Ancient homeobox gene loss and the evolution of chordate brain and pharynx development: deductions from amphioxus gene expression

Homeobox genes encode a large superclass of transcription factors with widespread roles in animal development. Within chordates there are over 100 homeobox genes in the invertebrate cephalochordate amphioxus and over 200 in humans. Set against this general trend of increasing gene number in vertebrate evolution, some ancient homeobox genes that were present in the last common ancestor of chordates have been lost from vertebrates. Here, we describe the embryonic expression of four amphioxus descendants of these genes— AmphiNedxa, AmphiNedxb, AmphiMsxlx and AmphiNKx7 . All four genes are expressed with a striking asymmetry about the left–right axis in the pharyngeal region of neurula embryos, mirroring the pronounced asymmetry of amphioxus embryogenesis. AmphiMsxlx and AmphiNKx7 are also transiently expressed in an anterior neural tube region destined to become the cerebral vesicle. These findings suggest significant rewiring of developmental gene regulatory networks occurred during chordate evolution, coincident with homeobox gene loss. We propose that loss of otherwise widely conserved genes is possible when these genes function in a confined role in development that is subsequently lost or significantly modified during evolution. In the case of these homeobox genes, we propose that this has occurred in relation to the evolution of the chordate pharynx and brain.

Histochemical localization of constitutive nitric oxide synthases in amphioxus Branchiostoma belcheri tsingtauense

The present study demonstrated histochemically that the enzyme activity was present in the cerebral vesicle, epidermis, muscles, endostyle and anus of amphioxus Branchiostoma belcheri tsingtaunese. This is the first noted report on localization of constitutive nitric oxide synthases in a celphalochordate.

Isolation and developmental expression of the amphioxus Pax-6 gene (AmphiPax-6): insights into eye and photoreceptor evolution

Pax-6 genes have been identified from a broad range of invertebrate and vertebrate animals and shown to be always involved in early eye development. Therefore, it has been proposed that the various types of eyes evolved from a single eye prototype, by a Pax-6-dependent mechanism. Here we describe the characterization of a cephalochordate Pax-6 gene. The single amphioxus Pax-6 gene (AmphiPax-6) can produce several alternatively spliced transcripts, resulting in proteins with markedly different amino and carboxy termini. The amphioxus Pax-6 proteins are 92% identical to mammalian Pax-6 proteins in the paired domain and 100% identical in the homeodomain. Expression of AmphiPax-6 in the anterior epidermis of embryos may be related to development of an olfactory epithelium. Expression is also detectable in Hatschek's left diverticulum as it forms the preoral ciliated pit, part of which gives rise to the homolog of the vertebrate anterior pituitary. A zone of expression in the anterior neural plate of early embryos is carried into the cerebral vesicle (a probable diencephalic homolog) during neurulation. This zone includes cells that will differentiate into the lamellar body, a presumed homolog of the vertebrate pineal eye. In neurulae, AmphiPax-6 is also expressed in ventral cells at the anterior tip of the nerve cord; these cells are precursors of the photoreceptive neurons of the frontal eye, the presumed homolog of the vertebrate paired eyes. However, AmphiPax-6 expression was not detected in two additional types of photoreceptors, the Joseph cells or the organs of Hesse, which are evidently relatively recent adaptations (ganglionic photoreceptors) and appear to be rare exceptions to the general rule that animal photoreceptors develop from a genetic program triggered by Pax-6.

Sequence and embryonic expression of the amphioxus engrailed gene (AmphiEn): the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila

Vertebrate segmentation has been proposed as an evolutionary inheritance either from some metameric protostome or from a more closely related deuterostome. To address this question, we studied the developmental expression of AmphiEn, the engrailed gene of amphioxus, the closest living invertebrate relative of the vertebrates. In neurula embryos of amphioxus, AmphiEn is expressed along the anteroposterior axis as metameric stripes, each located in the posterior part of a nascent or newly formed segment. This pattern resembles the expression stripes of the segment-polarity gene engrailed, which has a key role in establishing and maintaining the metameres in embryos of Drosophila and other metameric protostomes. Later, amphioxus embryos express AmphiEn in non-metameric patterns - transiently in the embryonic ectoderm and dorsal nerve cord. Nerve cord expression occurs in a few cells approximately midway along the rostrocaudal axis and also in a conspicuous group of anterior cells in the cerebral vesicle at a level previously identified as corresponding to the vertebrate diencephalon. Compared to vertebrate engrailed expression at the midbrain/hindbrain boundary, AmphiEn expression in the cerebral vesicle is relatively late. Thus, it is uncertain whether the cerebral vesicle expression marks the rostral end of the amphioxus hindbrain; if it does, then amphioxus may have little or no homolog of the vertebrate midbrain. The segmental expression of AmphiEn in forming somites suggests that the functions of engrailed homologs in establishing and maintaining a metameric body plan may have arisen only once during animal evolution. If so, the protostomes and deuterostomes probably shared a common segmented ancestor.

Sequence and developmental expression of AmphiDll, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous system: insights into evolution of craniate forebrain and neural crest

The dynamic expression patterns of the single amphioxus Distal-less homolog (AmphiDll) during development are consistent with successive roles of this gene in global regionalization of the ectoderm, establishment of the dorsoventral axis, specification of migratory epidermal cells early in neurulation and the specification of forebrain. Such a multiplicity of Distal-less functions probably represents an ancestral chordate condition and, during craniate evolution, when this gene diversified into a family of six or so members, the original functions evidently tended to be parcelled out among the descendant genes. In the amphioxus gastrula, AmphiDll is expressed throughout the animal hemisphere (presumptive ectoderm), but is soon downregulated dorsally (in the presumptive neural plate). During early neurulation, AmphiDll-expressing epidermal cells flanking the neural plate extend lamellipodia, appear to migrate over it and meet mid-dorsally. Midway in neurulation, cells near the anterior end of the neural plate begin expressing AmphiDll and, as neurulation terminates, these cells are incorporated into the dorsal part of the neural tube, which forms by a curling of the neural plate. This group of AmphiDll-expressing neural cells and a second group expressing the gene a little later and even more anteriorly in the neural tube demarcate a region that comprises the anterior three/fourths of the cerebral vesicle; this region of the amphioxus neural tube, as judged by neural expression domains of craniate Distal-less-related genes, is evidently homologous to the craniate forebrain. Our results suggest that craniates evolved from an amphioxus-like creature that had the beginnings of a forebrain and possibly a precursor of neural crest - namely, the cell population leading the epidermal overgrowth of the neural plate during early neurulation.

Expression of AmphiHox-1 and AmphiPax-1 in amphioxus embryos treated with retinoic acid: insights into evolution and patterning of the chordate nerve cord and pharynx

Excess all-trans retinoic acid (RA) causes severe craniofacial malformations in vertebrate embryos: pharyngeal arches are fused or absent, and a rostrad expansion of Hoxb-1 expression in the hindbrain shows that anterior rhombomeres are homeotically respecified to a more posterior identity. As a corollary, neural crest migration into the pharyngeal arches is abnormal. We administered excess RA to developing amphioxus, the closest invertebrate relative of the vertebrates and thus a key organism for understanding evolution of the vertebrate body plan. In normal amphioxus, the nerve cord has only a slight anterior swelling, the cerebral vesicle, and apparently lacks migratory neural crest. Nevertheless, excess RA similarly affects amphioxus and vertebrates. The expression domain of AmphiHox-1 (homologous to mouse Hoxb-1) in the amphioxus nerve cord is also extended anteriorly. For both the amphioxus and mouse genes, excess RA causes either (1) continuous expression throughout the preotic hindbrain (mouse) and from the level of somite 7 to the anterior end of the nerve cord (amphioxus) or (2) discontinuous expression with a gap in rhombomere 3 (mouse) and a gap at the posterior end of the cerebral vesicle (amphioxus). A comparison of these expression patterns suggests that amphioxus has a homolog of the vertebrate hindbrain, both preotic and postotic. Although RA alters the expression of AmphiHox-1 expression in the amphioxus nerve cord, it does not alter the expression of AmphiHox-1 in presomitic mesoderm or of alkali myosin light chain (AmphiMlc-alk) in somites, and the axial musculature and notochord develop normally. The most striking morphogenetic effect of RA on amphioxus larvae is the failure of mouth and gill slits to form. In vertebrates effects of excess RA on pharyngeal development have been attributed solely to the abnormal migratory patterns of Hox-expressing cranial neural crest cells. This cannot be true for amphioxus because of the lack of migratory neural crest. Furthermore, expression of Hox genes in pharyngeal tissues of amphioxus has not yet been detected. However, the absence of gill slits in RA-treated amphioxus embryos correlates with an RA-induced failure of AmphiPax-1 to become down-regulated in regions of pharyngeal endoderm that would normally fuse with the overlying ectoderm. In vertebrates, RA might similarly act via Pax-1/9, also expressed in pharyngeal endoderm, to impair pharyngeal patterning.

Frontal eye circuitry, rostral sensory pathways and brain organization in amphioxus larvae: evidence from 3D reconstructions

The cells comprising the frontal eye of a 12.5 day amphioxus larva are described based on 3D reconstructions from serial electron micrographs, along with the fibre tracts and more caudal groupings of cells in the nerve cord to which the frontal eye appears to be linked. The frontal eye consists of a pigment cup, two transverse rows of receptor cells, and clusters of neurons whose close association with the medial receptor cells suggests they may function as an integral part of the eye complex. Neurites from both the receptor cells and neurons supply the ventrolateral nerve tracts, which consist mainly of axons arising from sensory cells located at the rostral tip of the larva. A core group of 3—4 rostral fibres on each side innervate two ventral giant cells located just behind the cerebral vesicle in the primary motor centre (PMC). The circuitry suggests these cells may be responsible for triggering the larval startle response. The ventrolateral tracts also include two types of axial dendrite-like fibres: (i) a single unpaired fibre, a forward continuation of the principal dendrite of the left giant cell, which is the main target for synapses from neurons in the frontal eye; and (ii) sets of paired fibres from cells in the tectum, a dorsal cortex-like structure located at the back of the cerebral vesicle through which the dorsal sensory nerves pass in transit to the PMC. Recent behavioural studies show that larvae feed in a hovering posture that maximally shades the frontal eye. They also orient to light in this position. The shape and orientation of the frontal eye suggests it could be responsible for this response. The existence of separate pathways from lateral and medial receptor cells, both directly and indirectly to the PMC, suggests the frontal eye may also be involved in modulating locomotory behaviour during hovering. The visual ‘system’ described here for amphioxus larvae is more like that of vertebrates than has previously been recognized. Specifically: ( i )the medial nerve cells of the frontal eye appear to form local circuits with relay and integrative functions similar to those of the retina, involving cell types that resemble specific retinal interneurons; and (ii) output is directed to a region at the back of the posterior c.v. that resembles the vertebrate midbrain, and which may be its homologue. This region has a dorsal tectum and, like the midbrain, includes the anterior part of a ventral zone of motoneurons and reticulospinal interneurons. The morphological evidence supports the idea that the ‘brain’ of amphioxus is sufficiently like that of vertebrates to provide important clues concerning the basic organization and subdivision of the vertebrate brain.

Mapping of cortical histogenesis in the ferret

The columnar organization of forebrain cortical neuron production allows the neuroepithelium to be analysed in terms of a simple growth model. The pattern of neuron release within each column is repeated with great regularity across the surface of the developing cerebral hemisphere, resulting in the accumulation of cortical neurons in radial stacks above the proliferative ventricular epithelium. The time course of recruitment of adjacent tissue into neuron production accounts for the observed rostrocaudal and laterodorsal gradients of cortical neuron release. The present study plots the history of this front of neuron production across the surface of the developing cerebral vesicle in the ferret and interprets the resulting maps of cortical development stages in terms of a unified growth model that links the radial and tangential aspects of neuron production. These observations are discussed in relation to other studies of gradients in developing nervous tissue and their implications for the study of experimentally induced abnormalities of brain development.