Ronit Yelin

The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4

Specific signaling molecules play a pivotal role in the induction and specification of tissues during early vertebrate embryogenesis. BMP-4 specifies ventral mesoderm differentiation and inhibits neural induction in Xenopus, whereas three molecules secreted from the organizer, noggin, follistatin and chordin dorsalize mesoderm and promote neural induction. Here we report that follistatin antagonizes the activities of BMP-4 in frog embryos and mouse teratocarcinoma cells. In Xenopus embryos follistatin blocks the ventralizing effect of BMP-4. In mouse P19 cells follistatin promotes neural differentiation. BMP-4 antagonizes the action of follistatin and prevents neural differentiation. In addition we show that the follistatin and BMP-4 proteins can interact directly in vitro. These data provide evidence that follistatin might play a role in modulating BMP-4 activity in vivo.

Optical frequency domain imaging with a rapidly swept laser in the 815-870 nm range

Optical frequency domain imaging (OFDI) in the 800-nm biological imaging window is demonstrated by using a novel wavelength-swept laser source. The laser output is tuned continuously from 815 to 870 nm at a 43.2-kHz repetition rate with 7-mW average power. Axial resolution of 10-mum in biological tissue and peak sensitivity of 96 dB are achieved. In vivo imaging of Xenopus laevis is demonstrated with an acquisition speed of 84 frames per second (512 axial lines per frame). This new imaging technique may prove useful in comprehensive retinal screening for medical diagnosis and contrast-agent-based imaging for biological investigations.

Ultrahigh-resolution full-field optical coherence microscopy using InGaAs camera

Full-field optical coherence microscopy (FFOCM) is an interferometric technique for obtaining wide-field microscopic images deep within scattering biological samples. FFOCM has primarily been implemented in the 0.8 mum wavelength range with silicon-based cameras, which may limit penetration when imaging human tissue. In this paper, we demonstrate FFOCM at the wavelength range of 0.9 - 1.4 mum, where optical penetration into tissue is presumably greater owing to decreased scattering. Our FFOCM system, comprising a broadband spatially incoherent light source, a Linnik interferometer, and an InGaAs area scan camera, provided a detection sensitivity of 86 dB for a 2 sec imaging time and an axial resolution of 1.9 mum in water. Images of phantoms, tissue samples, and Xenopus Laevis embryos were obtained using InGaAs and silicon camera FFOCM systems, demonstrating enhanced imaging penetration at longer wavelengths.

Spectrally-modulated full-field optical coherence microscopy for ultrahigh-resolution endoscopic imaging

Full-field optical coherence microscopy (FFOCM) utilizes coherence gating to obtain high-resolution optical sections in thick tissues. FFOCM is an attractive technology for endoscopic microscopy at the cellular level since it does not require a high NA objective lens or beam scanning and is therefore particularly amenable to miniaturization. In this manuscript, we present a novel scheme for conducting FFOCM that utilizes spectrally modulated, spatially incoherent illumination and a static Linnik interferometer. This approach is advantageous for endoscopic microscopy since it allows FFOCM to be conducted through a single multimode fiber optic imaging bundle and does not require moving parts in the endoscope probe. Images acquired from biological samples in free space demonstrate that this new method provides the same detailed microscopic structure as that of conventional FFOCM. High-resolution images were also obtained through a multimode fiber bundle, further supporting the potential of this method for endoscopic microscopy.

The Xcad-2 gene can provide a ventral signal independent of BMP-4

Patterning of the marginal zone in the Xenopus embryo has been attributed to interactions between dorsal genes expressed in the organizer and ventral-specific genes. In this antagonistic interplay of activities, BMP-4, a gene that is not expressed in the organizer, provides a strong ventralizing signal. The Xenopus caudal type homeobox gene, Xcad-2, which is expressed around the blastopore with a gap over the dorsal lip, was analyzed as part of the ventral signal. Xcad-2 was shown to efficiently repress during early gastrula stages the dorsal genes gsc, Xnot-2, Otx-2, XFKH1 and Xlim-1, while it positively regulates the ventral genes, Xvent-1 and Xvent-2, with Xpo exhibiting a strong positive response to Xcad-2 overexpression. Xcad-2 was also capable of inducing BMP-4 expression in the organizer region. Support for a ventralizing role for Xcad-2 was obtained from co-injection experiments with the dominant negative BMP receptor which was used to block BMP-4 signaling. Under lack-of-BMP-signaling conditions Xcad-2 could still regulate dorsal and ventral gene expression and restore normal development, suggesting that it can act downstream of BMP-4 signaling or independently of it. Xcad-2 could also inhibit secondary axis formation and dorsalization induced by the dominant negative BMP receptor. Xcad-2 was also shown to efficiently reverse the dorsalizing effects of LiCl. These results place Xcad-2 as part of the ventralizing gene program which acts during early gastrula stages and can execute its ventralizing function in the absence of BMP signaling.

Multimodality optical imaging of embryonic heart microstructure

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

A role for the homeobox gene Xvex-1 as part of the BMP-4 ventral signaling pathway.

BMP-4 is believed to play a central role in the patterning of the mesoderm by providing a strong ventral signal. As part of this ventral patterning signal, BMP-4 has to activate a number of transcription factors to fulfill this role. Among the transcription factors regulated by BMP-4 are the Xvent and the GATA genes. A novel homeobox gene has been isolated termed Xvex-1 which represents a new class of homeobox genes. Transcription of Xvex-1 initiates soon after the midblastula transition. Xvex-1 transcripts undergo spatial restriction from the onset of gastrulation to the ventral marginal zone, and the transcripts will remain in this localization including at the tailbud stage in the proctodeum. Expression of Xvex-1 during gastrula stages requires normal BMP-4 activity as evidenced from the injection of BMP-4, Smad1, Smad5 and Smad6 mRNA and antisense BMP-4 RNA. Xvex-1 overexpression ventralizes the Xenopus embryo in a dose dependent manner. Partial loss of Xvex-1 activity induced by antisense RNA injection results in the dorsalization of embryos and the induction of secondary axis formation. Xvex-1 can rescue the effects of overexpressing the dominant negative BMP receptor. These results place Xvex-1 downstream of BMP-4 during gastrulation and suggest that it represents a novel homeobox family in Xenopus which is part of the ventral signaling pathway.

The Xvex-1 antimorph reveals the temporal competence for organizer formation and an early role for ventral homeobox genes

The organizer in vertebrate embryos has been shown to play a central role in their development by antagonizing ventralizing signals and promoting dorsal development. The ventral homeobox gene, Xvex-1, is capable of fulfilling some of the functions of BMP-4. By fusion to activation and repression domains, Xvex-1 was shown to function as a repressor of transcription. The activator version of Xvex-1, the antimorph, was made inducible by fusion to the ligand binding domain of the glucocorticoid receptor. The organizer genes, gsc and Otx-2, were identified as direct targets of Xvex-1. The XVEX-1 antimorph can induce the formation of secondary axes. Temporal analysis of secondary axis induction revealed that the competence to induce a secondary organizer ends with the onset of gastrulation. The same temporal competence window was exhibited by an inducible gsc construct. Partial loss of Xvex-1 activity was able to improve the efficiency of secondary axis induction by the dominant negative BMP receptor or Smad6. These observations together with the early widespread expression of Xvex-1 throughout the embryo prior to gastrulation encoding a homeodomain repressor protein, suggest that elements of the ventral signaling pathway play an important role during late blastula in restricting the formation of Spemanns organizer.

Ventral midline formation in the chick embryo is assisted by a temporary local extracellular matrix structure

a reduced progenitor pool able to contribute to the correct growth and lengthening of the outflow tract. This suggests cardiac neural crest cells signal to the second heart field to maintain these cells in a progenitor state. Current work is focussed on dissecting the interaction between neural crest cells and the second heart field in orchestrating outflow tract development, and what signalling pathways are involved in this communication.