Hindbrain Segmentation Research Articles

Hox gene misexpression and cell-specific lesions reveal functionality of homeotically transformed neurons.

Hox genes are critical for establishing the segmental pattern of the vertebrate hindbrain. Changes in their expression can alter neural organization of hindbrain segments and may be a mechanism for brain evolution. To test the hypothesis that neurons induced through changes in Hox gene expression can integrate into functional neural circuits, we examined the roles of ectopic Mauthner cells (M-cells) in the escape response of larval zebrafish. The activity of the paired Mauthner cells in rhombomere 4 (r4) has been shown to be critical for generating a high-performance startle behavior in response to stimulation of the tail (Liu and Fetcho, 1999). Previous studies have found that misexpression of particular Hox genes causes ectopic M-cells to be generated in r2 in addition to the r4 cells (Alexandre et al., 1996; McClintock et al., 2001). With calcium imaging, we found that the homeotically transformed neurons respond to startle stimuli. To determine the roles of ectopic and endogenous M-cells in the behavior, we lesioned the r2, r4, or both M-cells with cell-specific laser lesion and examined the effect on startle performance. Lesion of the normal M-cells did not decrease escape performance when the ectopic cells were present. These results indicate that the homeotically transformed Mauthner cells are fully functional in the escape circuit and are functionally redundant with normal M-cells. We suggest that such functional redundancy between neurons may provide a substrate for evolution of neural circuits.

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.

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation.

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers (foxb1.2/mariposa, krox20, pax2.1 and pax6.1), including anteriorly expressed Hox genes (hoxb1a, hoxa2 and hoxb2), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1, but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development.

Segmental identity and cerebellar granule cell induction in rhombomere 1

BackgroundCerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment?ResultsWe show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2.ConclusionsDifferent overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a subset of coextensive nuclei is refractory to ectopic Hoxa2 and is presumably induced solely by isthmic organiser activity. Thus, graded (isthmic) and segmental mechanisms may operate exclusively of one another in the specification of different neuronal populations within rhombomere 1. The early designation of an Otx2-negative, Hoxa2-negative region, prior to the appearance of the isthmic organiser, is a key initial step in the specification of the cerebellum.

Relationship between hindbrain segmentation, neural crest cell migration and branchial arch abnormalities in rat embryos exposed to fluconazole and retinoic acid in vitro

Fluconazole (FLUCO) and retinoic acid (RA) can perturb morphogenesis of the branchial apparatus in rodent embryos exposed in vitro. The aim of the present study was to compare the effects induced by in vitro exposure to FLUCO or to RA on rhombomere organisation, neural crest cell (NCC) migration and cranial nerve differentiation using specific antibodies. For this purpose 9.5 d.p.c. rat embryos were exposed to teratogenic concentrations of FLUCO or RA; another group was exposed to no-effect concentrations of both agents. Expression of Hox-b1 and Krox20 (markers of specific rhombomeres) was altered after FLUCO and RA exposure. Furthermore, FLUCO and RA showed a synergistic effect. These results suggest that the observed branchial abnormalities are due to anomalous NCC migration related to incorrect organisation of specific rhombomeres.

Vhnf1 and Fgf signals synergize to specify rhombomere identity in the zebrafish hindbrain.

Vertebrate hindbrain segmentation is a highly conserved process but the mechanism of rhombomere determination is not well understood. Recent work in the zebrafish has shown a requirement for fibroblast growth factor (Fgf) signaling and for the transcription factor variant hepatocyte nuclear factor 1 (vhnf1) in specification of rhombomeres 5 and 6 (r5+r6). We show here that vhnf1 functions in two ways to subdivide the zebrafish caudal hindbrain domain (r4-r7) into individual rhombomeres. First, vhnf1 promotes r5+r6 identity through an obligate synergy with Fgf signals to activate valentino and krox20 expression. Second, vhnf1 functions independently of Fgf signals to repress hoxb1a expression. Although vhnf1 is expressed in a broad posterior domain during gastrulation, it promotes the specification of individual rhombomeres. This is achieved in part because vhnf1 gives cellular competence to respond to Fgf signals in a caudal hindbrain-specific manner.

Regulation of Hoxb2 by APL-associated PLZF protein.

The PLZF gene is translocated in a subset of all-trans-retinoic acid resistant acute promyelocytic leukaemia (APL) cases, encodes a DNA binding transcription factor and is expressed highly in haematopoietic progenitor cells as well-developing central nervous system (CNS). The spatially restricted and temporally dynamic pattern of PLZF expression in the developing CNS suggested that it might play a role in the circuitry regulating hindbrain segmentation. We have now identified a PLZF binding site (PLZF-RE) in an enhancer region of Hoxb2 that itself is required for directing high-level expression in rhombomers 3 and 5 of the developing hindbrain. The wild-type r3/r5 enhancer linked to a heterologous promoter was responsive to regulation by PLZF, and this activity was lost in variants containing a mutated PLZF-RE. Compared with the wild-type protein, the binding of the APL-associated reciprocal RARalpha-PLZF fusion to PLZF-RE was much stronger, suggesting that the N-terminal PLZF sequences missing from the fusion may play a role in the regulation of DNA binding. Consistent with this, the N-terminal POZ domain was required for cooperative binding of PLZF to a multimerized PLZF-RE. In the context of the r3/r5 enhancer, the PLZF-RE cooperated for PLZF binding with an additional A/T-rich motif positioned downstream of the PLZF-RE. This A/T motif was previously shown to be essential for the regulation of Hoxb2 expression in r3 and r5 in cooperation with another Krüppel-like zinc finger protein Krox 20. The presence of both the PLZF-RE and the A/T-rich motif was required for a maximal effect of PLZF on a heterologous promoter and was essential in vivo to direct the expression of a lacZ reporter in the chick neural tube. Hence, both PLZF and Krox20 cooperate with a common A/T motif in mediating in vivo activity of the Hoxb2 enhancer. Our findings indicate that Hoxb2 is a direct target for regulation by PLZF in the developing CNS and suggest that deregulation of Hox gene expression may contribute to APL pathogenesis.

C- jun regulation and function in the developing hindbrain

Hindbrain development is a well-characterised segmentation process in vertebrates. The bZip transcription factor MafB/kreisler is specifically expressed in rhombomeres (r) 5 and 6 of the developing vertebrate hindbrain and is required for proper caudal hindbrain segmentation. Here, we provide evidence that the mouse protooncogene c- jun, which encodes a member of the bZip family, is coexpressed with MafB in prospective r5 and r6. Analysis of mouse mutants suggests that c- jun expression in these territories is dependent on MafB but independent of the zinc-finger transcription factor Krox20, another essential determinant of r5 development. Loss- and gain-of-function studies, performed in mouse and chick embryos, respectively, demonstrate that c-Jun participates, together with MafB and Krox20, in the transcriptional activation of the Hoxb3 gene in r5. The action of c-Jun is likely to be direct, since c-Jun homodimers and c-Jun/MafB heterodimers can bind to essential regulatory elements within the transcriptional enhancer responsible for Hoxb3 expression in r5. These data indicate that c-Jun acts both as a downstream effector and a cofactor of MafB and belongs to the complex network of factors governing hindbrain patterning.

Analysis of hindbrain patterning defects caused by the kreisler(enu) mutation reveals multiple roles of Kreisler in hindbrain segmentation.

The embryonic hindbrain is subdivided into eight subunits, termed rhombomeres (r1-r8). The Kreisler (Krml1/MafB/val) transcription factor is expressed in and essential for patterning rhombomeres 5 and 6. Here, we have shown that in the chemically induced kreisler(enu) (kr(enu)) allele, a point mutation in the DNA binding domain abolishes or severely reduces Kreisler-dependent transcription. Comparison of kr(enu)/kr(enu) embryos with those homozygous for the classic kreisler (kr) mutation has reconciled past discrepancies and revealed multiple roles of Kreisler in hindbrain segmentation. These analyses demonstrate that Kreisler is required for maintenance and expansion but not initiation of the Krox20 expressing r5 domain. The differences in the "r5-like" phenotype of kr(enu)/kr(enu) and kr/kr mouse embryos, and zebrafish carrying mutations in the Kreisler orthologue valentino (val) suggest that Kreisler performs many of its r5-specific functions by associating with other proteins. By contrast, kr/kr and kr(enu)/kr(enu) mouse and val-/- zebrafish embryos all exhibit indistinguishable defects in r6 specification. Thus, transcriptionally active Kreisler is required for r6 specification. Unlike mouse kr(enu)/kr(enu) and zebrafish val-/- embryos, kr/kr embryos exhibited anterior defects. We determined that the kr chromosomal inversion caused ectopic Kreisler expression in r3 of kr/kr and kr/+ embryos. Hence, Kreisler regulates maintenance and expansion of r5 and specification of r6 but is not required for r3 development.

Neural crest patterning: autoregulatory and crest-specific elements co-operate for Krox20 transcriptional control.

Neural crest patterning constitutes an important element in the control of the morphogenesis of craniofacial structures. Krox20, a transcription factor gene that plays a critical role in the development of the segmented hindbrain, is expressed in rhombomeres (r) 3 and 5 and in a stream of neural crest cells migrating from r5 toward the third branchial arch. We have investigated the basis of the specific neural crest expression of Krox20 and identified a cis-acting enhancer element (NCE) located 26 kb upstream of the gene that is conserved between mouse, man and chick and can recapitulate the Krox20 neural crest pattern in transgenic mice. Functional dissection of the enhancer revealed the presence of two conserved Krox20 binding sites mediating direct Krox20 autoregulation in the neural crest. In addition, the enhancer included another essential element containing conserved binding sites for high mobility group (HMG) box proteins and which responded to factors expressed throughout the neural crest. Consistent with this the NCE was strongly activated in vitro by Sox10, a crest-specific HMG box protein, in synergism with Krox20, and the inactivation of Sox10 prevented the maintenance of Krox20 expression in the migrating neural crest. These results suggest that the dependency of the enhancer on both crest- (Sox10) and r5- (Krox20) specific factors limits its activity to the r5-derived neural crest. This organisation also suggests a mechanism for the transfer and maintenance of rhombomere-specific gene expression from the hindbrain neuroepithelium to the emerging neural crest and may be of more general significance for neural crest patterning.

From hindbrain segmentation to breathing after birth: developmental patterning in rhombomeres 3 and 4.

Respiration is a rhythmic motor behavior that appears in the fetus and acquires a vital importance at birth. It is generated within central pattern-generating neuronal networks of the hindbrain. This region of the brain is of particular interest since it is the most understood part with respect to the cellular and molecular mechanisms that underlie its development. Hox paralogs and Hox-regulating genes kreisler/mafB and Krox20 are required for the normal formation of rhombomeres in vertebrate embryos. From studies of rhombomeres r3 and r4, the authors review mechanisms whereby these developmental genes may govern the early embryonic development of para-facial neuronal networks and specify patterns of motor activities operating throughout life. A model whereby the regional identity of progenitor cells can be abnormally specified in r3 and r4 after a mutation of these genes is proposed. Novel neuronal circuits may develop from some of these misspecified progenitors while others are eliminated, eventually affecting respiration and survival after birth.

Hindbrain and Cranial Nerve Dysmorphogenesis Result from Acute Maternal Ethanol Administration

Acute exposure of mouse embryos to ethanol during stages of hindbrain segmentation results in excessive cell death in specific cell populations. This study details the ethanol-induced cell loss and defines the subsequent effects of this early insult on rhombomere and cranial nerve development. Ethanol at a teratogenic dosage (2.9 g/kg) or a comparable volume of vehicle was administered in each of two intraperitoneal injections to pregnant C57BL/6J mice on gestational day (GD) 8, 8 h, and GD 8, 12 h (defined hereafter as GD 8.5). Ethanol-exposed GD 9 embryos, visualized in three dimensions using laser scanning confocal microscopy of LysoTracker Red fluorescence or Nile blue sulphate vital staining, displayed excessive apoptosis in the rostral hindbrain, specifically within rhombomeres 1–3, as well as in cranial neural crest cells and ectodermal placodes. Comparably treated embryos examined on GD 10.5–11 illustrated a disproportionate reduction in the length of the rostral hindbrain. Examination of plastic histological sections of GD 9 embryos and via scanning electron microscopy on GD 10 revealed deficiencies in the hindbrain, with a phenotype including abnormal rhombomere segmentation and an extremely small fourth ventricular roofplate. Whole-mount antineurofilament immunohistochemistry on GD 10.5 and GD 11 illustrated a variety of cranial nerve abnormalities ranging from fused or absent ganglia to ectopic or disorganized fibers. In addition, a delay in the development of the glossopharyngeal (IX) nerve/ganglia complex was observed. These hindbrain and cranial nerve abnormalities are discussed in the context of the genesis of human alcohol-related birth defects and neurodevelopmental disorder.

Novel Activities of Mafb Underlie Its Dual Role in Hindbrain Segmentation and Regional Specification

The bZip transcription factor Mafb is expressed in two segments of the developing vertebrate hindbrain: the rhombomeres 5 and 6. Loss of Mafb expression in the mouse mutant kreisler leads to elimination of r5 and to alterations of r6 regional identity. Here, we further investigated the role of Mafb in hindbrain patterning using gain-of-function experiments in the chick embryo. Our work has revealed novel functions for Mafb, including a positive autoregulatory activity, the capacity to repress Hoxb1 expression, and the capacity to synergise with or antagonise Krox20 activity. These different activities appear to be spatially restricted in the hindbrain, presumably due to interactions with other factors. Reinvestigation of the kreisler mutation indicated that it also results in an ectopic activation of Mafb in rhombomere 3, accounting for the previously described molecular alterations of this rhombomere in the mutant. Together, these data allow us to refine our view of the dual function of Mafb in both segmentation and specification of anteroposterior identity in the hindbrain.

Eliminating Zebrafish Pbx Proteins Reveals a Hindbrain Ground State

The vertebrate hindbrain is divided into serially homologous segments, the rhombomeres (r). Pbx and Hox proteins are hypothesized to form heterodimeric, DNA binding transcription complexes which specify rhombomere identities. Here, we show that eliminating zebrafish Lzr/Pbx4 and Pbx2 function prevents hindbrain segmentation and causes a wholesale anterior homeotic transformation of r2–r6, to r1 identity. We demonstrate that Pbx proteins interact with Hox paralog group 1 proteins to specify segment identities broadly within the hindbrain, and that this process involves the Pbx:Hox-1-dependent induction of Fgf signals in r4. We propose that in the absence of Pbx function, r2–r6 acquire a homogeneous ground state identity, that of r1, and that Pbx proteins, functioning primarily with their Hox partners, function to modify this ground state identity during normal hindbrain development.

Coincident iterated gene expression in the amphioxus neural tube.

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.

Constructing the hindbrain: insights from the zebrafish.

The hindbrain is responsible for controlling essential functions such as respiration and heart beat that we literally do not think about most of the time. In addition, cranial nerves projecting from the hindbrain control muscles in the jaw, eye, and face, and receive sensory input from these same areas. In all vertebrates that have been studied, the hindbrain passes through a segmented phase shortly after the neural tube has formed, with a series of seven bulges--the rhombomeres--forming along the anterior-posterior extent of the neural tube. Our current understanding of vertebrate hindbrain development comes from integrating data from several model systems. Work on the chick has helped us to understand the cell biology of the rhombomeres, whereas the power of mouse molecular genetics has allowed investigation of the molecular mechanisms underlying their development. This review focuses on the special insights that the zebrafish system has provided to our understanding of hindbrain development. As we will discuss, work in the zebrafish has elucidated inductive events that specify the presumptive hindbrain domain and has identified genes required for hindbrain segmentation and the specification of segment identities.

A novel zinc finger transcription factor resembles krox-20 in structure and in expression pattern in zebrafish

Krx-20, a zinc finger protein, plays an essential role in hindbrain segmentation of vertebrates. Zebrafish gene frb35 encodes a novel zinc finger protein that shares a high homology with Krx-20. The expression domains of frb35 overlap those of krx-20. The expression of frb35 first occurs in the 3rd rhombomere primordium at the onset of segmentation, which is soon followed by expression in the 5th rhombomere. From mid-segmentation onward, the 5th rhombomere has a higher frb35 expression level than the 3rd rhombomere. Unlike krx20, frb35 expression persists in the 5th rhombomere until the end of pharyngula period.

EphA4 is not required for Purkinje cell compartmentation

The Purkinje cells of both the adult and the developing cerebellar cortex are organized into parasagittal stripes or ‘segments’ expressing a variety of biochemical markers. We show that in the developing mouse cerebellar cortex, members of the Eph receptor gene family are expressed in mediolaterally alternating Purkinje cell segments. Since members of the Eph receptors family have been shown to play a role in hindbrain segmentation and boundary formation (Philos. Trans. R. Soc. Lond. B: Biol. Sci. 355 (2000) 993), we analyzed the effect of a null mutation of the EphA4 gene on Purkinje cell compartmentation. Using well characterized markers of Purkinje cell compartmentation in both the developing and the adult cerebellum, we observed no significant alteration in the banding pattern of these markers between the EphA4 knockout mice and their wild type controls. The ribboned pattern of migrating granule cells in the developing cerebellum also appears unaltered. The expression of other members of this gene family, including ephrin-B2, EphA2, and ephrin-A1, in a compartmentalized pattern within the Purkinje cell layer suggests a possible redundancy and/or a compensation of EphA4 function in the segmental patterning of cerebellar Purkinje cells.

Conditional, floxed allele of the Krox20 gene.

Krox20, initially identified as an immediate early serum response gene, encodes a transcription factor with a C2H2-type zinc finger DNA-binding domain (Chavrier et al., 1988, 1990). During mouse development, Krox20 is expressed in two rhombomeres (r3 and r5) of the segmented hindbrain and the neural crest derived from r5 (Schneider-Maunoury et al., 1993; Wilkinson et al., 1989), in immature and myelinating Schwann cells (Topilko et al., 1994), in hypertrophic chondrocytes and differentiating osteoblasts (Levi et al., 1996), and in hair follicles (Gambardella et al., 2000). In the adult, Krox20 expression has been shown to be maintained in myelinating Schwann cells and in the bones and has been observed in various other tissues, including specific neuronal populations of the cortex (Herdegen et al., 1993; Williams et al., 1995). Two Krox20 null mutant alleles, Krox20 and Krox20, have been previously generated by insertion of the E. coli lacZ gene and of the Cre recombinase gene, respectively, into the Krox20 locus (Schneider-Maunoury et al., 1993; Voiculescu et al., 2000). In homozygous mutants, hindbrain segments, r3 and r5, are eliminated (Schneider-Maunoury et al., 1993, 1997; Voiculescu et al., 2001), myelination is impaired in the peripheral nervous system (Topilko et al., 1994), and endochondral bone formation is affected (Levi et al., 1996). However, postnatal lethality prevented the analysis of late functions of Krox20. To investigate the role of Krox20 in the adult, we have generated a floxed allele (Sauer, 1998) that will be used to create conditional mutations. The second exon of the Krox20 gene encodes the largest part of the protein, including the DNA-binding domain. This exon was floxed by inserting a loxP site within the preceding intron and a floxed neoR cassette 3 of the gene (Fig. 1A). The targeting vector was electroporated into embryonic stem (ES) cells (CK35 line derived from the 129/SV Pasteur strain; Camus et al., 1996) and two out of 350 neo resistant ES cell clones were shown to contain a targeted allele (Krox20) by Southern blotting analysis (Fig. 1B). After injection of the recombinant ES cells into C57Bl6 blastocysts, mouse germ line transmission was obtained for one of them. The Krox20 line was established in a mixed C57Bl6/DBA2 background. As the neoR cassette could potentially interfere with Krox20 expression, Krox20 compound heterozygotes were generated. Indeed, analysis of lacZ expression in the hindbrain of 8.5 dpc embryos revealed that r3 is smaller in Krox20 embryos than in Krox20 embryos, whereas r5 is not affected (Fig.

Zebrafish Meis functions to stabilize Pbx proteins and regulate hindbrain patterning.

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr(-) phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.