Auto/cross-regulation of Hoxb3 expression in posterior hindbrain and spinal cord

The complex and dynamic pattern of Hoxb3 expression in the developing hindbrain and the associated neural crest of mouse embryos is controlled by three separate cis-regulatory elements: element I (region A), element IIIa, and the r5 enhancer (element IVa). We have examined the cis-regulatory element IIIa by transgenic and mutational analysis to determine the upstream trans-acting factors and mechanisms that are involved in controlling the expression of the mouse Hoxb3 gene in the anterior spinal cord and hindbrain up to the r5/r6 boundary, as well as the associated neural crest which migrate to the third and posterior branchial arches and to the gut. By deletion analysis, we have identified the sequence requirements within a 482-bp element III482. Two Hox binding sites are identified in element III482 and we have shown that in vitro both Hoxb3 and Hoxb4 proteins can interact with these Hox binding sites, suggesting that auto/cross-regulation is required for establishing the expression of Hoxb3 in the neural tube domain. Interestingly, we have identified a novel GCCAGGC sequence motif within element III482, which is also required to direct gene expression to a subset of the expression domains except for rhombomere 6 and the associated neural crest migrating to the third and posterior branchial arches. Element III482 can direct a higher level of reporter gene expression in r6, which led us to investigate whether kreisler is involved in regulating Hoxb3 expression in r6 through this element. However, our transgenic and mutational analysis has demonstrated that, although kreisler binding sites are present, they are not required for the establishment or maintenance of reporter gene expression in r6. Our results have provided evidence that the expression of Hoxb3 in the neural tube up to the r5/r6 boundary is auto/cross-regulated by Hox genes and expression of Hoxb3 in r6 does not require kreisler.     

Independent regulation of initiation and maintenance phases of Hoxa3 expression in the vertebrate hindbrain involve auto- and cross-regulatory mechanisms

During development of the vertebrate hindbrain, Hox genes play multiple roles in the segmental processes that regulate anteroposterior (AP) patterning. Paralogous Hox genes, such as Hoxa3, Hoxb3 and Hoxd3, generally have very similar patterns of expression, and gene targeting experiments have shown that members of paralogy group 3 can functionally compensate for each other. Hence, distinct functions for individual members of this family may primarily depend upon differences in their expression domains. The earliest domains of expression of the Hoxa3 and Hoxb3 genes in hindbrain rhombomeric (r) segments are transiently regulated by kreisler, a conserved Maf b-Zip protein, but the mechanisms that maintain expression in later stages are unknown. In this study, we have compared the segmental expression and regulation of Hoxa3 and Hoxb3 in mouse and chick embryos to investigate how they are controlled after initial activation. We found that the patterns of Hoxa3 and Hoxb3 expression in r5 and r6 in later stages during mouse and chick hindbrain development were differentially regulated. Hoxa3 expression was maintained in r5 and r6, while Hoxb3 was downregulated. Regulatory comparisons of cis-elements from the chick and mouse Hoxa3 locus in both transgenic mouse and chick embryos have identified a conserved enhancer that mediates the late phase of Hoxa3 expression through a conserved auto/cross-regulatory loop. This block of similarity is also present in the human and horn shark loci, and contains two bipartite Hox/Pbx-binding sites that are necessary for its in vivo activity in the hindbrain. These HOX/PBC sites are positioned near a conserved kreisler-binding site (KrA) that is involved in activating early expression in r5 and r6, but their activity is independent of kreisler. This work demonstrates that separate elements are involved in initiating and maintaining Hoxa3 expression during hindbrain segmentation, and that it is regulated in a manner different from Hoxb3 in later stages. Together, these findings add further strength to the emerging importance of positive auto- and cross-regulatory interactions between Hox genes as a general mechanism for maintaining their correct spatial patterns in the vertebrate nervous system.     

Retinoids regulate the anterior expression boundaries of 5' Hoxb genes in posterior hindbrain

We describe the regulatory interactions that cause anterior extension of the mouse 5' Hoxb expression domains from spinal cord levels to their definitive boundaries in the posterior hindbrain between embryonic day E10 and E11.5. This anterior expansion is retinoid dependent since it does not occur in mouse embryos deficient for the retinoic acid-synthesizing enzyme retinaldehyde dehydrogenase 2. A retinoic acid response element (RARE) was identified downstream of Hoxb5 and shown to be essential for expression of Hoxb5 and Hoxb8 reporter transgenes in the anterior neural tube. The spatio-temporal activity of this element overlaps with rostral extension of the expression domain of endogenous Hoxb5, Hoxb6 and Hoxb8 into the posterior hindbrain. The RARE and surrounding sequences are found at homologous positions in the human, mouse and zebrafish genome, which supports an evolutionarily conserved regulatory function.     

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.     

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.     

Hoxb1 enhancer and control of rhombomere 4 expression: complex interplay between PREP1-PBX1-HOXB1 binding sites

The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.     

Generation of a transgenic mouse line expressing GFP-Cre protein from a Hoxb4 neural enhancer

Here, we describe a transgenic mouse line, in which expression of green fluorescent protein fused to Cre recombinase (GFP-Cre) is directed by the early neuronal enhancer (ENE) of Hoxb4. In E9.0-13.5 transgenic embryos, Cre activity coincided with endogenous Hoxb4 throughout the neural tube up to the r6/r7 boundary in the hindbrain, the dorsal root ganglia, and the Xth cranial ganglia. Unexpectedly, Cre activity was also consistently detected in the trigeminal (Vth) cranial nerve, which is devoid of endogenous Hoxb4 expression. Strong GFP dependent fluorescence appeared slightly later in E9.5-E11.5 embryos, and reflected the later expression pattern expected for Hoxb4-ENE directed expression in the neural tube up to the r7/r8 not r6/r7 boundary. Thus, with the exception of the trigeminal nerve, this reporter faithfully reproduces endogenous embryonic neural Hoxb4 expression, and provides an excellent reagent for in vivo gene manipulations in neuronal Hoxb4 positive cells as well as the developing trigeminal nerve. This transgenic mouse line should prove especially useful for determining the fate map of neuronal populations arising in rhombomeres 7 and 8 on its own and in combination with the small set of other existing rhombomere-specific Cre recombinase expressing lines.     

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.     

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.     

Analysis of Fibroblast growth factor 15 cis-elements reveals two conserved enhancers which are closely related to cardiac outflow tract development

Fibroblast growth factor 15 (Fgf15) is expressed in the developing mouse central nervous system and pharyngeal arches. Fgf15 mutant mice showed defects of the cardiac outflow tract probably because of aberrant behavior of the cardiac neural crest cells. In this study, we examined cis-elements of the Fgf15 gene by transient transgenic analysis using lacZ as a reporter. We identified two enhancers: one directed lacZ expression in the hindbrain/spinal cord and the other in the posterior midbrain (pmb), rhombomere1 (r1) and pharyngeal epithelia. Interestingly, human genomic regions which are highly homologous to these two mouse enhancers showed almost the same enhancer activities as those of mice in transgenic mouse embryos, indicating that the two enhancers are conserved between humans and mice. We also showed that the mouse and human pmb/r1 enhancer can regulate lacZ expression in chick embryos in almost the same way as in mouse embryos. We found that the lacZ expression domain with this enhancer was expanded by ectopic Fgf8b expression, suggesting that this enhancer is regulated by Fgf8 signaling. Moreover, over-expression of Fgf15 resulted in up-regulation of Fgf8 expression in the isthmus/r1. These findings suggest that a reciprocal positive regulation exists between Fgf15 and Fgf8 in the isthmus/r1. Together with cardiac outflow tract defects in Fgf15 mutants, the conservation of enhancers in the hindbrain/spinal cord and pharyngeal epithelia suggests that human FGF19 (ortholog of Fgf15) is involved in early development and the distribution of cardiac neural crest cells and is one of the candidate genes for congenital heart defects.     

Expression of Hoxa2 in rhombomere 4 is regulated by a conserved cross-regulatory mechanism dependent upon Hoxb1

The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.