Parker B Antin

PMID: 15950601;Abstract:

Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, ∼40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils. © 2005 Elsevier Inc. All rights reserved.

PMID: 3958057;PMCID: PMC2114158;Abstract:

When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated 'myosheets': extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembraneous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform. When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d. By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic retriculum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.

PMID: 8875073;Abstract:

An avian cDNA homologue of human and rat glypicans has been cloned from a stage 17 chicken heart cDNA library and used to analyze the distribution of this proteoglycan during development by Northern analysis and whole mount in situ hybridization. At stages 7-12, strong signals were detected in the cephalic region of the neural folds, rostral portion of paraxial mesoderm, and newly formed epithelial somites. At stages 2025, strong expression was observed in the mantle zone of the telencephalon, the apical epidermal ridge and proximal region of developing limb. Transcripts also were found in the truncus arteriosus and arteriovenous-canal region of the heart, but not in the myocardium. This distribution pattern suggests that the avian glypican may be involved in the morphogenesis of limb, somite, heart, and brain. The expression of glypican also overlaps FGFs in limb bud, FGF receptors in heart and somite, and NGF receptors in forebrain. The affinity of heparan sulfate proteoglycans for growth factors and the distribution of the avian glypican are consistent with a role for this molecule in growth factor-mediated signals.

PMID: 14991723;Abstract:

Despite the increasing quality and quantity of genomic sequence that is available to researchers, predicting gene function from sequence information remains a challenge. One method for obtaining rapid insight into potential functional roles of novel genes is through gene expression mapping. We have performed a high throughput whole-mount in situ hybridization (ISH) screen with chick embryos to identify novel, differentially expressed genes. Approximately 1,200 5′ expressed sequence tags (ESTs) were generated from cDNA clones of a Hamburger and Hamilton (HH) stage 4-7 (late gastrula) chick embryo endoderm-mesoderm library. After screening to remove ubiquitously expressed cDNAs and internal clustering and after comparison to GenBank sequences, remaining cDNAs (representing both characterized and uncharacterized genes) were screened for expression in HH stage 3-14 embryos by automated high throughput ISH. Of 786 cDNAs for which ISH was successfully performed, approximately 30% showed ubiquitous expression, 40% were negative, and approximately 30% showed a restricted expression pattern. cDNAs were identified that showed restricted expression in every embryonic region, including the primitive streak, somites, developing cardiovascular system and neural tube/neural crest. A relational database was developed to hold all EST sequences, ISH images, and corresponding BLAST report information, and to enable browsing and querying of data. A user interface is freely accessible at http://geisha.biosci.arizona.edu. Results show that high throughput whole-mount ISH provides an effective approach for identifying novel genes that are differentially expressed in the developing chicken embryo. © 2004 Wiley-Liss, Inc.