Rb deficiency, neuronal survival and neurodegeneration: In search of the perfect mouse model.

Three decades following the introduction of the first Rb knockout (KO) mouse model, the role of this critical protein in regulating brain development during embryogenesis and beyond remains a major scientific interest. Rb is a tumor suppressor gene known as the master regulator of the G1/S checkpoint and control of cell cycle progression in stem and progenitor cells, but also their differentiated progeny. Here, we review the recent literature about the various Rb conditional Knockout (cKO) and inducible Knockout (iKO) models studied thus far, highlighting how findings should always be interpreted in light of the model and context under inquiry especially when studying the role of Rb in neuronal survival. There is indeed evidence of age-specific, cell type-specific and region-specific effects following Rb KO in the embryonic and the adult mouse brain. In terms of modeling neurodegenerative processes in human diseases, we discuss cell cycle re-entry (CCE) as a candidate mechanism underlying the increased vulnerability of Rb-deficient neurons to cell death. Notably, mouse models may limit the extent to which CCE due to Rb inactivation can mimic the pathological course of these disorders, such as Alzheimer's disease. These remarks ought to be considered in future research when studying the consequences of Rb inactivation on neuronal generation and survival in rodents and their corresponding clinical significance in humans.

The Rb/E2F axis is a key regulator of the molecular signatures instructing the quiescent and activated adult neural stem cell state.

Long-term maintenance of the adult neurogenic niche depends on proper regulation of entry and exit from quiescence. Neural stem cell (NSC) transition from quiescence to activation is a complex process requiring precise cell-cycle control coordinated with transcriptional and morphological changes. How NSC fate transitions in coordination with the cell-cycle machinery remains poorly understood. Here we show that the Rb/E2F axis functions by linking the cell-cycle machinery to pivotal regulators of NSC fate. Deletion of Rb family proteins results in activation of NSCs, inducing a transcriptomic transition toward activation. Deletion of their target activator E2Fs1/3 results in intractable quiescence and cessation of neurogenesis. We show that the Rb/E2F axis mediates these fate transitions through regulation of factors essential for NSC function, including REST and ASCL1. Thus, the Rb/E2F axis is an important regulator of NSC fate, coordinating cell-cycle control with NSC activation and quiescence fate transitions.

Aging entails distinct requirements for Rb at maintaining adult neurogenesis.

Cell cycle proteins play essential roles in regulating embryonic and adult neurogenesis in the mammalian brain. A key example is the Retinoblastoma protein (Rb) whose loss disrupts the whole neurogenic program during brain development, but only results in increased progenitor proliferation in the adult subventricular zone (SVZ) and compromised long-term neuronal survival in the adult olfactory bulb (OB). Whether this holds true of neurogenesis in the aged brain remains unknown. In this study, we find no evidence of irregular proliferation or early commitment defects in the mid-aged (12-month-old) and old-aged (20-month-old) SVZ following tamoxifen-inducible Rb knockout (Rb iKO) in mice. However, we highlight a striking defect in early maturation of Rb-deficient migrating neuroblasts along the rostral migratory stream (RMS), followed by massive decline in neuronal generation inside the aged OB. In the absence of Rb, we also show evidence of incomplete cell cycle re-entry (CCE) along with DNA damage in the young OB, while we find a similar trend towards CCE but no clear signs of DNA damage or neurodegenerative signatures (pTau or Synuclein accumulation) in the aged OB. However, such phenotype could be masked by the severe maturation defect reported above in addition to the natural decline in adult neurogenesis with age. Overall, we show that Rb is required to prevent CCE and DNA damage in adult-born OB neurons, hence maintain neuronal survival. Moreover, while loss of Rb alone is insufficient to trigger seeding of neurotoxic species, this study reveals age-dependent non-monotonic dynamics in regulating neurogenesis by Rb.

Exogenous Flupirtine as Potential Treatment for CLN3 Disease.

CLN3 disease is a fatal neurodegenerative disorder affecting children. Hallmarks include brain atrophy, accelerated neuronal apoptosis, and ceramide elevation. Treatment regimens are supportive, highlighting the importance of novel, disease-modifying drugs. Flupirtine and its new allyl carbamate derivative (compound 6) confer neuroprotective effects in CLN3-deficient cells. This study lays the groundwork for investigating beneficial effects in Cln3Δex7/8 mice. WT/Cln3Δex7/8 mice received flupirtine/compound 6/vehicle for 14 weeks. Short-term effect of flupirtine or compound 6 was tested using a battery of behavioral testing. For flupirtine, gene expression profiles, astrogliosis, and neuronal cell counts were determined. Flupirtine improved neurobehavioral parameters in open field, pole climbing, and Morris water maze tests in Cln3Δex7/8 mice. Several anti-apoptotic markers and ceramide synthesis/degradation enzymes expression was dysregulated in Cln3Δex7/8 mice. Flupirtine reduced astrogliosis in hippocampus and motor cortex of male and female Cln3Δex7/8 mice. Flupirtine increased neuronal cell counts in male mice. The newly synthesized compound 6 showed promising results in open field and pole climbing. In conclusion, flupirtine improved behavioral, neuropathological and biochemical parameters in Cln3Δex7/8 mice, paving the way for potential therapies for CLN3 disease.

Histological Assessment of Cre-loxP Genetic Recombination in the Aging Subventricular Zone of Nestin-CreERT2/Rosa26YFP Mice.

The use of inducible transgenic Nestin-CreERT2 mice has proved to be an essential research tool for gene targeting and studying the molecular pathways implicated in adult neurogenesis, namely, inside the adult subgranular zone (SGZ) of the dentate gyrus and the adult subventricular zone (SVZ) lining the lateral ventricles. Several lines of Nestin-CreER-expressing mice were generated and used in adult neurogenesis research in the past two decades; however, their suitability for studying neurogenesis in aged mice remains elusive. Here, we assessed the efficiency of Cre-loxP genetic recombination in the aging SVZ using the Nestin-CreERT2/Rosa26YFP line designed by Lagace et al. (J Neurosci 27(46):12623-12629, 2007). This analysis was performed in 12-month-old (middle-aged) mice and 20-month-old (old) mice compared to 2-month-old (young adult) mice. To evaluate successful recombination, our approach relies on the histological assessment of Cre mRNA level of expression and the YFP reporter gene's expression inside the aging SVZ by combining in situ hybridization and immunohistochemistry. Using co-immunolabeling, this approach also provides the advantage of estimating the percentage of recombined progeny [(GFP+Nestin+)/Nestin+] and the rate of cell proliferation [(GFP+Ki67+)/GFP+] inside the aging SVZ niche.

Combination of drug and stem cells neurotherapy: Potential interventions in neurotrauma and traumatic brain injury.

Traumatic brain injury (TBI) has been recognized as one of the major public health issues that leads to devastating neurological disability. As a consequence of primary and secondary injury phases, neuronal loss following brain trauma leads to pathophysiological alterations on the molecular and cellular levels that severely impact the neuropsycho-behavioral and motor outcomes. Thus, to mitigate the neuropathological sequelae post-TBI such as cerebral edema, inflammation and neural degeneration, several neurotherapeutic options have been investigated including drug intervention, stem cell use and combinational therapies. These treatments aim to ameliorate cellular degeneration, motor decline, cognitive and behavioral deficits. Recently, the use of neural stem cells (NSCs) coupled with selective drug therapy has emerged as an alternative treatment option for neural regeneration and behavioral rehabilitation post-neural injury. Given their neuroprotective abilities, NSC-based neurotherapy has been widely investigated and well-reported in numerous disease models, notably in trauma studies. In this review, we will elaborate on current updates in cell replacement therapy in the area of neurotrauma. In addition, we will discuss novel combination drug therapy treatments that have been investigated in conjunction with stem cells to overcome the limitations associated with stem cell transplantation. Understanding the regenerative capacities of stem cell and drug combination therapy will help improve functional recovery and brain repair post-TBI. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".

"Till Death Do Us Part": A Potential Irreversible Link Between Aberrant Cell Cycle Control and Neurodegeneration in the Adult Olfactory Bulb.

Adult neurogenesis (AN) is an ongoing developmental process that generates newborn neurons in the olfactory bulb (OB) and the hippocampus (Hi) throughout life and significantly contributes to brain plasticity. Adult neural stem and progenitor cells (aNSPCs) are relatively limited in number and fate and are spatially restricted to the subventricular zone (SVZ) and the subgranular zone (SGZ). During AN, the distinct roles played by cell cycle proteins extend beyond cell cycle control and constitute key regulatory mechanisms involved in neuronal maturation and survival. Importantly, aberrant cell cycle re-entry (CCE) in post-mitotic neurons has been strongly linked to the abnormal pathophysiology in rodent models of neurodegenerative diseases with potential implications on the etiology and progression of such diseases in humans. Here, we present an overview of AN in the SVZ-OB and olfactory epithelium (OE) in mice and humans followed by a comprehensive update of the distinct roles played by cell cycle proteins including major tumors suppressor genes in various steps during neurogenesis. We also discuss accumulating evidence underlining a strong link between abnormal cell cycle control, olfactory dysfunction and neurodegeneration in the adult and aging brain. We emphasize that: (1) CCE in post-mitotic neurons due to loss of cell cycle suppression and/or age-related insults as well as DNA damage can anticipate the development of neurodegenerative lesions and protein aggregates, (2) the age-related decline in SVZ and OE neurogenesis is associated with compensatory pro-survival mechanisms in the aging OB which are interestingly similar to those detected in Alzheimer's disease and Parkinson's disease in humans, and (3) the OB represents a well suitable model to study the early manifestation of age-related defects that may eventually progress into the formation of neurodegenerative lesions and, possibly, spread to the rest of the brain. Such findings may provide a novel approach to the modeling of neurodegenerative diseases in humans from early detection to progression and treatment as well.

Postnatal Neural Stem Cells in Treating Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disabilities worldwide. It affects approximately 1.5 million people each year and is associated with severe post-TBI symptoms such as sensory and motor deficits. Several neuro-therapeutic approaches ranging from cell therapy interventions such as the use of neural stem cells (NSCs) to drug-based therapies have been proposed for TBI management. Successful cell-based therapies are tightly dependent on reproducible preclinical animal models to ensure safety and optimal therapeutic benefits. In this chapter, we describe the isolation of NSCs from neonatal mouse brain using the neurosphere assay in culture. Subsequently, dissociated neurosphere-derived cells are used for transplantation into the ipsilateral cortex of a controlled cortical impact (CCI) TBI model in C57BL/6 mice. Following intra-cardiac perfusion and brain removal, the success of NSC transplantation is then evaluated using immunofluorescence in order to assess neurogenesis along with gliosis in the ipsilateral coronal brain sections. Behavioral tests including rotarod and pole climbing are conducted to evaluate the motor activity post-treatment intervention.

Role of Rb during Neurogenesis and Axonal Guidance in the Developing Olfactory System.

The Retinoblastoma protein, Rb, was shown to regulate distinct aspects of neurogenesis in the embryonic and adult brain besides its primary role in cell cycle control. It is still unknown, however, whether Rb is required for tissue morphogenesis and the establishment of synaptic connections between adjacent tissues during development. We have investigated here the role of Rb during development of the olfactory system (OS), which heavily relies on reciprocal interactions between the olfactory epithelium (OE) and the olfactory bulb (OB). We show that mice carrying a telencephalic-specific deletion of Rb display several neurogenic defects in the OS during late development. In the OE, loss of Rb leads to ectopic proliferation of late-born progenitors (Tuj-1+), abnormal radial migration and terminal maturation of olfactory sensory neurons (OSNs). In the OB, deletion of Rb causes severe lamination defects with loss of clear boundaries between distinct layers. Importantly, starting around E15.5 when OB glomerulogenesis is initiated, many OSNs axons that project along the olfactory nerve layer (ONL) fail to properly innervate the nascent bulb, thus resulting in partial loss of connectivity between OE-OB and gradual neuronal degeneration in both tissues peaking at birth. This deficiency correlates with deregulated expressions of two key chemo-repellant molecules, Robo2/Slit1 and Nrp2/Sema3F that control the formation of dorsal-ventral topographic map of OSNs connections with OB glomeruli. This study highlights a critical requirement for Rb during neurogenesis and the establishment of proper synaptic connections inside the OS during development.

RB regulates the production and the survival of newborn neurons in the embryonic and adult dentate gyrus.

In mammals, hippocampal dentate gyrus granule cells (DGCs) constitute a particular neuronal population produced both during embryogenesis and adult life, and play key roles in neural plasticity and memory. However, the molecular mechanisms regulating neurogenesis in the dentate lineage throughout development and adulthood are still not well understood. The Retinoblastoma protein (RB), a transcriptional repressor primarily involved in cell cycle control and cell death, plays crucial roles during cortical development but its function in the formation and maintenance of DGCs remains unknown. Here, we show that loss of RB during embryogenesis induces massive ectopic proliferation and delayed cell cycle exit of young DGCs specifically at late developmental stages but without affecting stem cells. This phenotype was partially counterbalanced by increased cell death. Similarly, during adulthood, loss of RB causes ectopic proliferation of newborn DGCs and dramatically impairs their survival. These results demonstrate a crucial role for RB in the generation and the survival of DGCs in the embryonic and the adult brain. © 2016 Wiley Periodicals, Inc.

Role of the Retinoblastoma protein, Rb, during adult neurogenesis in the olfactory bulb.

Adult neural stem cells (aNSCs) are relatively quiescent populations that give rise to distinct neuronal subtypes throughout life, yet, at a very low rate and restricted differentiation potential. Thus, identifying the molecular mechanisms that control their cellular expansion is critical for regeneration after brain injury. Loss of the Retinoblastoma protein, Rb, leads to several defects in cell cycle as well as neuronal differentiation and migration during brain development. Here, we investigated the role of Rb during adult neurogenesis in the olfactory bulb (OB) by inducing its temporal deletion in aNSCs and progenitors. Loss of Rb was associated with increased proliferation of adult progenitors in the subventricular zone (SVZ) and the rostral migratory stream (RMS) but did not alter self-renewal of aNSCs or neuroblasts subsequent migration and terminal differentiation. Hence, one month after their birth, Rb-null neuroblasts were able to differentiate into distinct subtypes of GABAergic OB interneurons but were gradually lost after 3 months. Similarly, Rb controlled aNSCs/progenitors proliferation in vitro without affecting their differentiation capacity. This enhanced SVZ/OB neurogenesis associated with loss of Rb was only transient and negatively affected by increased apoptosis indicating a critical requirement for Rb in the long-term survival of adult-born OB interneurons.

CDK2 transcriptional repression is an essential effector in p53-dependent cellular senescence-implications for therapeutic intervention.

UNLABELLED: Cellular senescence, a form of cell-cycle arrest, is a tumor-suppressor mechanism triggered by multiple tumor-promoting insults, including oncogenic stress and DNA damage. The role of cyclin-dependent kinase 2 (CDK2) regulation has been evaluated in models of replicative senescence, but little is known regarding its role in other senescence settings. Using in vitro and in vivo models of DNA damage-and oncogene-induced cellular senescence, it was determined that activation of the tumor-suppressor protein p53 (TP53) resulted in repression of the CDK2 transcript that was dependent on intact RB. Ectopic CDK2 expression was sufficient to bypass p53-dependent senescence, and CDK2-specific inhibition, either pharmacologically (CVT313) or by use of a dominant-negative CDK2, was sufficient to induce early senescence. Pharmacologic inhibition of CDK2 in an in vivo model of pineal tumor decreased proliferation and promoted early senescence, and it also decreased tumor penetrance and prolonged time to tumor formation in animals lacking p53. In conclusion, for both oncogene- and DNA damage-induced cellular senescence, CDK2 transcript and protein are decreased in a p53- and RB-dependent manner, and this repression is necessary for cell-cycle exit during senescence. IMPLICATIONS: These data show that CDK2 inhibition may be useful for cancer prevention in premalignant hyperproliferative lesions, as well as established tumors.

The Rb/E2F pathway modulates neurogenesis through direct regulation of the Dlx1/Dlx2 bigene cluster.

During brain morphogenesis, the mechanisms through which the cell cycle machinery integrates with differentiation signals remain elusive. Here we show that the Rb/E2F pathway regulates key aspects of differentiation and migration through direct control of the Dlx1 and Dlx2 homeodomain proteins, required for interneuron specification. Rb deficiency results in a dramatic reduction of Dlx1 and Dlx2 gene expression manifested by loss of interneuron subtypes and severe migration defects in the mouse brain. The Rb/E2F pathway modulates Dlx1/Dlx2 regulation through direct interaction with a Dlx forebrain-specific enhancer, I12b, and the Dlx1/Dlx2 proximal promoter regions, through repressor E2F sites both in vitro and in vivo. In the absence of Rb, we demonstrate that repressor E2Fs inhibit Dlx transcription at the Dlx1/Dlx2 promoters and Dlx1/2-I12b enhancer to suppress differentiation. Our findings support a model whereby the cell cycle machinery not only controls cell division but also modulates neuronal differentiation and migration through direct regulation of the Dlx1/Dlx2 bigene cluster during embryonic development.

Characterization of a distinct subpopulation of striatal projection neurons expressing the Dlx genes in the basal ganglia through the activity of the I56ii enhancer.

Regulation of region-specific neuronal differentiation and migration in the embryonic forebrain is a complex mechanism that involves a variety of transcription factors such as the Dlx genes. At least four cis-acting regulatory elements (CREs) are responsible for the Dlx transcriptional regulation in the subcortical telencephalon and the rostral diencephalon. These include I12b and URE2 in the Dlx1/2 bigene cluster, and, I56i and I56ii in the Dlx5/6 cluster. We previously reported that URE2, I12b, and I56i, mark different progenitor cell populations in the ganglionic eminences as well as different subtypes of adult cortical interneurons. Here, we carried out a detailed spatial and temporal analysis of the I56ii CRE activity in the developing telencephalon between E10.5 and E15.5, and compared its activity with the other three Dlx CREs using lacZ reporter genes in transgenic mice. We show that I56ii marks distinct group(s) of neurons located in the superficial mantle of the LGE and MGE between E11.5 and E13.5. The I56ii-positive cells are Dlx- and GABA-immunoreactive. However, unlike the other CREs, I56ii does not label interneuron progenitors in the basal ganglia, nor tangentially migrating cells to the cortex at E13.5. Instead, I56ii-positive cells mark a subpopulation(s) of post-mitotic projection neurons that tangentially migrate from the LGE to the deep mantle of the MGE and reside between the subventricular zone and the globus pallidus during midgestation. The majority of these neurons express the striatal markers Meis2 and Islet1. Moreover, both Meis2 and Islet1 activate transcription of a reporter gene containing the I56ii sequence in co-transfection assays, indicating that these transcriptional factors may be potential upstream modulators of the Dlx genes in vivo.

The Retinoblastoma family member p107 regulates the rate of progenitor commitment to a neuronal fate.

The Retinoblastoma protein p107 regulates the neural precursor pool in both the developing and adult brain. As p107-deficient mice exhibit enhanced levels of Hes1, we questioned whether p107 regulates neural precursor self-renewal through the repression of Hes1. p107 represses transcription at the Hes1 promoter. Despite an expanded neural precursor population, p107-null mice exhibit a striking reduction in the number of cortical neurons. Hes1 deficiency rescues neurosphere numbers in p107-null embryos. We find that the loss of a single Hes1 allele in vivo restores the number of neural precursor cells at the ventricular zone. Neuronal birthdating analysis reveals a dramatic reduction in the rate of neurogenesis, demonstrating impairment in p107(-/-) progenitors to commit to a neuronal fate. The loss of a single Hes1 allele restores the number of newly generated neurons in p107-deficient brains. Together, we identify a novel function for p107 in promoting neural progenitor commitment to a neuronal fate.

Distinct cis-regulatory elements from the Dlx1/Dlx2 locus mark different progenitor cell populations in the ganglionic eminences and different subtypes of adult cortical interneurons.

Distinct subtypes of cortical GABAergic interneurons provide inhibitory signals that are indispensable for neural network function. The Dlx homeobox genes have a central role in regulating their development and function. We have characterized the activity of three cis-regulatory sequences involved in forebrain expression of vertebrate Dlx genes: upstream regulatory element 2 (URE2), I12b, and I56i. The three regulatory elements display regional and temporal differences in their activities within the lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE), and caudal ganglionic eminence (CGE) and label distinct populations of tangentially migrating neurons at embryonic day 12.5 (E12.5) and E13.5. We provide evidence that the dorsomedial and ventral MGE are distinct sources of tangentially migrating neurons during midgestation. In the adult cortex, URE2 and I12b/I56i are differentially expressed in parvalbumin-, calretinin-, neuropeptide Y-, and neuronal nitric oxide synthase-positive interneurons; I12b and I56i were specifically active in somatostatin-, vasoactive intestinal peptide-, and calbindin-positive interneurons. These data suggest that interneuron subtypes use distinct combinations of Dlx1/Dlx2 enhancers from the time they are specified through adulthood.

The proneural determinant MASH1 regulates forebrain Dlx1/2 expression through the I12b intergenic enhancer.

Establishment of neuronal networks is an extremely complex process involving the interaction of a diversity of neuronal cells. During mammalian development, these highly organized networks are formed through the differentiation of multipotent neuronal progenitors into multiple neuronal cell lineages. In the developing forebrain of mammals, the combined function of the Dlx1, Dlx2, Dlx5 and Dlx6 homeobox genes is necessary for the differentiation of the GABAergic interneurons born in the ventricular and subventricular zones of the ventral telencephalon, as well as for the migration of these neurons to the hippocampus, cerebral cortex and olfactory bulbs. The 437 bp I12b enhancer sequence in the intergenic region of the Dlx1/2 bigene cluster is involved in the forebrain regulation of Dlx1/2. Using DNase I footprinting, we identified six regions of I12b potentially bound by transcription factors. Mutagenesis of each binding site affected the expression of reporter constructs in transgenic mice. However, the effects of impairing protein-DNA interactions were not uniform across the forebrain Dlx1/2 expression domains, suggesting that distinct regulatory interactions are taking place in the different populations of neuronal precursors. Analyses of protein-DNA interactions provide evidence of a direct role for MASH1 in Dlx1/2 regulation in the forebrain. DLX proteins play a crucial role in the maintenance of their own expression, as shown by transgenic and co-transfection experiments. These studies suggest that the seemingly continuous domains of Dlx gene expression in the telencephalon and diencephalon are in fact the combination of distinct cell populations within which different genetic regulatory interactions take place.

Analysis of four DLX homeobox genes in autistic probands.

BACKGROUND: Linkage studies in autism have identified susceptibility loci on chromosomes 2q and 7q, regions containing the DLX1/2 and DLX5/6 bigene clusters. The DLX genes encode homeodomain transcription factors that control craniofacial patterning and differentiation and survival of forebrain inhibitory neurons. We investigated the role that sequence variants in DLX genes play in autism by in-depth resequencing of these genes in 161 autism probands from the AGRE collection. RESULTS: Sequencing of exons, exon/intron boundaries and known enhancers of DLX1, 2, 5 and 6 identified several nonsynonymous variants in DLX2 and DLX5 and a variant in a DLX5/6 intragenic enhancer. The nonsynonymous variants were detected in 4 of 95 families from which samples were sequenced. Two of these four SNPs were not observed in 378 undiagnosed samples from North American populations, while the remaining 2 were seen in one sample each. CONCLUSION: Segregation of these variants in pedigrees did not generally support a contribution to autism susceptibility by these genes, although functional analyses may provide insight into the biological understanding of these important proteins.

Intergenic enhancers with distinct activities regulate Dlx gene expression in the mesenchyme of the branchial arches.

The vertebrate Dlx genes, generally organized as tail-to-tail bigene clusters, are expressed in the branchial arch epithelium and mesenchyme with nested proximodistal expression implicating a code that underlies the fates of jaws. Little is known of the regulatory architecture that is responsible for Dlx gene expression in developing arches. We have identified two distinct cis-acting regulatory sequences, I12a and I56i, in the intergenic regions of the Dlx1/2 and Dlx5/6 clusters that act as enhancers in the arch mesenchyme. LacZ transgene expression containing I12a is restricted to a subset of Dlx-expressing ectomesenchyme in the first arch. The I56i enhancer is active in a broader domain in the first arch mesenchyme. Expression of transgenes containing either the I12a or the I56i enhancers is dependent on the presence of epithelium between the onset of their expression at E9-10 until independence at E11. Both enhancers positively respond to FGF8 and FGF9; however, the responses of the reporter transgenes were limited to their normal domain of expression. BMP4 had a negative effect on expression of both transgenes and counteracted the effects of FGF8. Furthermore, bosentan, a pharmacological inhibitor of Endothelin-1 signaling caused a loss of I56i-lacZ expression in the most distal aspects of the expression domain, corresponding to the area of Dlx-6 expression previously shown to be under the control of Endothelin-1. Thus, the combinatorial branchial arch expression of Dlx genes is achieved through interactions between signaling pathways and intrinsic cellular factors. I56i drives the entire expression of Dlx5/6 in the first arch and contains necessary sequences for regulation by at least three separate pathways, whereas I12a only replicates a small domain of endogenous expression, regulated in part by BMP-4 and FGF-8.

Regulatory roles of conserved intergenic domains in vertebrate Dlx bigene clusters.

Dlx homeobox genes of vertebrates are generally arranged as three bigene clusters on distinct chromosomes. The Dlx1/Dlx2, Dlx5/Dlx6, and Dlx3/Dlx7 clusters likely originate from duplications of an ancestral Dlx gene pair. Overlaps in expression are often observed between genes from the different clusters. To determine if the overlaps are a result of the conservation of enhancer sequences between paralogous clusters, we compared the Dlx1/2 and the Dlx5/Dlx6 intergenic regions from human, mouse, zebrafish, and from two pufferfish, Spheroides nephelus and Takifugu rubripes. Conservation between all five vertebrates is limited to four sequences, two in Dlx1/Dlx2 and two in Dlx5/Dlx6. These noncoding sequences are >75% identical over a few hundred base pairs, even in distant vertebrates. However, when compared to each other, the four intergenic sequences show a much more limited similarity. Each intergenic sequence acts as an enhancer when tested in transgenic animals. Three of them are active in the forebrain with overlapping patterns despite their limited sequence similarity. The lack of sequence similarity between paralogous intergenic regions and the high degree of sequence conservation of orthologous enhancers suggest a rapid divergence of Dlx intergenic regions early in chordate/vertebrate evolution followed by fixation of cis-acting regulatory elements.