Interaction of Labial homologs with PBX

Dimerization with Extradenticle or the mammalian Exd homologs, the PBX homeoproteins, dramatically improves DNA binding by HOX transcription factors, indicating that recognition by such complexes is important for HOX specificity. For HOX monomeric binding, a major determinant of specificity is the flexible N-terminal arm. It makes base-specific contacts via the minor groove, including one to the 1st position of a 5'-TNAT-3' core by a conserved arginine (Arg-5). Arg-5 also contributes to the stability of HOX.PBX complexes, apparently by forming the same DNA contact. Heterodimers of PBX with HOXA1 (Drosophila homolog: labial) or HOXD4 (Drosophila homolog: Deformed) proteins have different specificities at another position recognized by the N-terminal arm (the 2nd position in the TNAT core). Significantly, N-terminal arm residues 2 and 3, which distinguish the binding of HOXA1 and HOXD4 monomers, play no role in the specificity of their complexes with PBX. In addition, HOXD9 and HOXD10,(Abdominal-B homologs) which are capable of binding both TTAT and TAAT sites as monomers, can cooperate with PBX1A only on a TTAT site. These data suggest that some DNA contacts made by the N-terminal arm are altered by interaction with PBX (Phelan, 1997).

The labial group protein HOXA-1 has intrinsically weak DNA-binding activity due to residues in the N-terminal arm of its homeodomain. This observation, among others, suggests that HOX and HOM proteins require cofactors for stable interactions with DNA. A putative HOX cofactor, PBX1A, participates in cooperative DNA binding with HOXA-1 and the Deformed group protein HOXD-4. Three Abdominal-B class HOX proteins fail to cooperate with PBX1A. The interacting domain of HOXD-4 maps to the YPWMK pentapeptide motif, a conserved sequence found N terminal to the homeodomain of HOXA-1 and many other homeoproteins, but one that is absent from the Abdominal-B class. The naturally occurring fusion of the transcriptional activation domain of E2A with PBX1 creates an oncoprotein implicated in human pre-B-cell leukemias. A pentapeptide mutation that abolished cooperative interaction with PBX1A in vitro also abrogates synergistic transcriptional activation with the E2A/PBX oncoprotein. The direct contact of PBX family members by the HOX pentapeptide is likely to play an important role in developmental and oncogenic processes (Phelan, 1995).

Autoregulation of vertebrate homeobox cluster genes requires the vertebrate homolog of Extradenticle. Hoxb-1 has a labial class homeodomain, and its expression was studied in transgenic mice and Drosophila embryos. Comparison of Hoxb-1 regulatory regions from different vertebrates identified three related sequence motifs critical for rhombomere 4 (r4) expression in the hindbrain. The conserved elements are involved in a positive autoregulatory loop dependent on Labial family members. Binding of HOXB-1 to these elements in vitro requires cofactors, and the motifs closely resemble the consensus binding site for PBX1, a homolog of the Drosophila Extradenticle. In vitro EXD/PBX serves as a Hoxb-1 cofactor in cooperative binding and in Drosophila expression mediated by the r4 enhancer is dependent on both lab and exd. This provides in vivo and in vitro evidence that r4 expression involves direct autoregulation dependent on cooperative interactions of HOXB-1 with EXD/PBX proteins as cofactors (Popperl, 1995).

The homeodomain proteins encoded by the Hox complex genes do not bind DNA with high specificity. In vitro, Hox specificity can be increased by binding to DNA cooperatively with the homeodomain protein Extradenticle or its vertebrate homologs, the PBX proteins (when considered together, known as the PBC family). One of the best characterized Hox-PBC binding sites is present in a 20 bp oligonucleotide repeat 3, which was identified in the 5' promoter region of the mouse Hoxb-1 gene. Hoxb-1 protein or its Drosophila ortholog Labial are both able to bind cooperatively with Exd to the binding site whereas other Hox proteins, such as Ultrabithorax or Hoxb-4 cannot. A two basepair change in a Hox-PBC binding site, from GG to TA, switches the Hox-dependent expression pattern generated in vivo from labial to Deformed. The change in vivo correlates with an altered Hox binding specificity in vitro. Similar Deformed-PBC binding sites were identified in the Deformed and Hoxb-4 genes. The Deformed sites include well characterized epidermal (EAE) and neural (NAE) autoregulatory enhancers. Two repeats containing TA sequence binding sites were found in the 2.7 kb EAE and two were found in the 600 bp NAE. These sites generate Deformed or Hoxb-4 expression patterns in Drosophila and mouse embryos, respectively. These results suggest a model in which Hox-PBC binding sites play an instructive role in Hox specificity by promoting the formation of different Hox-PBC heterodimers in vivo. Thus, the choice of Hox partner, and therefore Hox target genes, depends on subtle differences between Hox-PBC binding sites (Chan, 1997).

Human Pbx1 and HOXB1 proteins can cooperatively activate transcription through a genetically characterized Hox target, i.e. an autoregulatory element directing spatially restricted expression of the murine Hoxb-1 gene (b1-ARE) in rhombdomere 4 of the developing hindbrain. On the b1-ARE, only a restricted subset of HOX proteins (HOXA1, HOXB1, HOXA2) are able to bind cooperatively with Pbx1 and activate transcription. Selective recognition of the b1-ARE is mediated by the N-terminal region of the HOX homeodomain. The DNA-binding and protein-protein interaction functions of HOXB1 and Pbx1 are all necessary for the assembly of a transcriptionally active complex on the b1-ARE. Functional dissection of the complex shows that the localization of the main activation domain is in the HOXB1 N-terminal region, and that an additional activation domain is located in the C-terminal region of Pbx1, contained in the Pbx1a isoform, but not in the alternatively spliced Pbx1b isoform. These results indicate that Pbx1 acts as a transcriptional co-factor of Hox proteins, allowing selective recognition and cooperative activation of regulatory target sequences (Di Rocco, 1997).

HOX proteins are dependent on cofactors of the PBX family for the specificity of their DNA binding. Two regions that have been implicated in HOX/PBX cooperative interactions are the YPWM motif, found N-terminal to the HOX homeodomain, and the GKFQ domain (also known as the Hox cooperativity motif) immediately C-terminal to the PBX homeodomain. Using derivatives of the E2A-PBX oncoprotein, it was found that the GKFQ domain is not essential for cooperative interaction with HOXA1 but contributes to the stability of the complex. In contrast, the YPWM motif is strictly required for cooperative interactions in vitro and in vivo, even with mutants of E2A-PBX that lack the GKFQ domain. Using truncated PBX proteins, it was shown that the YPWM motif contacts the PBX homeodomain. The presence of the GKFQ domain increases monomer binding by the PBX homeodomain 5-fold, and the stability of the HOXA1.E2A-PBX complex 2-fold. These data suggest that the GKFQ domain acts mainly to increase DNA binding by PBX, rather than providing a primary contact site for the YPWM motif of HOXA1. Two residues, Glu-301 and Tyr-305, have been identified that are required for GKFQ function and it is suggested that this is dependent on alpha-helical character (Green, 1998).

Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. The 2.35 A structure of a ternary complex containing a human HoxB1-Pbx1 heterodimer bound to DNA is reported. Cooperative binding of Hox proteins with Pbx1/exd is dependent on a conserved hexapeptide sequence located N-terminal to the Hox homeodomain. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three-amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional alpha helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. This C-terminal portion of Pbx1 is an integral part of the Pbx1 DNA-binding domain and does not contact either the DNA or HoxB1. Rather, the C-terminal residues form part of the enlarged, four-helix DNA-binding domain of Pbx1 and may play a role in maintaining the structure of the DNA recognition helix and the hexapeptide binding pocket. The observed DNA contacts mediated by both HoxB1 and Pbx1 with the DNA provide a basis for understanding differential DNA sequence discrimination by Hox/Pbx heterodimers (Piper, 1999).

Human PREP1, a novel homeodomain protein of the TALE superfamily (see Drosophila Homothorax), forms a stable DNA-binding complex with PBX proteins in solution, a ternary complex with PBX and HOXB1 on DNA, and is able to act as a co-activator in the transcription of PBX-HOXB1 activated promoters. DNA-binding PREP1-PBX complexes are present in murine cells. In vivo, PREP1 is a predominant partner of PBX proteins in various murine tissues. However, the choice of PBX family member associated with PREP1 is largely tissue-type specific. The cloning and expression domain of the murine Prep1 gene is reported. Murine PREP1 shares 100% identity with human PREP1 in the homeodomain and 95% similarity throughout the whole protein. In the adult mouse, PREP1 is expressed ubiquitously, with peaks in testis and thymus. Murine Prep1 mRNA and protein and multiple DNA-binding PREP1-PBX complexes are present in mouse embryos from at least 9.5 days p.c. PREP1 is present in all embryonic tissues from at least 7.5-17.5 days of development, with a predominantly nuclear staining. PREP1 is able to superactivate the PBX-HOXB-1 autoregulated Hoxb-1 promoter, and all three proteins, PREP1, PBX and HOXB-1, are present together in the mouse rhombomere 4 domain in vivo, compatible with a role for PREP1 as a regulator of PBX and HOXB-1 proteins activity during development (Ferretti, 1999).

Direct auto- and cross-regulatory interactions between Hox genes serve to establish and maintain segmentally restricted patterns in the developing hindbrain. Rhombomere r4-specific expression of both Hoxb1 and Hoxb2 depends upon bipartite cis Hox response elements for the group 1 paralogous proteins, Labial-like Hoxa1 and Hoxb1. The DNA-binding ability and selectivity of these proteins depend upon the formation of specific heterodimeric complexes with members of the PBC homeodomain protein family (Pbx genes). The r4 enhancers from Hoxb1 and Hoxb2 have the same activity, but differ with respect to the number and organisation of bipartite Pbx/Hox (PH) sites required, suggesting the intervention of other components/sequences. Another family of homeodomain proteins, TALE (Three-Amino acids-Loop- Extension: Prep1, Meis, HTH), capable of dimerizing with Pbx/EXD, is involved in the mechanisms of r4- restricted expression. TALE/Pbx complexes bind both PH and specific Prep/Meis (PM) motifs. TALE/Pbx and Pbx/Hox interactions are not mutually exclusive, since they utilize different dimerization surfaces, allowing the formation of ternary Prep1/Pbx/Hoxb1 complexes in vitro on bipartite PH motifs. The interaction between Pbx and Hox proteins requires both homeodomains, a stretch of 20 amino acids C-terminal to the Pbx homeodomain, and the conserved pentapeptide sequence YPWMX or a similar ANW amino acid motif N-terminal to the Hox homeodomain. In contrast, Prep1 or Meis1 interaction with Pbx requires conserved amino-terminal sequences in both proteins. Therefore, by combining with Hox and Pbx, TALE proteins may also play an in vivo role in the mechanisms that serve to establish and maintain control of r4 identity. It has been shown that: (1) the r4-specific Hoxb1 and Hoxb2 enhancers are complex elements containing separate PH and Prep/Meis (PM) sites; (2) the PM site of the Hoxb2 enhancer, but not that of the Hoxb1 enhancer, is essential in vivo for r4 expression and also influences other sites of expression; (3) both PM and PH sites are required for in vitro binding of Prep1-Pbx and formation and binding of a ternary Hoxb1-Pbx1a (or 1b)-Prep1 complex. (4) A similar ternary association forms in nuclear extracts from embryonal P19 cells, but only upon retinoic acid induction. This requires synthesis of Hoxb1 and also contains Pbx with either Prep1 or Meis1. Together these findings highlight the fact that PM sites are found in close proximity to bipartite PH motifs in several Hox responsive elements shown to be important in vivo and that such sites play an essential role in potentiating regulatory activity in combination with the PH motifs (Ferretti, 2000).

Many Hox proteins are thought to require Pbx and Meis co-factors to specify cell identity during embryogenesis. Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish. Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere 2 of the hindbrain. In contrast, Hoxb1b and Pbx4 acting together with Meis3 induce hoxb1a, hoxb2, krox20 and valentino expression rostrally and cause extensive transformation of forebrain and midbrain fates to hindbrain fates, including differentiation of excess rhombomere 4-specific Mauthner neurons. This synergistic effect requires that Hoxb1b and Meis3 have intact Pbx-interaction domains, suggesting that their in vivo activity is dependent on binding to Pbx4. In the case of Meis3, binding to Pbx4 is also required for nuclear access. These results are consistent with Hoxb1b and Meis3 interacting with Pbx4 to form complexes that regulate hindbrain development during zebrafish embryogenesis (Vlachakis, 2001).

Notably, these experiments do not indicate the composition of Hoxb1b-, Pbx4- and Meis3-containing complexes, and several issues remain to be resolved. (1) It is not clear if all complexes contain a Meis family member. Ectopic expression of Hoxb1b by itself induces hoxb1a expression in r2. To perform this function, Hoxb1b needs to interact with an endogenous Pbx protein (most likely Pbx4 as this is the predominant Pbx protein at this stage), but does not require exogenous Meis3. While this is consistent with Hoxb1b and Pbx4 acting in the absence of a Meis protein, it leaves open the possibility that an endogenous Meis protein is involved. Indeed, the zebrafish prep1 gene appears to be ubiquitously expressed and endogenous Prep1 may interact with Hoxb1b and Pbx4 in these experiments. However, while the Pbx and Hox binding sites appear to be required for in vivo expression of most Hox-dependent genes, a Meis/Prep1 binding site is only required for some genes. Meis proteins may therefore not always be required (at least not as a DNA binding component) for Pbx and Hox proteins to function in vivo (Vlachakis, 2001).

(2) Different Hox proteins have different effects in vivo, but it is not clear if different Meis family members differ functionally. For instance, if Hoxb1b and Pbx4 require Prep1 to activate hoxb1a expression, the synergistic effect seen following co-expression of Meis3 could be due to Prep1 being limiting in vivo. In this scenario, Prep1 and Meis3 would be functionally equivalent. An alternative explanation to the synergistic effect is that exogenously supplied Meis3 provides a unique function, perhaps by replacing Prep1, thus playing an instructive role. The latter model is favored, primarily because published experiments suggest that Prep1 cannot substitute for Homothorax in Drosophila (Vlachakis, 2001 and references therein).

(3) Although both Hoxb1b and Meis3 appear to require Pbx4 interaction to be functional in vivo, it is not known whether Hoxb1b and Meis3 interact with the same Pbx4 molecule to form a trimeric complex, or whether they interact with separate Pbx4 molecules to form a pair of dimers. However, several pieces of data indicate the formation of trimeric complexes: (1) both the hoxb1 and hoxb2 r4 enhancers contain adjacent Hox/Pbx binding sites and a more distant Meis site, but there is no Pbx site near the Meis site; consistent with this, DNA fragments containing these sequences support formation of trimeric Hox/Pbx/Meis complexes, but not of a pair of dimers, in vitro; (2) a DNA-binding mutant Prep1 forms dimers with Pbx that bind DNA only very weakly. Therefore, if Meis3, Pbx4 and Hoxb1b act as a pair of dimers, a DNA-binding mutant of Meis3 (MutMeis3) should not be able to form a functional dimer with Pbx4 and should not have any in vivo activity. However, MutMeis3 still functions in vivo, as does a DNA-binding mutant Hth (Vlachakis, 2001).

(3) Misexpression of Xenopus Meis3 by itself has a minimal effect on krox20 and hoxb1 expression, but nevertheless mediates anterior deletions in Xenopus. This is in contrast with the analysis reported here, where zebrafish Meis3 requires Pbx4 and Hoxb1b for the transformation of anterior fates. Since lineage labeling was not utilized to analyze the deletions in Xenopus, it is not known if a distinct mechanism is at work, or if some Meis family members may be able to function independently of Pbx and Hox (Vlachakis, 2001 and references therein).

The effects mediated by Hoxb1b, Pbx4 and Meis3 co-expression are likely to be causally related and to occur in sequence. Since murine hoxb1 and hoxb2 have Pbx, Hox and Meis binding sites in their enhancers it is likely that zebrafish hoxb1a and hoxb2 are directly induced by Hoxb1b, Pbx4 and Meis3. Ectopic expression of hoxb2 induces krox20 and valentino expression in zebrafish, suggesting that these genes may be activated subsequent to hoxb2. Thus, expression of Hoxb1b, Pbx4 and Meis3 is sufficient to promote the differentiation of hindbrain fates, particularly r4 fates, and it is speculated that they normally perform this function within the caudal hindbrain during zebrafish embryogenesis (Vlachakis, 2001).

Expression of Labial homologs

Early sequential expression of mouse Hox genes is essential for their later function. Analysis of the relationship between early Hox gene expression and the laying down of anterior to posterior structures during and after gastrulation is therefore crucial for understanding the ontogenesis of Hox-mediated axial patterning. Using explants from gastrulation stage embryos, it has been shown that the ability to express 3' and 5' Hox genes develops sequentially in the primitive streak region, from posterior to anterior as the streak extends, about 12 hours earlier than overt Hox expression. The ability to express autonomously the earliest Hox gene, Hoxb1, is present in the posterior streak region at the onset of gastrulation, but not in the anterior region at this stage. However, the posterior region can induce Hoxb1 expression in these anterior region cells. It is concluded that tissues are primed to express Hox genes early in gastrulation, concomitant with primitive streak formation and extension, and that Hox gene inducibility is transferred by cell to cell signalling. Axial structures that will later express Hox genes are generated in the node region in the period that Hox expression domains arrive there and continue to spread rostrally. However, lineage analysis shows that definitive Hox codes are not fixed at the node, but must be acquired later and anterior to the node in the neurectoderm, and independently in the mesoderm. It is conclude that the rostral progression of Hox gene expression must be modulated by gene regulatory influences from early on in the posterior streak, until the time cells have acquired their stable positions along the axis well anterior to the node (Forlani, 2003).

Analyses using amphibian embryos have proposed that induction and anteroposterior patterning of the central nervous system is initiated by signals that are produced by the organizer and organizer-derived axial mesoderm. However, here it is shown that the initial anteroposterior pattern of the zebrafish central nervous system depends on the differential competence of the epiblast and is not imposed by organizer-derived signals. This anteroposterior information is present throughout the epiblast in ectodermal cells that normally give rise both to neural and non-neural derivatives. Because of this information, organizer tissues transplanted to the ventral side of the embryo induce neural tissue but the anteroposterior identity of the induced neural tissue is dependent on the position of the induced tissue within the epiblast. Thus, otx2, an anterior neural marker, was only induced in the anterior regions of the embryo, irrespective of the position of the grafts. Similarly, hoxa-1 (Drosophila homolog: labial), a posterior neural marker is induced only in the posterior regions. The boundary of each ectopic expression domain on the ventral side is always at an equivalent latitude to that of the endogenous expression of the dorsal side of the embryo. The anteroposterior specification of the epiblast is independent of the dorsoventral specification of the embryo because neural tissues induced in the ventralized embryos also show anteroposterior polarity. Cell transplantation and RNA injection experiments show that non-axial marginal mesoderm and FGF signaling is required for anteroposterior specification of the epiblast. However, the requirement for FGF signaling is indirect because cells with compromised ability to respond to FGF can still respond to anteroposterior positional information (Koshida, 1998).

The traditional view of neural inductions is that the dorsal mesoderm (the Organizer) comes to lie beneath the overlying ectoderm during gastrulation and that signals are passed vertically (dorsally) from the mesoderm to the ectoderm, where they induce the nervous system. An alternative explanation, one not mutually exclusive with the traditional view, holds that neural inducing signals pass to the animal pole from the Organizer and into the ectoderm in a planar fashion, not involving vertical transmission. Independent of either perspective, at stage 10-, at the first sign of the formation of the constricted bottle shaped cell, no mesoderm has come to lie beneath the overlying prospective neural ectoderm, but by stage 10+ and beyond, progressively more contact and potential signaling has occurred, since by then mesoderm underlies the neural ectoderm. Stage 10 is intermediate between these two, with some embryos having vertical contact and some not (Poznanski, 1997).

Is the expression of the Xenopus homeobox gene Hoxb-1 (a homolog of Drosophila Labial) regulated by instruction from the mesoderm and/or endoderm (vertical induction signals) or by a planar route involving transduction of the signal through intervening tissue? Sandwich explants of the dorsal marginal zone putatively allow only planar signals to pass from the mesodermal and endodermal tissue (Spemann's organizer) to the prospective neural tissue. Significant variability of expression was found with this type of explant. Observations during dissections suggest that variable degrees of invasion of the mesodermal-endodermal tissue at the leading edge of the mesodermal mantle might be the cause of this variability. Alternatively, differing lengths of time that the prospective neural region spends in planar contact with tissues of the lateral or ventral regions of the embryo could also contribute to this variability (Poznanski, 1997).

Analysis of staged Keller sandwich explants, "skewered" sandwiches, in which the degree of contact with underlying, involuted mesoderm-endodermal tissues was marked, and "over-the-pole" and "giant" sandwich explants, in which the degree of planar contact with lateral or ventral tissues was normalized, suggests that both planar and vertical signals are involved in induction and patterning of Hoxb-1 expression. Keller sandwich explants made from stage 10- embryos faintly express Hoxb-1 in the region of the prospective spinal cord. This diffuse expression must of necessity be derived from planar signals. The shift in Hoxb-1 expression from a broad, diffuse pattern to a local, focused pattern, characteristic of the ultimate expression pattern in vivo, does not reflect variable degrees of contact with ventral or lateral tissues, but rather reflects early vertical contact with underlying mesodermal-endodermal tissues. Such contact is observed at early gastrula stages (stages 10 to 10+), stages commonly assumed not to have the potential for vertical signaling. As the bottle cells first begin to form, at stage 10-, a massive rotation of the lower involuting marginal zone occurs around an internal lip. This rotation initiates the formation of the Cleft of Brachet from the floor of the blastocoele and brings the prospective mesoderm and endoderm at the leading edge of the marginal zone into vertical apposition with the prospective neural region quite early in gastrulation. The consequence and importance of recognizing these early internal rearrangements are that it pushes backward the time at which potential vertical inductive interactions between mesoderm and neurectoderm can occur. This means that a purely planar inductive situation can cease to exist as early as the inception of bottle cell formation and that neural patterning through vertical induction starts at the very beginning of gastrulation (Poznanski, 1997).

Xenopus has two labial homeodomain genes, HoxA1 and HoxD1. HoxA1 was expressed around the dorsoventral circumference of the trunk in neurula embryos, with later expression in spinal cord, midbrain, hindbrain, and endolymphatic duct. By mid gastrula, HoxD1 was predominantly expressed in dorsolateral and ventral ectoderm, with a gap in expression at the dorsal midline. By neurula, ventral expression had declined with most expression restricted to dorsolateral mesoderm and ectoderm. Retinoic acid strongly induces HoxA1 and HoxD1 throughout the ectoderm and mesendoderm of gastrula stages. Surprisingly, HoxA1 is expressed at high levels in isolated animal caps in the absence of retinoic acid. The peptide growth factors bFGF and activin A strongly induced expression of HoxD1, but not HoxA1, in animal caps; however, RNA accumulated only many hours after the application of these factors (Kolm, 1995).

Posterior neuropore (PNP) closure coincides with the end of gastrulation, marking the end of primary neurulation and primary body axis formation. Secondary neurulation and axis formation involve differentiation of the tail bud mesenchyme. Genetic control of the primary-secondary transition is not understood. A detailed analysis of gene expression in the caudal region of day 10 mouse embryos during primary neuropore closure is reported. Embryos were collected at the 27-32 somite stage, fixed, processed for whole mount in situ hybridization, and subsequently sectioned for a more detailed analysis. Genes selected for study include those involved in the key events of gastrulation and neurulation at earlier stages and more cranial levels. Patterns of expression within the tail bud, neural plate, recently closed neural tube, notochord, hindgut, mesoderm, and surface ectoderm are illustrated and described. Specifically, continuity of expression of the genes Wnt5a, Wnt5b, Evx1, Fgf8, RARgamma, Brachyury, and Hoxb1 from primitive streak and node into subpopulations of the tail bud and caudal axial structures is reported. Within the caudal notochord, developing floorplate, and hindgut, HNF3alpha, HNF3beta, Shh, and Brachyury expression domains correlate directly with known genetic roles and predicted tissue interdependence during induction and differentiation of these structures. The patterns of expression of Wnt5a, Hoxb1, Brachyury, RARgamma, and Evx1, together with observations on proliferation, reveal that the caudal mesoderm is organized at a molecular level into distinct domains delineated by longitudinal and transverse borders before histological differentiation. Expression of Wnt5a in the ventral ectodermal ridge supports previous evidence that this structure is involved in epithelial-mesenchymal interaction. These results provide a foundation for understanding the mechanisms facilitating transition from primary to secondary body axis formation, as well as the factors involved in defective spinal neurulation (Gofflot, 1999).

A mouse Hox 2.9 (HoxB1) homeodomain is similar to that of the Drosophila gene labial showing 80% identity. The equivalent gene in the Hox 1 cluster is Hox 1.6 which shows extensive similarity to Hox 2.9 both within and outside the homeodomain. Hox 2.9 has previously been shown to be expressed in a single segmental unit of the developing hindbrain (rhombomere) and has been predicted to be involved in conferring rhombomere identity. There are extensive similarities in the temporal and spatial expression of Hox 2.9 and Hox 1.6, throughout the period that both genes are expressed in the embryo (7 1/2 to 10 days). At 8 days the genes occupy identical domains in the neuroectoderm and mesoderm with the same sharp anterior boundary in the presumptive hindbrain. By 9 days the neuroectoderm expression of both genes retreats posteriorly along the anteroposterior (AP) axis. The difference at this stage between the expression patterns is the persistence of Hox 2.9 in a specific region of the hindbrain (Murphy, 1991).

The molecular basis of restricted expression of Hoxb2 (Drosophila homolog: proboscipedia) in rhombomere 4 (r4) of the hindbrain has been examined by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments show that the vertebrate Labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrates that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with Labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments it is concluded that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene (Maconochie, 1997).

The murine Hoxa1 gene is a member of the vertebrate Hox complex and plays a role in defining the body plan during development. At day 8.0-9.0 post coitus, Hoxa1 transcripts are detected extensively throughout the embryo in the neural tube, adjacent mesenchyme, paraxial mesoderm, somites and gut epithelium; expression extends from the most caudal region of the embryo to the border between rhombomeres 3 and 4. This spatiotemporal expression of Hoxa1 mRNA is critical for normal embryonic development. A 10 bp element, called CE2, has been identified that is located approximately 3 kilobases 3' to the Hoxa1 coding region in the RAIDR5 enhancer, and which binds to an approximately 170 kd protein in retinoic acid treated P19 embryonal carcinoma cells. CE2 elements have also been identified 3' to the murine Hoxb1 gene, the chicken Hoxb1 gene and the human Hoxa1 gene. To examine the role of this CE2 element in regulating Hoxa1 expression in vivo, transgenic mice were generated that express a Hoxa1 beta-galactosidase reporter gene that contains a mutation in the CE2 element. Relative to transgenic mice bearing a wild type CE2 element, the mutant CE2 construct recapitulates rhombomeric, neural, and gut epithelium expression but fails to show beta-galactosidase expression in somites and adjacent mesenchymal tissue. Gel shift analysis shows that binding activity similar to that detected in extracts prepared from retinoic acid treated P19 cells is present in nuclear extracts prepared from day 9.0 embryos. However, an additional binding complex not detected in P19 cells is also observed. These results indicate that in transgenic animals, the evolutionarily conserved CE2 element is a somite and adjacent mesenchymal enhancer of Hoxa1 expression (Thompson, 1998).

Recent evidence suggests that specific families of homeodomain transcription factors control the generation and survival of distinct neuronal types. The homeobox gene Phox2a is expressed in differentiating neurons of the central and peripheral autonomic nervous system as well as in motor nuclei of the hindbrain. Targeted deletion of the Phox2a gene affects parts of the structures in which it is expressed: the locus coeruleus, visceral sensory and parasympathetic ganglia and the nuclei of the IIIrd and IVth cranial nerves. Phox2b is a close relative of Phox2a and has an identical homeodomain. Phox2a and Phox2b are co-expressed at most sites, suggesting therefore, a broader role for Phox2 genes in the specification of the autonomic nervous system and cranial motor nuclei than revealed by the Phox2a knock-out mice. A detailed analysis of the relative timing of Phox2a and Phox2b expression at various sites suggests positive cross-regulation, which are substantiated by the loss of Phox2b expression in cranial ganglia of Phox2a-deficient mice. In the major part of the rhombencephalon, Phox2b expression precedes that of Phox2a and starts in the proliferative neuroepithelium, in a pattern strikingly restricted on the dorsoventral axis and at rhombomeric borders. This suggests that Phox2b links early patterning events to the differentiation of defined neuronal populations in the hindbrain. Phox2b is expressed most prominently in r4 at stages when the hindbrain expression of Hoxb1 is restricted to r4. This pattern prefigures the later expression of Hoxb1 and Phox2b in presumptive migrating facial motoneurons, which are known to depend on Hoxb-1 and which express Phox2 proteins up to postnatal stages. The borders of Phox2b expression correspond to the rostral expression of the Hox-2 and Hox-4 paralogs. These correlations suggest that the primary pattern of Phox2b in the CNS is under the control of Hox genes, whether direct or indirect, thus providing a link between Hox-directed patterning along the anterior/posterior axis and neurogenesis in the hindbrain (Pattyn, 1997).

The sympathetic, parasympathetic and enteric ganglia are the main components of the peripheral autonomic nervous system, and are all derived from the neural crest. The factors needed for these structures to develop include the transcription factor Mash1, the glial-derived neurotrophic factor GNDF and its receptor subunits, and the neuregulin signaling system, each of which is essential for the differentiation and survival of subsets of autonomic neurons. All autonomic ganglia fail to form properly and degenerate in mice lacking the homeodomain transcription factor Phox2b, as do the three cranial sensory ganglia that are part of the autonomic reflex circuits. In the anlagen of the enteric nervous system and the sympathetic ganglia, Phox2b is needed for the expression of the GDNF-receptor subunit Ret and for maintaining Mash1 expression. Mutant ganglionic anlagen also fail to switch on the genes that encode two enzymes needed for the biosynthesis of the neurotransmitter noradrenaline, dopamine-beta-hydroxylase and tyrosine hydroxylase, demonstrating that Phox2b regulates the noradrenergic phenotype in vertebrates (Pattyn, 1999).

During development, Hox gene transcription is activated in presomitic mesoderm with a time sequence that follows the order of the genes along the chromosome. Hoxd1 and other Hox genes display dynamic stripes of expression within presomitic mesoderm. The underlying transcriptional bursts may reflect the mechanism that coordinates Hox gene activation with somitogenesis. This mechanism appears to depend upon Notch signaling, because mice deficient for RBPJk, the effector of the Notch pathway, show severely reduced Hoxd gene expression in presomitic mesoderm. These results suggest a molecular link between Hox gene activation and the segmentation clock. Such a linkage would efficiently keep in phase the production of novel segments with their morphological specification (Zakany, 2001).

Transcriptional bursts in Hox gene expression in forming somites suggest how Hox complexes integrate a temporal parameter. Cells reaching the region where epithelial somites form (S-I) may respond to a localized signal by activating all Hox genes transcriptionally available. Consequently, the earliest burst would activate only group 1 genes; the subsequent burst (one somite-time later) would activate both group 1 and group 2 genes, etc., leading to a temporal coordination between somite formation and Hox gene activation. This view, however, doesn't suggest any mechanism whereby this oscillating time signal could be transformed into a linear activation of the clusters, i.e., how successive bursts would progressively activate more genes in a colinear fashion. Cells located at the more posterior level at time t3 would thus activate two more Hox genes than cells which were at the same more posterior level but at t1 (two segmentation cycles earlier). This requires that the accessibility of Hox genes be progressively increased within posterior presomitic mesoderm, rather than in the region of the stripes. This increased accessibility must occur either soon after gastrulation, i.e., within the pool of mesoderm cells that will produce the PSM, or during the time cells stay in PSM before reaching the more posterior level. Because many Hox genes are already expressed throughout the PSM, a view is favored whereby mesoderm cells are acquiring their 'state of opening' early on during gastrulation. This would uncouple the transcriptional activation of Hox genes from the mechanism that would regulate their accessibility and would give two temporal components to colinearity: (1) a progressive opening, which may rely upon the release of a silencing mechanism, followed by (2) time-dependent bursts of activation. In this context, PSM cells would express some background level of Hox gene products, due to the opening of the complex, and this expression would be coordinated in time by strong bursts of activation, whenever cells would approach the PSM to SM transition. These bursts would genetically 'label' the newly formed somite and imprint its morphological fate (Zakany, 2001).

In the absence of RBPJk function, expression of both Hoxd1 and Hoxd3 could hardly be detected in presomitic and somitic mesoderm, whereas expression in lateral plate mesoderm (for Hoxd1) and in the CNS (for Hoxd3) remain unchanged. This result is reminiscent of the loss of Lfng transcription in these same mutants and suggests that Hox gene may be under the control of the Notch pathway. Since the first establishment of the segmental pattern seems to occur at the level of Mesp2 (for mesoderm posterior 2), and because this latter gene is controlled by Notch signaling, the possibility exists that Hoxd1 activation be downstream of bHLH protein Mesp2 in S-I. This is supported by the apparent coordination of both expression patterns. In this view, the recurrent activation of the Hox system at the presomitic to somitic boundary would respond to a cyclic exposure to the outcome of the Notch pathway. Accordingly, the Notch-dependent coordination of the segmental pattern would be linked to the timing of activation, or enhanced transcription, of the Hox gene family (Zakany, 2001).

The recurrent activation of anterior Hox genes in PSM indicates that the segmentation mechanism, or the mechanism involved in somite boundary formation, may trigger or coordinate the activation of the Hox system. Interestingly, the expression of Lfng, a modulator of Notch signaling cycles in a way resembling the transcriptional oscillations of the chicken c-hairy-1 and -2 genes. These latter genes were proposed to be part of, or tightly linked to, the molecular oscillator underlying the segmental clock. Mutations of genes in the Notch pathway, such as the Notch genes themselves, Delta-like genes, RBPJk, Lfng, Mesp-2, and Presenilin-1, induce strong alterations of the segmental pattern, confirming their function in this fundamental process. In addition, cyclic expression of Lfng in RBPJk mutants is drastically reduced, and so is that of Hes1 in Dll1 mutants, suggesting a causal role for Notch signaling in the segmentation clock itself (Zakany, 2001).

To study the relationship between DNA replication and transcription in vivo, Hox gene activation was studied in two vertebrate systems: the embryogenesis of Xenopus and the retinoic acid-induced differentiation of pluripotent mouse P19 cells. The first cell cycles following the mid-blastula transition in Xenopus are necessary and sufficient for HoxB activation, whereas later cell cycles are necessary for the correct expression pattern. In P19 cells, HoxB expression requires proliferation, and the entire locus is activated within one cell cycle. Using synchronous cultures, it was found that activation of HoxB genes is colinear within a single cell cycle, occurs during S phase and requires S phase. The HoxB locus replicates early, whereas replication is still required for maximal expression later in S phase. Thus, induction of HoxB genes occurs in a DNA replication-dependent manner and requires only one cell cycle. It is proposed that S-phase remodelling licenses the locus for transcriptional regulation (Fisher, 2003).

Temporal colinearity of HoxB expression in P19 cells is clear only when cells are synchronized within the cell cycle, suggesting that HoxB activation is linked to progression through the cell cycle. If HoxB genes are activated in a defined order during the cell cycle, each successive gene will be activated at the same time in all cells in synchronous cultures, which would explain why differences in timing of activation (i.e., temporal colinearity) are clearly visible (Fisher, 2003).

The colinearity of expression of the HoxB domain within a single cell cycle has a striking relationship with its sensitivity to inhibition of DNA replication. Thus, the later the genes are expressed, the more they are sensitive to inhibition of DNA synthesis. The peak of sensitivity is for paralogues 6-8, whereas the border paralogs Hoxb9 and Hoxb13 at the 5' end and Hoxb1 at the 3' end are less sensitive. All HoxB genes, other than Hoxb13 (which does not obey temporal colinearity in these cells) and Hoxb1, are activated in S phase when stimulated in G2. Hoxb1 and Hoxb13 are activated before S phase and thus not surprisingly do not require S phase for correct activation (Fisher, 2003).

G1-S progression is not sufficient for Hoxb2-Hoxb9 expression, since aphidicolin, which specifically blocks DNA polymerase function, prevents HoxB expression in G2-synchronized cells. This is not due to the requirement for RA itself to act at a particular cell-cycle stage, since a transient pulse of RA can induce neural differentiation from any phase of the cell cycle, and in these experiments the control gene Cyp26, as well as Hoxb1 and Hoxb13, are activated normally in replication-inhibited cells (Fisher, 2003).

These results are compatible with current models for temporal colinearity. Colinearity of HoxD genes in the mouse embryo appears to be due in part to a polar release from transcriptional repression by a distal enhancer. Furthermore, HoxB activation along the anterior-posterior axis in chick embryos suggests that progressive opening of the HoxB locus occurs in all regions of the neural tube but genes are only expressed if cis-acting factors are present. Thus, colinearity might be achieved by two component steps: regulated derepression of the locus by DNA replication, making it permissive to regulated expression of specific transcription factors (Fisher, 2003).

It is proposed that the gradient of sensitivity to aphidicolin observed according to the position of HoxB paralogs on the chromatin domain is related to polar competition between transcriptional repressor and activator elements, in which DNA replication strongly favours expression of repressed chromatin by creating nascent DNA upon which new chromatin is formed or by restructuring the domain during S phase. In P19 cells the HoxB locus may be in a predetermined state, since it replicates early and is mainly unmethylated and activation of the entire locus is rapid, yet DNA replication is still required to allow normal Hox gene expression. Possibly, the HoxB locus in P19 cells is organized into chromatin in such a way that passage through a single cell cycle allows relief of repression over the entire locus in much the same way that a single DNA replication derepresses chromatin to allow long-range enhancer function in mouse embryos(Fisher, 2003).

In some vertebrates, cell-cycle lengths show an anterior-posterior gradient (long to short), and there is a caudal-rostral wave of initiation of HoxB expression in the neural tube in these embryos. Furthermore, cell-cycle synchrony occurs in developing somites, which also show waves of HoxB expression. As such, the spatial and temporal organization of proliferation could provide a template for developmental gene expression in which staggered DNA replication along a developing axis relieves gene expression from transcriptional silencing (Fisher, 2003).

The spatial and temporal co-linear expression of Hox genes during development is an exquisite example of programmed gene expression. The precise mechanisms underpinning this are not known. Analysis of Hoxb chromatin structure and nuclear organisation, during the differentiation of murine ES cells, has lent support to the idea that there is a progressive 'opening' of chromatin structure propagated through Hox clusters from 3' to 5', which contributes to the sequential activation of gene expression. Similar events occur in vivo in at least two stages of development. The first changes in chromatin structure and nuclear organisation are detected during gastrulation in the Hoxb1-expressing posterior primitive streak region: Hoxb chromatin is decondensed and the Hoxb1 locus loops out from its chromosome territory, in contrast to non-expressing Hoxb9, which remains within the chromosome territory. At E9.5, when differential Hox expression along the anteroposterior axis is being established, concomitant changes are found in the organisation of Hoxb. Hoxb organisation differs between regions of the neural tube that never express Hoxb [rhombomeres (r) 1 and 2], strongly express Hoxb1 but not b9 (r4), have downregulated Hoxb1 (r5), express Hoxb9 but not Hoxb1 (spinal cord), and express both genes (tail bud). It is concluded that Hoxb chromatin decondensation and nuclear re-organisation is regulated in different parts of the developing embryo, and at different developmental stages. The differential nuclear organisation of Hoxb along the anteroposterior axis of the developing neural tube is coherent with co-linear Hox gene expression. In early development nuclear re-organisation is coupled to Hoxb expression, but does not anticipate it (Chambeyron, 2005).

Retinoic acid (RA) generated by Raldh2 in paraxial mesoderm is required for specification of the posterior hindbrain, including restriction of Hoxb1 expression to presumptive rhombomere 4 (r4). Hoxb1 expression requires 3' and 5' RA response elements for widespread induction up to r4 and for r3/r5 repression, but RA has previously been detected only from r5-r8, and vHnf1 is required for repression of Hoxb1 posterior to r4 in zebrafish. In mouse embryos an RA signal initially travels from the paraxial mesoderm to r3, forming a boundary next to the r2 expression domain of Cyp26a1 (which encodes an RA-degrading enzyme). After Hoxb1 induction, the RA boundary quickly shifts to r4/r5, coincident with induction of Cyp26c1 in r4. A functional role for Cyp26c1 in RA degradation was established through examination of RA-treated embryos. Analysis of Raldh2–/– and vHnf1–/– embryos supports a direct role for RA in Hoxb1 induction up to r4 and repression in r3/r5, as well as an indirect role for RA in Hoxb1 repression posterior to r4 via RA induction of vHnf1 up to the r4/r5 boundary. These findings suggest that Raldh2 and Cyp26 generate shifting boundaries of RA activity, such that r3-r4 receives a short pulse of RA and r5-r8 receives a long pulse of RA. These two pulses of RA activity function to establish expression of Hoxb1 and vHnf1 on opposite sides of the r4/r5 boundary (Sirbu, 2005).

Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation

The relocalisation of some genes to positions outside chromosome territories, and the visible decondensation or unfolding of interphase chromatin, are two striking facets of nuclear reorganisation linked to gene activation that have been assumed to be related to each other. Here, in a study of nuclear reorganisation around the Hoxd cluster, it is suggested that this may not be the case. Despite its very different genomic environment from Hoxb, Hoxd also loops out from its chromosome territory, and unfolds, upon activation in differentiating embryonic stem (ES) cells and in the tailbud of the embryo. However, looping out and decondensation are not simply two different manifestations of the same underlying change in chromatin structure. In the limb bud of the embryonic day 9.5 embryo, where Hoxd is also activated, there is visible decondensation of chromatin but no detectable movement of the region out from the chromosome territory. During ES cell differentiation, decondensed alleles can also be found inside of chromosome territories, and loci that have looped out of the territories can appear to still be condensed. It is concluded that evolutionarily conserved chromosome remodelling mechanisms, predating the duplication of mammalian Hox loci, underlie Hox regulation along the rostrocaudal embryonic axis. However, it is suggested that separate modes of regulation can modify Hoxd chromatin in different ways in different developmental contexts (Morey, 2007).

To dissect the events of nuclear reorganisation, an ES cell differentiation system was used. Gene expression and nuclear reorganisation could be induced at Hoxb by triggering the differentiation of murine ES cells with retinoic acid (RA). To determine whether similar activation occurs at Hoxd RT-PCR was used to analyse the expression of Hoxd genes in undifferentiated OS25 ES cells, and during 18 days after the withdrawal of LIF and the addition of RA. As for Hoxb, there was no detectable expression of Hoxd genes in undifferentiated cells. The extinction of Oct4 expression upon differentiation is accompanied by the rapid induction (by day 2) of Hoxd1 expression, but not of the more 5' genes Hoxd3 through to Hoxd12. Expression of Hoxd3 and Hoxd4 were detected by day 6, Hoxd8 by day 8, Hoxd9 and Hoxd10 by day 10 and Hoxd12 expression was not detected until day 18. Hoxd1 expression declined at later stages of differentiation, but not as rapidly as seen for Hoxb1 (Morey, 2007).

Hoxd is flanked by structurally and functionally unrelated genes. Expression of Mtx2, located 3' of Hoxd1, is induced by day 2, suggesting that this gene might also be subject to temporal colinearity. However, at the 5' end of Hoxd, the early detection of Hoxd13 expression (by day 2) suggests a break in the temporal colinearity at this end of the cluster in this system. A large conserved noncoding region 5' of Hoxd, termed a global control region (GCR), contains digit enhancers that act on Hoxd13, Lnp and Evx2. Neural enhancers in the GCR also act on Evx2 and Lnp. As Evx2 is also activated early in the timecourse of differentiation, this suggests that the GCR may have some activity in ES cells. Lnp expression in ES cells is constitutive. This analysis shows that the colinear activation of the Hoxd cluster is mostly recapitulated upon RA-induced ES cell differentiation (Morey, 2007).

Mutation of Labial homologs

Continued: Evolutionary Homologs part 3/3 | back to part 1/3

labial: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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