Differential requirement for BMP signaling in atrial and ventricular lineages establishes cardiac chamber proportionality

AbstractThe function of an organ relies upon the proper relative proportions of its individual operational components. For example, effective embryonic circulation requires the appropriate relative sizes of each of the distinct pumps created by the atrial and ventricular cardiac chambers. Although the differences between atrial and ventricular cardiomyocytes are well established, little is known about the mechanisms regulating production of proportional numbers of each cell type. We find that mutation of the zebrafish type I BMP receptor gene alk8 causes reduction of atrial size without affecting the ventricle. Loss of atrial tissue is evident in the lateral mesoderm prior to heart tube formation and results from the inhibition of BMP signaling during cardiac progenitor specification stages. Comparison of the effects of decreased and increased BMP signaling further demonstrates that atrial cardiomyocyte production correlates with levels of BMP signaling while ventricular cardiomyocyte production is less susceptible to manipulation of BMP signaling. Additionally, mosaic analysis provides evidence for a cell-autonomous requirement for BMP signaling during cardiomyocyte formation and chamber fate assignment. Together, our studies uncover a new role for BMP signaling in the regulation of chamber size, supporting a model in which differential reception of cardiac inductive signals establishes chamber proportion

Creating Online Lessons: A Faculty Development Seminar Series

The World Wide Web is being used increasingly to deliver instruction in medical education. Consequently, there is a need to train faculty in developing and implementing online instruction. We developed and implemented a seminar series to teach faculty to create educationally sound, well designed online instruction. Instruction was delivered to 15 participants via a six session seminar on developing web based lessons, supplemented with web-based instruction. First, the participants learned the basics of instructional design via a web based module. They then completed content outlines for their online lessons prior to the first seminar. Lesson development, web site development and the use of a web based instructional shell to implement the online lessons were each taught in two two hour sessions. Eight participants developed online lessons and four actually implemented them. Feedback was mostly positive, with suggestions for improvement. All eight participants who completed the series said they would recommend it to their colleagues. Because a longitudinal workshop type of seminar series requires a large amount of participant time outside of class, a six month seminar series may be too long. It is important at the beginning of the series to help participants select topics suitable for online instruction and to help them narrow their topics. We may change the attendance guidelines so faculty would attend only the session on instructional design and have their staff attend the technical sessions on web site design, HTML editing and online course delivery systems. This would better match the actual practice of faculty designing the instruction and staff developing it

Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics

The use of the zebrafish as a model organism for the analysis of cardiac development is no longer proof-of-principle science. Over the last decade, the identification of a variety of zebrafish mutations and the subsequent cloning of mutated genes have revealed many critical regulators of cardiogenesis. More recently, increasingly sophisticated techniques for phenotypic characterization have facilitated analysis of the specific mechanisms by which key genes drive cardiac specification, morphogenesis, and function. Future enrichment of the arsenal of experimental strategies available for zebrafish should continue the yield of high returns from such a small source

Reiterative roles for FGF signaling in the establishment of size and proportion of the zebrafish heart

AbstractDevelopment of a functional organ requires the establishment of its proper size as well as the establishment of the relative proportions of its individual components. In the zebrafish heart, organ size and proportion depend heavily on the number of cells in each of its two major chambers, the ventricle and the atrium. Heart size and chamber proportionality are both affected in zebrafish fgf8 mutants. To determine when and how FGF signaling influences these characteristics, we examined the effect of temporally controlled pathway inhibition. During cardiac specification, reduction of FGF signaling inhibits formation of both ventricular and atrial cardiomyocytes, with a stronger impact on ventricular cells. After cardiomyocyte differentiation begins, reduction of FGF signaling can still result in a deficiency of ventricular cardiomyocytes. Consistent with two temporally distinct roles for FGF, we find that increased FGF signaling induces a cardiomyocyte surplus only before cardiac differentiation begins. Thus, FGF signaling first regulates heart size and chamber proportionality during cardiac specification and later refines ventricular proportion by regulating cell number after the onset of differentiation. Together, our data demonstrate that a single signaling pathway can act reiteratively to coordinate organ size and proportion

Hand2 elevates cardiomyocyte production during zebrafish heart development and regeneration

Embryonic heart formation requires the production of an appropriate number of cardiomyocytes; likewise, cardiac regeneration following injury relies upon the recovery of lost cardiomyocytes. The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in promoting cardiomyocyte formation. It is unclear, however, whether Hand2 plays an instructive or permissive role during this process. Here, we find that overexpression of hand2 in the early zebrafish embryo is able to enhance cardiomyocyte production, resulting in an enlarged heart with a striking increase in the size of the outflow tract. Our evidence indicates that these increases are dependent on the interactions of Hand2 in multimeric complexes and are independent of direct DNA binding by Hand2. Proliferation assays reveal that hand2 can impact cardiomyocyte production by promoting division of late-differentiating cardiac progenitors within the second heart field. Additionally, our data suggest that hand2 can influence cardiomyocyte production by altering the patterning of the anterior lateral plate mesoderm, potentially favoring formation of the first heart field at the expense of hematopoietic and vascular lineages. The potency of hand2 during embryonic cardiogenesis suggested that hand2 could also impact cardiac regeneration in adult zebrafish; indeed, we find that overexpression of hand2 can augment the regenerative proliferation of cardiomyocytes in response to injury. Together, our studies demonstrate that hand2 can drive cardiomyocyte production in multiple contexts and through multiple mechanisms. These results contribute to our understanding of the potential origins of congenital heart disease and inform future strategies in regenerative medicine

Myocardial Lineage Development

The myocardium of the heart is composed of multiple highly specialized myocardial lineages, including those of the ventricular and atrial myocardium, and the specialized conduction system. Specification and maturation of each of these lineages during heart development is a highly ordered, ongoing process involving multiple signaling pathways and their intersection with transcriptional regulatory networks. Here, we attempt to summarize and compare much of what we know about specification and maturation of myocardial lineages from studies in several different vertebrate model systems. To date, most research has focused on early specification, and while there is still more to learn, less is known about factors that promote subsequent maturation of myocardial lineages required to build the functioning adult heart

Combinatorial roles for zebrafish retinoic acid receptors in the hindbrain, limbs and pharyngeal arches

AbstractRetinoic acid (RA) signaling regulates multiple aspects of vertebrate embryonic development and tissue patterning, in part through the local availability of nuclear hormone receptors called retinoic acid receptors (RARs) and retinoid receptors (RXRs). RAR/RXR heterodimers transduce the RA signal, and loss-of-function studies in mice have demonstrated requirements for distinct receptor combinations at different stages of embryogenesis. However, the tissue-specific functions of each receptor and their individual contributions to RA signaling in vivo are only partially understood. Here we use morpholino oligonucleotides to deplete the four known zebrafish RARs (raraa, rarab, rarga, and rargb). We show that while all four are required for anterior–posterior patterning of rhombomeres in the hindbrain, there are unique requirements for rarga in the cranial mesoderm for hindbrain patterning, and rarab in lateral plate mesoderm for specification of the pectoral fins. In addition, the alpha subclass (raraa, rarab) is RA inducible, and of these only raraa expression is RA-dependent, suggesting that these receptors establish a region of particularly high RA signaling through positive-feedback. These studies reveal novel tissue-specific roles for RARs in controlling the competence and sensitivity of cells to respond to RA

The Spinster Homolog, Two of Hearts, Is Required for Sphingosine 1-Phosphate Signaling in Zebrafish

SummaryThe bioactive lipid sphingosine 1-phosphate (S1P) and its G protein-coupled receptors play critical roles in cardiovascular, immunological, and neural development and function [1–6]. Despite its importance, many questions remain about S1P signaling, including how S1P, which is synthesized intracellularly, is released from cells. Mutations in the zebrafish gene encoding the S1P receptor Miles Apart (Mil)/S1P2 disrupt the formation of the primitive heart tube [5]. We find that mutations of another zebrafish locus, two of hearts (toh), cause phenotypes that are morphologically indistinguishable from those seen in mil/s1p2 mutants. Positional cloning of toh reveals that it encodes a member of the Spinster-like family of putative transmembrane transporters. The biological functions of these proteins are poorly understood, although phenotypes of the Drosophila spinster and zebrafish not really started mutants suggest that these proteins may play a role in lipid trafficking [7, 8]. Through gain- and loss-of-function analyses, we show that toh is required for signaling by S1P2. Further evidence indicates that Toh is involved in the trafficking or cellular release of S1P