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.     

Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development.

The analysis of mice mutant for both Hoxa1 and Hoxb1 suggests that these two genes function together to pattern the hindbrain. Separately, mutations in Hoxa1 and Hoxb1 have profoundly different effects on hindbrain development. Hoxa1 mutations disrupt the rhombomeric organization of the hindbrain, whereas Hoxb1 mutations do not alter the rhombomeric pattern, but instead influence the fate of cells originating in rhombomere 4. We suggest that these differences are not the consequences of different functional roles for these gene products, but rather reflect differences in the kinetics of Hoxa1 and Hoxb1 gene expression. In strong support of the idea that Hoxa1 and Hoxb1 have overlapping functions, Hoxa1/Hoxb1 double mutant homozygotes exhibit a plethora of defects either not seen, or seen only in a very mild form, in mice mutant for only Hoxa1 or Hoxb1. Examples include: the loss of both rhombomeres 4 and 5, the selective loss of the 2(nd) branchial arch, and the loss of most, but not all, 2(nd) branchial arch-derived tissues. We suggest that the early role for both of these genes in hindbrain development is specification of rhombomere identities and that the aberrant development of the hindbrain in Hoxa1/Hoxb1 double mutants proceeds through two phases, the misspecification of rhombomeres within the hindbrain, followed subsequently by size regulation of the misspecified hindbrain through induction of apoptosis.     

Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention

We have used a morpholino-based knockdown approach to investigate the functions of a pair of zebrafish Hox gene duplicates, hoxb1a and hoxb1b, which are expressed during development of the hindbrain. We find that the zebrafish hoxb1 duplicates have equivalent functions to mouse Hoxb1 and its paralogue Hoxa1. Thus, we have revealed a 'function shuffling' among genes of paralogue group 1 during the evolution of vertebrates. Like mouse Hoxb1, zebrafish hoxb1a is required for migration of the VIIth cranial nerve branchiomotor neurons from their point of origin in hindbrain rhombomere 4 towards the posterior. By contrast, zebrafish hoxb1b, like mouse Hoxa1, is required for proper segmental organization of rhombomere 4 and the posterior hindbrain. Double knockdown experiments demonstrate that the zebrafish hoxb1 duplicates have partially redundant functions. However, using an RNA rescue approach, we reveal that these duplicated genes do not have interchangeable biochemical functions: only hoxb1a can properly pattern the VIIth cranial nerve. Despite this difference in protein function, we provide evidence that the hoxb1 duplicate genes were initially maintained in the genome because of complementary degenerative mutations in defined cis-regulatory elements.     

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.     

Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish

Many Hox proteins are thought to require Pbx and Meis co-factors to specify cell identity during embryogenesis. Here we demonstrate that Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish. We find that Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere 2 of the hindbrain. In contrast, Hoxb1b and Pbx4 acting together with Meis3 induce hoxb1a, hoxb2, krox20 and valentino expression rostrally and cause extensive transformation of forebrain and midbrain fates to hindbrain fates, including differentiation of excess rhombomere 4-specific Mauthner neurons. This synergistic effect requires that Hoxb1b and Meis3 have intact Pbx-interaction domains, suggesting that their in vivo activity is dependent on binding to Pbx4. In the case of Meis3, binding to Pbx4 is also required for nuclear access. Our results are consistent with Hoxb1b and Meis3 interacting with Pbx4 to form complexes that regulate hindbrain development during zebrafish embryogenesis.     

Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the rostral hindbrain

Little is known about how the generation of specific neuronal types at stereotypic positions within the hindbrain is linked to Hox gene-mediated patterning. Here, we show that during neurogenesis, Hox paralog group 2 genes control both anteroposterior (A-P) and dorsoventral (D-V) patterning. Hoxa2 and Hoxb2 differentially regulate, in a rhombomere-specific manner, the expression of several genes in broad D-V-restricted domains or narrower longitudinal columns of neuronal progenitors, immature neurons, and differentiating neuronal subtypes. Moreover, Hoxa2 and Hoxb2 can functionally synergize in controlling the development of ventral neuronal subtypes in rhombomere 3 (r3). Thus, in addition to their roles in A-P patterning, Hoxa2 and Hoxb2 have distinct and restricted functions along the D-V axis during neurogenesis, providing insights into how neuronal fates are assigned at stereotypic positions within the hindbrain.     

Specification of distinct motor neuron identities by the singular activities of individual Hox genes.

Hox genes have been implicated in specifying positional values along the anteroposterior axis of the caudal central nervous system, but their nested and overlapping expression has complicated the understanding of how they confer specific neural identity. We have employed a direct gain-of-function approach using retroviral vectors to misexpress Hoxa2 and Hoxb1 outside of the normal Hox expression domains, thereby avoiding complications resulting from possible interactions with endogenous Hox genes. Misexpression of either Hoxa2 or Hoxb1 in the anteriormost hindbrain (rhombomere1, r1) leads to the generation of motor neurons in this territory, even though it is normally devoid of this cell type. These ectopic neurons have the specific identity of branchiomotor neurons and, in the case of Hoxb1-induced cells, their axons leave the hindbrain either by fasciculating with the resident cranial motor axons at isthmic (trochlear) or r2 (trigeminal) levels of the axis or via novel ectopic exit points in r1. Next, we have attempted to identify the precise branchiomotor subtypes that are generated after misexpression and our results suggest that the ectopic motor neurons generated following Hoxa2 misexpression are trigeminal-like, while those generated following Hoxb1 misexpression are facial-like. Our data demonstrate, therefore, that at least to a certain extent and for certain cell types, the singular activities of individual Hox genes (compared to a combinatorial mode of action, for example) are sufficient to impose on neuronal precursor cells the competence to generate distinctly specified cell types. Moreover, as these particular motor neuron subtypes are normally generated in the most anterior domains of Hoxa2 and Hoxb1 expression, respectively, our data support the idea that the main site of individual Hox gene action is in the anteriormost subdomain of their expression, consistent with the phenomenon of posterior dominance.     

Knockdown of the complete Hox paralogous group 1 leads to dramatic hindbrain and neural crest defects

The Hox paralogous group 1 (PG1) genes are the first and initially most anterior Hox genes expressed in the embryo. In Xenopus, the three PG1 genes, Hoxa1, Hoxb1 and Hoxd1, are expressed in a widely overlapping domain, which includes the region of the future hindbrain and its associated neural crest. We used morpholinos to achieve a complete knockdown of PG1 function. When Hoxa1, Hoxb1 and Hoxd1 are knocked down in combination, the hindbrain patterning phenotype is more severe than in the single or double knockdowns, indicating a degree of redundancy for these genes. In the triple PG1 knockdown embryos the hindbrain is reduced and lacks segmentation. The patterning of rhombomeres 2 to 7 is lost, with a concurrent posterior expansion of the rhombomere 1 marker, Gbx2. This effect could be via the downregulation of other Hox genes, as we show that PG1 function is necessary for the hindbrain expression of Hox genes from paralogous groups 2 to 4. Furthermore, in the absence of PG1 function, the cranial neural crest is correctly specified but does not migrate into the pharyngeal arches. Embryos with no active PG1 genes have defects in derivatives of the pharyngeal arches and, most strikingly, the gill cartilages are completely missing. These results show that the complete abrogation of PG1 function in Xenopus has a much wider scope of effect than would be predicted from the single and double PG1 knockouts in other organisms.     

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.     

Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia

Complex genetic and biochemical interactions between HOX proteins and members of the TALE (i.e., PBX and MEIS) family have been identified in embryonic development, and some of these interactions also appear to be important for leukemic transformation. We have previously shown that HOXA9 collaborates with MEIS1 in the induction of acute myeloid leukemia (AML). In this report, we demonstrate that HOXB3, which is highly divergent from HOXA9, also genetically interacts with MEIS1, but not with PBX1, in generating AML. In addition, we show that the HOXA9 and HOXB3 genes play key roles in establishing all the main characteristics of the leukemias, while MEIS1 functions only to accelerate the onset of the leukemic transformation. Contrasting the reported functional similarities between PREP1 and MEIS1, such as PBX nuclear retention, we also show that PREP1 overexpression is incapable of accelerating the HOXA9-induced AML, suggesting that MEIS1 function in transformation must entail more than PBX nuclear localization. Collectively, these data demonstrate that MEIS1 is a common leukemic collaborator with two structurally and functionally divergent HOX genes and that, in this collaboration, the HOX gene defines the identity of the leukemia.     

Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes

Hox genes are instrumental in assigning segmental identity in the developing hindbrain. Auto-, cross- and para-regulatory interactions help establish and maintain their expression. To understand to what extent such regulatory interactions shape neuronal patterning in the hindbrain, we analysed neurogenesis, neuronal differentiation and motoneuron migration in Hoxa1, Hoxb1 and Hoxb2 mutant mice. This comparison revealed that neurogenesis and differentiation of specific neuronal subpopulations in r4 was impaired in a similar fashion in all three mutants, but with different degrees of severity. In the Hoxb1 mutants, neurons derived from the presumptive r4 territory were re-specified towards an r2-like identity. Motoneurons derived from that territory resembled trigeminal motoneurons in both their migration patterns and the expression of molecular markers. Both migrating motoneurons and the resident territory underwent changes consistent with a switch from an r4 to r2 identity. Abnormally migrating motoneurons initially formed ectopic nuclei that were subsequently cleared. Their survival could be prolonged through the introduction of a block in the apoptotic pathway. The Hoxa1 mutant phenotype is consistent with a partial misspecification of the presumptive r4 territory that results from partial Hoxb1 activation. The Hoxb2 mutant phenotype is a hypomorph of the Hoxb1 mutant phenotype, consistent with the overlapping roles of these genes in facial motoneuron specification. Therefore, we have delineated the functional requirements in hindbrain neuronal patterning that follow the establishment of the genetic regulatory hierarchy between Hoxa1, Hoxb1 and Hoxb2.