Analysis of lamprey meis genes reveals that conserved inputs from Hox, Meis and Pbx proteins control their expression in the hindbrain and neural tube.

Meis genes are known to play important roles in the hindbrain and neural crest cells of jawed vertebrates. To explore the roles of Meis genes in head development during evolution of vertebrates, we have identified four meis genes in the sea lamprey genome and characterized their patterns of expression and regulation, with a focus on the hindbrain and pharynx. Each of the lamprey meis genes displays temporally and spatially dynamic patterns of expression, some of which are coupled to rhombomeric domains in the developing hindbrain and select pharyngeal arches. Studies of Meis loci in mouse and zebrafish have identified enhancers that are bound by Hox and TALE (Meis and Pbx) proteins, implicating these factors in the direct regulation of Meis expression. We examined the lamprey meis loci and identified a series of cis-elements conserved between lamprey and jawed vertebrate meis genes. In transgenic reporter assays we demonstrated that these elements act as neural enhancers in lamprey embryos, directing reporter expression in appropriate domains when compared to expression of their associated endogenous meis gene. Sequence alignments reveal that these conserved elements are in similar relative positions of the meis loci and contain a series of consensus binding motifs for Hox and TALE proteins. This suggests that ancient Hox and TALE-responsive enhancers regulated expression of ancestral vertebrate meis genes in segmental domains in the hindbrain and have been retained in the meis loci during vertebrate evolution. The presence of conserved Meis, Pbx and Hox binding sites in these lamprey enhancers links Hox and TALE factors to regulation of lamprey meis genes in the developing hindbrain, indicating a deep ancestry for these regulatory interactions prior to the divergence of jawed and jawless vertebrates.

Hox genes: Downstream "effectors" of retinoic acid signaling in vertebrate embryogenesis.

One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.

Retinoid-Sensitive Epigenetic Regulation of the Hoxb Cluster Maintains Normal Hematopoiesis and Inhibits Leukemogenesis.

Hox genes modulate the properties of hematopoietic stem cells (HSCs) and reacquired Hox expression in progenitors contributes to leukemogenesis. Here, our transcriptome and DNA methylome analyses revealed that Hoxb cluster and retinoid signaling genes are predominantly enriched in LT-HSCs, and this coordinate regulation of Hoxb expression is mediated by a retinoid-dependent cis-regulatory element, distal element RARE (DERARE). Deletion of the DERARE reduced Hoxb expression, resulting in changes to many downstream signaling pathways (e.g., non-canonical Wnt signaling) and loss of HSC self-renewal and reconstitution capacity. DNA methyltransferases mediate DNA methylation on the DERARE, leading to reduced Hoxb cluster expression. Acute myeloid leukemia patients with DNMT3A mutations exhibit DERARE hypomethylation, elevated HOXB expression, and adverse outcomes. CRISPR-Cas9-mediated specific DNA methylation at DERARE attenuated HOXB expression and alleviated leukemogenesis. Collectively, these findings demonstrate pivotal roles for retinoid signaling and the DERARE in maintaining HSCs and preventing leukemogenesis by coordinate regulation of Hoxb genes.

Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells.

Homeobox a1 (Hoxa1) is one of the most rapidly induced genes in ES cell differentiation and it is the earliest expressed Hox gene in the mouse embryo. In this study, we used genomic approaches to identify Hoxa1-bound regions during early stages of ES cell differentiation into the neuro-ectoderm. Within 2 h of retinoic acid treatment, Hoxa1 is rapidly recruited to target sites that are associated with genes involved in regulation of pluripotency, and these genes display early changes in expression. The pattern of occupancy of Hoxa1 is dynamic and changes over time. At 12 h of differentiation, many sites bound at 2 h are lost and a new cohort of bound regions appears. At both time points the genome-wide mapping reveals that there is significant co-occupancy of Nanog (Nanog homeobox) and Hoxa1 on many common target sites, and these are linked to genes in the pluripotential regulatory network. In addition to shared target genes, Hoxa1 binds to regulatory regions of Nanog, and conversely Nanog binds to a 3' enhancer of Hoxa1 This finding provides evidence for direct cross-regulatory feedback between Hoxa1 and Nanog through a mechanism of mutual repression. Hoxa1 also binds to regulatory regions of Sox2 (sex-determining region Y box 2), Esrrb (estrogen-related receptor beta), and Myc, which underscores its key input into core components of the pluripotential regulatory network. We propose a model whereby direct inputs of Nanog and Hoxa1 on shared targets and mutual repression between Hoxa1 and the core pluripotency network provides a molecular mechanism that modulates the fine balance between the alternate states of pluripotency and differentiation.

Lrp4 and Wise interplay controls the formation and patterning of mammary and other skin appendage placodes by modulating Wnt signaling.

The future site of skin appendage development is marked by a placode during embryogenesis. Although Wnt/ß-catenin signaling is known to be essential for skin appendage development, it is unclear which cellular processes are controlled by the signaling and how the precise level of the signaling activity is achieved during placode formation. We have investigated roles for Lrp4 and its potential ligand Wise (Sostdc1) in mammary and other skin appendage placodes. Lrp4 mutant mice displayed a delay in placode initiation and changes in distribution and number of mammary precursor cells leading to abnormal morphology, number and position of mammary placodes. These Lrp4 mammary defects, as well as limb defects, were associated with elevated Wnt/ß-catenin signaling and were rescued by reducing the dose of the Wnt co-receptor genes Lrp5 and Lrp6, or by inactivating the gene encoding ß-catenin. Wise-null mice phenocopied a subset of the Lrp4 mammary defects and Wise overexpression reduced the number of mammary precursor cells. Genetic epistasis analyses suggest that Wise requires Lrp4 to exert its function and that, together, they have a role in limiting mammary fate, but Lrp4 has an early Wise-independent role in facilitating placode formation. Lrp4 and Wise mutants also share defects in vibrissa and hair follicle development, suggesting that the roles played by Lrp4 and Wise are common to skin appendages. Our study presents genetic evidence for interplay between Lrp4 and Wise in inhibiting Wnt/ß-catenin signaling and provides an insight into how modulation of Wnt/ß-catenin signaling controls cellular processes important for skin placode formation.

Regulation of tooth number by fine-tuning levels of receptor-tyrosine kinase signaling.

Much of our knowledge about mammalian evolution comes from examination of dental fossils, because the highly calcified enamel that covers teeth causes them to be among the best-preserved organs. As mammals entered new ecological niches, many changes in tooth number occurred, presumably as adaptations to new diets. For example, in contrast to humans, who have two incisors in each dental quadrant, rodents only have one incisor per quadrant. The rodent incisor, because of its unusual morphogenesis and remarkable stem cell-based continuous growth, presents a quandary for evolutionary biologists, as its origin in the fossil record is difficult to trace, and the genetic regulation of incisor number remains a largely open question. Here, we studied a series of mice carrying mutations in sprouty genes, the protein products of which are antagonists of receptor-tyrosine kinase signaling. In sprouty loss-of-function mutants, splitting of gene expression domains and reduced apoptosis was associated with subdivision of the incisor primordium and a multiplication of its stem cell-containing regions. Interestingly, changes in sprouty gene dosage led to a graded change in incisor number, with progressive decreases in sprouty dosage leading to increasing numbers of teeth. Moreover, the independent development of two incisors in mutants with large decreases in sprouty dosage mimicked the likely condition of rodent ancestors. Together, our findings indicate that altering genetic dosage of an antagonist can recapitulate ancestral dental characters, and that tooth number can be progressively regulated by changing levels of activity of a single signal transduction pathway.

Dynamic transcriptional events in embryonic stem cells mediated by the super elongation complex (SEC).

Transcriptional regulation of developmentally controlled genes is at the heart of differentiation and organogenesis. In this study, we performed global genomic analyses in murine embryonic stem (ES) cells and in human cells in response to activation signals. We identified an essential role for the ELL (eleven-nineteen lysine-rich leukemia gene)/P-TEFb (positive transcription elongation factor)-containing super elongation complex (SEC) in the regulation of gene expression, including several genes bearing paused RNA polymerase II (Pol II). Paused Pol II has been proposed to be associated with loci that respond rapidly to environmental stimuli. However, our studies in ES cells also identified a requirement for SEC at genes without paused Pol II, which also respond dynamically to differentiation signals. Our findings suggest that SEC is a major class of active P-TEFb-containing complexes required for transcriptional activation in response to environmental cues such as differentiation signals.

Global analysis of H3K4 methylation defines MLL family member targets and points to a role for MLL1-mediated H3K4 methylation in the regulation of transcriptional initiation by RNA polymerase II.

A common landmark of activated genes is the presence of trimethylation on lysine 4 of histone H3 (H3K4) at promoter regions. Set1/COMPASS was the founding member and is the only H3K4 methylase in Saccharomyces cerevisiae; however, in mammals, at least six H3K4 methylases, Set1A and Set1B and MLL1 to MLL4, are found in COMPASS-like complexes capable of methylating H3K4. To gain further insight into the different roles and functional targets for the H3K4 methylases, we have undertaken a genome-wide analysis of H3K4 methylation patterns in wild-type Mll1(+/+) and Mll1(-)(/)(-) mouse embryonic fibroblasts (MEFs). We found that Mll1 is required for the H3K4 trimethylation of less than 5% of promoters carrying this modification. Many of these genes, which include developmental regulators such as Hox genes, show decreased levels of RNA polymerase II recruitment and expression concomitant with the loss of H3K4 methylation. Although Mll1 is only required for the methylation of a subset of Hox genes, menin, a component of the Mll1 and Mll2 complexes, is required for the overwhelming majority of H3K4 methylation at Hox loci. However, the loss of MLL3/MLL4 and/or the Set1 complexes has little to no effect on the H3K4 methylation of Hox loci or their expression levels in these MEFs. Together these data provide insight into the redundancy and specialization of COMPASS-like complexes in mammals and provide evidence for a possible role for Mll1-mediated H3K4 methylation in the regulation of transcriptional initiation.

Non-cell-autonomous effects of Ret deletion in early enteric neurogenesis.

Neural crest cells (NCCs) form at the dorsal margin of the neural tube and migrate along distinct pathways throughout the vertebrate embryo to generate multiple cell types. A subpopulation of vagal NCCs invades the foregut and colonises the entire gastrointestinal tract to form the enteric nervous system (ENS). The colonisation of embryonic gut by NCCs has been studied extensively in chick embryos, and genetic studies in mice have identified genes crucial for ENS development, including Ret. Here, we have combined mouse embryo and organotypic gut culture to monitor and experimentally manipulate the progenitors of the ENS. Using this system, we demonstrate that lineally marked intestinal ENS progenitors from E11.5 mouse embryos grafted into the early vagal NCC pathway of E8.5 embryos colonise the entire length of the gastrointestinal tract. By contrast, similar progenitors transplanted into Ret-deficient host embryos are restricted to the proximal foregut. Our findings establish an experimental system that can be used to explore the interactions of NCCs with their cellular environment and reveal a previously unrecognised non-cell-autonomous effect of Ret deletion on ENS development.

Cranial neural crest cells regulate head muscle patterning and differentiation during vertebrate embryogenesis.

In the vertebrate head, mesoderm cells fuse together to form a myofiber, which is attached to specific cranial neural crest (CNC)-derived skeletal elements in a highly coordinated manner. Although it has long been recognized that CNC plays a role in the formation of the head musculature, the precise molecular underpinnings of this process remain elusive. In the present study we explored the nature of the crosstalk between CNC and mesoderm cells during head muscle development, employing three models for genetic perturbations of CNC development in mice, as well as experimental ablation of CNC in chick embryos. We demonstrate that although early myogenesis is CNC-independent, the migration, patterning and differentiation of muscle precursors are regulated by CNC. In the absence of CNC cells, accumulated myoblasts are kept in a proliferative state, presumably because of an increase of Fgf8 in adjacent tissues, which leads to abnormalities in both differentiation and subsequent myofiber organization in the head. These results have uncovered a surprising degree of complexity and multiple distinct roles for CNC in the patterning and differentiation of muscles during craniofacial development. We suggest that CNC cells control craniofacial development by regulating positional interactions with mesoderm-derived muscle progenitors that together shape the cranial musculoskeletal architecture in vertebrate embryos.

Bone density ligand, Sclerostin, directly interacts with LRP5 but not LRP5G171V to modulate Wnt activity.

UNLABELLED: We compared and contrasted the mechanism of action for the cysteine knot protein subfamily, Wise and Sost (Sclerostin). Our data suggest that functional interactions between Sost or Wise and LRP5/LRP6 have the potential to regulate bone deposition by modulating the Wnt pathway. INTRODUCTION: The human disease sclerosteosis exhibits an increase in bone mass thought to be caused by hyperactive osteoblasts. Sclerostin, SOST, the gene affected in this disease, has been postulated to exert its activity by functioning as a BMP antagonist. However, recent evidence indicates that SOST is highly related to Wise, which can also modulate the Wnt pathway by binding to LRP5 and LRP6. MATERIALS AND METHODS: For this study, we used cell culture to test the BMP and Wnt activity function of both Wise and Sost. In addition, we used Xenopus in vivo Wnt assays along with Xenopus in vitro Wnt assays to support our cell culture results. Epitope tagged cell supernatants containing either Sost or soluble mutant or wildtype LRP5/LRP6 were used for immunoprecipitation. Sost immunoprecipitation results were confirmed in vivo using cell culture. Finally, to support our in vitro data, we co-localized Sost, Wise, LRP5, and LRP6 in mouse long bone sections. RESULTS: In this study, we report in vitro and in vivo evidence to show that Sost physically interacts with Lrp5 and Lrp6 and inhibits the canonical Wnt signaling pathway. Furthermore, using in vitro and in vivo assays, we showed that a variant of LRP5 (LRP5(G171V)) known to cause the human high bone mass (HBM) trait and a homologous change in LRP6 (LRP6(G158V)) abolished protein interactions with Sost. We used variants of Sost amino acids to further identify the contact points between Sost and LRP6. In Xenopus and mammalian cell culture assays, we showed that SOST is able to attenuate Wnt signaling and that this attenuation can be rescued by the addition of alpha-Sost antibodies or by the introduction of single amino acid substitution that alter its binding to LRP6. Sost differs from Wise in that it is unable to stimulate Wnt signaling. Using immunohistochemistry, we found that Sost and Wise are co-localized to osteoblasts, along with LRP5 and LRP6. CONCLUSIONS: Our data suggest that functional interactions between Sost or Wise and LRPs have the potential to regulate bone deposition by modulating Wnt signaling.

Bone formation: The nuclear matrix reloaded.

In this issue of Cell, Grosschedl and colleagues (Dobreva et al., 2006) report that the nuclear matrix protein Satb2 represses Hoxa2 expression and acts with other regulatory proteins to promote osteoblast differentiation. This work suggests a molecular mechanism that enables the integration of patterning and differentiation during bone formation.

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.

Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex.

Connective-tissue growth factor (CTGF) is a member of the CCN family of secreted proteins. CCN family members contain four characteristic domains and exhibit multiple activities: they associate with the extracellular matrix, they can mediate cell adhesion, cell migration and chemotaxis, and they can modulate the activities of peptide growth factors. Many of the effects of CTGF are thought to be mediated by binding to integrins, whereas others may be because of its recently identified ability to interact with BMP4 and TGF beta. We demonstrate, using Xenopus embryos, that CTGF also regulates signalling through the Wnt pathway, in accord with its ability to bind to the Wnt co-receptor LDL receptor-related protein 6 (LRP6). This interaction is likely to occur through the C-terminal (CT) domain of CTGF, which is distinct from the BMP- and TGF beta-interacting domain. Our results define new activities of CTGF and add to the variety of routes through which cells regulate growth factor activity in development, disease and tissue homeostasis.

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.

Wise, a context-dependent activator and inhibitor of Wnt signalling.

We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of beta-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.

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.

Requirement for downregulation of kreisler during late patterning of the hindbrain.

Pattern formation in the hindbrain is governed by a segmentation process that provides the basis for the organisation of cranial motor nerves. A cascade of transcriptional activators, including the bZIP transcription factor encoded by the kreisler gene controls this segmentation process. In kreisler mutants, r5 fails to form and this correlates with abnormalities in the neuroanatomical organisation of the hindbrain. Studies of Hox gene regulation suggest that kreisler may regulate the identity as well as the formation of r5, but such a role cannot be detected in kreisler mutants since r5 is absent. To gain further insights into the function of kreisler we have generated transgenic mice in which kreisler is ectopically expressed in r3 and for an extended period in r5. In these transgenic mice, the Fgf3, Krox20, Hoxa3 and Hoxb3 genes have ectopic or prolonged expression domains in r3, indicating that it acquires molecular characteristics of r5. Prolonged kreisler expression subsequently causes morphological alterations of r3/r5 that are due to an inhibition of neuronal differentiation and migration from the ventricular zone to form the mantle layer. We find that these alterations in r5 correlate with an arrest of facial branchiomotor neurone migration from r4 into the caudal hindbrain, which is possibly due to the deficiency in the mantle layer through which they normally migrate. We propose that the requirement for the downregulation of segmental kreisler expression prior to neuronal differentiation reflects the stage-specific roles of this gene and its targets.

Krox20 and kreisler co-operate in the transcriptional control of segmental expression of Hoxb3 in the developing hindbrain.

In the segmented vertebrate hindbrain, the Hoxa3 and Hoxb3 genes are expressed at high relative levels in the rhombomeres (r) 5 and 6, and 5, respectively. The single enhancer elements responsible for these activities have been identified previously and shown to constitute direct targets of the transcription factor kreisler, which is expressed in r5 and r6. Here, we have analysed the contribution of the transcription factor Krox20, present in r3 and r5. Genetic analyses demonstrated that Krox20 is required for activity of the Hoxb3 r5 enhancer, but not of the Hoxa3 r5/6 enhancer. Mutational analysis of the Hoxb3 r5 enhancer, together with ectopic expression experiments, revealed that Krox20 binds to the enhancer and synergizes with kreisler to promote Hoxb3 transcription, restricting enhancer activity to their domain of overlap, r5. These analyses also suggested contributions from an Ets-related factor and from putative factors likely to heterodimerize with kreisler. The integration of multiple independent inputs present in overlapping domains by a single enhancer is likely to constitute a general mechanism for the patterning of subterritories during vertebrate development.