BarH1 and BarH2
The conservation of developmental functions exerted by Antp-class
homeoproteins in protostomes and deuterostomes has suggested that homologs
with related functions are present in diploblastic animals, in particular, in Hydra. Phylogenetic analyses show that Antp-class homeodomains belong either
to non-Hox or to Hox/paraHox families. See Phylogenetic relationships among 200 Antp-class genes. Among the 13 non-Hox families,
9 reported here have diploblastic homologs: Msx, Emx,
Barx, Evx, Tlx,
NK-2, and Prh/Hex, Not,
and Dlx. Among the Hox/paraHox,
poriferan sequences are not found, and the cnidarian sequences form at least five distinct cnox families. Cnox-1 shows some affinity to paralogous group (PG) 1; this group includes Drosophila Labial. Cnox-2 is related to Drosophila Intermediate neuroblast defective. Cnox-3 and 5 show some affinity to PG9-10; this group includes Drosophila AbominalB. Cnox-4 has no counterparts in Drosophila or vertebrates. Intermediate
Hox/paraHox genes (PG 3 to 8 and lox) do not have clear cnidarian counterparts. In Hydra,
cnox-1, cnox-2, and cnox-3
are not found chromosomally linked within a 150-kb range and display
specific expression patterns in the adult head. During regeneration,
cnox-1 is expressed as an early gene whatever the
polarity, whereas cnox-2 is up-regulated later during head but not foot regeneration. Finally, cnox-3
expression is reestablished in the adult head once the head is fully
formed. These results suggest that the Hydra genes
related to anterior Hox/paraHox genes are involved at
different stages of apical differentiation. However, the positional
information defining the oral/aboral axis in Hydra
cannot be correlated strictly to that characterizing the
anterior-posterior axis in vertebrates or arthropods (Gauchat, 2000)
In the process of cloning murine proteins capable of binding to a regulatory module of the Ncam
promoter, a homeobox gene, Barx1, was isolated; it is the first vertebrate member of the structural
subclass defined by Drosophila BarH1. Barx1 is strongly expressed in restricted areas of head and neck
mesenchyme and in the wall of the developing stomach and at weaker levels in the proximal fore- and
hindlimbs. At embryonic day 10.5, expression in the head region is detected in spatially restricted areas
of the first and second branchial arches, before any apparent cellular or morphological differentiation.
Later in development, all expressing tissues in this region, which include the mesenchyme underlying
the olfactory epithelium, the primary and secondary palate, the molar tooth papillae and the stroma of
the submandibular gland, appear to be derived from ectomesenchyme of neural crest origin. At day 16.5, all
locations other than the developing molars had become Barx1-negative. An intriguing feature is the
restriction of Barx1 expression to the molars, suggesting a role in the differentiation of molars from
incisors. Barx1 already marks the future stomach region of the primitive gut at embryonic day 9.5 and
is present in the mesenchymal wall of the stomach up to day 16.5. These results thus direct a search
for its function to a number of inductive epithelial-mesenchymal interactions during craniofacial
development and to stomach organogenesis (Tissier-Seta, 1995).
Mammalian dentitions are highly patterned, with different types of teeth positioned in different regions of the jaws. BMP4 is an early oral epithelial protein
signal that directs odontogenic gene expression in mesenchyme cells of the developing mandibular arch. BMP4 was shown to inhibit expression of the
homeobox gene Barx-1 and to restrict expression to the proximal, presumptive molar mesenchyme of mouse embryos at embryonic day 10. The inhibition
of BMP signaling early in mandible development by the action of exogenous Noggin protein results in ectopic Barx-1 expression in the distal, presumptive
incisor mesenchyme and a transformation of tooth identity from incisor to molar (Tucker, 1998).
Vertebrate neurogenesis involves sequential actions of transcription factors. Neurogenins, encoding Atonal-related bHLH transcription factors, function as neuronal determination genes in Xenopus. Neurogenins and another bHLH factor gene, Mash1, are expressed in distinct subsets or areas of cells giving rise to neurons, suggesting that these genes play important roles to generate distinct populations of neurons. A mammalian homolog of BarH (MBH1) is expressed in a complementary pattern to Mash1 expression in the developing nervous system like neurogenins. Forced expression of MBH1 down-regulates expression of Mash1 and up-regulates neurogenin2/Math4A, a member of neurogenins, in P19 cells during neuronal differentiation. This suggests that MBH1 is a potential regulator of mammalian neural bHLH genes, thereby establishing distinct pathways of neuronal differentiation (Saito, 1998).
The BARX genes 1 and 2 are Bar class homeobox genes expressed in craniofacial structures during development. This report presents the genomic structure, chromosomal localization, and polymorphic markers in BARX2. The gene has four exons, ranging in size from 85 to 1099 bp. BARX2 is localized on human chromosome 11q25, as determined by radiation hybrid mapping. In the mouse, Barx2 is coexpressed with Pitx2 in several tissues. Based on the coexpression, BARX2 was assumed to be a candidate gene for those cases of Rieger syndrome that cannot be associated with mutations of PITX2. Mutations in PITX2 cause some cases of Rieger syndrome, an autosomal dominant disorder affecting eyes, teeth, and umbilicus. DNA from Rieger patients was subjected to single-strand conformation polymorphism screening of the BARX2 coding region. Three single nucleotide polymorphisms were found in a normal population, although no etiologic mutations were detectable in over 100 cases of Rieger syndrome or in individuals with related ocular disorders (Hjalt, 1999).
Two mouse and human homeobox genes highly related to the Bar Drosophila genes, Barhl1 and Barhl2, have been identified. While Barhl1 represents a novel gene, Barhl2 turned out to correspond to the mBH1 cDNA recently described in rat. The full-length mouse Barhl1 have been isolated and sequenced and the human BARHL1 and BARHL2 genes have been mapped to chromosomes 9q34 and 1p22, respectively. Detailed analysis of the murine Barhl1 expression pattern by in situ hybridization has revealed that this transcript is exclusively expressed in restricted domains of the developing CNS, which suggests that this gene, similar to its Drosophila counterparts BarH1 and BarH2, may play a crucial role in cell fate determination of neural structures. In particular, Barhl1 show specific domains of expression in the diencephalon and in the rhombencephalon where it was found to be expressed in migrating cells giving rise to the cerebellar external granular layer and to specific populations of dorsal sensory interneurons of the spinal cord. Thus, Barhl1 function may be required for the generation of these specific subtypes of neuronal progenitors. Furthermore, the mapping assignment and the expression pattern make BARHL1 an attractive positional candidate gene for a form of Joubert syndrome, a rare developmental anomaly of the cerebellum in humans (Bulfone, 2000).
Barx1 and Barx2 are homeodomain proteins originally identified using regulatory elements of genes encoding certain cell adhesion molecules (CAMs). In the present study, regions of Barx2 were characterized that bind to regulatory elements of genes encoding three CAMs, L1, neuron-glia CAM (Ng-CAM), and neural CAM (N-CAM); domains of Barx2 were identified that regulate N-CAM transcription. The homeodomain of Barx2 is sufficient for binding to homeodomain binding sites (HBS) from all three CAM genes. The presence of a 17-amino acid Barx basic region resulted in a 2-fold decrease in binding to HBS sequences from the Ng-CAM and L1 genes, whereas it led to a 6.5-fold increase in binding to the HBS from the N-CAM promoter. Thus, the Barx basic region influences the strength and specificity of Barx2 binding to DNA. In co-transfection experiments, Barx2 repressed N-CAM promoter activity. A 24-residue N-terminal region of Barx2 is essential for repression. When this region is absent, Barx2 activates the N-CAM promoter. A 63-residue C-terminal domain is required for this activation. In GST pull-down experiments, Barx2 binds to proteins of the CREB family, CREB1 and ATF2. Overall, these findings provide a framework for understanding developmental and physiological contexts that influence repressor or activator functions of Barx2 (Edelman, 2000).
The cochlea of the mammalian inner ear contains three rows of outer hair cells and a single row of inner hair cells. These hair cell receptors reside in the organ of Corti and function to transduce mechanical stimuli into electrical signals that mediate hearing. To date, the molecular mechanisms underlying the maintenance of these delicate sensory hair cells are unknown. Targeted disruption of Barhl1, a mouse homolog of the Drosophila BarH homeobox genes, results in severe to profound hearing loss, providing a unique model for the study of age-related human deafness disorders. Barhl1 is expressed in all sensory hair cells during inner ear development, 2 days after the onset of hair cell generation. Loss of Barhl1 function in mice results in age-related progressive degeneration of both outer and inner hair cells in the organ of Corti, following two reciprocal longitudinal gradients. These data together indicate an essential role for Barhl1 in the long-term maintenance of cochlear hair cells, but not in the determination or differentiation of these cells (Li, 2002).
The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Antisense-oligonucleotide inhibition of Barx2 expression has been used in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, the Barx2 gene promoter was isolated and DNA regulatory elements were identified that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contains binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors (Meech, 2003).
Among the many factors involved in regulation of chondrogenesis, bone morphogenetic proteins (BMPs) and members of the Sox and homeobox transcription factor families have been shown to have crucial roles. Of these regulators, the homeobox transcription factors that function during chondrogenesis have been the least well defined. The homeobox transcription factor Barx2 is expressed in primary mesenchymal condensations, digital rays, developing joints and articular cartilage of the developing limb, suggesting that it plays a role in chondrogenesis. Using retroviruses and antisense oligonucleotides to manipulate Barx2 expression in limb bud micromass cultures, it was determined that Barx2 is necessary for mesenchymal aggregation and chondrogenic differentiation. In accordance with these findings, Barx2 regulates the expression of several genes encoding cell-adhesion molecules and extracellular matrix proteins, including NCAM and collagen II (Col2a1) in the limb bud. Barx2 binds to elements within the cartilage-specific Col2a1 enhancer, and this binding is reduced by addition of Barx2 or Sox9 antibodies, or by mutation of a HMG box adjacent to the Barx2-binding element, suggesting cooperation between Barx2 and Sox proteins. Moreover, both Barx2 and Sox9 occupy Col2a1 enhancer during chondrogenesis in vivo. It was also found that two members of the BMP family that are crucial for chondrogenesis, GDF5 and BMP4, regulate the pattern of Barx2 expression in developing limbs. Based on these data, it is suggested that Barx2 acts downstream of BMP signaling and in concert with Sox proteins to regulate chondrogenesis (Meech, 2005).
Targeted disruption of effectors molecules of the apoptotic pathway have demonstrated the occurrence and magnitude of early programmed cell death (EPCD), a form of apoptosis that affects proliferating and newly differentiated cells in vertebrates, and most dramatically cells of the central nervous system (CNS). Little is known about the molecular pathways controlling apoptosis at these early developmental stages, like the roles of EPCD during patterning of the developing nervous system. A new function, in Xenopus neurodevelopment, is described for a highly conserved homeodomain protein Barhl2. Barhl2 promotes apoptosis in the Xenopus neuroectoderm and mesoderm, acting as a transcriptional repressor, through a mechanism that cannot be attributed to an unspecific cellular stress response. The pro-apoptotic activity of Barhl2 is essential during normal neural plate formation since it limits the number of chordin- and Xshh-expressing cells in the prospective notochord and floorplate, which act as organizing centers. These findings show that Barhl2 is part of a pathway regulating EPCD. They also provide evidence that apoptosis plays an important role in regulating the size of organizing centers (Offner, 2005).
The mouse homeobox gene Barhl1 plays a central role in cerebellum
development and its expression is activated by the transcription factor
Math1 which is involved in bone morphogenetic protein response pathways.
The human ortholog BARHL1 was studied and it was found that human, mouse,
monkey, rat, and zebrafish orthologs were highly conserved and are members of
the BarH homeogene family, containing Drosophila BarH1 and
BarH2. The N-terminus of BARHL1 protein presents two FIL domains and an
acidic domain rich in serine/threonine and proline, while the C-terminus
contains a canonical proline-rich domain. Secondary structure analysis showed
that outside the three helixes of the homeodomain, BARHL1 protein has
essentially a random coil structure. BARHL1 was isolated and its expression
pattern was examined in human embryonic and fetal central nervous system (CNS)
and the expression was compared to that of mouse Barhl1. BARHL1
mRNA was found exclusively in the CNS restricted to p1-p4 prosomeres of the
diencephalon, to the dorsal cells of the mesencephalon, to the dorsal dl1
sensory neurons of the spinal cord, and to the rhombic lips yielding the
cerebellar anlage. Detailed analysis of BARHL1 expression in fetal
cerebellar cell layers using optic microscopy technology showed BARHL1
expression in external and internal granular cells and also in mouse adult
granular cells, in agreement to Barhl1 null mouse phenotype affecting the
differentiation and migration of granular cells. These findings indicate that
the regional and cellular specificities of BARHL1 transcriptional control
correspond well to the mouse Barhl1 transcription and suggest a potential
role for this gene in the differentiation of BARHL1-expressing neuronal
progenitors involved in the pattern formation of human cerebral and cerebellar
structures (Lopes, 2006).
Apoptosis is essential for proper development and tissue homeostasis in metazoans. It plays a critical role in generating sexual dimorphism by eliminating structures that are not needed in a specific sex. The molecular mechanisms that regulate sexually dimorphic apoptosis are poorly understood. This study reports the identification of the ceh-30 gene as a key regulator of sex-specific apoptosis in Caenorhabditis elegans. Loss-of-function mutations in ceh-30 cause the ectopic death of male-specific CEM neurons. ceh-30 encodes a BarH homeodomain protein that acts downstream from the terminal sex determination gene tra-1, but upstream of, or in parallel to, the cell-death-initiating gene egl-1 to protect CEM neurons from undergoing apoptosis in males. The second intron of the ceh-30 gene contains two adjacent cis-elements that are binding sites for TRA-1A and a POU-type homeodomain protein UNC-86 and acts as a sensor to regulate proper specification of the CEM cell fate. Surprisingly, the N terminus of CEH-30 but not its homeodomain is critical for CEH-30's cell death inhibitory activity in CEMs and contains a conserved eh1/FIL domain that is important for the recruitment of the general transcriptional repressor UNC-37/Groucho. This study suggests that ceh-30 defines a critical checkpoint that integrates the sex determination signal TRA-1 and the cell fate determination and survival signal UNC-86 to control the sex-specific activation of the cell death program in CEMs through the general transcription repressor UNC-37 (Peden, 2007).
Proneural basic helix-loop-helix (bHLH) proteins are key regulators of
neurogenesis. However, downstream target genes of the bHLH proteins remain
poorly defined. Mbh1 (Barhl2 - Mouse Genome Informatics) confers commissural neuron identity in the
spinal cord. Enhancer analysis using transgenic mice reveals that
Mbh1 expression requires an E-box 3' of the Mbh1 gene.
Mbh1 expression is lost in Math1 knockout mice, whereas
misexpression of Math1 induces ectopic expression of Mbh1.
Moreover, Math1 binds the Mbh1 enhancer containing the E-box in vivo and activated gene expression. Generation of commissural neurons by Math1 is inhibited by a dominant negative form of Mbh1. These findings indicate that Mbh1 is necessary and sufficient for the specification of commissural neurons, as a direct downstream target of Math1 (Saba, 2005).
Two distinct
vertebrate Bar-related homeobox genes, XBH1 and
XBH2, have been identified in Xenopus. XBH1 is highly related in sequence and expression
pattern to a mammalian gene, MBH1, suggesting
that they are orthologs. XBH2 has not previously been
identified but is clearly related to the Drosophila Bar
genes. During early Xenopus embryogenesis XBH1 and
XBH2 are expressed in overlapping regions of the central
nervous system. XBH1, but not XBH2, is expressed in
the developing retina. By comparing the expression of
XBH1 with that of hermes, a marker of differentiated retinal
ganglion cells, it has been shown that XBH1 is expressed in
retinal ganglion cells during the differentiation process,
but is down-regulated as cells become terminally differentiated (Patterson, 2000).
The mammalian retina contains numerous morphological and physiological subtypes of amacrine cells necessary for integrating and modulating visual signals presented to the output neurons. Among subtypes of amacrine cells grouped by neurotransmitter phenotypes, the glycinergic and gamma-aminobutyric acid (GABA)ergic amacrine cells constitute two major subpopulations. To date, the molecular mechanisms governing the specification of subtype identity of amacrine cells remain elusive. During mouse development, the Barhl2 homeobox gene displays an expression pattern in the nervous system that is distinct from that of its homolog Barhl1. In the developing retina, Barhl2 expression is found in postmitotic amacrine, horizontal and ganglion cells, while Barhl1 expression is absent. Forced expression of Barhl2 in retinal progenitors promotes the differentiation of glycinergic amacrine cells, whereas a dominant-negative form of Barhl2 has the opposite effect. By contrast, they exert no effect on the formation of GABAergic neurons. Moreover, misexpressed Barhl2 inhibits the formation of bipolar and Müller glial cells, indicating that Barhl2 is able to function both as a positive and negative regulator, depending on different types of cells. Taken together, these data suggest that Barhl2 may function to specify the identity of glycinergic amacrine cells from competent progenitors during retinogenesis (Mo, 2004).
Recent studies on vertebrate eye development have focused on the molecular mechanisms of specification of different retinal cell types during development. Only a limited number of genes involved in this process has been identified. In Drosophila, BarH genes are necessary for the correct specification of R1/R6 eye photoreceptors. Vertebrate Bar homologs have been identified and are expressed in vertebrate retinal ganglion cells during differentiation; however, their retinal function has not yet been addressed. The roles have been examined of the Xenopus Bar homolog Xbh1 in retinal ganglion cell development and its interaction with the proneural genes Xath5 and Xath3, whose ability to promote ganglion cell fate has been demonstrated. XHB1 plays a crucial role in retinal cell determination, acting as a switch towards ganglion cell fate. Detailed expression analysis, animal cap assays and in vivo lipofection assays, indicate that Xbh1 acts as a late transcriptional repressor downstream of the atonal genes Xath3 and Xath5. However, the action of Xbh1 on ganglion cell development is different and more specific than that of the Xath genes, and accounts for only a part of their activities during retinogenesis (Poggi, 2004).
To elucidate in detail how Xbh1 expression is related to retinal
neurogenesis in time and space, its expression was compared with that of
Xath5. Xath5 expression starts in the retina at around stage 24,
preceding the reported onset of retinal differentiation.
Expression is initially present throughout most of the neural retina, but
displays a dorsal to ventral gradient that is consistent with neurogenesis
commencing slightly earlier in the dorsal retina than in the ventral retina. When RGC, inner nuclear, and photoreceptor cell layers become distinct, Xath5 expression is downregulated in differentiated neurons, but remains in the ciliary marginal zone (CMZ), where retinoblasts are generated throughout life. Xbh1 expression in the retina is first detected in the dorsal inner optic cup around stage 26-27, shortly after the onset of Xath5 expression, and subsequently spreads from dorsal to ventral until it covers the entire retina, thus
following the wave of retinal differentiation. At stage
38, when the three main retinal cell layers become distinct, Xbh1 is detected in the ganglion cell layer, in some scattered cells in the inner part of the inner nuclear layer (INL), and also in the most central part of the CMZ. At stage 42,
Xbh1 expression is almost completely restricted to cells of the
central differentiated ganglion cell layer, and to the central CMZ; a few
cells in the INL also showed expression.
At stage 42, double in situ hybridization shows expression of Xbh1
and Xath5 in the central most part of the CMZ. High magnification
of these sections shows that, in spite of some superposition, Xbh1
does not extend as far peripherally as Xath5. Xbh1
expression in the CMZ is also more central than that of XNotch1,
predominantly restricted to proliferating cells. Interestingly, a few cells of the ventral-most central retina still express both Xath5 and Xbh1. These cells may be in a similar commitment state as those co-expressing the two genes in the CMZ, and may reflect the delay in differentiation in the ventral retina with respect to the dorsal retina. After stage 42, when differentiation occurs almost exclusively in the CMZ,
Xbh1 is also progressively downregulated in the ganglion cell layer, but persists in the CMZ. Thus, both in the retina and in the CMZ, Xbh1 expression strictly follows, in time and space, the dynamics of Xath5 expression (Poggi, 2004).
The fact that Xath5 expression precedes and later partially
overlaps Xbh1 expression in the CMZ, suggests a possible
regulatory interaction. The animal cap assay was used to investigate whether Xbh1 can be transcriptionally regulated by Xath5.
One-cell-stage embryos were injected into the animal pole with 1 ng of
Xath5 RNA. Animal caps were cut at blastula stage, harvested at stage 28, and processed for RT-PCR assays to detect possible activation of
Xbh1, and of the ganglion cell markers Xbrn3.0 and
Xbrn3d, the earliest markers of RGCs, known as Xath5
downstream genes. Xath5 is able to activate Xbh1, Xbrn3d
and Xbrn3.0 transcription in injected animal caps, whereas none of
these genes was transcribed in control caps. It was also found that
Xath3 is able to activate Xbh1, as well as Xbrn3.0
and Xbrn3d. Interestingly, injection of 500 pg of XneuroD mRNA, although able to trigger Xbrn3d in animal caps,
is not able to activate Xbh1 expression. This suggests that
Xbh1 transcription may be specifically controlled by atonal-like
factors, but not by any bHLH factor (Poggi, 2004).
To test whether Xbh1 could activate Xbrn3.0 and
Xbrn3d, one ng of RNA encoding Xbh1 was injected into
1-cell-stage embryos and assayed for the expression of Xbrn3 genes in stage 28 animal caps. Xbh1 was found to trigger both
Xbrn3.0 and Xbrn3d transcription in animal caps. Whether Xbh1 was able to activate Xath5 and/or
Xath3 was also tested in animal caps. Xbh1 does not activate
Xath5, but does activate Xath3 transcription (Poggi, 2004).
A homeobox protein has been identfied that binds to a regulatory element common to the genes for two neural CAMs, Ng-CAM and L1 (see Fasciclin2). The homeodomain portion of this protein has an 87% sequence identity to that of Barx1, and both genes are related to genes at the bar locus of Drosophila. Barx1 and Barx2 also encode an identical stretch of 17 residues downstream of the homeobox; otherwise, they share no appreciable homology. Barx2 stimulates in vitro activity of an L1 promoter construct containing the CCATTAGPyGA motif, but represses activity when this sequence is deleted. Localization studies show that expression of Barx1 and Barx2 overlap in the nervous system, particularly in the telencephalon, spinal cord, and dorsal root ganglia. Barx2 is also prominently expressed in the floor plate and in Rathke's pouch. During craniofacial development, Barx1 and Barx2 show complementary patterns of expression; whereas Barx1 appears in the mesenchyme of the mandibular and maxillary processes, Barx2 is observed in the ectodermal lining of these tissues. Intense expression of Barx2 is observed in small groups of cells undergoing tissue remodeling, such as ectodermal cells within indentations surrounding the eye and maxillo-nasal groove and in the first branchial pouch, lung buds, precartilagenous condensations and mesenchyme of the limb. The localization data, combined with Barx2's dual function as activator and repressor, suggest that Barx2 may differentially control the expression of L1 and other target genes during embryonic development (Jones, 1997).
BARX2 is a homeobox transcription factor that influences cellular differentiation in various developmental contexts. To begin to identify the gene targets that mediate its effects, chromatin immunoprecipitation (ChIP) was used to isolate BARX2 binding sites from the human MCF7 breast cancer cell line. Cloning and sequencing of BARX2-ChIP-derived DNA fragments identified 60 potential BARX2 target loci that were proximal to or within introns of genes involved in cytoskeletal organization, cell adhesion, growth factor signaling, transcriptional regulation, and RNA metabolism. The sequences of over half of the fragments showed homology with the mouse genome, and several sequences could be mapped to orthologous human and mouse genes. Binding of BARX2 to 21 genomic loci examined was confirmed quantitatively by replicate ChIP assays. A combination of sequence analysis and electrophoretic mobility shift assays revealed homeodomain binding sites within several fragments that bind to BARX2 in vitro. The majority of BARX2 binding fragments tested (14/19), also affected transcription in luciferase reporter gene assays. Mutation analyses of three fragments showed that their transcriptional activities required the homeodomain binding sites, and suggested that BARX2 regulates gene expression by binding to DNA elements containing paired TAAT motifs that are separated by a poly(T) sequence. Inhibition of BARX2 expression in MCF7 cells led to reduced expression of eight genes associated with BARX2 binding sites, indicating that BARX2 directly regulates their expression. The data suggest that BARX2 can coordinate the expression of a network of genes that influence the growth of MCF7 cells (Stevens, 2004).
Inductive interactions between gut endoderm and the underlying mesenchyme
pattern the developing digestive tract into regions with specific morphology and
functions. The molecular mechanisms behind these interactions remain largely
unknown. Expression of the conserved homeobox gene Barx1 is restricted to the
stomach mesenchyme during gut organogenesis. Using recombinant tissue cultures,
it has been shown that Barx1 loss in the mesenchyme prevents stomach epithelial
differentiation of overlying endoderm and induces intestine-specific genes
instead. Additionally, Barx1 null mouse embryos show visceral homeosis, with
intestinal gene expression within a highly disorganized gastric epithelium.
Barx1 directs mesenchymal cell expression of two secreted Wnt antagonists, sFRP1
and sFRP2, and these factors are sufficient replacements for Barx1 function.
Canonical Wnt signaling is prominent in the prospective gastric endoderm prior
to epithelial differentiation, and its inhibition by Barx1-dependent signaling
permits development of stomach-specific epithelium. These results define a
transcriptional and signaling pathway of inductive cell interactions in
vertebrate organogenesis (Kim, 2005).
Barhl1, a mouse homologous gene of Drosophila BarH class homeobox genes, is highly expressed within the inner ear and crucial for the long-term maintenance of auditory hair cells that mediate hearing and balance, yet little is known about the molecular events underlying Barhl1 regulation and function in hair cells. Through data mining and in vitro report assay, this study firstly found that Barhl1 is a direct target gene of Atoh1 and one E-box (E3) in the Barhl1 3' enhancer that is crucial for Atoh1-mediated Barhl1 activation. Then a mouse embryonic stem cell (mESC) line was generated carrying disruptions on this E3 site E-box (CAGCTG) using CRISPR/Cas9 technology and this E3 mutated mESC line was further subjected to an efficient stepwise hair cell differentiation strategy in vitro. Disruptions on this E3 site caused dramatic loss of Barhl1 expression and significantly reduced the number of induced hair cell-like cells, while no changes were identified in differentiation toward early primitive ectoderm-like cells and otic progenitors. Finally, through RNA-seq profiling and gene ontology (GO) enrichment analysis, this E3 box was found to be indispensable for Barhl1 expression to maintain hair cell development and normal functions. The transcriptional profiles were compared of induced cells from CDS mutated and E3 mutated mESCs, respectively, and very consistent results were obtained except the Barhl1 transcript itself. These observations indicated that Atoh1-mediated Barhl1 expression could have important roles during auditory hair cell development. In brief, these findings delineate the detail molecular mechanism of Barhl1 expression regulation in auditory hair cell differentiation (Hou, 2019).
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