Rina Shah

Function and regulation of FoxF1 during Xenopus gut development

Development of the visceral mesoderm is a critical process in the organogenesis of the gut. Elucidation of function and regulation of genes involved in the development of visceral mesoderm is therefore essential for an understanding of gut organogenesis. One of the genes specifically expressed in the lateral plate mesoderm, and later in its derivative, the visceral mesoderm, is the Fox gene FoxF1. Its function is critical for Xenopus gut development, and embryos injected with FoxF1 morpholino display abnormal gut development. In the absence of FoxF1 function, the lateral plate mesoderm, and later the visceral mesoderm, does not proliferate and differentiate properly. Region- and stage-specific markers of visceral mesoderm differentiation, such as Xbap and α-smooth muscle actin, are not activated. The gut does not elongate and coil. These experiments provide support for the function of FoxF1 in the development of visceral mesoderm and the organogenesis of the gut. At the molecular level, FoxF1 is a downstream target of BMP4 signaling. BMP4 can activate FoxF1 transcription in animal caps and overexpression of FoxF1 can rescue twinning phenotypes, which results from the elimination of BMP4 signaling. The cis-regulatory elements of FoxF1 are located within a 2 kb DNA fragment upstream of the coding region. These sequences can drive correct temporal-spatial expression of a GFP reporter gene in transgenic Xenopus tadpoles. These sequences represent a unique tool, which can be used to specifically alter gene expression in the lateral plate mesoderm.

Cell-Autonomous Requirement for Rx Function in the Mammalian Retina and Posterior Pituitary

Rx is a paired-like homeobox gene that is required for vertebrate eye formation. Mice lacking Rx function do not develop eyes or the posterior pituitary. To determine whether Rx is required cell autonomously in these tissues, we generated embryonic chimeras consisting of wild type and Rx−/− cells. We found that in the eye, Rx-deficient cells cannot participate in the formation of the neuroretina, retina pigment epithelium and the distal part of the optic stalk. In addition, in the ventral forebrain, Rx function is required cell autonomously for the formation of the posterior pituitary. Interestingly, Rx−/− and wild type cells segregate before the morphogenesis of these two tissues begins. Our observations suggest that Rx function is not only required for the morphogenesis of the retina and posterior pituitary, but also prior to morphogenesis, for the sorting out of cells to form distinct fields of retinal/pituitary cells.

Pitx3 controls multiple aspects of lens development

The transcription factor Pitx3 is critical for lens formation. Deletions in the promoter of this gene cause abnormal lens development in the aphakia (ak) mouse mutant, which has only rudimentary lenses. In this study, we investigated the role of Pitx3 in lens development and differentiation. We found that reduced expression of Pitx3 leads to changes in the proliferation, differentiation and survival of lens cells. The genetic interactions between Pitx3 and Foxe3 were investigated, as these two transcription factors are expressed at the same time in lens development and their absence has similar consequences for lens development. We found no evidence that these two genes genetically interact. In general, our study shows that the abnormal phenotype of the ak lenses is not due to just one molecular pathway, rather in the absence of Pitx3 expression multiple aspects of lens development are disrupted. Developmental Dynamics 238:2193–2201, 2009.

Expression of complement components coincides with early patterning and organogenesis in Xenopus laevis

The complement system is the central component of innate immunity and an important player in the adaptive immunity of vertebrates. We analyzed the expression patterns of several key members of the complement cascade during Xenopus development. We found extensive expression of these molecules already during gastrula/early neurula stage. Remarkably, several genes also showed an organ-specific expression pattern during early organogenesis. Early expression is notable for two different expression patterns in the neuroectoderm. In one group, there is early strong neural plate and neural precursor expression. This is the case of properdin, C1qA, C3 and C9. The second pattern, seen with C1qR and C6, is noteworthy for its expression at the periphery of the neural plate, in the presumptive neural crest. Two genes stand out for their predominantly mesodermal expression. C3aR, the message for the cognate receptor for C3 in the complement cascade, is expressed at the same time as C3, but in a complementary, reciprocal pattern in the mesoderm. C1qA expression also predominates in somites, pronephros, visceral mesoderm and ventral blood islands. Finally, several genes are characterized by later expression in developing organs. C1qR displays a reticular pattern consistent with expression in the developing vasculature. The late expression of C1qA and C3bC4b is strongest in the pronephros. Finally, the expression of properdin in the hindbrain and in the developing lens are novel findings. The expression patterns of these molecules suggest that these components of the complement system may have in Xenopus a so far undefined developmental role.

E-selectin ligand-1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-β

The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-beta signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-beta signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-beta/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-beta during skeletal development and homeostasis.

Eye formation in the absence of retina.

Eye development is a complex process that involves the formation of the retina and the lens, collectively called the eyeball, as well as the formation of auxiliary eye structures such as the eyelid, lacrimal gland, cornea and conjunctiva. The developmental requirements for the formation of each individual structure are only partially understood. We have shown previously that the homeobox-containing gene Rx is a key component in eye formation, as retinal structures do not develop and retina-specific gene expression is not observed in Rx-deficient mice. In addition, Rx-/- embryos do not develop any lens structure, despite the fact that Rx is not expressed in the lens. This demonstrates that during normal mammalian development, retina-specific gene expression is necessary for lens formation. In this paper we show that lens formation can be restored in Rx-deficient embryos experimentally, by the elimination of beta-catenin expression in the head surface ectoderm. This suggests that beta-catenin is involved in lens specification either through Wnt signaling or through its function in cell adhesion. In contrast to lens formation, we demonstrate that the development of auxiliary eye structures does not depend on retina-specific gene expression or retinal morphogenesis. These results point to the existence of two separate developmental processes involved in the formation of the eye and its associated structures. One involved in the formation of the eyeball and the second involved in the formation of the auxiliary eye structures.

Regulation and function of foxe3 during early zebrafish development.

In this article, we investigate the expression, regulation, and function of the zebrafish forkhead gene foxe3. In wild type embryos, foxe3 is first expressed in a crescent‐shaped area at the anterior end of the prechordal plate, corresponding to the polster. At later stages, the hatching gland, the lens, and the anterior pituitary express this gene. Using morpholinos against the zinc finger Kruppel‐like factor 4 (KLF4) we show that foxe3 is regulated differently in the polster and in the lens. In the absence of KLF4, expression of foxe3 in the polster is not activated, whereas in the lens placode the expression of KLF4 is not required for the transcription of foxe3. The expression of foxe3 is also regulated by the hedgehog and nodal signaling pathways. foxe3 expression is altered in the hedgehog pathway mutants iguana and you‐too and the nodal pathway mutant cyclops. foxe3 function is necessary for the execution of lens‐specific gene expression and lens morphogenesis, as the knockdown of foxe3 results in a loss of platelet‐derived growth factor receptor alpha (pdgfrα) expression and in the vacuolization of the lens. 46:177–183, 2008.

Identification and gastrointestinal expression of Xenopus laevis FoxF2.

FoxF genes are essential for visceral mesoderm development from Drosophila to human. However, part of the difficulty of studying the visceral mesoderm is its relative inaccessibility during early development. Owing to its external development Xenopus laevis presents considerable advantages for the study of visceral mesoderm formation, yet FoxF2 has not been identified in this system. Here, we describe the cloning and expression pattern of XFoxF2 during embryonic development, metamorphosis and adulthood, and compare and contrast it to the expression of FoxF1 in Xenopus laevis and FoxF2 in mouse.

Role of Fox Genes During Xenopus Embryogenesis

Fox genes encode a remarkably conserved family of nuclear proteins that can act as transcriptional activators or repressors. Their high level of conservation is probably due to the critical roles they play in embryonic pattern formation and tissue-specific gene expression (Dirksen and Jamrich 1992; Sasaki and Hogan 1993; Hatini et al. 1994; Dirksen and Jamrich 1995; Kaufmann and Knochel 1996; Martinez et al. 1997; Kenyon et al. 1999; Brownell et al. 2000; Carlsson and Mahlapuu 2002). Fox genes encode proteins that contain a highly conserved 110 amino acid long DNA-binding domain that was originally described in the Drosophila mutant fork head (Lai et al. 1990; Weigel and Jackie 1990). Because of this, they were called the forkhead genes. The structure of these proteins resembles a winged helix, and because of their structure, they are also referred to as winged helix proteins (Clark et al. 1993). Eventually, a unified nomenclature was established, and currently these genes are called Fox genes (Kaestner et al. 2000).