A Gbx homeobox gene in amphioxus: insights into ancestry of the ANTP class and evolution of the midbrain/hindbrain boundary

In the vertebrate central nervous system (CNS), mutual antagonism between posteriorly expressed Gbx2 and anteriorly expressed Otx2 positions the midbrain/hindbrain boundary (MHB), but does not induce MHB organizer genes such as En, Pax2/5/8 and Wnt1. In the CNS of the cephalochordate amphioxus, Otx is also expressed anteriorly, but En, Pax2/5/8 and Wnt1 are not expressed near the caudal limit of Otx, raising questions about the existence of an MHB organizer in amphioxus. To investigate the evolutionary origins of the MHB, we cloned the single amphioxus Gbx gene. Fluorescence in situ hybridization showed that, as in vertebrates, amphioxus Gbx and the Hox cluster are on the same chromosome. From analysis of linked genes, we argue that during evolution a single ancestral Gbx gene duplicated fourfold in vertebrates, with subsequent loss of two duplicates. Amphioxus Gbx is expressed in all germ layers in the posterior 75% of the embryo, and in the CNS, the Gbx and Otx domains abut at the boundary between the cerebral vesicle (forebrain/midbrain) and the hindbrain. Thus, the genetic machinery to position the MHB was present in the protochordate ancestors of the vertebrates, but is insufficient for induction of organizer genes. Comparison with hemichordates suggests that anterior Otx and posterior Gbx domains were probably overlapping in the ancestral deuterostome and came to abut at the MHB early in the chordate lineage before MHB organizer properties evolved.     

The evolution of chordate neural segmentation

Amphioxus is the closest relative to vertebrates but lacks key vertebrate characters, like rhombomeres, neural crest cells, and the cartilaginous endoskeleton. This reflects major differences in the developmental patterning of neural and mesodermal structures between basal chordates and vertebrates. Here, we analyse the expression pattern of an amphioxus FoxB ortholog and an amphioxus single-minded ortholog to gain insight into the evolution of vertebrate neural segmentation. AmphiFoxB expression shows cryptic segmentation of the cerebral vesicle and hindbrain, suggesting that neuromeric segmentation of the chordate neural tube arose before the origin of the vertebrates. In the forebrain, AmphiFoxB expression combined with AmphiSim and other amphioxus gene expression patterns shows that the cerebral vesicle is divided into several distinct domains: we propose homology between these domains and the subdivided diencephalon and midbrain of vertebrates. In the Hox-expressing region of the amphioxus neural tube that is homologous to the vertebrate hindbrain, AmphiFoxB shows the presence of repeated blocks of cells along the anterior-posterior axis, each aligned with a somite. This and other data lead us to propose a model for the evolution of vertebrate rhombomeric segmentation, in which rhombomere evolution involved the transfer of mechanisms regulating neural segmentation from vertical induction by underlying segmented mesoderm to horizontal induction by graded retinoic acid signalling. A consequence of this would have been that segmentation of vertebrate head mesoderm would no longer have been required, paving the way for the evolution of the unsegmented head mesoderm seen in living vertebrates.     

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.     

The evolutionary origin of the vertebrate neural crest and its developmental gene regulatory network – insights from amphioxus

The neural crest is an embryonic cell population unique to vertebrates. During vertebrate embryogenesis, neural crest cells are first induced from the neural plate border; subsequently, they delaminate from the dorsal neural tube and migrate to their destination, where they differentiate into a wide variety of derivatives. The emergence of the neural crest is thought to be responsible for the evolution of many complex novel structures of vertebrates that are lacking in invertebrate chordates. Despite its central importance in understanding the origin of vertebrates, the evolutionary origin of the neural crest remains elusive. The basal chordate amphioxus (Branchiostoma floridae) occupies an outgroup position that is useful for investigating this question. In this review, I summarize recent genomic and comparative developmental studies between amphioxus and vertebrates and discuss their implications for the evolutionary origin of neural crest cells. I focus mainly on the origin of the gene regulatory network underlying neural crest development, and suggest several hypotheses regarding how this network could have been assembled during early vertebrate evolution.     

Origins of anteroposterior patterning and Hox gene regulation during chordate evolution

All chordates share a basic body plan and many common features of early development. Anteroposterior (AP) regions of the vertebrate neural tube are specified by a combinatorial pattern of Hox gene expression that is conserved in urochordates and cephalochordates. Another primitive feature of Hox gene regulation in all chordates is a sensitivity to retinoic acid during embryogenesis, and recent developmental genetic studies have demonstrated the essential role for retinoid signalling in vertebrates. Two AP regions develop within the chordate neural tube during gastrulation: an anterior 'forebrain-midbrain' region specified by Otx genes and a posterior 'hindbrain-spinal cord' region specified by Hox genes. A third, intermediate region corresponding to the midbrain or midbrain-hindbrain boundary develops at around the same time in vertebrates, and comparative data suggest that this was also present in the chordate ancestor. Within the anterior part of the Hox-expressing domain, however, vertebrates appear to have evolved unique roles for segmentation genes, such as Krox-20, in patterning the hindbrain. Genetic approaches in mammals and zebrafish, coupled with molecular phylogenetic studies in ascidians, amphioxus and lampreys, promise to reveal how the complex mechanisms that specify the vertebrate body plan may have arisen from a relatively simple set of ancestral developmental components.     

Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region

On the basis of developmental gene expression, the vertebrate central nervous system comprises: a forebrain plus anterior midbrain, a midbrain-hindbrain boundary region (MHB) having organizer properties, and a rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Wada and others (Wada, H., Saiga, H., Satoh, N. and Holland, P. W. H. (1998) Development 125, 1113-1122) suggested that ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking. We, therefore, isolated and characterized AmphiPax2/5/8, the sole member of this subfamily in amphioxus. AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous - support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits; although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologues of vertebrate otic placodes or otic vesicles. In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region. Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus. The alternative is that the MHB is a vertebrate innovation.     

Expression of estrogen-receptor related receptors in amphioxus and zebrafish: implications for the evolution of posterior brain segmentation at the invertebrate-to-vertebrate transition

Summary The evolutionary origin of vertebrate hindbrain segmentation is unclear since the amphioxus, the closest living invertebrate relative to the vertebrates, possesses a hindbrain homolog that displays no gross morphological segmentation. Three of the estrogen-receptor related (ERR) receptors are segmentally expressed in the zebrafish hindbrain, suggesting that their common ancestor was expressed in a similar, reiterated manner. We have also cloned and determined the developmental expression of the single homolog of the vertebrate ERR genes in the amphioxus (AmphiERR). This gene is also expressed in a segmented manner in a region considered homologous to the vertebrate hindbrain. In contrast to the expression of amphioxus islet (a LIM-homeobox gene that also labels motoneurons), AmphiERR expression persists longer in the hindbrain homolog and does not later extend to additional posterior cells. In addition, AmphiERR and one of its vertebrate homologs (ERRalpha) are expressed in the developing somitic musculature of amphioxus and zebrafish, respectively. Altogether, our results are consistent with fine structural evidence suggesting that the amphioxus hindbrain is segmented, and indicate that chordate ERR gene expression is a marker for both hindbrain and muscle segmentation. Furthermore, our data support an evolution model of chordate brain segmentation: originally, the program for anterior segmentation in the protochordate ancestors of the vertebrates resided in the developing axial mesoderm which imposed reiterated patterning on the adjacent neural tube; during early vertebrate evolution, this segmentation program was transferred to and controlled by the neural tube.     

Gbx2 and Fgf8 are sequentially required for formation of the midbrain-hindbrain compartment boundary

In vertebrates, the common expression border of two homeobox genes, Otx2 and Gbx2, demarcates the prospective midbrain-hindbrain border (MHB) in the neural plate at the end of gastrulation. The presence of a compartment boundary at the MHB has been demonstrated, but the mechanism and timing of its formation remain unclear. We show by genetic inducible fate mapping using a Gbx2(CreER) knock-in mouse line that descendants of Gbx2(+) cells as early as embryonic day (E) 7.5 do not cross the MHB. Without Gbx2, hindbrain-born cells abnormally populate the entire midbrain, demonstrating that Gbx2 is essential for specifying hindbrain fate. Gbx2(+) and Otx2(+) cells segregate from each other, suggesting that mutually exclusive expression of Otx2 and Gbx2 in midbrain and hindbrain progenitors is responsible for cell sorting in establishing the MHB. The MHB organizer gene Fgf8, which is expressed as a sharp transverse band immediately posterior to the lineage boundary at the MHB, is crucial in maintaining the lineage-restricted boundary after E7.5. Partial deletion of Fgf8 disrupts MHB lineage separation. Activation of FGF pathways has a cell-autonomous effect on cell sorting in midbrain progenitors. Therefore, Fgf8 from the MHB may signal the nearby mesencephalic cells to impart distinct cell surface characteristics or induce local cell-cell signaling, which consequently prevents cell movements across the MHB. Our findings reveal the distinct function of Gbx2 and Fgf8 in a stepwise process in the development of the compartment boundary at the MHB and that Fgf8, in addition to its organizer function, plays a crucial role in maintaining the lineage boundary at the MHB by restricting cell movement.     

An amphioxus Msx gene expressed predominantly in the dorsal neural tube

 Genomic and cDNA clones of an Msx class homeobox gene were isolated from amphioxus (Branchiostoma floridae). The gene, AmphiMsx, is expressed in the neural plate from late gastrulation; in later embryos it is expressed in dorsal cells of the neural tube, excluding anterior and posterior regions, in an irregular reiterated pattern. There is transient expression in dorsal cells within somites, reminiscent of migrating neural crest cells of vertebrates. In larvae, mRNA is detected in two patches of anterior ectoderm proposed to be placodes. Evolutionary analyses show there is little phylogenetic information in Msx protein sequences; however, it is likely that duplication of Msx genes occurred in the vertebrate lineage. Received: 12 October 1998 / Accepted: 26 December 1998     

An orphan PRD class homeobox gene expressed in mouse brain and limb development

We report the cDNA sequence and expression of a mouse homeobox gene, Dmbx1, from the PRD class and comparison to its human orthologue. The gene defines a new homeobox gene family, Dmbx, phylogenetically distinct from the Ptx, Alx, Prx Otx, Gsc, Otp and Pax gene families. The Dmbx1 gene is expressed in the developing mouse diencephalon, midbrain and hindbrain, and has dynamic expression during forelimb and hindlimb development. Unusually for homeobox genes, there is no orthologue in the Drosophila or Caenorhabditis genomes; we argue this reflects secondary loss.     

The Mnx homeobox gene class defined by HB9, MNR2 and amphioxus AmphiMnx

The HB9 homeobox gene has been cloned from several vertebrates and is implicated in motor neuron differentiation. In the chick, a related gene, MNR2, acts upstream of HB9 in this process. Here we report an amphioxus homologue of these genes and show that it diverged before the gene duplication yielding HB9 and MNR2. AmphiMnx RNA is detected in two irregular punctate stripes along the developing neural tube, comparable to the distribution of 'dorsal compartment' motor neurons, and also in dorsal endoderm and posterior mesoderm. We propose a new homeobox class, Mnx, to include AmphiMnx, HB9, MNR2 and their Drosophila and echinoderm orthologues; we suggest that vertebrate HB9 is renamed Mnx1 and MNR2 be renamed Mnx2.     

Enhancer evolution in chordates: Lessons from functional analyses of cephalochordate cis-regulatory modules

Chordates comprise three major groups, cephalochordates (amphioxus), tunicates (urochordates), and vertebrates. Since cephalochordates were the early branching group, comparisons between amphioxus and other chordates help us to speculate about ancestral chordates. Here, I summarize accumulating data from functional studies analyzing amphioxus cis-regulatory modules (CRMs) in model systems of other chordate groups, such as mice, chickens, clawed frogs, fish, and ascidians. Conservatism and variability of CRM functions illustrate how gene regulatory networks have evolved in chordates. Amphioxus CRMs, which correspond to CRMs deeply conserved among animal phyla, govern reporter gene expression in conserved expression domains of the putative target gene in host animals. In addition, some CRMs located in similar genomic regions (intron, upstream, or downstream) also possess conserved activity, even though their sequences are divergent. These conservative CRM functions imply ancestral genomic structures and gene regulatory networks in chordates. However, interestingly, if expression patterns of amphioxus genes do not correspond to those of orthologs of experimental models, some amphioxus CRMs recapitulate expression patterns of amphioxus genes, but not those of endogenous genes, suggesting that these amphioxus CRMs are close to the ancestral states of chordate CRMs, while vertebrates/tunicates innovated new CRMs to reconstruct gene regulatory networks subsequent to the divergence of the cephalochordates. Alternatively, amphioxus CRMs may have secondarily lost ancestral CRM activity and evolved independently. These data help to solve fundamental questions of chordate evolution, such as neural crest cells, placodes, a forebrain/midbrain, and genome duplication. Experimental validation is crucial to verify CRM functions and evolution.     

Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns

The neural crest is a uniquely vertebrate cell type present in the most basal vertebrates, but not in cephalochordates. We have studied differences in regulation of the neural crest marker AP-2 across two evolutionary transitions: invertebrate to vertebrate, and agnathan to gnathostome. Isolation and comparison of amphioxus, lamprey and axolotl AP-2 reveals its extensive expansion in the vertebrate dorsal neural tube and pharyngeal arches, implying co-option of AP-2 genes by neural crest cells early in vertebrate evolution. Expression in non-neural ectoderm is a conserved feature in amphioxus and vertebrates, suggesting an ancient role for AP-2 genes in this tissue. There is also common expression in subsets of ventrolateral neurons in the anterior neural tube, consistent with a primitive role in brain development. Comparison of AP-2 expression in axolotl and lamprey suggests an elaboration of cranial neural crest patterning in gnathostomes. However, migration of AP-2-expressing neural crest cells medial to the pharyngeal arch mesoderm appears to be a primitive feature retained in all vertebrates. Because AP-2 has essential roles in cranial neural crest differentiation and proliferation, the co-option of AP-2 by neural crest cells in the vertebrate lineage was a potentially crucial event in vertebrate evolution.     

Early development of the cerebellum in teleost fishes: a study based on gene expression patterns and histology in the medaka embryo

The cerebellar structures of teleosts are markedly different from those of other vertebrates. The cerebellum continues rostrally into the midbrain ventricle, forming the valvula cerebelli, only in ray-finned fishes among vertebrates. To analyze the ontogenetic processes that underlie this morphological difference, we examined the early development of the cerebellar regions, including the isthmus (mid/hindbrain boundary, MHB), of the medaka (Oryzias latipes), by histology and in-situ hybridization using two gene (wnt1 and fgf8) probes. Isthmic wnt1 was expressed stably in the caudalmost mesencephalic region in the neural tube at all developmental stages examined, defining molecularly the caudal limit of the mesencephalon. The wnt1-positive mesencephalic cells became located rostrally to the isthmic constriction at Iwamatsu's stages 25-26. Isthmic fgf8 expression changed dynamically and became restricted to the rostralmost metencephalic region at stage 24. The rostralmost part (prospective valvula cerebelli) of the fgf8-positive rostral metencephalon protruded rostrally into the midbrain ventricle, bypassing the isthmic constriction, at stages 25-26. Thus, the isthmic constriction shifted caudally with respect to the molecularly defined MHB at stages 25-26. Paired cerebellar primordia were formed from the alar plates of the fgf8-positive rostral metencephalon and the fgf8-negative caudal metencephalon in the medaka neural tube. Our results show that cerebellar development differs between teleosts and murines: both the rostral and caudal metencephalic alar plates develop into the cerebellum in medaka, whereas in the murines only the caudal metencephalic alar plate develops into the cerebellum, and the rostral plate is reduced to a thin membrane.     

Evolution of CUT class homeobox genes: insights from the genome of the amphioxus, Branchiostoma floridae

CUT class homeobox genes, including CUX/CASP, ONECUT, SATB and COMPASS family genes, are known to exhibit diverse features in the homeodomain and the domain architecture. Furthermore, the intron/exon organization of CUX/CASP is different between vertebrates and protostomes, and SATB genes are only known for vertebrates, whereas COMPASS genes have only been found in protostomes. These observations suggest a complex evolutionary history for the CUT class homeobox genes, but the evolution of CUT class homeobox genes in the lineage to vertebrates remained largely unknown. To obtain clearer insights into this issue, we searched the genome of amphioxus, Branchiostoma floridae, a lower chordate, for CUT class homeobox genes by extensive BLAST survey and phylogenetic analyses. We found that the genome of Branchiostoma floridae encodes each single orthologue of CUX/CASP, ONECUT, and COMPASS, but not the SATB gene, and one atypical CUT gene likely specific to this species. In addition, the genomic structure of the amphioxus CUX/CASP gene turned out to be protostome-type, but not vertebrate-type. Based on these observations, we propose a model in which SATB is suggested to evolve at the expense of COMPASS and this change, together with the structural change in CUX/CASP, is supposed to take place in the lineage to vertebrates after divergence of the amphioxus and vertebrate ancestors. The present study provides an example of dramatic evolution among homeobox gene groups in the vertebrate lineage and highlights the ancient character of amphioxus, retaining genomic features shared by protostomes.     

Dmbx1, a novel evolutionarily conserved paired-like homeobox gene expressed in the brain of mouse embryos.

To identify novel homeobox genes expressed during mouse embryogenesis, we searched the databases and found a novel mouse paired-like homeobox gene, Dmbx1(diencephalon/mesencephalon-expressed brain homeobox gene 1), that is also conserved in zebrafish and human. Linkage analysis mapped mouse Dmbx1 to the mid-portion of chromosome 4 that is the homologous gene cluster region of human chromosome 1, where human DMBX1 is located. Both mouse and human Dmbx1/DMBX1 have four coding exons and their gene structures are conserved. Whole-mount in situ hybridization revealed that Dmbx1 expression is detected in 7.5-9.5 dpc mouse embryos. At 7.5 and 8.5 dpc, Dmbx1 is expressed in a sub-region of the anterior head folds. At 9.5 dpc, expression is observed in the caudal diencephalon as well as in the mesencephalon and is restricted to the neuroepithelium. Expression in adult tissues was detected in brain, stomach, and testis. Dmbx1 provides a unique marker of the developing anterior nervous system and should provide a useful molecular resource to elucidate the mechanisms that pattern the vertebrate brain.     

Colinear and segmental expression of amphioxus Hox genes.

The cephalochordate amphioxus has a single Hox gene cluster. Here we describe the genomic organization of four adjacent amphioxus genes, AmphiHox-1 to AmphiHox-4, together with analysis of their spatiotemporal expression patterns. We demonstrate that these genes obey temporal colinearity and that three of the genes also obey spatial colinearity in the developing neural tube. AmphiHox-1, AmphiHox-3, and AmphiHox-4 show segmental modulation of their expression levels, a two-segment phasing of spatial colinearity, and, at least for AmphiHox-4, asymmetrical expression. AmphiHox-2 is unlike other amphioxus Hox genes: it does not obey spatial colinearity and it has no positional expression in the neural tube. AmphiHox-2 is expressed in the preoral pit of larvae, from which the homologue of the anterior pituitary develops. We suggest that the ancestral role of chordate Hox genes was primarily in the neural tube and that chordate Hox genes can functionally diverge in a manner analogous to that of Drosophila ftz or zen.