Mutation of Labial homologs

Mice with a disruption in the hoxb-2 locus (homolog: Drosophila proboscipedia) were generated by gene targeting. 75% of the hoxb-2 mutant homozygotes died within 24 hours of birth. While a majority of these mice have severe sternal defects that compromise their ability to breathe, some have relatively normal sternum morphology, suggesting that one or more additional factor(s) contribute to neonatal lethality. At 3-3.5 weeks of age, half of the remaining hoxb-2 homozygotes become weak and subsequently die. All of the mutants that survive to 3 weeks of age show marked facial paralysis similar to, but more severe than, that reported for hoxb-1 mutant homozygotes (homolog: Drosophila labial). As for the hoxb-1 mutations, the facial paralysis observed in mice homozygous for the hoxb-2 mutation results from a failure to form the somatic motor component of the VIIth (facial) nerve which controls the muscles of facial expression. Features of this phenotype closely resemble the clinical signs associated with Bell's Palsy and Moebius Syndrome in humans. The sternal defects seen in hoxb-2 mutant mice are similar to those previously reported for hoxb-4 mutant mice (homolog: Drosophila Deformed). The above results suggest that the hoxb-2 mutant phenotype may result in part from effects of the hoxb-2 mutation on the expression of both hoxb-1 and hoxb-4. Consistent with this proposal, it was found that the hoxb-2 mutation disrupts the expression of hoxb-1 in cis. In addition, the hoxb-2 mutation changes the expression of hoxb-4 and the hoxb-4 mutation, in turn, alters the pattern of hoxb-2 expression. hoxb-2 and hoxb-4 appear to function together to mediate proper closure of the ventral thoracic body wall. Failure in this closure results in severe defects of the sternum (Barrow, 1996).

The rhombencephalic neural crest play several roles in craniofacial development. This structure gives rise to the cranial sensory ganglia and much of the craniofacial skeleton, and is vital for patterning of the craniofacial muscles. The loss of Hoxa1 or Hoxa2 function affects the development of multiple neural crest-derived structures. To understand how these two genes function together in craniofacial development, an allele was generated that disrupts both of these linked genes. Some of the craniofacial defects observed in the double mutants are additive combinations of those that exist in each of the single mutants, indicating that each gene functions independently in the formation of these structures. Other defects are found only in the double mutants, demonstrating overlapping or synergistic functions. Multiple defects were uncovered in the attachments and trajectories of the extrinsic tongue and hyoid muscles in Hoxa2 mutants. Interestingly, the abnormal trajectory of two of these muscles, the styloglossus and the stylohyoideus, blocks the attachment of the hyoglossus to the greater horn of the hyoid, which in turn correlates exactly with the presence of cleft palate in Hoxa2 mutants. It is suggested that the hyoglossus, whose function is to depress the lateral edges of the tongue, when unable to make its proper attachment to the greater horn of the hyoid, forces the tongue to adopt an abnormal posture which blocks closure of the palatal shelves. Unexpectedly, in Hoxa1/Hoxa2 double mutants, the penetrance of cleft palate is dramatically reduced. Two compensatory defects, associated with the loss of Hoxa1 function, restore normal attachment of the hyoglossus to the greater horn, thereby allowing the palatal shelves to lift and fuse above the flattened tongue (Barrow, 1999).

The external ear or pinna is derived from six mesenchymal swellings that flank the first branchial cleft. Three of the swellings are located on the first arch adjacent to the cleft, whereas the other three are found on the second arch. All mutant classes exhibit defects in the pinnae. Mutants lacking Hoxa1, but possessing at least one copy of Hoxa2, demonstrated reductions (usually unilaterally) of the external ear, although there are no significant changes in the patterning of the pinna. Hoxa2-/- mutants and Hoxa1 +/-:Hoxa2-/- mutants possess severe external ear anomalies. The pinnae are almost entirely absent, leaving the underlying external auditory meatus exposed. Interestingly, at 100% penetrance two folds of tissue dorsal and posterior to the external auditory meatus that correspond to the pinnae have been found. It has been reported that Hoxa2 mutants demonstrate several duplications of first arch structures. It is possible therefore that these two outgrowths represent duplications of the first arch portions of the external ear. As further evidence that these outgrowths represent duplications, a single ectopic vibrissum was observed to be associated with each pinna structure. Hoxa1/Hoxa2 double mutant mice do not possess any remnants of the pinnae or ectopic vibrissae. The only structure that is visible is the external auditory meatus (Barrow, 1999).

The middle ear ossicles are derived from neural crest that populates the first and second branchial arches. The proximal first arch structures, the malleus and incus, are derived from neural crest that originates from rhombomeres (r) 1 and 2. The gonial bone and tympanic ring are also derived from neural crest that migrates to the first arch. The stapes, however, is derived from r4 neural crest that populates the second arch. Hoxa1 -/- and Hoxa1 -/- /Hoxa2 +/- mutants exhibit reductions in the middle ear apparatus. Similar to the situation with the external ear, these reductions are not associated with major changes in patterning. In most cases, the stapes is absent; however, in one specimen, it was fused to the styloid process. In contrast, Hoxa2 (and Hoxa1+/- ::Hoxa2 -/-) mutants possess severe patterning defects of the middle ear ossicles and the associated skeleton. Mutants of these classes exhibit mirror-image duplications of Meckel’s cartilage, the malleus and incus, as well as the tympanic ring and gonial bone. In addition, the orthotopic and duplicated gonial bones are connected by an ossified bridge. The second arch-derived stapes is not present. The collection of these defects is identical to those previously reported for two other Hoxa2 alleles. The middle ear ossicles of Hoxa1/Hoxa2 double mutants exhibit the patterning defects of the Hoxa2 mutants and the hypoplastic characteristics of Hoxa1 mutants. For example, the double mutants possess mirror-image duplications of Meckel’s cartilage and the malleus and incus; however, both the orthotopic and duplicated structures are markedly reduced in size. The stapes and gonial bone are always absent. The tympanic ring is almost always absent although a small vestige could sometimes be found. Thus, Hoxa2 appears to not only affect the patterning of the tympanic ring and gonial bone but synergizes with Hoxa1 in controlling the growth of these structures as well (Barrow, 1999).

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. It has been suggested 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 second branchial arch, and the loss of most, but not all, second branchial arch-derived tissues. It is suggested that the early role for both of these genes in hindbrain development is the 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 (Rossel, 1999).

It is proposed that the function of both genes in the early hindbrain is to specify rhombomere identity and that the differences in the mutant phenotypes resulting from individual disruption of these genes arise because Hoxa1, in addition to its normal role in specifying rhombomere identities, is also required to extend the anterior boundary of Hoxb1 to the presumptive r3/r4 boundary. In the absence of Hoxa1 function, the anterior boundary of Hoxb1 is within the normal r4 territory rather than at the r3/r4 presumptive boundary. This in turn leads to misspecification of the normal rhombomere 3 and rhombomere 5 territories. A main feature of the model is that the unique roles attributable to Hoxa1 and Hoxb1 do not arise from separate protein functions, but rather from differences in the kinetics of their gene expression. Hoxa1 is expressed first and reaches the presumptive r3/r4 boundary first. Having reached this anterior boundary, it participates in activating Hoxb1 expression at this anterior level. In the absence of Hoxa1 activity, Hoxb1 expression never reaches this anterior boundary and specification of rhombomere 4 is incomplete and occurs at a more caudal level than normal (Rossel, 1999).

Homeobox genes are expressed with a specific spatial and temporal order, which is essential for pattern formation during the early development of both invertebrates and vertebrates. Widespread ectopic expression of the Hoxa-1 (Hox 1.6) gene (Drosophila homolog: Labial) directed by a human beta-actin promoter in transgenic mice is embryolethal and produces abnormal phenotypes in a subset of domains primarily located in anterior regions. Interestingly, this abnormal development in the Hoxa-1 transgenic mice is associated with ectopic expression of the Hoxb-1 (Hox 2.9) gene in select hindbrain regions. At gestation day 9.5, two domains of strong Hoxb-1 expression are found in the anterior region of the hindbrains of Hoxa-1 transgenic embryos. One region represents the normal pattern of Hoxb-1 expression in rhombomere 4 and its associated migrating neural crest cells, while another major domain of Hoxb-1 expression consistently appears in rhombomere 2. Similar ectopic domains are detected in dual transgenic embryos containing both beta-actin/Hoxa-1 transgene and a Hoxb-1/lacZ reporter construct. Expression of another lacZ reporter gene that directs beta-galactosidase activity predominantly in rhombomere 2 is suppressed in the Hoxa-1 transgenic embryos. Weaker and variable ectopic Hoxb-1 expression iss detected in rhombomeres 1, 3 and 6. No ectopic Hoxb-1 expression is detected in rhombomere 5 and the expression of Hoxa-3 and Krox-20 in this region is unchanged in the Hoxa-1 transgenic embryos. While no obvious change in the morphology of the trigeminal or facial-acoustic ganglia is evident, phenotypic changes do occur in neurons that emanate from rhombomeres 2 and 3 in the Hoxa-1 transgenic embryos. Additionally, alterations in the pattern of Hoxa-2 and Hoxb-1 expression in a subpopulation of neural crest cells migrating from the rhombomere 2 region are detected in these transgenics. Taken together, these data suggest that ectopic Hoxa-1 expression can reorganize select regions of the developing hindbrain by inducing partial transformations of several rhombomeres into a rhombomere-4-like identity (Zhang, 1994).

The transcription factor GATA3 is dynamically expressed during hindbrain development. Function of GATA3 in ventral rhombomere (r) 4 is dependent on functional GATA2, which in turn is under the control of Hoxb1. In particular, the absence of Hoxb1 results in the loss of GATA2 expression in r4 and the absence of GATA2 results in the loss of GATA3 expression. The lack of GATA3 expression in r4 inhibits the projection of contralateral vestibuloacoustic efferent neurons and the migration of facial branchiomotor neurons similar to Hoxb1-deficient mice. Ubiquitous expression of Hoxb1 in the hindbrain induces ectopic expression of GATA2 and GATA3 in ventral r2 and r3. These findings demonstrate that GATA2 and GATA3 lie downstream of Hoxb1 and provide the first example of Hox pathway transcription factors within a defined population of vertebrate motor neurons (Pata, 1999).

Retinoids are essential for normal development and both deficiency and excess of retinoic acid (RA) are teratogenic. Retinoic acid response elements (RAREs) have been identified in Hox gene promoters suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. In order to test this hypothesis, Hoxa-1 3'RARE was mutated using the Cre-loxP targeting strategy, and its functional role during mouse development was studied. The RARE enhancer plays an important role in the early establishment of the Hoxa-1 anterior expression boundary in the neural plate. This early disturbance in Hoxa-1 activation results in rhombomere and cranial nerve abnormalities, reminiscent of those obtained in the Hoxa-1 total knockout, although their severity and penetrance are lower, thus providing strong evidence for direct control of Hox gene function by retinoids during normal development. Interestingly, the Hoxa-1 expression response to RA treatment is not entirely controlled by the RARE, suggesting the existence of other retinoid-induced factors mediating the Hoxa-1 response to RA and/or the presence of additional RAREs. Although the RARE is not required for the spatiotemporal control of colinear expression of the Hoxa genes, it is absolutely required for correct Hoxa-2 expression in rhombomere 5 (Dupe, 1997).

In the developing vertebrate hindbrain Hoxa1 and Hoxb1 play important roles in patterning segmental units (rhombomeres). In this study, genetic analysis of double mutants demonstrates that both Hoxa1 and Hoxb1 participate in the establishment and maintenance of Hoxb1 expression in rhombomere 4 through auto- and para-regulatory interactions. The generation of a targeted mutation in a Hoxb1 3' retinoic acid response element (RARE) shows that it is required for establishing early high levels of Hoxb1 expression in neural ectoderm. Double mutant analysis with this Hoxb13'RARE allele and other targeted loss-of-function alleles from both Hoxa1 and Hoxb1 reveals synergy between these genes. In the absence of both genes, a territory appears in the region of r4, but the earliest r4 marker, the Eph tyrosine kinase receptor EphA2, fails to be activated. This suggests a failure to initiate rather than maintain the specification of r4 identity and defines new roles for both Hoxb1 and Hoxa1 in early patterning events in r4. This genetic analysis shows that individual members of the vertebrate labial-related genes have multiple roles in different steps governing segmental processes in the developing hindbrain (Studer, 1998).

The transcription factor genes Hoxa1 and Krox-20 have been shown to play important roles in vertebrate hindbrain segmentation: Hoxa1 is required for maintenance and/or generation of parts of r4 and r5, and Krox-2 plays a similar role for r3 and r5. Evidence is presented for novel functions of these genes, which co-operate in specifying cellular identity in rhombomere (r) 3. Although Hoxa1 has not been observed to be expressed rostrally to the prospective r3/r4 boundary, its inactivation results in (1) the appearance of patches of cells presenting an r2-like molecular identity within r3; (2) early neuronal differentiation in r3, normally characteristic of even-numbered rhombomeres, and (3) abnormal navigation of r3 motor axons, similar to that observed in even-numbered rhombomeres. These phenotypic manifestations become more severe in the context of the additional inactivation of one allele of the Krox-20 gene, demonstrating that Hoxa1 and Krox-20 synergize in a dosage-dependent manner to specify r3 identity and odd- versus even-numbered rhombomere characters. In addition, these data suggest that the control of the development of r3 may not be autonomous but dependent on interactions with Hoxa1-expressing cells. It is thought that a signal originating from r4 or dependent on Hoxa1 function would prevent the proliferation of the early Krox-20-negative cells present in r3. One of the roles of Krox-20 would be to counteract this signal in an autonomous manner and allow for normal proliferation (Helmbacher 1998).

Functional synergy between Hoxa1 and Hoxb1 has been investigated. The generation of Hoxa13'RARE/Hoxb13'RARE compound mutants results in mild facial motor nerve defects reminiscent of those present in the Hoxb1 null mutants. Strong genetic interactions between Hoxa1 and Hoxb1 were uncovered by introducing the Hoxb13'RARE and Hoxb1 null mutations into the Hoxa1 null genetic background. Hoxa1null/Hoxb13'RARE and Hoxa1null/Hoxb1null double homozygous embryos show additional patterning defects in the r4-r6 region but maintain a molecularly distinct r4-like territory. Neurofilament staining and retrograde labelling of motor neurons indicate that Hoxa1 and Hoxb1 synergize in patterning the VIIth through XIth cranial nerves. The second arch expression of neural crest cell markers is abolished or dramatically reduced, suggesting a defect in this cell population. Strikingly, the second arch of the double mutant embryos involutes by 10.5 dpc; this results in the loss of all second arch-derived elements and complete disruption of external and middle ear development. Additional defects, most notably the lack of tympanic ring, are found in first arch-derived elements, suggesting that interactions between first and second arch take place during development. Taken together, the results unveil an extensive functional synergy between Hoxa1 and Hoxb1 that was not anticipated based on the phenotypes of the simple null mutants (Gavalas, 1998).

The inner ear is a complex sensory organ responsible for balance and sound detection in vertebrates. It originates from a transient embryonic structure, the otic vesicle, which contains all of the information to develop autonomously into the mature inner ear. The development of the otic vesicle is reviewed here, bringing together classical embryological experiments and recent genetic and molecular data. The specification of the prospective ectoderm and its commitment to the otic fate are very early events and can be related to the expression of genes with restricted expression domains. A combinatorial gene expression model for placode specification and diversification, based on classical embryological evidence and gene expression patterns, is discussed. The formation of the otic vesicle is dependent on inducing signals from endoderm, mesoderm and neuroectoderm. Ear induction consists of a sequence of discrete instructions from those tissues that confer the final identity on the otic field, rather than a single all-or-none process. The head ectoderm develops three pairs of sensory placodes from anterior to posterior -- nose, lens and ear -- along with those placodes that generate the neurons of some cranial sensory ganglia. Homeobox genes of the Sine oculis (six) and Distal-less related Dlx families are expressed but these factors are expressed in more than one sensory placode. Specific combinations of genes rather than single gene expression thus appears to be characteristic for each placode. The important role of the neural tube in otic development is highlighted by the abnormalities observed in mouse mutants for the Hoxa1, kreisler and fgf3 genes and those reported in retinoic acid-deficient quail. Still, the nature of the relation between the neural tube and otic development remains unclear. Gene targeting experiments in the mouse have provided evidence for genes potentially involved in regional and cell-fate specification in the inner ear. The disruption of the mouse Brn3.1 gene identifies the first mutation affecting sensory hair-cell specification; mutants for the Pax2 and Nkx5.1 genes show that these two genes are required for the the development of specific regions of the otic vesicle. Several growth-factors contribute to the patterned cell proliferation of the otic vesicle. Among these, IGF-I and FGF-2 are expressed in the otic vesicle and may act in an autocrine manner. Little is known about early mechanisms involved in guiding ear innervation. However, targeted disruption of genes coding for neurotrophins and Trk receptors have shown that once synaptic contacts are established, they depend on specific trophic interactions that involve these two gene families. The accessibility of new cellular and molecular approaches are opening new perspectives in vertebrate developmental biology (Torres, 1998).

Hoxa1 and Hoxb1 have overlapping synergistic roles in patterning the hindbrain and cranial neural crest cells. The combination of an ectoderm-specific regulatory mutation in the Hoxb1 locus and the Hoxa1 mutant genetic background results in an ectoderm-specific double mutation, leaving the other germ layers impaired only in Hoxa1 function. This has allowed an examination of neural crest and arch patterning defects that originate exclusively from the neuroepithelium as a result of the simultaneous loss of Hoxa1 and Hoxb1 in this tissue. Using molecular and lineage analysis in this double mutant background it has been demonstrated that presumptive rhombomere 4, the major site of origin of the second pharyngeal arch neural crest, is reduced in size and has lost the ability to generate neural crest cells. Grafting experiments using wild-type cells in cultured normal or double mutant mouse embryos demonstrate that this is a cell-autonomous defect, suggesting that the formation or generation of cranial neural crest has been uncoupled from segmental identity in these mutants. Furthermore, loss of the second arch neural crest population does not have any adverse consequences on early patterning of the second arch. Signaling molecules are expressed correctly and pharyngeal pouch and epibranchial placode formation are unaffected. There are no signs of excessive cell death or loss of proliferation in the epithelium of the second arch, suggesting that the neural crest cells are not the source of any indispensable mitogenic or survival signals. These results illustrate that Hox genes are not only necessary for proper axial specification of the neural crest but that they also play a vital role in the generation of this population itself. Furthermore, they demonstrate that early patterning of the separate components of the pharyngeal arches can proceed independently of neural crest cell migration (Gavalas, 2001).

Formation of neuronal circuits in the head requires the coordinated development of neurons within the central nervous system (CNS) and neural crest-derived peripheral target tissues. Hoxb1, which is expressed throughout rhombomere 4 (r4), has been shown to be required for the specification of facial branchiomotor neuron progenitors that are programmed to innervate the muscles of facial expression. In this study, additional roles have been uncovered for Hoxb1-expressing cells in the formation and maintenance of the VIIth cranial nerve circuitry. By conditionally deleting the Hoxb1 locus in neural crest, it has been demonstrated that Hoxb1 is also required in r4-derived neural crest to facilitate and maintain formation of the VIIth nerve circuitry. Genetic lineage analysis reveals that a significant population of r4-derived neural crest is fated to generate glia that myelinate the VIIth cranial nerve. Neural crest cultures show that the absence of Hoxb1 function does not appear to affect overall glial progenitor specification, suggesting that a later glial function is critical for maintenance of the VIIth nerve. Taken together, these results suggest that the molecular program governing the development and maintenance of the VIIth cranial nerve is dependent upon Hoxb1, both in the neural crest-derived glia and in the facial branchiomotor neurons (Arenkiel, 2004).

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. Morpholinos were used 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, since it is shown 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 (McNulty, 2005).

Transcriptional targets of Labial homologs

Vertebrate Hox and Otx genes encode homeodomain-containing transcription factors thought to transduce positional information along the body axis in the segmental portion of the trunk and in the rostral brain, respectively. Moreover, Hox and Otx2 genes show a complementary spatial regulation during embryogenesis. A 1821-base pair (bp) upstream DNA fragment of the Otx2 gene is positively regulated by co-transfection with expression vectors for the human HOXB1, HOXB2, and HOXB3 proteins in an embryonal carcinoma cell line (NT2/D1) and a shorter fragment of only 534 bp is able to drive this regulation. The HOXB1, HOXB2, and HOXB3 DNA-binding region on the 534-bp Otx2 genomic fragment has been demonstrated using nuclear extracts from Hox-transfected COS cells and 12.5 days postcoitum mouse embryos or HOXB3 homeodomain-containing bacterial extracts. HOXB1, HOXB3, and nuclear extracts from 12.5 day mouse embryos bind to a sequence containing two palindromic TAATTA sites, which bear four copies of the ATTA core sequence, a common feature of most HOM-C/HOX binding sites. HOXB2 protects an adjacent site containing a direct repeat of an ACTT sequence, quite divergent from the ATTA consensus. The region bound by the three homeoproteins is strikingly conserved through evolution and necessary (at least for HOXB1 and HOXB3) to mediate the up-regulation of the Otx2 transcription. Taken together, the data support the hypothesis that anteriorly expressed Hox genes might play a role in the refinement of the Otx2 early expression boundaries in vivo (Guazzi, 1998).

Labial homologs and the subdivision of the brain

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. A direct gain-of-function approach was employed 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 this region is normally devoid of this cell type. Depending on the target tissue they innervate, motor neurons in the hindbrain can be classified into three different subtypes: visceromotor, branchiomotor and somatomotor. An attempt was made to determine whether misexpression of Hoxb1 leads to the induction of a particular subtype of motor neuron or rather to the induction of a generic type of motor neuron. The migratory behaviour displayed by the ectopic motor neurons in r1 indicate that they might be of the branchiomotor subtype. To test this at the molecular level, use was made of the observation that in the hindbrain only somatomotor neurons but not branchiomotor neurons express the LIM-homeobox gene Isl2. When analysed at HH25 stage, somatic trochlear and abducens motor neurons coexpress Isl1 and Isl2, whereas branchiomotor neurons express Isl1 but not Isl2. Ectopic motor neurons in r1 are never found to express Isl2 in addition to Isl1, consistent with their being of the branchiomotor subtype. This result demonstrated, therefore, that the activity of Hoxb1 was sufficient to selectively induce the generation of a distinct specified subtype of motor neurons (Jungbluth, 1999).

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. To determine the subclass of motor neurons generated, the expression profiles of Isl1 and Isl2 were examined. The ectopic motor neurons generated following Hoxa2 misexpression were found to express Isl1 but not Isl2. Therefore, as with Hoxb1, Hoxa2 misexpression also resulted in the generation of motor neurons of branchiomotor subtype identity, as shown by their lateral migration behaviour and by their Isl gene expression patterns. Next, an attempt was made to identify the precise branchiomotor subtypes that are generated after misexpression: the results suggest that the ectopic motor neurons generated following Hoxa2 misexpression are trigeminal-like, while those generated following Hoxb1 misexpression are facial-like. The data demonstrate 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, since these particular motor neuron subtypes are normally generated in the most anterior domains of Hoxa2 and Hoxb1 expression, respectively, the 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 (Jungbluth, 1999).

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, mice simultaneously mutant at both loci were generated. 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 (Barrow, 2000).

Hoxa1 and Hoxb1 are coexpressed up to the presumptive r3/4 boundary. Hoxa1 is required to establish Hoxb1 expression in anterior r4. Hoxa1 and Hoxb1 activate the transcription of r4-specific downstream targets including a signal that, in turn, induces Krox 20 expression in cells just anterior to the r3/4 boundary (in cells that are not expressing Hoxa1 or Hoxb1). Krox 20 is repressed, however, in r4 and r5 cells that are expressing Hoxa1 and Hoxb1. Hoxa1 is required for kreisler expression in r5. Without Hoxa1, the anterior limit of Hoxb1 is established in the posterior region of r4. Because of this posterior shift, neither Hoxa1 nor Hoxb1 is expressed in the anterior portion of r4 and Krox 20 is no longer repressed there. Furthermore, the signal downstream of Hoxb1 must be propagated a longer distance causing a delay in the induction of Krox 20 expression in presumptive r3. Due to the absence of Hoxa1, kreisler expression is not activated in r5 (Barrow, 2000).

Without Hoxa1 and Hoxb1 expression, Krox 20 expression is no longer repressed in r4 and r5. In addition, the signal downstream from Hoxa1 and Hoxb1 required to induce Krox 20 expression in r3 is not activated. By E8.5, Hoxa1 expression has completely retreated from the hindbrain. Hoxb1 has also retreated with the exception of the strong autoregulatory expression in r4. Once Hoxa1 and Hoxb1 expression has fully retreated from r5, Krox 20 expression commences at this level. Krox 20 expression also expands into r3. This expansion requires activation of its downstream target(s) Hoxa2 and possibly Hoxb2. Strong kreisler expression in r5 maintains Hoxb1 autoregulated expression at the r4/5 boundary. In Hoxa1 mutants Hoxb1 expression retreats from the caudal hindbrain leaving autoregulated expression in caudal r4. Because kreisler is not activated in r5, autoregulated Hoxb1 expression extends into r5 as well. Krox 20 expansion into r3 although delayed (due to the fewer number of cells that were induced at E8.0) occurs somewhat normally due to the fact that Krox 20 and its downstream target(s) Hoxa2 (and perhaps Hoxb2) are functioning. As a consequence of the larger expression domains of follistatin (r2 and part of r3) and Krox 20 (part of r3 and r4), a regulatory event driven by apoptosis commences in these regions of the neural tube. The hindbrain is similar to that of Hoxa1 single mutants except that Krox 20 expansion into r3 is severely delayed. Hoxa2 is a downstream target of Krox 20 and if absent, cripples the expansion of Krox 20-expressing cells into r3. In double Hoxa1/Hoxa2 mutants Krox 20 is never induced in r3 and thus never expands into r3. As a result, follistatin expression extends to the r3/r4 boundary. Due to enlarged follistatin and Krox 20-expressing domains, apoptosis is activated in the neural tube at this level. Due to the apoptosis at the levels of r2 and r3 in HoxA1 mutants, there is not only a reduction in the number of neural crest cells that will populate the first arch, but also the abnormally large r3 is reduced to almost normal proportions. There is also a reduction in the number of neural crest cells that reach the second arch due to the reduced size of r4 and the fact that the otocyst may act as a barrier to prevent normal migration of the crest. The otocysts do not shift anteriorly to the level of r4; instead, r4 is specified more posteriorly. Double HoxA1/HoxA2 mutants are very similar to Hoxa1 single mutants except that, due to the lack of Hoxa2, the r4 neural crest takes on an r1/r2 identity. In addition, the lack of Hoxa1 causes a reduction in r4 neural crest contributing to the second arch. In HoxA1/HoxA2 double mutants r4 is never specified. Therefore, there is no r4 neural crest to populate the second arch (Barrow, 2000).

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). vhnf1 functions in two ways to subdivide the zebrafish caudal hindbrain domain (r4-r7) into individual rhombomeres: (1) vhnf1 promotes r5+r6 identity through an obligate synergy with Fgf signals to activate valentino and krox20 expression; (2) 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 (Wiellette, 2003).

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, neurogenesis, neuronal differentiation and motoneuron migration were examined in Hoxa1, Hoxb1 and Hoxb2 mutant mice. This comparison revealsthat neurogenesis and differentiation of specific neuronal subpopulations in r4 are 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 are re-specified towards an r2-like identity. Motoneurons derived from that territory resemble trigeminal motoneurons in both their migration patterns and the expression of molecular markers. Both migrating motoneurons and the resident territory undergo changes consistent with a switch from an r4 to r2 identity. Abnormally migrating motoneurons initially form ectopic nuclei that are subsequently cleared. Their survival can 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, the functional requirements in hindbrain neuronal patterning that follow the establishment of the genetic regulatory hierarchy between Hoxa1, Hoxb1 and Hoxb2 have been functionally delineated (Gavalas, 2003).

Rhombomeric and neuronal patterning defects are milder in Hoxb2 mutants, compared with Hoxb1–/– embryos. Furthermore, there are no r4-derived phenotypes in Hoxb2 mutants that are not detected in Hoxb1 mutants. This is consistent with Hoxb2 being a direct transcriptional target of Hoxb1 and raises a number of possibilities concerning the precise regulatory and functional relationships between Hoxb1 and Hoxb2. Hoxb2 may act synergistically with Hoxb1 by regulating either distinct target genes or a set of common target genes in r4, so that their combined activities are required for the normal differentiation of r4-derived motoneurons. An alternative mechanism whereby Hoxb2 may synergise with Hoxb1 would be through a role for Hoxb2 in maintaining Hoxb1 expression (Gavalas, 2003).

To begin to distinguish between these possibilities, the r4 status was monitored in the Hoxb2 mutants by assaying Hoxb1 expression and the expression of a the r4-specific transgene (HL5/lacz), which is known to be a direct target of Hoxb1. Endogenous Hoxb1 expression and staining for the HL5/lacZ transgene are initiated in the r4 of Hoxb2 mutants but are not maintained at appropriate levels in later stages. This demonstrates a direct or indirect requirement for Hoxb2 in maintaining Hoxb1 expression in r4. The observation that Hoxb1 expression is initiated normally in Hoxb2 mutants, but is not maintained properly could explain the mixed behavior of facial motoneurons. Those r4 motoneuron progenitors that retain sufficient Hoxb1 activity adopt a normal fbm identity, while the rest adopt trigeminal motoneuron characteristics. The idea is favored that the effect of Hoxb2 on Hoxb1 expression is most probably indirect, through regulation of general aspects of r4 identity. Hoxb2 cannot bind the Hoxb1 r4 regulatory element in vitro, although it is possible that Hoxb2 may bind to an as yet unidentified Hoxb1 r4 regulatory element. In vivo, ectopic expression of Hoxb2 does not ectopically activate Hoxb1, whereas Hoxb1 does transactivate Hoxb2 (Gavalas, 2003).

Little is known about the molecular mechanisms that integrate anteroposterior (AP) and dorsoventral (DV) positional information in neural progenitors that specify distinct neuronal types within the vertebrate neural tube. In ventral rhombomere (r)4 of Hoxb1 and Hoxb2 mutant mouse embryos, Phox2b expression is not properly maintained in the visceral motoneuron progenitor domain (pMNv), resulting in a switch to serotonergic fate. Phox2b has been shown to be a direct target of Hoxb1 and Hoxb2. A highly conserved Phox2b proximal enhancer has been found that mediates rhombomere-restricted expression and contains separate Pbx-Hox (PH) and Prep/Meis (P/M) binding sites. Both the PH and P/M sites are essential for Hox-Pbx-Prep ternary complex formation and regulation of the Phox2b enhancer activity in ventral r4. Moreover, the DV factor Nkx2.2 enhances Hox-mediated transactivation via a derepression mechanism. Induction of ectopic Phox2b-expressing visceral motoneurons in the chick hindbrain requires the combined activities of Hox and Nkx2 homeodomain proteins. This study takes an important first step to understand how activators and repressors, induced along the AP and DV axes in response to signaling pathways, interact to regulate specific target gene promoters, leading to neuronal fate specification in the appropriate developmental context (Samad, 2004)

Sequencing of the Phox2b enhancer revealed the presence of a putative bipartite PH-binding site (TGATTGAA). Notably, its nucleotide sequence was identical to that of the low-affinity PH binding site of repeat 2 (R2) of the Hoxb1 autoregulatory (b1-ARE) r4 enhancer. Moreover, it shared fairly high conservation with the PH site present in the Hoxb2 r4 enhancer, also regulated by Hoxb1. Similar to the Hoxb1 and Hoxb2 r4 enhancers, a conserved P/M site (TTGTCATG), was found downstream of the PH site. The Phox2b P/M site and its flanking nucleotides exactly matched the sequence found in the Hoxb1 r4 enhancer and shared six out of eight nucleotides with that in the Hoxb2 r4 enhancer. Interestingly, unlike the previously identified PH and P/M sites lying in relative proximity to each other, the Phox2b P/M site was 147 nucleotides distant from the PH site (Samad, 2004).

There are functional differences between PH-P/M modules in the Phox2b and other Hox-regulated r4 enhancers. Similar to the Hoxb1 and Hoxb2 r4 enhancers, separate PH and P/M sites were found embedded within the Phox2b enhancer. Nevertheless, the in vivo output of Hox regulation on these three enhancers is rather different, since the Phox2b PH or P/M sites mediate a transcriptional response restricted to ventral progenitors, despite widespread Hoxb1 and Hoxb2 distribution throughout r4. This is in keeping with the observation that endogenous Phox2b expression is upregulated in sharp columns of selected progenitor domains at distinct DV levels. Comparing the nature and function of bipartite PH and P/M sites in the context of the Hoxb1, Hoxb2 and Phox2b enhancers may therefore provide clues of how Phox2b regulation is spatially constrained (Samad, 2004).

In the Hoxb2 enhancer, only one PH site is present that shows cooperative binding of Hoxb1 and Pbx/Exd proteins in vitro and is required for r4 expression in vivo. By contrast, the Hoxb1 autoregulatory (b1-ARE) r4 enhancer contains three PH motifs (R1-R3). Mutational analysis in the mouse indicates that all three PH sites are cooperatively required for high levels of r4 expression, although with distinct individual contributions. Among the three Hoxb1 PH sites, the R2 sequence precisely matches that of the Phox2b PH octamer core. Like the Phox2b PH site, the R2 repeat did not bind Hoxb1/Exd heterodimers in vitro, nor Hoxb1 or Exd alone, although it is necessary for optimal r4 activity. Thus, the Hoxb1 R2 repeat requires cooperative interactions with adjacent sequences in the b1-ARE to fully function in vivo. Similarly, a trimerized Phox2b PH site is not sufficient on its own to direct r4 restricted expression in the chick hindbrain, unlike the sufficiency for r4 expression of multimerized Hoxb1 R3 or Hoxb2 high-affinity PH sites. Nonetheless, the PH motif is necessary, in the context of the Phox2b enhancer, for mediating the transcriptional cooperation of Hox, Pbx and Prep/Meis co-factors and for in vivo regulation in ventral r4 both in chick and mouse hindbrain. Thus, the Phox2b low-affinity PH site, while representing a necessary site of integration of r4 activity, operates in vivo mainly through cooperative interactions with its surrounding regulatory environment, even in the presence of high endogenous levels of binding factors (Samad, 2004).

Rostral hindbrain patterning involves the direct activation of a Krox20 transcriptional enhancer by Hox/Pbx and Meis factors

The morphogenesis of the vertebrate hindbrain involves the generation of metameric units called rhombomeres (r), and Krox20 encodes a transcription factor that is expressed in r3 and r5 and plays a major role in this segmentation process. Knowledge of the basis of Krox20 regulation in r3 is rather confusing, especially concerning the involvement of Hox factors. This paper describes a study of one of the Krox20 hindbrain cis-regulatory sequences, element C, which is active in r3-r5 and which is the only initiator element in r3. Element C is shown to contains multiple binding sites for Meis and Hox/Pbx factors; these proteins synergize to activate the enhancer. Mutation of these binding sites showed that Krox20 is under the direct transcriptional control of both Meis (presumably Meis2) and Hox/Pbx factors in r3. Furthermore, the data indicate that element C functions according to multiple modes, in Meis-independent or -dependent manners and with different Hox proteins, in r3 and r5. Finally, it was shown that the Hoxb1 and Krox20 expression domains transiently overlap in prospective r3, and that Hoxb1 binds to element C in vivo, supporting a cell-autonomous involvement of Hox paralogous group 1 proteins in Krox20 regulation. Altogether, these data clarify the molecular mechanisms of an essential step in hindbrain patterning. A model is proposed for the complex regulation of Krox20, involving a novel mode of initiation, positive and negative controls by Hox proteins, and multiple direct and indirect autoregulatory loops (Wassef, 2008).

Krox20 regulation appears to constitute a complex process and this study attempted to amalgamate the observations collected in the present work with previous data to develop a molecular model. The consistent observations in mouse, chick and zebrafish allow combination of data obtained in different vertebrate species. First the regulation in r3 will be envisaged. It is proposed that, in contrast to what was previously thought, at around E8 in the mouse, when Hoxa1/Hoxb1 neural domains reach their maximal rostral extensions, their limits are located within prospective r3. This point is consistent with recent tracing data indicating that derivatives of Hoxa1-expressing cells are found in r3, and is supported by the observation of an overlap between Krox20 and Hoxb1 expression domains in r3. In addition, the existence of another factor (X, unknown) is postulated, whose expression domain extends caudally and will start to overlap with the Hox paralog group (PG) 1 domain around E8. This defines a transversal, narrow stripe of cells where Krox20 is specifically activated under the synergistic transcriptional activities of factor X, Hox PG 1, Pbx and Meis2 proteins, acting through element C. Interestingly, an essential role of Iroquois transcription factors in the activation of krox20 in r3 has been recently uncovered. Factor X might therefore be an Iroquois transcription factors or it might lie downstream to them in the regulatory cascade. A complementary involvement of Hox PG 2 proteins is also likely, although loss-of-function analyses suggest that the major role is played by PG 1 factors. An important feature of this hypothesis is that it provides an explanation for the characteristic initial expression pattern of Krox20, restricted to a very narrow stripe of cells. Krox20 activation will have multiple consequences. (1) It will lead to the progressive retraction of the rostral limit of Hox PG 1 gene expression to the future r3/r4 boundary. This is consistent with the observations that the Hoxb1-positive domain extends within prospective r3 in a Krox20-null mutant and that ectopic Krox20 expression results in Hoxb1 repression. (2) Krox20 initiates several transcriptional autoregulatory loops that are necessary for the maintenance of its own expression. One of them is direct and relies on the binding of Krox20 to element A, whereas the others involve the activation of Hoxa2 and Hoxb2, which will replace Hox PG 1 proteins on element C. These autoregulatory mechanisms are likely to be redundant, as the double mutation of Hoxa2 and Hoxb2 only marginally affects the r3 domain of Krox20 expression. (3) Expression of Krox20 also results in its activation in neighbouring Krox20-negative cells by non-cell autonomous autoregulation, a process thought to participate in the extension of r3. The caudal extension of r3 might also rely on the progression of the front of gene X expression. These processes will give rise to a moving stripe of cells co-expressing Krox20 and Hoxb1 at the caudal edge of developing r3, as was observed in mouse and zebrafish embryos. At some point (around E8.5), these processes of extension of r3 at the expense of adjacent rhombomeres will stop, delimiting the final extensions of r2, r3 and r4 (Wassef, 2008).

In r5, Krox20 is under the control of two initiation enhancer elements, B and C. The severe loss of Krox20 expression in r5 upon mutation of Mafb or vHnf1, and the fact that these factors are likely to act only via element B, suggests that element B is predominant. In r5, element C functions according to a different mode than in r3: although it still requires binding of a Hox protein, Meis factors are not necessary (Wassef, 2008).

Finally, what happens in r4, where element C is active but Krox20 is not expressed? To explain this apparent contradiction, it is proposed that Krox20, in addition to the positive regulatory mechanisms discussed above, is subject to a negative regulation, which may lie downstream of the Hox PG 1 genes and prevent Krox20 expression in r4. The existence of such a negative regulation is consistent with the inactivation of Hoxa1, which results in an extension of the anterior domain of Krox20 into prospective r4, and with the repressive activity of Nlz family members on Krox20 expression (Wassef, 2008).

In conclusion, a particularly interesting feature of this model resides in the initial phase of Krox20 expression in r3. It is proposed that a narrow band of cells is defined by the encounter of two domains extending in opposite directions. In these cells, Krox20 is very transiently activated by Hox PG 1 proteins, which disappear rapidly while Krox20 expression is maintained and propagated by different molecular mechanisms. It is proposed to use the term 'ignition' to refer to the role of Hox PG 1 proteins in this novel type of initiation of gene expression, which may occur in other developmental processes (Wassef, 2008).

Labial homologs responsiveness to FGF and retinoic acid

Initiation of Hox genes requires interactions between numerous factors and signaling pathways in order to establish their precise domain boundaries in the developing nervous system. There are distinct differences in the expression and regulation of members of the Hox gene family within a complex, suggesting that multiple competing mechanisms are used to initiate Hox gene expression domains in early embryogenesis. In this study, by analyzing the response of HoxB genes to both RA and FGF signaling in neural tissue during early chick embryogenesis (HH stages 7-15), two distinct groups of Hox genes have been defined based on their reciprocal sensitivity to RA or FGF during this developmental period. The sharp reciprocal transition from RA to FGF responsiveness in moving from the 3' (Hoxb1 to Hoxb5) to the 5' (Hoxb6-Hoxb9) Hox genes is surprising. In mouse the 3' Hox genes do not respond uniformly to RA treatment, since there is a progressive temporal shift in their competence or ability to respond to RA during gastrulation, such that successively more 5' genes respond in later time windows. Hence, it had been suggested that the most posterior 5' Hox genes might also be progressively sensitive to RA in later stages at the end of or after gastrulation. The expression domain of 5' members from the HoxB complex (Hoxb6-Hoxb9) can be expanded anteriorly in the chick neural tube up to the level of the otic vesicle following FGF treatment and these same genes are refractory to RA treatment at these stages (Bel-Vialar, 2002).

Labial homologs and Hox gene colinearity

Transposition of Hoxd genes to a more posterior (5') location within the HoxD complex suggests that colinearity in the expression of these genes is due, in part, to the existence of a silencing mechanism originating at the 5' end of the cluster and extending toward the 3' direction. To assess the strength and specificity of this repression, as well as to challenge available models on colinearity, a Hoxb1/lacZ transgene was inserted within the posterior HoxD complex, thereby reconstructing a cluster with a copy of the most anterior gene inserted at the most posterior position. Analysis of Hoxb1 expression after ectopic relocation reveals that Hoxb1-specific activity in the fourth rhombomere is totally abolished. Treatment with retinoic acid, or subsequent relocations toward more 3' positions in the HoxD complex, does not release this silencing in hindbrain cells. In contrast, however, early and anterior transgene expression in the mesoderm is unexpectedly not suppressed. Furthermore, the transgene induces a transient ectopic activation of the neighboring Hoxd13 gene, without affecting other genes of the complex. Such a local and transient break in colinearity has also been observed after transposition of the Hoxd9/lacZ reporter gene, indicating that it may be a general property of these transgenes when transposed at an ectopic location (Kmita, 2000).

A surprising finding in this study, which contrasts with the suppression of r4-specific transcription, is that expression of the relocated Hoxb1 transgene in presomitic and somitic mesoderm is not dramatically affected by its new genomic location, as if the surrounding HoxD environment has little impact on its regulation in this tissue. Furthermore, the transposed Hoxb1, whose expression is anteriorized, causes changes in Hoxd13 since Hoxd13 is ectopically expressed, in a manner following a Hoxb1-like pattern and well before 5' Hoxd genes are normally activated. This is not seen if the selection cassette is left in place, indicating that this cassette can behave as a potent enhancer block. Interestingly, premature and anterior activation is restricted to Hoxd13 and does not affect other Hoxd genes. Therefore, Hoxb1 induces a local break in both spatial and temporal colinearities rather than a global change in expression (Kmita, 2000).

Hoxd9/lacZ recombined at the same position, after the PGK-neo cassette has been removed, also induces the neighboring Hoxd13 gene to be expressed too early and anteriorly, in a way somewhat reminiscent of endogenous Hoxd9 expression. However, even though Hoxd13 regulation is clearly perturbed, the Hoxd9 transgene itself, unlike Hoxb1, is not expressed until all posterior Hoxd genes have been activated. This suggests that the integration of this foreign locus near Hoxd13 leads to the deregulation of this latter gene before the transgene itself is activated. In both cases, premature activation of Hoxd13 as well as the observed early expression of Hoxb1/lacZ are at odds with a model whereby a repression over the posterior HoxD complex would prevent any 5'-located genes from being activated early on. Therefore, whereas the block in r4 expression is consistent with this view, an extreme interpretation of this model whereby repression occurs in all tissues and genes through the existence of a tight global silencing mechanism seems unlikely, and a more dynamic mechanism, capable of some discrimination between tissues and, perhaps, activating and maintenance complexes must be operating (Kmita, 2000).

One possibility is that factors necessary for Hoxb1 activation can recognize the ectopic locus and they access the Hoxb1 promoter by inducing a local opening within an otherwise refractory structure. Because of the proximity of the Hoxd13 promoter, this latter gene would also be activated, either through the Hoxb1 machinery itself or as a response to posterior factors that would normally not have access to the locus but which, in this particular context, would take advantage of a local disorganization. In this model (Model I), cells currently engaged in Hox gene activation would, over time, make posterior genes progressively accessible to transcription. Cells with a given state of Hox gene activation, that is, cells that have reached a particular AP level as a result of gastrulation, would leave this mode of activation and maintain their current state by the same silencing system, preventing posterior genes from being activated. In the case in which a transgene is introduced, factors normally required for the transcription of the endogenous copies could locally unwind the repressive structure and allow for transcription to occur for both the transgene and the nearby located Hox gene (Kmita, 2000).

In this scheme, transcriptional activation is tightly linked to the linear retraction of the repressive mechanism, and silencing over the ectopic transgene would be competed by transgene-specific activating complexes. Under this model, expression mediated by the r4 enhancer does not occur, either due to the lack of access, or the weak remodeling capacity of the Hox/Pbx complex. Another potential explanation is that Hox gene activation in mesoderm strictly depends on combinations or gradients of upstream regulators acting in cis, without any contribution of a higher type of regulation. In such a view, clustering would have no critical function in the time course of activation. Whereas this simple scheme explains several results obtained by conventional transgenesis, it fails to account for observations made on recombining the HoxD complex. This possibility is therefore considered as unlikely (Kmita, 2000).

Alternatively, silencing may occur transiently and exclusively in those cells in which Hox gene activation occurs, that is, in a restricted cellular population during gastrulation. In this view, although cells activate their Hox genes through a linear release of silencing, subsequent maintenance would rely on the interplay between gene-specific enhancer sequences (Model II). Accordingly, particular states of activation would be maintained without the requirement for a high-order type of regulation, for example, by relying on cross- and auto-regulation as well as the action of local enhancers. In this context, it is possible that the Hoxb1 transgene, even though expressed early on, is properly silenced during the activation phase. However, it might subsequently be able to respond to a maintenance phase before posterior Hoxd's have been activated, through the early enhancers that were transferred together with the relocated locus. The difference between Models I and II is that the transgene would respond either to genuine activating signals (Model I) or to the maintenance machinery (Model II). Nevertheless, both models imply that Hox transgenes carry with them regulatory information regarding their expression in a defined domain and at a defined developmental stage. As it is difficult to discriminate between regulatory elements responding to either cross- or auto-regulatory loops (directly or indirectly) and elements responding to the original activating signal, the activation versus maintenance question is difficult to address. A genetic approach should help in investigating this problem, but the redundancy of the system requires that multiple inactivation strategies be implemented (Kmita, 2000).

Ectopic nuclear reorganisation driven by a Hoxb1 transgene transposed into Hoxd

The extent to which the nuclear organisation of a gene impacts on its ability to be expressed, or whether nuclear organisation merely reflects gene expression states, remains an important but unresolved issue. A model system that has been instrumental in investigating this question is the murine Hox clusters. Nuclear reorganisation and chromatin decondensation, initiated towards the 3' end of the clusters, accompanies activation of Hox genes in both differentiation and development, and may be linked to mechanisms underlying colinearity. To investigate this, and to delineate the cis-acting elements involved, the nuclear behaviour was used of a 3' Hoxb1 transgene transposed to the 5' end of the Hoxd cluster. This transgene contains the cis-acting elements sufficient to initiate ectopic local nuclear reorganisation and chromatin decondensation, and to break Hoxd colinearity, in the primitive streak region of the early embryo. Significantly, in rhombomere 4 the transgene is able to induce attenuated nuclear reorganisation and decondensation of Hoxd even though there is no detectable expression of the transgene at this site. This shows that chromosome territory reorganisation and chromatin decondensation can be uncoupled from transcription itself, and suggests that they can therefore operate upstream of gene expression (Morey, 2008).

Several facets of nuclear organisation have been correlated with gene expression at both constitutively active regions of the genome and at regions subject to co-ordinate regulation. Firstly, when active, these regions appear decondensed at a cytological level. Secondly, even though transcription and transcription factories can be seen within chromosome territories (CTs), many active genomic loci have been seen outside of their CTs. A key issue remains whether these forms of nuclear organisation are just a consequence of transcriptional activation or whether they have a causative role. The fact that the incidence of localisation outside of CTs decreases when transcription is blocked suggests that relocalisation is, at least in part, driven by transcription itself. On the other hand, looping out from the CT is not just a downstream consequence of a specific gene's activation. Along the primary (rostro-caudal) axis of the developing mouse embryo, activation of murine Hoxb and Hoxd loci is accompanied by both their decondensation and looping out from the CT. However, in the limb bud, Hoxd activation and chromatin decondensation occur without relocalisation of this locus to the outside of its CT. It was suggested that this difference in nuclear behaviour of the same locus, when it is activated in different developmental contexts, maybe due to the different regulatory pathways and cis-acting sequences acting upon it (Morey, 2007; Morey, 2008 and references therein).

This latter example illustrates how useful the study of the Hox gene clusters is in providing insights into the dynamic repositioning of a locus in the nucleus during spatial and temporal patterning of gene expression. Within mammalian Hox clusters there is a correspondence between the linear order of the genes and their sequence of activation in development: colinearity. Genes located at the 3' end of the clusters are expressed earlier, and more anteriorly, than genes located more 5'. It has been proposed that a transition from an inactive to an active chromatin state, propagated through Hox clusters from 3' to 5', might underlie colinear activation. The relocalisation of Hox genes outside of their CTs does indeed initiate toward the 3' end of the clusters (Morey, 2008).

Transgenic experiments have revealed both local and more distant cis-regulatory elements involved in the control of Hox gene expression. Manipulation of mouse Hox clusters, transferring genes from one position to another, has also indicated that part of this regulation depends on the position of a given gene in the cluster. To determine whether cis-acting elements responsible for initiating large-scale changes in chromatin structure are located at the 3' end of Hox clusters, this study analysed the nuclear behaviour of the anterior (3') Hoxb1 gene transposed to the posterior (5') end of Hoxd. Transgenic mouse embryos were used carrying a Hoxb1/LacZ reporter inserted by homologous recombination upstream of Hoxd13. This transgene has the regulatory elements necessary for its autonomous expression when randomly integrated in the genome, including 5' and 3' retinoic acid response elements (RAREs) and the rhombomere 4 (r4) autoregulatory enhancer. When transposed this transgene breaks the colinearity of Hoxd, inducing ectopic Hoxd13 expression in the primitive streak region (PS) of E7.5 embryos in a manner reminiscent of endogenous Hoxb1 expression. However, ectopic activation of the Hoxb1/lacZ transgene occurs in the distal part of the E10.5 developing limb bud, where Hoxb1 is not normally expressed. Conversely expression of the transgene is absent in r4 of the E9.5 embryo, a site where both endogenous Hoxb1 and Hoxb1/lacZ transgenes inserted randomly into the genome, are expressed (Morey, 2008).

Using fluorescence in situ hybridisation (FISH) on wild-type and transgenic embryos, this study shows that the transposed Hoxb1/LacZ recapitulates some of the behaviour of endogenous Hoxb1 in the PS of E7.5 embryos, suggesting that the transgene contains the minimal DNA elements necessary to initiate nuclear reorganisation and chromatin decondensation early in embryonic development, and that these elements can initiate nuclear reorganisation at an inappropriate genomic location (the 5' end of Hoxd) that is normally still silent at this early stage of embryogenesis. Moreover, the transgene can also initate these movements when ectopically activated in the limb bud later in development, where the rest of Hoxd does not loop out from the CT even though the Hoxd cluster is now active. In both of these cases (early embryo and limb bud) the nuclear organisation of the transposed Hoxb1/Lacz transgene completely correlates with its expression, so that transcription and nuclear organisation cannot be uncoupled from one another. However, in r4, the transgene is able to induce some attenuated, but still significant, nuclear reorganisation and chromatin decondensation of surrounding Hoxd regions, even though it is not itself detectably expressed. This suggests that chromosome territory reorganisation is not, in this case, simply a downstream consequence of transcription of the transgene (Morey, 2008).

This paper has shown the contrasting behaviours of a Hoxb1/lacZ transgene, which has been transposed into the 5' end of the Hoxd locus, at different embryonic stages and in different tissues. In E7.5, the transgene is able to recapitulate some of the nuclear reorganisation and chromatin condensation seen at the endogenous Hoxb1 locus. This indicates that the transgene contains the cis-acting elements needed to initiate these changes in higher-order chromatin structure during gastrulation, even though it is embedded in a Hoxd locus that is normally still silent at this stage. Whether the elements in the transgene responsible for chromatin decondensation are the same as those involved in CT reorganisation remains to be determined. The observation that looping out from the CT is restricted to the vicinity of the transgene, but that transgene-induced chromatin decondensation spreads to adjacent genomic regions, further serves to illustrate the fact that these two facets of nuclear reorganisation can be independent of one another (Morey, 2007). Interestingly, ectopic gene expression in the PS of the transgenic embryos is limited to the Hoxd13 gene immediately adjacent to the transgene, suggesting that this may be a consequence of the nuclear repositioning of Hoxb1/LacZ (Morey, 2008).

It has been shown that the normal Hoxd region, when activated in the E9.5 limb bud, decondenses but does not loop out from its CT. Therefore it is surprising in this study to find that the Hoxb1lacZ transgene does loop out from the CT in the limb bud. These results show that the transgene does not contain any DNA elements preventing its nuclear reorganisation and chromatin decondensation in tissues where Hoxb1 is normally silent, and that the absence of Hoxd looping out from the CT in the limb bud is not, as has been previously suggested just due to the pathway activating Hoxd expression in this tissue, but that it is also dependent on the sequence of the Hoxd locus itself (Morey, 2008).

So far these experiments have shown that the nuclear behaviour of the transposed transgene cannot be dissociated from its pattern of gene expression. In the r4 hindbrain region of E9.5 embryos, although the extensive looping out and chromatin decondensation seen at endogenous Hoxb1 are attenuated at the transgene, coincident with the suppression of its expression here, a discrete effect was noticed of the transgene on adjacent Hoxd regions, which are relocated significantly more toward the outside of the CT, and are more decondensed, in r4 of transgenic embryos than are these regions in wild-type embryos. Interestingly, this effect was more pronounced toward the 3' side of the transgene where the early mesoderm enhancer and the Hoxd cluster are located. This result suggests that the transgene, which does contain the regulatory elements necessary for its autonomous expression in r4 when randomly integrated in the genome, including the rhombomere 4 (r4) autoregulatory enhancer, contains the elements that can initiate some degree of nuclear reorganisation at 5' Hoxd in r4 even in the absence of expression, and that it is likely that the chromatin environment of the rest of Hoxd in this tissue prevents subsequent events that are necessary for gene expression per se (Morey, 2008).

The nuclear re-organisation at the transposed Hoxd locus in r4 therefore suggests that chromatin decondensation, and looping out from the CT, can occur upstream of gene expression and so are unlikely to just be passive consequences of, for example, RNA polymerase II activity (though it cannot be excluded that there may be non-coding transcription from the transgene). Similarly, a recent study has shown that decondensation and looping out from the CT of the human major histocompatibility complex, stimulated by interferon-γ via the JAK/STAT signalling pathway, also occurs upstream of transcriptional activity. Interestingly, given the presence of RAREs on the Hox1/LacZ transgene described in this study, this was correlated with the recruitment of the chromatin remodelling enzyme Brg1. Brg1 has been shown to interact with nuclear hormone receptors. A better understanding of the molecular mechanisms driving nuclear reorganisation and chromatin decondensation of the Hoxb and d loci at different stages of development, are now required (Morey, 2008).

Hox genes define distinct progenitor sub-domains within the second heart field

Much of the heart, including the atria, right ventricle and outflow tract (OFT) is derived from a progenitor cell population termed the second heart field (SHF) that contributes progressively to the embryonic heart during cardiac looping. Several studies have revealed anterior-posterior patterning of the SHF, since the anterior region (anterior heart field) contributes to right ventricular and OFT myocardium whereas the posterior region gives rise to the atria. Retinoic Acid (RA) signal has been shown to participate to this patterning. This study shows that Hoxb1, Hoxa1, and Hoxa3, as downstream RA targets, are expressed in distinct sub-domains within the SHF. Genetic lineage tracing analysis revealed that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells contribute to both atria and the inferior wall of the OFT, which subsequently gives rise to myocardium at the base of pulmonary trunk. By contrast to Hoxb1Cre, the contribution of Hoxa1-enhIII-Cre and Hoxa3Cre-labeled cells is restricted to the distal regions of the OFT suggesting that proximo-distal patterning of the OFT is related to SHF sub-domains characterized by combinatorial Hox genes expression. Manipulation of RA signaling pathways showed that RA is required for the correct deployment of Hox-expressing SHF cells. This report provides new insights into the regulatory gene network in SHF cells contributing to the atria and sub-pulmonary myocardium (Bertrand, 2011).

The four-chambered mammalian heart forms from a heterogeneous population of progenitor cells in anterior lateral mesoderm. Studies in mouse and chick have established that the heart forms from two sources of progenitor cells. As the embryo grows, cells of the cardiac crescent fuse at the midline to form the primitive heart tube. The primitive heart tube initially functions to support the embryonic circulation and provides a scaffold into which the cells from the second heart field (SHF) migrate prior to chamber morphogenesis. SHF cells are first located medially to the cardiac crescent, and subsequently reside in mesoderm underlying the pharynx before they accrue to the heart. The contribution of this population of cardiac progenitors to the heart was revealed by studies of the LIM transcription factor Islet1 (Isl1), which is a pan-marker of the SHF. The rostral part of the SHF, the anterior heart field (AHF), which is marked by Fgf10 expression contributes to the formation of right ventricular and outflow tract (OFT) myocardium, whereas cells in the posterior SHF expressing Isl1, but not AHF markers, contribute to atrial myocardium. These data indicate that the SHF is patterned along the anterior-posterior (AP) axis of the mouse embryo, however, a detailed understanding of the molecular regulatory pathways governing this process is lacking (Bertrand, 2011).

The retinoic acid (RA) signaling pathway plays a potent role in limiting cardiac specification. Mouse embryos lacking the RA synthesis enzyme Raldh2 have an expanded SHF, resulting in morphogenetic defects at both the arterial and venous poles. Consistent with this, zebrafish embryos lacking RA signaling exhibit an excess of cardiac progenitor cells in the lateral mesoderm. In the avian model, RA signaling promotes atrial cell identity within the heart field. It remains unknown whether the functions of RA signaling on SHF development and cardiac identity are distinct or overlapping. Identifying RA-target genes in cardiac progenitor cells will help to elucidate the mechanisms downstream of RA signaling that delimit the SHF. Studies in zebrafish embryos demonstrated that Hoxb5b, expressed in the forelimb field, acts downstream of RA signaling to restrict the number of cardiac progenitor cells. Thus, it is hypothesized that some of the homeobox (Hox) genes may be functional targets of RA in cardiac lineages in the mouse (Bertrand, 2011).

Hox genes are a large family of related genes that encode homeodomain transcription factors. Mammalian Hox genes are clustered in four chromosomal loci (the Hox clusters) and play an important role in regulating the specification of positional identities along the AP axis during development. Within each cluster, the genes are arranged in a sequence that reflects their sequential activation during development (temporal collinearity) and the position of the anterior boundary of their expression domains along the AP body axis (spatial collinearity). Initial Hox transcription and rostral expansion of Hox expression domains are regulated in part by events that are connected to the emergence and extension of the primitive streak. A contribution of RA signaling to the initial activation of Hox expression has been suggested, since at early developmental stages, embryos with impaired RA synthesis (Raldh2−/− mutants) exhibit abnormal initial 3' Hox gene expression domains. Moreover, RA was shown to regulate embryonic AP patterning, in particular by controlling the expression of specific Hox genes (Bertrand, 2011).

This study shows that the anterior Hox genes, Hoxb1, Hoxa1 and Hoxa3, are expressed in the SHF as early as embryonic day (E) 7.5 and define distinct sub-domains in the splanchnic mesoderm. Genetic (cre-mediated) lineage tracing reveals that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells give rise to the atria and the inferior wall of the OFT, which subsequently yields the myocardium at the base of the pulmonary trunk. Furthermore, Hoxb1IRES-Cre, Hoxa1-enhIII-Cre and Hoxa3IRES-Cre marked cells shows differential contributions to the proximal and distal regions of the OFT. Manipulation of the RA signaling pathway using Raldh2−/− embryos or injection of all-trans-RA demonstrates that expression of these Hox genes in the SHF and their cardiac contribution to the heart are sensitive to RA dosage. Comparison of transgenes expression in Raldh2 mutant embryos reveals that RA signaling is required for these Hox-expressing cardiac progenitor cell populations to contribute to the heart (Bertrand, 2011).

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