Hindbrain patterning: FGFs regulate Krox20 and mafB/kr expression in the otic/preotic region

Krox20 and mafB/kr are regulatory genes involved in hindbrain segmentation and anteroposterior (AP) patterning. They are expressed in rhombomeres (r) r3/r5 and r5/r6 respectively, as well as in the r5/r6 neural crest. Since several members of the fibroblast growth factor (FGF) family are expressed in the otic/preotic region (r2-r6), we investigated their possible involvement in the regulation of Krox20 and mafB/kr. Application of exogenous FGFs to the neural tube of 4- to 7-somite chick embryos led to ectopic expression in the neural crest of the somitic hindbrain (r7 and r8) and to the extension of the Krox20- or mafB/kr-positive areas in the neuroepithelium. Application of an inhibitor of FGF signalling led to severe and specific downregulation of Krox20 and mafB/kr in the hindbrain neuroepithelium and neural crest. These data indicate that FGFs are involved in the control of regional induction and/or maintenance of Krox20 and mafB/kr expression, thus identifying a novel function for these factors in hindbrain development, besides their proposed more general role in early neural caudalisation.     

Retinoic acid synthesis and hindbrain patterning in the mouse embryo

Targeted disruption of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene precludes embryonic retinoic acid (RA) synthesis, leading to midgestational lethality (Niederreither, K., Subbarayan, V., Dolle, P. and Chambon, P. (1999). Nature Genet. 21, 444-448). We describe here the effects of this RA deficiency on the development of the hindbrain and associated neural crest. Morphological segmentation is impaired throughout the hindbrain of Raldh2-/- embryos, but its caudal portion becomes preferentially reduced in size during development. Specification of the midbrain region and of the rostralmost rhombomeres is apparently normal in the absence of RA synthesis. In contrast, marked alterations are seen throughout the caudal hindbrain of mutant embryos. Instead of being expressed in two alternate rhombomeres (r3 and r5), Krox20 is expressed in a single broad domain, correlating with an abnormal expansion of the r2-r3 marker Meis2. Instead of forming a defined r4, Hoxb1- and Wnt8A-expressing cells are scattered throughout the caudal hindbrain, whereas r5/r8 markers such as kreisler or group 3/4 Hox genes are undetectable or markedly downregulated. Lack of alternate Eph receptor gene expression could explain the failure to establish rhombomere boundaries. Increased apoptosis and altered migratory pathways of the posterior rhombencephalic neural crest cells are associated with impaired branchial arch morphogenesis in mutant embryos. We conclude that RA produced by the embryo is required to generate posterior cell fates in the developing mouse hindbrain, its absence leading to an abnormal r3 (and, to a lesser extent, r4) identity of the caudal hindbrain cells.     

Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse

Early in its development, the vertebrate hindbrain is transiently subdivided into a series of compartments called rhombomeres. Genes have been identified whose expression patterns distinguish these cellular compartments. Two of these genes, Hoxa1 and Hoxa2, have been shown to be required for proper patterning of the early mouse hindbrain and the associated neural crest. To determine the extent to which these two genes function together to pattern the hindbrain, we generated mice simultaneously mutant at both loci. The hindbrain patterning defects were analyzed in embryos individually mutant for Hoxa1 and Hoxa2 in greater detail and extended to embryos mutant for both genes. From these data a model is proposed to describe how Hoxa1, Hoxa2, Hoxb1, Krox20 (Egr2) and kreisler function together to pattern the early mouse hindbrain. Critical to the model is the demonstration that Hoxa1 activity is required to set the anterior limit of Hoxb1 expression at the presumptive r3/4 rhombomere boundary. Failure to express Hoxb1 to this boundary in Hoxa1 mutant embryos initiates a cascade of gene misexpressions that result in misspecification of the hindbrain compartments from r2 through r5. Subsequent to misspecification of the hindbrain compartments, ectopic induction of apoptosis appears to be used to regulate the aberrant size of the misspecified rhombomeres.     

spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.     

Hindbrain patterning: Krox20 couples segmentation and specification of regional identity.

We have previously demonstrated that inactivation of the Krox20 gene led to the disappearance of its segmental expression territories in the hindbrain, the rhombomeres (r) 3 and 5. We now performed a detailed analysis of the fate of prospective r3 and r5 cells in Krox20 mutant embryos. Genetic fate mapping indicates that at least some of these cells persist in the absence of a functional Krox20 protein and uncovers the requirement for autoregulatory mechanisms in the expansion and maintenance of Krox20-expressing territories. Analysis of even-numbered rhombomere molecular markers demonstrates that in Krox20-null embryos, r3 cells acquire r2 or r4 identity, and r5 cells acquire r6 identity. Finally, study of embryonic chimaeras between Krox20 homozygous mutant and wild-type cells shows that the mingling properties of r3/r5 mutant cells are changed towards those of even-numbered rhombomere cells. Together, these data demonstrate that Krox20 is essential to the generation of alternating odd- and even-numbered territories in the hindbrain and that it acts by coupling the processes of segment formation, cell segregation and specification of regional identity.     

Conservation and diversity in the cis-regulatory networks that integrate information controlling expression of Hoxa2 in hindbrain and cranial neural crest cells in vertebrates

The Hoxa2 and Hoxb2 genes are members of paralogy group II and display segmental patterns of expression in the developing vertebrate hindbrain and cranial neural crest cells. Functional analyses have demonstrated that these genes play critical roles in regulating morphogenetic pathways that direct the regional identity and anteroposterior character of hindbrain rhombomeres and neural crest-derived structures. Transgenic regulatory studies have also begun to characterize enhancers and cis-elements for those mouse and chicken genes that direct restricted patterns of expression in the hindbrain and neural crest. In light of the conserved role of Hoxa2 in neural crest patterning in vertebrates and the similarities between paralogs, it is important to understand the extent to which common regulatory networks and elements have been preserved between species and between paralogs. To investigate this problem, we have cloned and sequenced the intergenic region between Hoxa2 and Hoxa3 in the chick HoxA complex and used it for making comparative analyses with the respective human, mouse, and horn shark regions. We have also used transgenic assays in mouse and chick embryos to test the functional activity of Hoxa2 enhancers in heterologous species. Our analysis reveals that three of the critical individual components of the Hoxa2 enhancer region from mouse necessary for hindbrain expression (Krox20, BoxA, and TCT motifs) have been partially conserved. However, their number and organization are highly varied for the same gene in different species and between paralogs within a species. Other essential mouse elements appear to have diverged or are absent in chick and shark. We find the mouse r3/r5 enhancer fails to work in chick embryos and the chick enhancer works poorly in mice. This implies that new motifs have been recruited or utilized to mediate restricted activity of the enhancer in other species. With respect to neural crest regulation, cis-components are embedded among the hindbrain control elements and are highly diverged between species. Hence, there has been no widespread conservation of sequence identity over the entire enhancer domain from shark to humans, despite the common function of these genes in head patterning. This provides insight into how apparently equivalent regulatory regions from the same gene in different species have evolved different components to potentiate their activity in combination with a selection of core components.     

Apoptosis of premigratory neural crest cells in rhombomeres 3 and 5: consequences for patterning of the branchial region

In the avian hindbrain, premigratory neural crest cells undergo programmed cell death (apoptosis) in rhombomeres 3 and 5 (r3, r5). Here, we have attempted to analyze the significance of the loss of neural crest cells from these odd-numbered rhombomeres. When apoptosis is prevented in r3 and r5, r3 crest migrate into the first arch and r5 into the third arch. Interestingly, these extra neural crest cells contributed to the formation of ectopic muscle attachment sites that are also found in those species in which r3 and r5 neural crest cells do not undergo apoptosis. Thus, apoptosis in the odd-numbered rhombomeres appears to be an evolutionarily derived mechanism that is required to eliminate r3 and r5 crest migration into first and third arches and thereby remove these muscle attachment sites.     

The WNT antagonist cSFRP2 modulates programmed cell death in the developing hindbrain

In the avian hindbrain, the loss of premigratory neural crest cells from rhombomeres 3 and 5 (r3, r5) through programmed cell death contributes to the patterning of emigrant crest cells into three discrete streams. Programmed cell death is induced by the upregulation of Bmp4 and Msx2 in r3 and r5. We show that cSFRP2, a WNT antagonist, is expressed in the even-numbered rhombomeres and that over-expression of cSfrp2 inhibits Bmp4 expression in r3 and r5, preventing programmed cell death. By contrast, depleting cSFRP2 function in r4 results in elevated levels of Msx2 expression and ectopic programmed cell death, as does overexpression of Wnt1. We propose that programmed cell death in the rhombencephalic neural crest is modulated by pre-patterned cSfrp2 expression and a WNT-BMP signalling loop.     

Differences in Krox20-dependent regulation of Hoxa2 and Hoxb2 during hindbrain development

During hindbrain development, segmental regulation of the paralogous Hoxa2 and Hoxb2 genes in rhombomeres (r) 3 and 5 involves Krox20-dependent enhancers that have been conserved during the duplication of the vertebrate Hox clusters from a common ancestor. Examining these evolutionarily related control regions could provide important insight into the degree to which the basic Krox20-dependent mechanisms, cis-regulatory components, and their organization have been conserved. Toward this goal we have performed a detailed functional analysis of a mouse Hoxa2 enhancer capable of directing reporter expression in r3 and r5. The combined activities of five separate cis-regions, in addition to the conserved Krox20 binding sites, are involved in mediating enhancer function. A CTTT (BoxA) motif adjacent to the Krox20 binding sites is important for r3/r5 activity. The BoxA motif is similar to one (Box1) found in the Hoxb2 enhancer and indicates that the close proximity of these Box motifs to Krox20 sites is a common feature of Krox20 targets in vivo. Two other rhombomeric elements (RE1 and RE3) are essential for r3/r5 activity and share common TCT motifs, indicating that they interact with a similar cofactor(s). TCT motifs are also found in the Hoxb2 enhancer, suggesting that they may be another common feature of Krox20-dependent control regions. The two remaining Hoxa2 cis-elements, RE2 and RE4, are not conserved in the Hoxb2 enhancer and define differences in some of components that can contribute to the Krox20-dependent activities of these enhancers. Furthermore, analysis of regulatory activities of these enhancers in a Krox20 mutant background has uncovered differences in their degree of dependence upon Krox20 for segmental expression. Together, this work has revealed a surprising degree of complexity in the number of cis-elements and regulatory components that contribute to segmental expression mediated by Krox20 and sheds light on the diversity and evolution of Krox20 target sites and Hox regulatory elements in vertebrates.     

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.