Epithelial-to-mesenchymal transition (EMT) is fundamental to both embryogenesis and tumor metastasis. The Notch intercellular signaling pathway regulates cell fate determination throughout metazoan evolution, and overexpression of activating alleles is oncogenic in mammals. Here we demonstrate that Notch activity promotes EMT during both cardiac development and oncogenic transformation via transcriptional induction of the Snail repressor, a potent and evolutionarily conserved mediator of EMT in many tissues and tumor types. In the embryonic heart, Notch functions via lateral induction to promote a selective transforming growth factor-␤ (TGF␤)-mediated EMT that leads to cellularization of developing cardiac valvular primordia. Embryos that lack Notch signaling elements exhibit severely attenuated cardiac snail expression, abnormal maintenance of intercellular endocardial adhesion complexes, and abortive endocardial EMT in vivo and in vitro. Accordingly, transient ectopic expression of activated Notch1 (N1IC) in zebrafish embryos leads to hypercellular cardiac valves, whereas Notch inhibition prevents valve development. Overexpression of N1IC in immortalized endothelial cells in vitro induces EMT accompanied by oncogenic transformation, with corresponding induction of snail and repression of VE-cadherin expression. Notch is expressed in embryonic regions where EMT occurs, suggesting an intimate and fundamental role for Notch, which may be reactivated during tumor metastasis.[Keywords: Notch; endocardium; lateral induction; EMT; snail; TGF␤] Supplemental material is available at http://www.genesdev.org. Epithelial-to-mesenchymal transition (EMT) is fundamental to both normal development and the progression of malignant epithelial tumors (for review, see Thiery 2002). During EMT, epithelial cells undergo sweeping alterations in gene expression to lose apical/basolateral polarity, sever intercellular adhesive junctions, degrade basement membrane components, and become migratory. Several signaling pathways seem to be common to EMT regulation during both development and tumor progression, leading to the notion that developmentally regulated EMT and tumor metastasis are under the control of common molecular mechanisms (Thiery 2002), and raising the hypothesis that tumor metastasis could be regarded as a reactivation of at least some aspects of the embryonic program of EMT.The snail family of Zinc-finger-containing transcriptional repressors is believed to play a pivotal role in the process of EMT (Nieto 2002). Expression of various snail family members has been tightly associated with cells undergoing both metastatic and developmental EMT (Nieto et al. 1992;Romano and Runyan 2000). One important target of Snail repression is the E-cadherin gene, the primary cadherin that is responsible for homotypic adhesion between members of an epithelial sheet (Batlle et al. 2000;Cano et al. 2000).A classical example of developmentally regulated EMT occurs during the initial stages of cardiac morphogenesis. At embryonic day 8.5 (E8.5...

Pitx1 and Pitx2 are highly homologous, bicoid-related transcription factors. Pitx2 was initially identified as the gene responsible for the human Rieger syndrome, an autosomal dominant condition that causes developmental abnormalities. Pitx2 is asymmetrically expressed in the left lateral-plate mesoderm, and mutant mice with laterality defects show altered patterns of Pitx2 expression that correlate with changes in the visceral symmetry (situs). Ectopic expression of Pitx2 in the right lateral-plate mesoderm alters looping of the heart and gut and reverses body rotation in chick and Xenopus embryos. Here we describe the phenotype of Pitx2 gene-deleted mice, characterized by defective body-wall closure, right pulmonary isomerism, altered cardiac position, arrest in turning and, subsequently, a block in the determination and proliferation events of anterior pituitary gland and tooth organogenesis. Thus, Pitx2 is a transcription factor that encodes 'leftness' of the lung.

IB kinases (IKKs) IKK1 and IKK2 are two putative IB␣ kinases involved in NF-B activation. To examine the in vivo functions of IKK1, we generated IKK1-deficient mice. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-B activation mediated by TNF␣ and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IB␣ and IB␤ in vitro. Our results support a role for IKK1 in NF-B activation and uncover its involvement in skin and skeleton development. We conclude further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other's functions. NF-B transcription factors are dimers composed of various combinations of structurally related proteins p50 (NF-B1), p52 (NF-B2), p65 (RelA), c-Rel, and RelB (for review, see Verma et al. 1995;Baeuerle and Baichwal 1997 ). In resting cells, NF-B complexes are retained in the cytoplasm in association with inhibitory proteins IBs (IB␣, I␤, I⑀). Upon stimulation by TNF␣, IL-1␣, UV, and ␥-irradiation, or bacterial and viral infection, IBs are phosphorylated at specific sites that lead to their ubiqitination, degradation by the proteosome, and release of NF-B proteins for translocation to the nucleus where they regulate expression of target genes.NF-B proteins play a major role in many physiological and pathological processes. Analyses of mice deficient in different members of the NF-B and IB families have revealed essential roles for these transcription factors in lymphocyte development and immune responses (for review, see Attar et al. 1997), fetal liver development (Beg et al. 1995), and osteoclast maturation (Franzoso et al. 1997;Iotsova et al. 1997). Rel/NF-B genes may also play a role in vertebrate limb development (Bushdid et al. 1998;Kanegae et al. 1998). Additionally, several groups have shown the involvement of NF-B proteins in antiapoptotic processes (Beg and Baltimore 1996; Van Antwerp et al. 1996;Wang et al. 1996). Lack of p65 (RelA) results in hepatocyte apoptosis and embryonic lethality at embryonic day 15 (E15), which may reflect its antiapoptotic function in hepatocytes during development (Beg et al. 1995).A central step to NF-B activation is the induced phosphorylation of IBs (Verma et al. 1995). Recently, the long-sought kinases for signal-induced phosphorylation of IB have been identified by three independent groups (DiDonato et al. 1997;Mercurio 1997;Regnier et al. 1997;Woronicz et al. 1997;Zandi et al. 1997). Two highly homologous IB kinases (IKKs), IKK1 (IKK␣) and IKK2 (IKK␤), are present in a large 700-900 kD complex and can specifically phosphorylate IB...

Pitx1 is a Bicoid-related homeodomain factor that exhibits preferential expression in the hindlimb, as well as expression in the developing anterior pituitary gland and first branchial arch. Here, we report that Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of the hindlimb, resulting in a limb that exhibits structural changes in tibia and fibula as well as patterning alterations in patella and proximal tarsus, to more closely resemble the corresponding forelimb structures. Deletion of the Pitx1 locus results in decreased distal expression of the hindlimb-specific marker, the T-box factor, Tbx4. On the basis of similar expression patterns in chick, targeted misexpression of chick Pitx1 in the developing wing bud causes the resulting limb to assume altered digit number and morphogenesis, with Tbx4 induction. We hypothesize that Pitx1 serves to critically modulate morphogenesis, growth, and potential patterning of a specific hindlimb region, serving as a component of the morphological and growth distinctions in forelimb and hindlimb identity. Pitx1 gene-deleted mice also exhibit reciprocal abnormalities of two ventral and one dorsal anterior pituitary cell types, presumably on the basis of its synergistic functions with other transcription factors, and defects in the derivatives of the first branchial arch, including cleft palate, suggesting a proliferative defect in these organs analogous to that observed in the hindlimb.

Several vertebrates display the ability to regenerate parts of their body after amputation. During this process, differentiated cells reenter the cell cycle and proliferate to generate a mass of undifferentiated cells. Repatterning mechanisms act on these cells to eventually shape a regenerated tissue or organ that replaces the amputated one. Experiments with regenerating limbs͞fins in newts and zebrafish have shown that members of the Msx family of homeodomain-containing transcription factors play key roles during blastema formation and patterning. Here we show that adult zebrafish have a remarkable capacity to regenerate the heart in a process that involves up-regulation of msxB and msxC genes. We present evidence indicating that heart regeneration involves the execution of a specific genetic program, rather than redeployment of a cardiac development program. Preceding Msx activation, there is a marked increase in the expression of notch1b and deltaC, which we show are also up-regulated during fin regeneration. These data suggest a role for the Notch pathway in the activation of the regenerative response. Taken together, our results underscore the use of zebrafish as a model for investigating the process of regeneration in particular and the biology of stem cells in general. Advances in these fields will undoubtedly aid in the implementation of strategies for regenerative medicine.