Timothy J. Mohun

MEF‐2 function is modified by a novel co‐repressor, MITR

The MEF‐2 proteins are a family of transcriptional activators that have been detected in a wide variety of cell types. In skeletal muscle cells, MEF‐2 proteins interact with members of the MyoD family of transcriptional activators to synergistically activate gene expression. Similar interactions with tissue or lineage‐specific cofactors may also underlie MEF‐2 function in other cell types. In order to screen for such cofactors, we have used a transcriptionally inactive mutant of Xenopus MEF2D in a yeast two‐hybrid screen. This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A. The MEF‐2 interacting transcription repressor (MITR) protein binds to the N‐terminal MADS/MEF‐2 region of the MEF‐2 proteins but does not bind to the related Xenopus MADS protein serum response factor. In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle. In functional assays, MITR negatively regulates MEF‐2‐dependent transcription and we show that this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1. Thus, we propose that MITR acts as a co‐repressor, recruiting a specific deacetylase to downregulate MEF‐2 activity.

Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.

The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) [2]. Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein [3] [4], and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5-7], and that SUMO-1 conjugation promotes RanGAP1s interaction with RanBP2 [2,5,6]. Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.

Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing.

We describe a technique suitable for routine three-dimensional (3-D) analysis of mouse embryos that is based on episcopic fluorescence images captured during serial sectioning of wax-embedded specimens. We have used this procedure to describe the cardiac phenotype and associated blood vessels of trisomic 16 (Ts16) and Cited2-null mutant mice, as well as the expression pattern of an Myf5 enhancer/β-galactosidase transgene. The consistency of the images and their precise alignment are ideally suited for 3-D analysis using video animations, virtual resectioning or commercial 3-D reconstruction software packages. Episcopic fluorescence image capturing (EFIC) provides a simple and powerful tool for analyzing embryo and organ morphology in normal and transgenic embryos.

High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology

We describe a new methodology for rapid 2D and 3D computer analysis and visualisation of gene expression and gene product pattern in the context of anatomy and tissue architecture. It is based on episcopic imaging of embryos and tissue samples, as they are physically sectioned, thereby producing inherently aligned digital image series and volume data sets, which immediately permit the generation of 3D computer representations. The technique uses resin as embedding medium, eosin for unspecific tissue staining, and colour reactions (β-galactosidase/Xgal or BCIP/NBT) for specific labelling of gene activity and mRNA pattern. We tested the potential of the method for producing high-resolution volume data sets of adult human and porcine tissue samples and of specifically and unspecifically stained mouse, chick, quail, frog, and zebrafish embryos. The quality of the episcopic images resembles the quality of digital images of true histological sections with respect to resolution and contrast. Specifically labelled structures can be extracted using simple thresholding algorithms. Thus, the method is capable of quickly and precisely detecting molecular signals simultaneously with anatomical details and tissue architecture. It has no tissue restrictions and can be applied for analysis of human tissue samples as well as for analysis of all developmental stages of embryos of a wide variety of biomedically relevant species.

Muscle-specific expression of SRF-related genes in the early embryo of Xenopus laevis

We have isolated two members of the RSRF protein family, SL‐1 and SL‐2, in Xenopus laevis. Both proteins contain SRF‐type DNA binding domains and are related to the human protein, RSRFC4. SL‐1 constitutes a novel member of the RSRF family whilst SL‐2 is similar to human RSRFC4 throughout its length. SL‐1 protein recognizes the consensus DNA sequence CTA(A/T)4TAR in vitro and can bind to the same regulatory sites as other A/T‐rich sequence‐specific binding activities, such as the muscle‐specific regulatory factor, MEF‐2. Transcription of both Xenopus genes is restricted to the somitic mesoderm of early embryos and subsequently to the body muscle (myotomes) of the tadpole. In contrast, both genes are expressed ubiquitously in the adult frog. A binding activity, antigenically related to both human RSRFC4 and the SL‐2 gene product, is detected in Xenopus embryos and after gastrulation is localized to embryonic muscle. An indistinguishable binding activity is detected in many adult frog tissues. We conclude that the RSRF genes undergo a dramatic switch in their patterns of expression during development. We suggest that RSRF proteins may regulate muscle‐specific transcription in embryos, but acquire other roles during the course of development.

Tbx4 and Tbx5 Acting in Connective Tissue Are Required for Limb Muscle and Tendon Patterning

Summary Proper functioning of the musculo-skeletal system requires the precise integration of bones, muscles and tendons. Complex morphogenetic events ensure that these elements are linked together in the appropriate 3D configuration. It has been difficult, however, to tease apart the mechanisms that regulate tissue morphogenesis. We find that deletion of Tbx5 in forelimb (or Tbx4 in hindlimbs) specifically affects muscle and tendon patterning without disrupting skeletal development thus suggesting that distinct cues regulate these processes. We identify muscle connective tissue as the site of action of these transcription factors and show that N-Cadherin and β-Catenin are key downstream effectors acting in muscle connective tissue regulating soft-tissue morphogenesis. In humans, TBX5 mutations lead to Holt-Oram syndrome, which is characterised by forelimb musculo-skeletal defects. Our results suggest that a focus on connective tissue is required to understand the aetiology of diseases affecting soft tissue formation.

Normal and abnormal development of the intrapericardial arterial trunks in humans and mice

AIMS The definitive cardiac outflow channels have three components: the intrapericardial arterial trunks; the arterial roots with valves; and the ventricular outflow tracts (OFTs). We studied the normal and abnormal development of the most distal of these, the arterial trunks, comparing findings in mice and humans. METHODS AND RESULTS Using lineage tracing and three-dimensional visualization by episcopic reconstruction and scanning electron microscopy, we studied embryonic day 9.5-12.5 mouse hearts, clarifying the development of the OFTs distal to the primordia of the arterial valves. We characterize a transient aortopulmonary (AP) foramen, located between the leading edge of a protrusion from the dorsal wall of the aortic sac and the distal margins of the two outflow cushions. The foramen is closed by fusion of the protrusion, with its cap of neural crest cells (NCCs), with the NCC-filled cushions; the resulting structure then functioning transiently as an AP septum. Only subsequent to this closure is it possible to recognize, more proximally, the previously described AP septal complex. The adjacent walls of the intrapericardial trunks are derived from the protrusion and distal parts of the outflow cushions, whereas the lateral walls are formed from intrapericardial extensions of the pharyngeal mesenchyme derived from the second heart field. CONCLUSIONS We provide, for the first time, objective evidence of the mechanisms of closure of an AP foramen that exists distally between the lumens of the developing intrapericardial arterial trunks. Our findings provide insights into the formation of AP windows and the variants of common arterial trunk.

XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus

Phagocytic myeloid cells provide the principle line of immune defence during early embryogenesis in lower vertebrates. They may also have important functions during normal embryo morphogenesis, not least through the phagocytic clearance of cell corpses arising from apoptosis. We have identified two cDNAs that provide sensitive molecular markers of embryonic leukocytes in the early Xenopus embryo. These encode a peroxidase (XPOX2) and a Ly-6/uPAR-related protein (XLURP-1). We show that myeloid progenitors can first be detected at an antero-ventral site in early tailbud stage embryos (a region previously termed the anterior ventral blood island) and transiently express the haematopoetic transcription factors SCL and AML. Phagocytes migrate from this site along consistent routes and proliferate, becoming widely distributed throughout the tadpole long before the circulatory system is established. This migration can be followed in living embryos using a 5 kb portion of the XLURP-1 promoter to drive expression of EGFP specifically in the myeloid cells. Interestingly, whilst much of this migration occurs by movement of individual cells between embryonic germ layers, the rostral-most myeloid cells apparently migrate in an anterior direction along the ventral midline within the mesodermal layer itself. The transient presence of such cells as a strip bisecting the cardiac mesoderm immediately prior to heart tube formation suggests that embryonic myeloid cells may play a role in early cardiac morphogenesis.

Xenopus eHAND: a marker for the developing cardiovascular system of the embryo that is regulated by bone morphogenetic proteins

The bHLH protein eHAND is a sensitive marker for cardiovascular precursors in the Xenopus embryo. The earliest site of expression is a broad domain within the lateral plate mesoderm of the tailbud embryo. This domain comprises precursors that contribute to the posterior cardinal veins in later stages. Surprisingly, expression is profoundly asymmetric at this stage and is random with respect to embryo side. XeHAND is also expressed in an anterior domain that encompasses the prospective heart region. Within the myocardium and pericardium, transcripts are also asymmetrically distributed, but in these tissues they are localised in a left-sided manner. Later in development XeHAND transcripts are largely restricted to the ventral aorta, aortic arches and venous inflow tract (sinus venosus) which flank the heart itself, but no expression is detected in neural crest derivatives at any stage. This demonstrates that patterns of XeHAND expression differ markedly amongst vertebrates and that in Xenopus, XeHAND expression identifies all of the earliest formed elements of the cardiovascular system. In animal cap explants, expression of XeHAND (but not other markers of cardiogenic differentiation) is strongly induced by ectopic expression of the TGFbeta family members, BMP-2 and BMP-4, but this can be blocked by coexpression of a dominant negative BMP receptor. This suggests that XeHAND expression in the embryo is regulated by the ventralising signals of bone morphogenetic proteins. High levels of expression are also detected in explants treated with high doses of activin A which induces cardiac muscle differentiation. No such effect is seen with lower doses of activin, indicating that a second pathway may regulate the XeHAND gene during cardiogenesis.

Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening

Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data.