hearing: mechanisms of otic placode induction

The entire inner ear, together with the neurons that innervate it, derive from a simple piece of ectoderm on the side of the embryonic head the otic placode. In this review, we describe the current state of the field of otic placode induction. Several lines of evidence suggest that all craniofacial sensory organs, including the inner ear, derive from a common "pre-placodal region" early in development. We review data showing that assumption of a pre-placodal cell state correlates with the competence of embryonic ectoderm to respond to otic placode inducing signals, such as members of the fibroblast growth factor (FGF) family. We also review evidence for FGF-independent signals that contribute to the induction of the otic placode. Finally, we review recent evidence suggesting that Wnt signals may act after FGF signaling to mediate a cell fate decision between otic placode and epidermis.     

Pitx homeobox genes in Ciona and amphioxus show left-right asymmetry is a conserved chordate character and define the ascidian adenohypophysis

All vertebrates have directional asymmetries in the organization of their internal organs. In jawed vertebrates, development of asymmetry is controlled by a conserved molecular pathway that includes Pitx2, which is expressed by lateral plate mesoderm cells on the left side of the embryo. Pitx2 is a member of the Pitx homeobox gene family, the expression of which also marks stomodeal ectoderm and the adenohypophysis. Here we report the characterization of Pitx genes from Branchiostoma floridae (an amphioxus) and Ciona intestinalis (a urochordate), representatives of two basal chordate lineages and successively deeper outgroups to the vertebrates. Expression of B. floridae Pitx is similar to that reported from B. belcheri, a different amphioxus species. Expression of the Ciona Pitx ortholog in the embryonic primordial pharynx and adult neural complex leads us to propose the Ciona primordial pharynx and ciliated funnel are homologous to the adenohypophyseal placode and adenohypophysis, respectively. Additionally, in both species we identify asymmetrical left-sided expression of Pitx genes during embryonic development. This shows that asymmetrical Pitx gene expression, and by inference directional asymmetry, evolved before the radiation of living chordates and should be considered a chordate character.     

Fgf3 and Fgf10 are required for mouse otic placode induction

The inner ear, which contains the sensory organs specialised for audition and balance, develops from an ectodermal placode adjacent to the developing hindbrain. Tissue grafting and recombination experiments suggest that placodal development is directed by signals arising from the underlying mesoderm and adjacent neurectoderm. In mice, Fgf3 is expressed in the neurectoderm prior to and concomitant with placode induction and otic vesicle formation, but its absence affects only the later stages of otic vesicle morphogenesis. We show here that mouse Fgf10 is expressed in the mesenchyme underlying the prospective otic placode. Embryos lacking both Fgf3 and Fgf10 fail to form otic vesicles and have aberrant patterns of otic marker gene expression, suggesting that FGF signals are required for otic placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the ectoderm, as double mutant embryos showed normal patterns of gene expression in the hindbrain. Cell proliferation and survival were not markedly affected in double mutant embryos, suggesting that the major role of FGF signals in otic induction is to establish normal patterns of gene expression in the prospective placode. Finally, examination of embryos carrying three out of the four mutant Fgf alleles revealed intermediate phenotypes, suggesting a quantitative requirement for FGF signalling in otic vesicle formation.     

Sprouty1 and Sprouty2 limit both the size of the otic placode and hindbrain Wnt8a by antagonizing FGF signaling

Multiple signaling molecules, including Fibroblast Growth Factor (FGF) and Wnt, induce two patches of ectoderm on either side of the hindbrain to form the progenitor cell population for the inner ear, or otic placode. Here we report that in Spry1, Spry2 compound mutant embryos (Spry1^−/−; Spry2^−/− embryos), the otic placode is increased in size. We demonstrate that the otic placode is larger due to the recruitment of cells, normally destined to become cranial epidermis, into the otic domain. The enlargement of the otic placode observed in Spry1^−/−; Spry2^−/− embryos is preceded by an expansion of a Wnt8a expression domain in the adjacent hindbrain. We demonstrate that both the enlargement of the otic placode and the expansion of the Wnt8a expression domain can be rescued in Spry1^−/−; Spry2^−/− embryos by reducing the gene dosage of Fgf10. Our results define a FGF-responsive window during which cells can be continually recruited into the otic domain and uncover SPRY regulation of the size of a putative Wnt inductive center.     

Key steps in the morphogenesis of a cranial placode in an invertebrate chordate, the tunicate Ciona savignyi

Tunicates and vertebrates share a common ancestor that possessed cranial neurogenic placodes, thickenings in embryonic head epidermis giving rise to sensory structures. Though orthology assignments between vertebrate and tunicate placodes are not entirely resolved, vertebrate otic placodes and tunicate atrial siphon primordia are thought to be homologous based on morphology and position, gene expression, and a common signaling requirement during induction. Here, we probe key points in the morphogenesis of the tunicate atrial siphon. We show that the siphon primordium arises within a non-dividing field of lateral-dorsal epidermis. The initial steps of atrial primordium invagination are similar to otic placode invagination, but a placode-derived vesicle is never observed as for the otic vesicle of vertebrates. Rather, confocal imaging reveals an atrial opening through juvenile stages and beyond. We inject a photoactivatable lineage tracer to show that the early atrial siphon of the metamorphic juvenile, including its aperture and lining, derives from cells of the atrial placode itself. Finally, we perturb the routing of the gut to the left atrium by laser ablation and pharmacology to show that this adaptation to a sessile lifestyle depends on left-right patterning mechanisms present in the free-swimming chordate ancestor.     

Analysis of FGF-Dependent and FGF-Independent Pathways in Otic Placode Induction

The inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the otic placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the otic placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent otic placode formation. To better understand the genetic pathways activated by FGF signaling during otic placode induction, we performed microarray experiments to estimate the proportion of chicken otic placode genes that can be up-regulated by the FGF pathway in a simple culture model of otic placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15% of chick otic placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of otic placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in otic placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for otic placode induction, it demonstrates that other unidentified signaling pathways are required to co-operate with FGF signaling to induce the full otic placode program.     

RA and FGF Signalling Are Required in the Zebrafish Otic Vesicle to Pattern and Maintain Ventral Otic Identities

During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.     

Neural ectoderm,neural crest,and placodes: Contribution of the otic placode to the ectodermal lining of the embryonic opercular cavity in Atlantic cod (Teleostei)

Development of neural ectoderm, neural crest, and otic placode with special reference to a new placodal derivative, the ectodermal lining of the opercular cavity, is described in a teleost fish, the Atlantic cod Gadus morhua, from a stage-by-stage examination of embryonic development. The ectodermal lining of the opercular cavity forms by invagination of the otic placode. The neural plate “infolds” by a wave of cellular rearrangement that transforms the neural plate into a neural rod. This transformation creates a distinct dorsal ectodermal cell layer. When the neural rod is arranged as monostratified columnar cells in the forebrain and midbrain, dorsal ectoderm at the midbrain level thickens lateral to the neural rod to form a cell cluster—the presumptive neural crest and placode. Upon migration of the neural crest from the postoptic midbrain, the dorsolateral area of the dorsal ectoderm thickens and segregates from the neural crest as a placode that is continuous with the presumptive lens placode. As the neural crest migrates from the hindbrain, this placode extends along the hindbrain as a single continuous cluster of cells. At the onset of formation of the lens placode, this continuous placode becomes the placode in the postoptic area of the midbrain and separates into the otic placode at the hindbrain. The otic placode gives rise to the otic neuromast and probably the otic lateral line nerves rostrally and to the ectodermal cell lining of the opercular cavity and otic vesicles caudally. The opercular cavity forms by invagination of the otic placode, creating an internal lumen lined by ectoderm that becomes continuous with evaginated endodermal pharyngeal cells. Free neuromasts are observed along the trailing edge of the external opening of the opercular cavity, which lies horizontally, ventral to the otic vesicles. As embryos develop to hatching, the opening rotates and takes up a vertical position. The adult opercular apparatus, including associated bones and muscles, forms during larval stages. The otic neuromast may be a remnant of neuromasts in the spiracle organ. The spiracle opening lies between the mandibular and hyoid arches, whereas the opercular cavity opens between the hyoid and the first branchial arches. The spiracle opening is, therefore, not homologous with the external opening of the opercular cavity, although the cell lining of the spiracle opening may be of placodal origin. J Morphol 231:231–252, 1997. © 1997 Wiley-Liss, Inc.     

Competence, specification and commitment in otic placode induction

The inner ear is induced from cranial ectoderm adjacent to the hindbrain. Despite almost a century of study, the molecular mechanisms of inner ear induction remain obscure. We have identified four genes expressed very early in the anlage of the inner ear, the otic placode. Pax-2, Sox-3, BMP-7 and Notch are all expressed in placodal ectoderm from the 4-5 somite stage (ss) onwards, well before the otic placode becomes morphologically visible at the 12-14ss. We have used these four molecular markers to show that cranial ectoderm becomes specified to form the otic placode at the 4-6ss, and that this ectoderm is committed to a placodal fate by the 10ss. We also demonstrate that much of the embryonic ectoderm is competent to generate an otic placode if taken at a sufficiently early age. We have mapped the location of otic placode-inducing activity along the rostrocaudal axis of the embryo, and have determined that this activity persists at least until the 10ss. Use of the four molecular otic placode markers suggests that induction of the otic placode in birds occurs earlier than previously thought, and proceeds in a series of steps that are independently regulated.