ABSTRACT A small network of spontaneously active Tbx3 + cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3 + cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3 + cells was restricted to the definitive CCS. We identified a Tbx3 + population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3 + cardiomyocytes also contributed to the adjacent Gja5 + atrial and ventricular chamber myocardium, embryonic Gja5 + chamber cardiomyocytes did not contribute to the Tbx3 + sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3 + cell population in the early developing heart, which implicates Tbx3 as a useful tool for developing strategies to study and treat CCS diseases.

The role of Hox genes in the formation of cutaneous accessory organs such as hair follicles and mammary glands has proved elusive, a likely consequence of overlapping function and expression among various homeobox factors. Lineage and immunohistochemical analysis of Hoxc8 in mice revealed that this midthoracic Hox gene has transient but strong regional expression in ventrolateral surface ectoderm at E10.5, much earlier than previously reported. Targeted mice were generated to conditionally misexpress Hoxc8 from the Rosa locus using select Cre drivers, which significantly expanded the domain of thoracic identity in mutant embryos. Accompanying this expansion was the induction of paired zones of ectopic mammary development in the cervical region, which generated between three and five pairs of mammary placodes anterior to the first wild-type mammary rudiment. These rudiments expressed the mammary placode markers Wnt10b and Tbx3 and were labeled by antibodies to the mammary mesenchyme markers ERα and androgen receptor. Somitic Fgf10 expression, which is required for normal mammary line formation, was upregulated in mutant cervical somites, and conditional ablation of ectodermal Tbx3 expression eliminated all normally positioned and ectopic mammary placodes. We present evidence that Hoxc8 participates in regulating the initiation stages of mammary placode morphogenesis, and suggest that this and other Hox genes are likely to have important roles during regional specification and initiation of these and other cutaneous accessory organs.

Facial somatosensory input is relayed by trigeminal ganglion (TG) neurons and serially wired to brainstem, thalamus and cortex. Spatially ordered sets of target neurons generate central topographic maps reproducing the spatial arrangement of peripheral facial receptors. Facial pattern provides a necessary template for map formation, but may be insufficient to impose a brain somatotopic pattern. In mice, lower jaw sensory information is relayed by the trigeminal nerve mandibular branch, whose axons target the brainstem dorsal principal sensory trigeminal nucleus (dPrV). Input from mystacial whiskers is relayed by the maxillary branch and forms a topographic representation of rows and whiskers in the ventral PrV (vPrV). To investigate peripheral organisation in imposing a brain topographic pattern, we analysed Edn1 −/− mice, which present ectopic whisker rows on the lower jaw. We found that these whiskers were innervated by mandibular TG neurons which initially targeted dPrV. Unlike maxillary TG neurons, the ectopic whisker-innervating mandibular neuron cell bodies and pre-target central axons did not segregate into a row-specific pattern nor target the dPrV with a topographic pattern. Following periphery-driven molecular repatterning to a maxillary-like identity, mandibular neurons partially redirected their central projections from dPrV to vPrV. Thus, while able to induce maxillary-like molecular features resulting in vPrV final targeting, a spatially ordered lower jaw ectopic whisker pattern is insufficient to impose row-specific pre-target organisation of the central mandibular tract or a whisker-related matching pattern of afferents in dPrV. These results provide novel insights into periphery-dependent versus periphery-independent mechanisms of trigeminal ganglion and brainstem patterning in matching whisker topography.

Although the T-box family of transcription factors function in many different tissues, their role in liver development is unknown. Here we show that Tbx3 , the T-box gene that is mutated in human ulnar-mammary syndrome, is specifically expressed in multipotent hepatic progenitor cells,`hepatoblasts', isolated from the developing mouse liver. Tbx3 -deficient hepatoblasts presented severe defects in proliferation as well as uncontrollable hepatobiliary lineage segregation, including the promotion of cholangiocyte (biliary epithelial cell) differentiation, which thereby caused abnormal liver development. Deletion of Tbx3 resulted in the increased expression of the tumor suppressor p19 ARF ( Cdkn2a ), which in turn induced a growth arrest in hepatoblasts and activated a program of cholangiocyte differentiation. Thus, Tbx3 plays a crucial role in controlling hepatoblast proliferation and cell-fate determination by suppressing p19 ARF expression and thereby promoting liver organogenesis.

A number of human congenital disorders present with both heart and limb defects, consistent with common genetic pathways. We have recently shown that the LIM homeodomain transcription factor islet 1 (Isl1) marks a subset of cardiac progenitors. Here, we perform lineage studies with an Isl1Cre mouse line to demonstrate that Isl1 also marks a subset of limb progenitors. In both cardiac and limb progenitors, Isl1 expression is downregulated as progenitors migrate in to form either heart or limb. To investigate common heart-limb pathways in Isl1-expressing progenitors, we ablated the Type I Bmp receptor,Bmpr1a utilizing Isl1Cre/+. Analysis of consequent heart and limb phenotypes has revealed novel requirements for Bmp signaling. Additionally, we find that Bmp signaling in Isl1-expressing progenitors is required for expression of T-box transcription factors Tbx2 and Tbx3 in heart and limb. Tbx3 is required for heart and limb formation, and is mutated in ulnar-mammary syndrome. We provide evidence that the Tbx3 promoter is directly regulated by Bmp Smads in vivo.

The limbs of the vertebrate embryo form at precise locations along the body and these positions are fixed across different species. The mechanisms that control this process are not understood. Ectopic expression of Tbx3 ,a transcriptional repressor that belongs to the Tbx2/3/4/5 subfamily of T-box transcriptional regulators, in the forelimb results in a rostral shift in the position of the limb along the main body axis. By contrast, a transcriptional activator form of Tbx3 shifts the limb to more caudal locations. We also show that dHand and Gli3 , genes previously implicated in anteroposterior pre-patterning of the limb-forming region, are also involved in refining the position of the limbs. Our data suggest a new role for Tbx3 in positioning the limb along the main body axis through a genetic interplay between dHand and Gli3 .

Spontanteous mutations in the T-box gene TBX3 , result in the human ulnar-mammary syndrome, a dominant developmental disorder characterized by abnormal forelimb and apocrine gland development. In order to develop a mouse model to study the role of this gene during development and disease, we produced a mutation in the mouse ortholog, Tbx3 . The phenotype of the mutant mice verifies the role of this gene in limb and mammary gland development, and, in addition, reveals a previously unknown role for the gene in the yolk sac, a fetal membrane that is the site of hematopoiesis and is essential for survival during gestation. In homozygous mutant embryos, the yolk sac undergoes cell death and degeneration at midgestation and the fetuses die over a range of several days; none survive to birth. Tbx3 is the first T-box gene implicated in yolk sac development. Homozygous embryos show a deficiency of mammary gland induction, and exhibit both forelimb and hindlimb abnormalities. Although heterozygous mice, unlike their heterozygous human counterparts, have no apparent phenotype in limb or mammary gland, the homozygous defects in the development of these organs represent more severe manifestations of the defects characteristic of the ulnar-mammary syndrome.