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Current Projects

The role of T-box genes in the regionalization of the murine heart

Funding: REBIRTH, EU

The complex multi-chambered heart of vertebrates arises from a short tubular structure through a coordinated program of cellular differentiation and proliferation, and tissue morphogenesis. Elongation of the simple tube is supported by recruitment of precursor cells and differentiation into cardiomyocytes at the two poles. Highly localized processes of further myocardial differentiation and increased proliferation within the growing heart tube mediate the out-bulging of the atrial and ventricular chambers on its dorsal and ventral side, respectively. Regions separating and bordering the developing chambers retain low proliferation rates and slow impulse conduction and resist differentiation in a working type of myocardium, resulting in the generation of primitive morphological constrictions, the AV canal and outflow tract, and a delay in AV conduction. Formation of mesenchymal cushions in the AV canal and outflow tract that are subsequently remodeled into thin valve leaflets and components of the septa ensure structural and functional compartmentalization of the mature heart.

Insight in the molecular control of myocardial patterning and differentiation has remained incomplete despite its relevance for congenital heart diseases. However, it has emerged that compartmentalization of the heart tube is regulated by a network of transcription factor activities including members of the evolutionary conserved family of T-box transcription factors consisting of Tbx1, Tbx2, Tbx3, Tbx5, Tbx18 and Tbx20. We and others have shown that Tbx20 and Tbx5 promote chamber formation. Mice homozygous mutant for Tbx20 establish a heart tube with a primary myocardial phenotype but fail to undergo looping morphogenesis and initiate chamber formation. Tbx5 acts independently of Tbx20 and maintains posterior domains of the heart. Both Tbx20 and Tbx5 synergize with cardiac transcription factors Nkx2-5 and Gata4 to activate expression of chamber specific genes such as Nppa and Cx40.

Transcriptional repressors Tbx2 and Tbx3 act downstream of BMP-signaling and regionally inhibit a chamber myocardial gene program in the AV canal by competing with activating T-box proteins such as Tbx5 for binding to conserved T-box binding elements (TBE)s in promoters of chamber specific genes. Tbx2 mutant embryos show septation defects in the outflow tract and partial expansion of chamber-specific gene expression into the AV canal. Tbx3 is required for the molecular specification of the sinus node, the AV bundle and bundle branches and for the development of the ventricular septum and outflow tract in the mouse. Tbx2 expression shows partial overlap with Tbx3 in the AV canal myocardium, suggesting that functional redundancy has prevented a full appreciation of their role in the development of this tissue to date. Embryos with ectopic expression of either Tbx2 or Tbx3 in the pre-chamber heart tube fail to form chambers, proving that the two genes are not only required but also sufficient to prevent differentiation of chamber myocardium. Notably, Tbx2 is up-regulated in the embryonic heart tube of Tbx20-deficient embryos and may be responsible for the observed block in chamber differentiation in Tbx20-deficient hearts, suggesting that Tbx20 is required to repress Tbx2 for the progression to a multi-chambered heart. Tbx18 is expressed in the developing sinus horn and is required for release and myocardialization of these vessels at the venous pole of the heart. In addition, we showed that Tbx18 is required for the formation of the pacemaker of the heart, the sinoatrial node.

We are currently engaged in further deciphering the molecular network regulated by the T-box genes Tbx2, Tbx3, Tbx20 and Tbx18 that controls the formation of cardiac chambers and the formation of the caval veins.

Analysis of regulation and function of Tbx18 in the ureteric mesenchyme

Funding: Deutsche Forschungsgemeinschaft

The ureter represents a pivotal connection between the upper and lower urinary system by ensures the unidirectional transport of urine from renal the pelvis to the bladder whilst preventing any reflux or efflux at the same time. The crucial importance of this simple tube for renal function is dramatically reflected by acquired and inherited defects that interfere with the efficient removal of the urine from the renal pelvis. Any kind of anatomical or functional obstruction along the ureter or at its junctions will result in fluid pressure-mediated dilation of the tubular system proximal to the side of constriction (hydroureter and hydronephrosis) with eventual destruction of the renal parenchyme.

Formation of the ureter with its two-layered tissue architecture and connectivity with bladder and pelvis relies on a multi-step developmental program that is characterized by the interaction of different mesenchymal and epithelial cell lineages of the early metanephric field. Our recent findings on the expression and function of the transcription factor gene Tbx18 in the ureteric mesenchyme have revealed the importance of this tissue and Tbx18 therein for the formation of a functional ureter tube. In Tbx18-deficient mice, the ureteric mesenchyme disperses and remains undifferentiated leading to hydroureter formation by functional obstruction.

We are currently interested to further explore the molecular pathways that confer the spatial restriction of Tbx18 expression in the early metanephric field and characterize the genetic circuits that act downstream of Tbx18 to mediate its function in the ureteric mesenchyme.

Functional redundancy of Tbx15 and Tbx18 in mouse limb development

Funding: Deutsche Forschungsgemeinschaft

Vertebrate limbs are body appendages with a stereotyped pattern of skeletal elements. These elements (girdle region, stylopod, zeugopod, autopod) arise by growth and patterning processes coordinated by specialized regions of the developing limb bud. While the molecular nature of the signaling systems establishing the main axes of the limb bud have been identified, much less is known about the mechanisms controlling regionalization along these axes. Transcription factors including members of the T-box gene family are likely to play pivotal roles in these processes.

Tbx15 and Tbx18, two closely related T-box genes, are expressed in a largely overlapping pattern in the limb bud mesenchyme. Null alleles of Tbx15 and Tbx18 cause minor and no defects, respectively, in limb development. In contrast, mice double homozygous for Tbx15 and Tbx18 mutant alleles show severe defects of specific limb skeletal elements. Conserved DNA-binding and transcriptional modulation activities point to biochemical equivalence of the two transcription factors.

We are currently engaged in further characterizing the phenotypic changes of limb development in double mutant Tbx15 and Tbx18 embryos at the histological, immunohistochemical and molecular level. We analyze the regulation of Tbx18 expression in to get insight into the mechanisms restricting Tbx18 to the proximal limb bud mesenchyme. In addition, we perform gain-of-function experiments in vivo to explore the cellular pathways regulated by Tbx18 in the developing limb.

The function of Tbx18 in patterning the otic mesenchyme

The cochlea of the inner ear is a sensory apparatus that converts the mechanical stimulation of sound into electrical activity. A crucial factor in sound transduction, and thus hearing, is the maintenance of the ionic homeostasis of the endolymph, the extracellular fluid of the cochlear duct. The importance of fibrocyte integrity in this process has become apparent by pathological changes caused by inherited disorders or environmental stress. Genetic ablation of certain fibrocyte-expressed genes is known to cause deafness, and noise-induced, as well as age-related, hearing deficits are initiated by changes in fibrocyte physiology.

Otic fibrocytes represent a heterogeneous population of cells with special structural and molecular adaptations according to their location and physiological properties. Fibrocytes are found in the spiral limbus at the proximal site of the cochlea, and in the spiral ligament in the cochlear lateral wall, where five subgroups can be distinguished. Type I fibrocytes underlie the stria vascularis, a specialized non-sensory epithelial thickening of the lateral wall, type II fibrocytes are situated under the spiral prominence, type III fibrocytes line, as a thin layer, the otic capsule, type IV fibrocytes are located lateral to the basilar membrane and anchor it to the lateral wall, and type V fibrocytes reside above the stria vascularis. Fibrocytes of subtypes I, II and V are highly interconnected, and form a mesenchymal gap junction network. This and an independent epithelial network couple non-sensory supporting cells of the Organ of Corti with basal and intermediate cells of the stria vascularis. Basal cells form a multi-layered epithelial barrier that separates the extracellular spaces of the stria vascularis and the spiral ligament. Neural crest-derived intermediate cells form a discontinuous layer between basal cells and marginal cells that constitutes an epithelial barrier facing the endolymph in the cochlear duct. The mesenchymal gap junction network plays a central role in ionic homeostasis. In fact, recycling of K+-ions through this network is pivotal for cochlear physiology. Strial marginal cells actively transport K+-ions into the endolymph to maintain a very high concentration in this compartment. A voltage gradient between the negative potential inside the sensory hair cells and the positive endocochlear potential (EP) in the endolymph, together with the concentration gradient in the same direction, drives the influx of K+- ions through apical mechano-sensitive channels and, thus causes the depolarization of hair cells. After secretion by hair cells and re-uptake by supporting cells, K+-ions are thought to travel through the epithelial and mesenchymal gap junction networks back to the stria
Vascularis  Despite the importance of otic fibrocytes for the physiology and pathology of hearing, little insight has been gained into the genetic circuits regulating fibrocyte development. Mice mutant for the transcription factor gene Pou3f4 (also known as Brn4) show ultrastructural alterations in fibrocyte morphology and exhibit a reduced EP and profound deafness. In mice mutant for otospiralin (Otos), a gene encoding a small extracellular matrix (ECM) protein of unknown function,fibrocytes type II and IV are degenerated. Similar to Pou3f4, the precise role of Otos in fibrocyte differentiation is unknown.
We have recently defined a critical role in the development of otic fibrocytes for Tbx18, a member of the evolutionary conserved family of T-box transcription factors. Tbx18 expression during inner ear development is restricted to the sub-region of otic mesenchyme that is fated to differentiate into fibrocytes. We rescued the somitic defect that underlies the perinatal lethality of Tbx18-mutant mice by a transgenic approach, and measured auditory brainstem responses. Adult Tbx18- deficient mice showed profound deafness and a complete disruption of the endocochlear potential that is essential for the transduction of sound by sensory hair cells. The differentiation of otic fibrocytes of the spiral ligament was severely compromised.

Tissue architecture of the stria vascularis of the lateral wall was disrupted, exhibiting an almost complete absence of the basal cell layer, and a reduction and changes of intermediate and marginal cells, respectively. Stria vascularis defects resulted from the failure of Tbx18-mutant otic fibrocytes to generate the basal cell layer by a mesenchymal-epithelial transition. Defects in otic fibrocyte differentiation may be subordinate to a primary role of Tbx18 in early compartmentalization of the otic mesenchyme, as lineage restriction and boundary formation between otic fibrocytes and the surrounding otic capsule were severely affected in the mutant. We are currently further exploring the molecular pathways regulated by Tbx18 critical for compartmentalization of the otic mesenchyme and the differentiation of otic fibrocytes.

The role of Uncx4.1 and Tbx18 in anterior-posterior polarization of somites

Funding: Deutsche Forschungsgemeinschaft

The metameric organization of the vertebral column derives from the somites, segmentally repeated units in the paraxial mesoderm. Somites form in a highly periodic and synchronized fashion by condensation and subsequent epithelialization of groups of mesenchymal cells at the anterior end of the presomitic mesoderm (PSM) on both sides of the neural tube.

Under the influence of signals from surrounding tissues, somites start to differentiate along their dorso-ventral axis. The ventral part undergoes an epithelial-mesenchymal transition to form the sclerotome, which contains precursors of the vertebral column and parts of the ribs. The dorsal part remains epithelial and generates the dermomyotome, from which skeletal muscles and the dermis of the skin will develop. In addition to differentiation along the dorso-ventral axis, somites become subdivided into distinct anterior and posterior compartments.

Anterior-posterior (AP) polarization of somites underlies the segmental arrangement of the peripheral nervous system, since trajectories of neural crest and spinal nerves are confined to anterior somite halves. On the level of the sclerotome, the differential contribution of either compartment to the forming vertebra affects the structure of the axial skeleton. Vertebral bodies, laminae with the spinal processes, the rib heads, and the distal ribs derive from both  omite halves, whereas pedicles with their transverse processes and proximal ribs derive from posterior somite halves only.

Establishment of somitic AP polarity occurs in the anterior presomitic mesoderm by the combined action of the Notch/Delta signaling pathway and the basic helix-loop-helix transcription factor Mesp2. The expression domain of Mesp2 thereby defines the anterior somite half, and its anterior limit demarcates the next segmental border to be formed.

Molecular players required for the further maintenance of somitic AP polarity have recently surfaced by our work. Genetic evidence from both loss- and gain-of-function studies in the mouse suggest that this process is controlled by the combined action of a pair of transcription factors, the T-box (Tbx) protein Tbx18 and the paired type homeobox protein Uncx4.1, which are expressed in anterior and posterior somite halves, respectively. Uncx4.1 is specifically required for the development of pedicles and proximal ribs, elements exclusively derived from the posterior lateral sclerotome. In contrast, loss of Tbx18 function results in expansion of pedicles and proximal ribs in the cervical and thoracic region of the axial skeleton. Notably, the forced misexpression of Tbx18 in posterior somite halves results in reduction of pedicles and proximal ribs, suggesting that Tbx18 is sufficient to specify anterior versus posterior somite fates. Opposing phenotypic consequences of loss of either factor are based on molecular cross-regulation. In Uncx4.1 mutants, Tbx18 expression is derepressed in posterior
somite halves, whereas in Tbx18 mutants, expression of Uncx4.1 progressively expands in anterior somite halves. We are currently further analying the molecular circuits regulated by Uncx4.1 and Tbx18 that maintaincompartmentalization of somites along the anterior-posterior axis.


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