Region specific gene expressions in the central nervous system of the ascidian embryo

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. Fibroblast growth factor 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.     

Region specific gene expressions in the central nervous system of the ascidian embryo

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. Fibroblast growth factor 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.     

Functional and structural diversity of the human Dickkopf gene family

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.     

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.     

Cloning,expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling

The organizer at the midbrain-hindbrain boundary (MHB) forms at the interface between Otx2 and Gbx2 expressing cell populations, but how these gene expression domains are set up and integrated with the remaining machinery controlling MHB development is unclear. Here we report the isolation, mapping, chromosomal synteny and spatiotemporal expression of gbx1 and gbx2 in zebrafish. We focus in particular on the expression of these genes during development of the midbrain-hindbrain territory. Our results suggest that these genes function in this area in a complex fashion, as evidenced by their highly dynamic expression patterns and relation to Fgf signaling. Analysis of gbx1 and gbx2 expression during formation of the MHB in mutant embryos for pax2.1, fgf8 and pou2 (noi, ace, spg), as well as Fgf-inhibition experiments, show that gbx1 acts upstream of these genes in MHB development. In contrast, gbx2 activation requires ace (fgf8) function, and in the hindbrain primordium, also spg (pou2). We propose that in zebrafish, gbx genes act repeatedly in MHB development, with gbx1 acting during the positioning period of the MHB at gastrula stages, and gbx2 functioning after initial formation of the MHB, from late gastrulation stages onwards. Transplantation studies furthermore reveal that at the gastrula stage, Fgf8 signals from the hindbrain primordium into the underlying mesendoderm. Apart from the general involvement of gbx genes in MHB development reported also in other vertebrates, these results emphasize that early MHB development can be divided into multiple steps with different genetic requirements with respect to gbx gene function and Fgf signaling. Moreover, our results provide an example for switching of a specific gene function of gbx1 versus gbx2 between orthologous genes in zebrafish and mammals.     

The midbrain-hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-eng2.

In vertebrates, the engrailed genes are expressed at early neurula stage in a narrow stripe encompassing the midbrain-hindbrain boundary (MHB), a region from which a peculiar structure, the isthmus, is formed. Knock-out experiments in mice demonstrated that these genes are essential for the development of this structure and of its derivatives. In contrast, little is known about the effect of an overexpression of engrailed genes in vertebrate development. Here we report the isolation of Ol-eng2, a medaka fish (Oryzias latipes) engrailed gene. We have monitored the effects of its widespread expression following mRNA injections in 1- and 2-cell medaka and Xenopus embryos. We found that the ectopic expression of Ol-eng2 predominantly results in an altered development of the anterior brain, including an inhibition of optic vesicle formation. No change in the patterns of mesencephalic and telencephalic markers were observed. In contrast, expressions of markers of the diencephalon were strongly repressed in injected embryos. Furthermore, the endogenous Ol-eng2, Pax2, Wnt1 and Fgf8, which are essential components of the MHB genetic cascade, were ectopically expressed in this region. Therefore, we propose that Ol-eng2 induces de novo formation of an isthmus-like structure, which correlates with the development of ectopic midbrain structures, including optic tectum. A competence of the diencephalon to change to a midbrain fate has been demonstrated in isthmic graft experiments. Our data demonstrate that this change can be mimicked by ectopic engrailed expression alone.     

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.     

Darmin is a novel secreted protein expressed during endoderm development in Xenopus

Endoderm development is an area of intense interest in developmental biology, but progress has been hampered by the lack of specific markers for differentiated endodermal cells. In an unbiased secretion cloning screen of Xenopus gastrula embryos we isolated a novel gene, designated Darmin. Darmin encodes a secreted protein of 56 kDa containing a peptidase M20 domain characteristic of the glutamate carboxypeptidase group of zinc metalloproteases. We also identified homologous Darmin genes in other eukaryotes and in prokaryotes suggesting that Darmin is the founding member of a family of evolutionarily conserved proteins. Xenopus Darmin showed zygotic expression in the early endoderm and later became restricted to the midgut. By secretion cloning of Xenopus cleavage-stage embryos we isolated another novel protein, designated Darmin-related (Darmin-r) due to its sequence similarity with Darmin. Darmin-r was maternally expressed and showed at later stages expression in the lens and pronephric glomus. The endoderm-specific expression of Darmin makes this gene a useful marker for the study of endoderm development.     

Overlapping and distinct functions provided by fgf17, a new zebrafish member of the Fgf8/17/18 subgroup of Fgfs

Members of the fibroblast growth factor (Fgf) family are important signaling molecules in several inductive and patterning processes, and act as brain organizer-derived signals during formation of the early vertebrate nervous system. We isolated a new member of the Fgf8/17/18 subgroup of Fgfs from the zebrafish, and studied its expression and function during somitogenesis, optic stalk and midbrain-hindbrain boundary (MHB) development. In spite of a slightly higher aminoacid similarity to Fgf8, expression analysis and mapping to a chromosome stretch that is syntenic with mammalian chromosomes shows that this gene is orthologous to mammalian Fgf17. These data provide a further example of conserved chromosomal organization between zebrafish and mammalian genomes. Using an mRNA injection assay, we show that fgf17 can act similar to fgf8 during gastrulation, when fgf17 is not normally expressed. Direct comparison of the expression patterns of fgf17 and fgf8 suggest however a possible cooperation of these Fgfs at later stages in several tissues requiring Fgf signaling. Analysis of zebrafish MHB mutants demonstrates a gene-dosage dependent requirement of fgf17 expression for the no isthmus// pax2.1 gene, showing that no isthmus/pax2.1 functions upstream of fgf17 at the MHB in a haplo-insufficient manner, similar to what has been reported for mammalian pax2 mutants. In contrast, only maintenance of fgf17 expression is disturbed at the MHB of acerebellar/fgf8 mutants. Consistent with a requirement for fgf8 function, implantation of FGF8-soaked beads induces fgf17 expression, and expression is upregulated in aussicht mutants, which display upregulation of the Fgf8 signaling pathway. Taken together, our results argue that Fgf8 and Fgf17 act as hierarchically organized signaling molecules during development of the MHB organizer and possibly other organizers in the developing nervous system.     

Exploration of the extracellular space by a large-scale secretion screen in the early Xenopus embryo

Secreted proteins play a crucial role in intercellular communication during embryogenesis and in the adult. We recently described a novel method, designated as secretion cloning, that allows identifying extracellular proteins exclusively based on their ability to be secreted by transfected cells. In this paper, we present the results of a large-scale screening of more than 90,000 clones from three cDNA expression libraries constructed from early Xenopus embryos. Of 170 sequenced clones, 65 appeared to encode secreted proteins; 26 clones (40%) were identical to previously known Xenopus genes, 25 clones (38%) were homologous to other genes identified in various organisms and 14 clones (22%) were novel. Apart from these bona fide secreted proteins, we also isolated lysosomal or other secretory pathway proteins and some cytoplasmic proteins commonly found in body fluids. Among the novel secreted proteins were two putative growth factors of the Granulin family, termed xGra1 and xGra2; they are structurally similar to EGF and TGFalpha and show a spotted expression pattern in the epidermis. Another secreted protein, designated xSOUL, belongs to the family of heme-binding proteins and exhibits distinct expression in the early brain. A third protein, termed Xystatin, is related to cysteine proteinase inhibitors. Our results indicate that secretion cloning is an effective and generally useful tool for the unbiased isolation of secreted proteins.     

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.