Transcription factor occupancy in differentiating skeletal muscle

With recent advances in high-throughput sequencing, mapping of genome-wide transcription factor occupancy has become feasible. To advance the understanding of skeletal muscle differentiation specifically and transcriptional regulation in general, I determined the genome-wide occupancy map for myogenin in differentiating C2C12 myocyte cells. I then analyzed the myogenin map for underlying sequence content and the association between occupied elements and expression trajectories of adjacent genes. Having determined that myogenin primarily associates with expressed genes, I performed a similar analysis on occupancy maps of other transcription factors active during skeletal muscle differentiation, including an extensive analysis of co-occupancy. This analysis provided strong motif evidence for protein-protein interactions as the primary driving force in the formation of Myogenin / Mef2 and MyoD / AP-1 complexes at jointly-occupied sites. Finally, factor occupancy analysis was extended to include bHLH transcription factors in tissues other than skeletal muscle. The cross-tissue analysis led to the emergence of a motif structure used by bHLH TFs to encode either tissue-specific or "general" (public) access in a variety of lineages.

Molecular genetics of the Drosophila eyes absent gene

The Drosophila compound eye has provided a genetic approach to understanding the specification of cell fates during differentiation. The eye is made up of some 750 repeated units or ommatidia, arranged in a lattice. The cellular composition of each ommatidium is identical. The arrangement of the lattice and the specification of cell fates in each ommatidium are thought to occur in development through cellular interactions with the local environment. Many mutations have been studied that disrupt the proper patterning and cell fating in the eye. The eyes absent (eya) mutation, the subject of this thesis, was chosen because of its eyeless phenotype. In eya mutants, eye progenitor cells undergo programmed cell death before the onset of patterning has occurred. The molecular genetic analysis of the gene is presented.

The eye arises from the larval eye-antennal imaginal disc. During the third larval instar, a wave of differentiation progresses across the disc, marked by a furrow. Anterior to the furrow, proliferating cells are found in apparent disarray. Posterior to the furrow, clusters of differentiating cells can be discerned, that correspond to the ommatidia of the adult eye. Analysis of an allelic series of eya mutants in comparison to wild type revealed the presence of a selection point: a wave of programmed cell death that normally precedes the furrow. In eya mutants, an excessive number of eye progenitor cells die at this selection point, suggesting the eya gene influences the distribution of cells between fates of death and differentiation.

In addition to its role in the eye, the eya gene has an embryonic function. The eye function is autonomous to the eye progenitor cells. Molecular maps of the eye and embryonic phenotypes are different. Therefore, the function of eya in the eye can be treated independently of the embryonic function. Cloning of the gene reveals two cDNA's that are identical except for the use of an alternatively-spliced 5' exon. The predicted protein products differ only at the N-termini. Sequence analysis shows these two proteins to be the first of their kind to be isolated. Trangenic studies using the two cDNA's show that either gene product is able to rescue the eye phenotype of eya mutants.

The eya gene exhibits interallelic complementation. This interaction is an example of an "allelic position effect": an interaction that depends on the relative position in the genome of the two alleles, which is thought to be mediated by chromosomal pairing. The interaction at eya is essentially identical to a phenomenon known as transvection, which is an allelic position effect that is sensitive to certain kinds of chromosomal rearrangements. A current model for the mechanism of transvection is the trans action of gene regulatory regions. The eya locus is particularly well suited for the study of transvection because the mutant phenotypes can be quantified by scoring the size of the eye.

The molecular genetic analysis of eya provides a system for uncovering mechanisms underlying differentiation, developmentally regulated programmed cell death, and gene regulation.

Dynamics of cell–matrix mechanical interactions in three dimensions

The forces cells apply to their surroundings control biological processes such as growth, adhesion, development, and migration. In the past 20 years, a number of experimental techniques have been developed to measure such cell tractions. These approaches have primarily measured the tractions applied by cells to synthetic two-dimensional substrates, which do not mimic in vivo conditions for most cell types. Many cell types live in a fibrous three-dimensional (3D) matrix environment. While studying cell behavior in such 3D matrices will provide valuable insights for the mechanobiology and tissue engineering communities, no experimental approaches have yet measured cell tractions in a fibrous 3D matrix.

This thesis describes the development and application of an experimental technique for quantifying cellular forces in a natural 3D matrix. Cells and their surrounding matrix are imaged in three dimensions with high speed confocal microscopy. The cell-induced matrix displacements are computed from the 3D image volumes using digital volume correlation. The strain tensor in the 3D matrix is computed by differentiating the displacements, and the stress tensor is computed by applying a constitutive law. Finally, tractions applied by the cells to the matrix are computed directly from the stress tensor.

The 3D traction measurement approach is used to investigate how cells mechanically interact with the matrix in biologically relevant processes such as division and invasion. During division, a single mother cell undergoes a drastic morphological change to split into two daughter cells. In a 3D matrix, dividing cells apply tensile force to the matrix through thin, persistent extensions that in turn direct the orientation and location of the daughter cells. Cell invasion into a 3D matrix is the first step required for cell migration in three dimensions. During invasion, cells initially apply minimal tractions to the matrix as they extend thin protrusions into the matrix fiber network. The invading cells anchor themselves to the matrix using these protrusions, and subsequently pull on the matrix to propel themselves forward.

Lastly, this thesis describes a constitutive model for the 3D fibrous matrix that uses a finite element (FE) approach. The FE model simulates the fibrous microstructure of the matrix and matches the cell-induced matrix displacements observed experimentally using digital volume correlation. The model is applied to predict how cells mechanically sense one another in a 3D matrix. It is found that cell-induced matrix displacements localize along linear paths. These linear paths propagate over a long range through the fibrous matrix, and provide a mechanism for cell-cell signaling and mechanosensing. The FE model developed here has the potential to reveal the effects of matrix density, inhomogeneity, and anisotropy in signaling cell behavior through mechanotransduction.

Expression of eph-family receptor tyrosine kinases and ephrins in the tadpole of the frog Xenopus laevis, and possible roles in the development of retinotectal topography

Assembling a nervous system requires exquisite specificity in the construction of neuronal connectivity. One method by which such specificity is implemented is the presence of chemical cues within the tissues, differentiating one region from another, and the presence of receptors for those cues on the surface of neurons and their axons that are navigating within this cellular environment.

Connections from one part of the nervous system to another often take the form of a topographic mapping. One widely studied model system that involves such a mapping is the vertebrate retinotectal projection-the set of connections between the eye and the optic tectum of the midbrain, which is the primary visual center in non-mammals and is homologous to the superior colliculus in mammals. In this projection the two-dimensional surface of the retina is mapped smoothly onto the two-dimensional surface of the tectum, such that light from neighboring points in visual space excites neighboring cells in the brain. This mapping is implemented at least in part via differential chemical cues in different regions of the tectum.

The Eph family of receptor tyrosine kinases and their cell-surface ligands, the ephrins, have been implicated in a wide variety of processes, generally involving cellular movement in response to extracellular cues. In particular, they possess expression patterns-i.e., complementary gradients of receptor in retina and ligand in tectum- and in vitro and in vivo activities and phenotypes-i.e., repulsive guidance of axons and defective mapping in mutants, respectively-consistent with the long-sought retinotectal chemical mapping cues.

The tadpole of Xenopus laevis, the South African clawed frog, is advantageous for in vivo retinotectal studies because of its transparency and manipulability. However, neither the expression patterns nor the retinotectal roles of these proteins have been well characterized in this system. We report here comprehensive descriptions in swimming stage tadpoles of the messenger RNA expression patterns of eleven known Xenopus Eph and ephrin genes, including xephrin-A3, which is novel, and xEphB2, whose expression pattern has not previously been published in detail. We also report the results of in vivo protein injection perturbation studies on Xenopus retinotectal topography, which were negative, and of in vitro axonal guidance assays, which suggest a previously unrecognized attractive activity of ephrins at low concentrations on retinal ganglion cell axons. This raises the possibility that these axons find their correct targets in part by seeking out a preferred concentration of ligands appropriate to their individual receptor expression levels, rather than by being repelled to greater or lesser degrees by the ephrins but attracted by some as-yet-unknown cue(s).

Isolation of the murine interleukin 2 gene and characterization of its regulatory architecture

Interleukin 2 (IL2) is the primary growth hormone used by mature T cells and this lymphokine plays an important role in the magnification of cell-mediated immune responses. Under normal circumstances its expression is limited to antigen-activated type 1 helper T cells (T_H1) and the ability to transcribe this gene is often regarded as evidence for commitment to this developmental lineage. There is, however, abundant evidence than many non-T_H1 T cells, under appropriate conditions, possess the ability to express this gene. Of paramount interest in the study of T-cell development is the mechanisms by which differentiating thymocytes are endowed with particular combinations of cell surface proteins and response repertoires. For example, why do most helper T cells express the CD4 differentiation antigen?

As a first step in understanding these developmental processes the gene encoding IL2 was isolated from a mouse genomic library by probing with a conspecific IL2 cDNA. The sequence of the 5' flanking region from + 1 to -2800 was determined and compared to the previously reported human sequence. Extensive identity exists between +1 and -580 (86%) and sites previously shown to be crucial for the proper expression of the human gene are well conserved in both sequence location in the mouse counterpart.

Transient expression assays were used to evaluate the contribution of various genomic sequences to high-level gene expression mediated by a cloned IL2 promoter fragment. Differing lengths of 5' flanking DNA, all terminating in the 5' untranslated region, were linked to a reporter gene, bacterial chloramphenicol acetyltransferase (CAT) and enzyme activity was measured after introduction into IL2-producing cell lines. No CAT was ever detected without stimulation of the recipient cells. A cloned promoter fragment containing only 321 bp of upstream DNA was expressed well in both Jurkat and EL4.El cells. Addition of intragenic or downstream DNA to these 5' IL2-CAT constructs showed that no obvious regulatory regions resided there. However, increasing the extent of 5' DNA from -321 to -2800 revealed several positive and negative regulatory elements. One negative region that was well characterized resided between -750 and -1000 and consisted almost exclusively of alternating purine and pyrimidines. There is no sequence resembling this in the human gene now, but there is evidence that there may have once been.

No region, when deleted, could relax either the stringent induction-dependence on cell-type specificity displayed by this promoter. Reagents that modulated endogenous IL2 expression, such as cAMP, cyclosporin A, and IL1, affected expression of the 5' IL2-CAT constructs also. For a given reagent, expression from all expressible constructs was suppressed or enhanced to the same extent. This suggests that these modulators affect IL2 expression through perturbation of a central inductive signal rather than by summation of the effects of discrete, independently regulated, negative and positive transcription factors.