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.     

Coincident iterated gene expression in the amphioxus neural tube

SUMMARY The segmental patterning of the vertebrate hindbrain has been intensely investigated, yet the evolutionary origin of hindbrain segmentation remains unclear. In the vertebrate sister group, amphioxus (Cephalochordata), the embryonic neural tube lacks obvious morphological segmentation, but comparative Hox gene expression analysis has suggested the presence of a region homologous to the vertebrate hindbrain. Does this region contain ancient segmental features shared with the vertebrate hindbrain? To help address this question we cloned the paired‐like amphioxus homeodomain gene shox and found that its expression is segmental in the amphioxus neural tube. We also uncovered a previously uncharacterized iterated neural tube expression pattern of the zinc‐finger gene AmphiKrox. We propose that these genes, along with amphioxus islet and AmphiMnx, share a one‐somite width periodicity of expression in the neural tube, the coincidence of which may reflect an underlying segmental organization. We hypothesize that the segmental patterning of neurons in the neural tube was present in the amphioxus/vertebrate ancestor, but the establishment of a bona fide segmented hindbrain may indeed have arisen in the vertebrate lineage.     

Hindbrain signals in otic regionalization: walk on the wild side

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this level of complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium and more particularly the adjacent segmented hindbrain, have been implicated in conferring signals required for inner ear development. In this review, we present the current view on the role of hindbrain signals in axial specification of the inner ear. The functional analysis of mutants of hindbrain segmentation genes, as well as the investigation of signaling pathways potentially involved, all point to an essential role of FGF, Wnt and Hh signaling in otic regionalization. However, these data provide conflicting evidence regarding the involvement of hindbrain signals in otic regionalization in fish and in amniotes. We discuss the possible origin of these differences.     

Cross-midline interactions between mouse commissural hindbrain axons contribute to their efficient decussation

Information from both sides of the brain is integrated by axons that project across the midline of the central nervous system via numerous commissures present at all axial levels. Despite the accumulated experimental evidence, questions remain regarding the formation of commissures in the presence of strong repulsive signals in the ventral midline. Studies from invertebrates suggest that interaction at the midline between homologous axons of specific decussating neurons contributes to efficient midline crossing, but such evidence is lacking in vertebrate systems. We performed experiments to determine whether commissural axons of the caudal region of the hindbrain interact with their contralateral counterparts at the ventral midline and to evaluate the relevance of this reciprocal interaction. Double anterograde axon labeling with lipophilic tracers revealed close apposition between growth cones of contralateral pioneer decussating axons at the midline. Later, we detected fasciculation between contralateral axons that is maintained even after they have crossed the midline. Blocking axon projections unilaterally with a solid mechanical barrier decreased dramatically the midline crossing of the equivalent population from the contralateral side. Decussation was also blocked by a unilateral barrier permeable to diffusible molecules but not by an axon-permeable barrier. These results suggest that in the caudal region of the hindbrain, midline crossing is facilitated by interactions between decussating contralateral axon partners.     

Screening from a subtracted embryonic chick hindbrain cDNA library: identification of genes expressed during hindbrain, midbrain and cranial neural crest development

The vertebrate hindbrain is segmented into a series of transient structures called rhombomeres. Despite knowing several factors that are responsible for the segmentation and maintenance of the rhombomeres, there are still large gaps in understanding the genetic pathways that govern their development. To find previously unknown genes that are expressed within the embryonic hindbrain, a subtracted chick hindbrain cDNA library has been made and 445 randomly picked clones from this library have been analysed using whole mount in situ hybridisation. Thirty-six of these clones (8%) display restricted expression patterns within the hindbrain, midbrain or cranial neural crest and of these, twenty-two are novel and eleven encode peptides that correspond to or are highly related to proteins with previously uncharacterised roles during early neural development. The large proportion of genes with restricted expression patterns and previously unknown functions in the embryonic brain identified during this screen provides insights into the different types of molecules that have spatially regulated expression patterns in cranial neural tissue.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

Paralog group 1 hox genes regulate rhombomere 5/6 expression of vhnf1, a repressor of rostral hindbrain fates, in a meis-dependent manner

The vertebrate hindbrain is segmented into an array of rhombomeres (r), but it remains to be fully understood how segmentation is achieved. Here we report that reducing meis function transforms the caudal hindbrain to an r4-like fate, and we exploit this experimental state to explore how r4 versus r5-r6 segments are set aside. We demonstrate that r4 transformation of the caudal hindbrain is mediated by paralog group 1 (PG1) hox genes and can be repressed by vhnf1, a gene expressed in r5-r6. We further find that vhnf1 expression is regulated by PG1 hox genes in a meis-dependent manner. This implies that PG1 hox genes not only induce r4 fates throughout the caudal hindbrain, but also induce expression of vhnf1, which then represses r4 fates in the future r5-r6. Our results further indicate that r4 transformation of the caudal hindbrain occurs at intermediate levels of meis function, while extensive removal of meis function produces a hindbrain completely devoid of segments, suggesting that different hox-dependent processes may have distinct meis requirements. Notably, reductions in the function of another Hox cofactor, pbx, have not been reported to transform the caudal hindbrain, suggesting that Meis and Pbx proteins may also function differently in their roles as Hox cofactors.     

Hox gene colinear expression in the avian medulla oblongata is correlated with pseudorhombomeric domains

The medulla oblongata (or caudal hindbrain) is not overtly segmented, since it lacks observable interrhombomeric boundaries. However, quail-chick fate maps showed that it is formed by 5 pseudorhombomeres (r7-r11) which were empirically found to be delimited consistently at planes crossing through adjacent somites (Cambronero and Puelles, 2000). We aimed to reexamine the possible segmentation or rostrocaudal regionalisation of this brain region attending to molecular criteria. To this end, we studied the expression of Hox genes from groups 3 to 7 correlative to the differentiating nuclei of the medulla oblongata. Our results show that these genes are differentially expressed in the mature medulla oblongata, displaying instances of typical antero-posterior (3′ to 5′) Hox colinearity. The different sensory and motor columns, as well as the reticular formation, appear rostrocaudally regionalised according to spaced steps in their Hox expression pattern. The anterior limits of the respective expression domains largely fit boundaries defined between the experimental pseudorhombomeres. Therefore the medulla oblongata shows a Hox-related rostrocaudal molecular regionalisation comparable to that found among rhombomeres, and numerically consistent with the pseudorhombomere list. This suggests that medullary pseudorhombomeres share some AP patterning mechanisms with the rhombomeres present in the rostral, overtly-segmented hindbrain, irrespective of variant boundary properties.     

Segmental development of reticulospinal and branchiomotor neurons in lamprey: insights into the evolution of the vertebrate hindbrain

During development, the vertebrate hindbrain is subdivided along its anteroposterior axis into a series of segmental bulges called rhombomeres. These segments in turn generate a repeated pattern of rhombomere-specific neurons, including reticular and branchiomotor neurons. In amphioxus (Cephalochordata), the sister group of the vertebrates, a bona fide segmented hindbrain is lacking, although the embryonic brain vesicle shows molecular anteroposterior regionalization. Therefore, evaluation of the segmental patterning of the central nervous system of agnathan embryos is relevant to our understanding of the origin of the developmental plan of the vertebrate hindbrain. To investigate the neuronal organization of the hindbrain of the Japanese lamprey, Lethenteron japonicum, we retrogradely labeled the reticulospinal and branchial motoneurons. By combining this analysis with a study of the expression patterns of genes identifying specific rhombomeric territories such as LjKrox20, LjPax6, LjEphC and LjHox3, we found that the reticular neurons in the lamprey hindbrain, including isthmic, bulbar and Mauthner cells, develop in conserved rhombomere-specific positions, similar to those in the zebrafish. By contrast, lamprey trigeminal and facial motor nuclei are not in register with rhombomere boundaries, unlike those of gnathostomes. The trigeminal-facial boundary corresponds to the rostral border of LjHox3 expression in the middle of rhombomere 4. Exogenous application of retinoic acid (RA) induced a rostral shift of both the LjHox3 expression domain and branchiomotor nuclei with no obvious repatterning of rhombomeric segmentation and reticular neurons. Therefore, whereas subtype variations of motoneuron identity along the anteroposterior axis may rely on Hox-dependent positional values, as in gnathostomes, such variations in the lamprey are not constrained by hindbrain segmentation. We hypothesize that the registering of hindbrain segmentation and neuronal patterning may have been acquired through successive and independent stepwise patterning changes during evolution.     

Fgfr2 and Fgfr3 are not required for patterning and maintenance of the midbrain and anterior hindbrain

The mid-/hindbrain organizer (MHO) is characterized by the expression of a network of genes, which controls the patterning and development of the prospective midbrain and anterior hindbrain. One key molecule acting at the MHO is the fibroblast growth factor (Fgf) 8. Ectopic expression of Fgf8 induces genes that are normally expressed at the mid-/hindbrain boundary followed by the induction of midbrain and anterior hindbrain structures. Inactivation of the Fgf receptor (Fgfr) 1 gene, which was thought to be the primary transducer of the Fgf8 signal at the MHO, in the mid-/hindbrain region, leads to a deletion of dorsal structures of the mid-/hindbrain region, whereas ventral tissues are less severely affected. This suggests that other Fgfrs might be responsible for ventral mid-/hindbrain region development. Here we report the analysis of Fgfr2 conditional knockout mice, lacking the Fgfr2 in the mid-/hindbrain region and of Fgfr3 knockout mice with respect to the mid-/hindbrain region. In both homozygous mouse mutants, patterning of the mid-/hindbrain region is not altered, neuronal populations develop normal and are maintained into adulthood. This analysis shows that the Fgfr2 and the Fgfr3 on their own are dispensable for the development of the mid-/hindbrain region. We suggest functional redundancy of Fgf receptors in the mid-/hindbrain region.     

The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio)

The development of the vertebrate head is a highly complex process involving tissues derived from all three germ layers. The endoderm forms pharyngeal pouches, the paraxial mesoderm gives rise to endothelia and muscles, and the neural crest cells, which originate from the embryonic midbrain and hindbrain, migrate ventrally to form cartilage, connective tissue, sensory neurons, and pigment cells. All three tissues form segmental structures: the hindbrain compartmentalizes into rhombomeres, the mesoderm into somitomeres, and the endoderm into serial gill slits. It is not known whether the different segmented tissues in the head develop by the same molecular mechanism or whether different pathways are employed. It is also possible that one tissue imposes segmentation on the others. Most recent studies have emphasized the importance of neural crest cells in patterning the head. Neural crest cells colonize the segmentally arranged arches according to their original position in the brain and convey positional information from the hindbrain into the periphery. During the screen for mutations that affect embryonic development of zebrafish, one mutant, called van gogh (vgo), in which segmentation of the pharyngeal region is absent, was isolated. In vgo, even though hindbrain segmentation is unaffected, the pharyngeal endoderm does not form reiterated pouches and surrounding mesoderm is not patterned correctly. Accordingly, migrating neural crest cells initially form distinct streams but fuse when they reach the arches. This failure to populate distinct pharyngeal arches is likely due to the lack of pharyngeal pouches. The results of our analysis suggest that the segmentation of the endoderm occurs without signaling from neural crest cells but that tissue interactions between the mesendoderm and the neural crest cells are required for the segmental appearance of the neural crest-derived cartilages in the pharyngeal arches. The lack of distinct patches of neural crest cells in the pharyngeal region is also seen in mutants of one-eyed pinhead and casanova, which are characterized by a lack of endoderm, as well as defects in mesodermal structures, providing evidence for the important role of the endoderm and mesoderm in governing head segmentation.     

EphA4 and EfnB2a maintain rhombomere coherence by independently regulating intercalation of progenitor cells in the zebrafish neural keel

During vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell-cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation.     

Vitamin A deficiency results in the dose-dependent acquisition of anterior character and shortening of the caudal hindbrain of the rat embryo

The developing nervous system is particularly vulnerable to vitamin A deficiency. Retinoid has been proposed to be a posteriorizing factor during hindbrain development, although direct evidence in the mammalian embryo is lacking. In the present study, pregnant vitamin A-deficient (VAD) rats were fed purified diets containing varying levels of all-trans-retinoic acid (atRA; 0, 0.5, 1.5, 6, 12, 25, 50, 125, or 250 microg/g diet) or were supplemented with retinol. Hindbrain development was studied from embryonic day 10 to 12.5 ( approximately 6 to 40 somites). Normal morphogenesis was observed in all embryos from groups fed 250 microg atRA/g diet or retinol. The most caudal region of the hindbrain was the most sensitive to retinoid insufficiency, as evidenced by a loss of the hypoglossal nerve (cranial nerve XII) in embryos from the 125 microg atRA/g diet group. Further reduction of atRA to 50 microg/g diet led to the loss of cranial nerves IX, X, XI, and XII and associated sensory ganglia IX and X in all embryos as well as the loss of hindbrain segmentation caudal to the rhombomere (r) 3/4 border in a subset of embryos. Dysmorphic orthotopic otic vesicles or immature otic-like vesicles in both orthotopic and caudally ectopic locations were also observed. As the level of atRA was reduced, a loss of caudal hindbrain segmentation was observed in all embryos and the incidence of otic vesicle abnormalities increased. Perturbations in hindbrain segmentation, cranial nerve formation, and otic vesicle development were associated with abnormal patterning of the posterior hindbrain. Embryos from VAD dams fed between 0.5 and 50 microg atRA/g diet exhibited Hoxb-1 protein expression along the entire neural tube caudal to the r3/r4 border at a time when it should be restricted to r4. Krox-20 protein expression was expanded in r3 but absent or reduced in presumptive r5. Hoxd-4 mRNA expression was absent in the posterior hindbrain, and the rostral limit of Hoxb-5 protein expression in the neural tube was anteriorized, suggesting that the most posterior hindbrain region (r7/r8) had been deleted and/or improperly patterned. Thus, when limiting amounts of atRA are provided to VAD dams, the caudal portion of the hindbrain is shortened and possesses r4/r5-like characteristics, with this region finally exhibiting r4-like gene expression when retinoid is restricted even more severely. Thus, regions of the anterior hindbrain (i.e., r3 and r4) appear to be greatly expanded, whereas the posterior hindbrain (r5-r8) is reduced or absent. This work shows that retinoid plays a critical role in patterning, segmentation, and neurogenesis of the caudal hindbrain and serves as an essential posteriorizing signal for this region of the central nervous system in the mammal.     

Scanning electron microscopy of the gut microflora of two earthworms: Lumbricus terrestris and Octolasion cyaneum

Scanning electron microscopy was used to investigate the presence of microorganisms, probably bacteria, on the gut surface of earthworms. The washed surfaces of the intestines of two earthworms, Lumbricus terrestris and Octolasion cyaneum, were examined. Numerous organisms resembling bacteria were observed throughout the gut, some in situations suggesting attachment. Compared with similar investigations in other invertebrates, there were fewer bacteria, showing less morphological diversity, on the earthworm gut surface. The majority of organisms viewed were coccoid, some were filamentous, and a few rod-shaped cells were observed. Cocci, often in chains, were seen in the foregut of both species. Although cocci were also numerous in the midgut region, particularly in the typhlosole, in O. cyaneum tufts of segmented, filamentous organisms were also seen with some segments resembling spores. Fewer organisms were found in the hindgut, but in L. terrestris there were segmented, filamentous organisms, attached to the epithelium by way of a socket-like structure, similar to that by which segmented, filamentous bacteria (SFBs) are attached to the ileum of rats and mice. Transmission electron microscopy of the hindgut of L. terrestris was undertaken to explore the structure and attachment of SFBs to the gut epithelium. However, although a few rod-shaped bacteria were observed, no SFBs were located. The observations reported here provide evidence that earthworms have an attached gut microflora of filamentous microorganisms which are probably indigenous, and as far as we are aware this is the first published report of such findings in these invertebrates. Offprint requests to: J.M. Jolly.     

Hox Genes and Segmental Patterning of the Vertebrate Hindbrain

SYNOPSIS. Pattern formation in the developing hindbrain andcranio-facial region has been studied in a range of vertebrateorganisms. The developing hindbrain is transiently segmentedinto units termed rhombomeres which correspond with domainsof gene expression, lineage restriction and neuronal organizationand serve to coordinate the migration of cranial neural crestinto the adjacent branchial arches. In this paper I review thecellular and molecular events underlying both hindbrain segmentationand the acquisition of segmental identity, consolidating recentresults from different model systems. Data suggesting that thevertebrate Hox genes play an important role in specifying positionalvalue to the rhombomeres and cranial neural crest are also examined.I compare expression patterns of the Hox genes between speciesand consider the mechanisms involved in controlling their appropriatespatial regulation. In addition I describe a recently characterizedzebraflsh hindbrain segmentation mutant, Valentino; morphological,cellular and gene expression data for this mutant are helpingto further our understanding of hindbrain patterning.