Michael K. Gross

Cranial muscle defects of Pitx2 mutants result from specification defects in the first branchial arch

Pitx2 expression is observed during all states of the myogenic progression in embryonic muscle anlagen and persists in adult muscle. Pitx2 mutant mice form all but a few muscle anlagen. Loss or degeneration in muscle anlagen could generally be attributed to the loss of a muscle attachment site induced by some other aspect of the Pitx2 phenotype. Muscles derived from the first branchial arch were absent, whereas muscles derived from the second branchial arch were merely distorted in Pitx2 mutants at midgestation. Pitx2 was expressed well before, and was required for, initiation of the myogenic progression in the first, but not second, branchial arch mesoderm. Pitx2 was also required for expression of premyoblast specification markers Tbx1, Tcf21, and Msc in the first, but not second, branchial arch. First, but not second, arch mesoderm of Pitx2 mutants failed to enlarge after embryonic day 9.5, well before the onset of the myogenic progression. Thus, Pitx2 contributes to specification of first, but not second, arch mesoderm. The jaw of Pitx2 mutants was vestigial by midgestation, but significant size reductions were observed as early as embryonic day 10.5. The diminutive first branchial arch of mutants could not be explained by loss of mesoderm alone, suggesting that Pitx2 contributes to the earliest specification of jaw itself.

Bcl11b represses a mature T-cell gene expression program in immature CD4+CD8+ thymocytes

Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T‐cell differentiation in newborn Bcl11b‐null mice, and for positive selection in the adult thymus of mice bearing a T‐cell‐targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4+CD8+CD3lo double‐positive (DP) thymocytes. Bcl11b‐deficient DP cells displayed a gene expression program associated with mature CD4+CD8− and CD4−CD8+ single‐positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage‐associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.

Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance during S Phase

Thioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that Δtrx1 Δtrx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and Δtrx1 Δtrx2 yeast. In contrast to wild-type cells, Δtrx1 Δtrx2 cells were unable to accumulate or maintain high levels of dNTPs when α-factor- or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in Δtrx1 Δtrx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.

Ctip2/Bcl11b controls ameloblast formation during mammalian odontogenesis

The transcription factor Ctip2/Bcl11b plays essential roles in developmental processes of the immune and central nervous systems and skin. Here we show that Ctip2 also plays a key role in tooth development. Ctip2 is highly expressed in the ectodermal components of the developing tooth, including inner and outer enamel epithelia, stellate reticulum, stratum intermedium, and the ameloblast cell lineage. In Ctip2−/− mice, tooth morphogenesis appeared to proceed normally through the cap stage but developed multiple defects at the bell stage. Mutant incisors and molars were reduced in size and exhibited hypoplasticity of the stellate reticulum. An ameloblast-like cell population developed ectopically on the lingual aspect of mutant lower incisors, and the morphology, polarization, and adhesion properties of ameloblasts on the labial side of these teeth were severely disrupted. Perturbations of gene expression were also observed in the mandible of Ctip2−/− mice: expression of the ameloblast markers amelogenin, ameloblastin, and enamelin was down-regulated, as was expression of Msx2 and epiprofin, transcription factors implicated in the tooth development and ameloblast differentiation. These results suggest that Ctip2 functions as a critical regulator of epithelial cell fate and differentiation during tooth morphogenesis.

The chicken thymidine kinase gene is transcriptionally repressed during terminal differentiation: the associated decline in TK mRNA cannot account fully for the disappearance of TK enzyme activity.

Thymidine kinase (TK) is representative of a class of enzymes involved in DNA precursor biosynthesis that declines as cells withdraw from the cell cycle. If TK activity is regulated exclusively by the availability of messenger RNA, changes in enzyme activity levels should not precede or excede changes in TK mRNA levels. This prediction was tested in several tissues during chicken embryogenesis and in differentiating muscle cells in culture. A sensitive method of determining absolute TK mRNA levels was developed. A synthetic complimentary RNA probe spanning an intron acceptor site in the chicken TK gene was hybridized with cellular RNA or synthetic colinear TK RNA of known concentration. After RNase digestion and gel electrophoresis, the intensity of the protected fragment was used to calculate absolute TK mRNA levels. As few as 0.02 molecules of TK mRNA per cell could be measured accurately. Depending on the tissue type, 8-day embryos contained between 3 and 12 TK mRNAs per cell. Proliferating mouse muscle cells transformed with the chicken TK gene contained between 30 and 150 TK mRNAs per cell. Both in vivo and in vitro, TK mRNA levels declined as cells withdrew from the cell cycle during differentiation. In vivo, the decline in TK activity never preceded or exceeded observed changes in TK mRNA. However, in the cell culture system, TK activity consistently declined to a greater extent than TK mRNA. Thus, a translational or a post-translational mechanism must also be operative in controlling TK activity levels. Estimation of transcription rates in nuclei isolated from proliferating and differentiated muscle cell transformants indicated that the TK gene was transcriptionally repressed in postreplicative cells.

Pitx2-dependent Occupancy by Histone Deacetylases Is Associated with T-box Gene Regulation in Mammalian Abdominal Tissue

The homeodomain transcription factor Pitx2 and the T-box transcription factors are essential for organogenesis. Pitx2 and T-box genes are induced by growth factors and function as transcriptional activators or repressors. Gene expression analyses on abdominal tissue were used to identify seven of the T-box genes of the genome as Pitx2 target genes in the abdomen at embryonic day.10.5. Pitx2 activated Tbx4, Tbx15, and Mga and repressed Tbx1, Tbx2, Tbx5, and Tbx6 expression. As expected, activated genes showed reduced expression patterns, and repressed T-box genes showed increased expression patterns in the abdomen of Pitx2 mutants. Pitx2 occupied chromatin sites near all of these T-box genes. Co-occupancy by coactivators, corepressors, and histone acetylation at these sites was frequently Pitx2-dependent. Genes repressed by Pitx2 generally showed increased histone acetylation and decreased histone deacetylase (HDAC)/corepressor occupancy in Pitx2 mutants. The lower N-CoR, HDAC1, and HDAC3 occupancy observed at multiple sites along Tbx1 chromatin in mutants is consistent with the model that increased histone acetylation and gene expression of Tbx1 may result from a loss of recruitment of corepressors by Pitx2. Genes activated by Pitx2 showed less consistent patterns in chromatin analyses. Reduced H4 acetylation and increased HDAC1/nuclear receptor corepressor (N-CoR) occupancy at some Tbx4 sites were accompanied by increased H3 acetylation and reduced HDAC3 occupancy at the same or other more distal chromatin sites in mutants. Pitx2-dependent occupancy by corepressors resulted in alteration of the acetylation levels of several T-box genes, whereas Pitx2-dependent occupancy by coactivators was more site-localized. These studies will provide the basic scientific underpinning to understand abdominal wall syndromes.

Introns are inconsequential to efficient formation of cellular thymidine kinase mRNA in mouse L cells.

TK mRNA levels were determined in mouse L cells transformed with intron deletion mutations of the chicken TK gene. Whether normalized per cell, per integrated gene, or per internal control signal, intron deletion did not diminish the efficiency of TK mRNA formation in transformed L cells. The results demonstrated that introns are not required for efficient biogenesis of cellular mRNA in transformed mouse L cells.

Pitx2-mediated cardiac outflow tract remodeling.

Background: Heart morphogenesis involves sequential anatomical changes from a linear tube of a single channel peristaltic pump to a four‐chamber structure with two channels controlled by one‐way valves. The developing heart undergoes continuous remodeling, including septation. Results: Pitx2‐null mice are characterized by cardiac septational defects of the atria, ventricles, and outflow tract. Pitx2‐null mice also exhibited a short outflow tract, including unseptated conus and deformed endocardial cushions. Cushions were characterized with a jelly‐like structure, rather than the distinct membrane‐looking leaflets, indicating that endothelial mesenchymal transition was impaired in Pitx2−/− embryos. Mesoderm cells from the branchial arches and neural crest cells from the otic region contribute to the development of the endocardial cushions, and both were reduced in number. Members of the Fgf and Bmp families exhibited altered expression levels in the mutants. Conclusions: We suggest that Pitx2 is involved in the cardiac outflow tract septation by promoting and/or maintaining the number and the remodeling process of the mesoderm progenitor cells. Pitx2 influences the expression of transcription factors and signaling molecules involved in the differentiation of the cushion mesenchyme during heart development. Developmental Dynamics 242:456–468, 2013.

Regulation of Motility of Myogenic Cells in Filling Limb Muscle Anlagen by Pitx2

Cells of the ventrolateral dermomyotome delaminate and migrate into the limb buds where they give rise to all muscles of the limbs. The migratory cells proliferate and form myoblasts, which withdraw from the cell cycle to become terminally differentiated myocytes. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed specifically in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Microarrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1EGFP/+, which resulted from the loss of Pitx2. Very few genes showed changes in expression. Many small-fold, yet significant, changes were observed in genes encoding cytoskeletal and adhesion proteins which play a role in cell motility. Myogenic cells from genetically-tagged mice were cultured and subjected to live cell-tracking analysis using time-lapse imaging. Myogenic cells lacking Pitx2 were smaller, more symmetrical, and had more actin bundling. They also migrated about half of the total distance and velocity. Decreased motility may prevent myogenic cells from filling pre-patterned regions of the limb bud in a timely manner. Altered shape may prevent proper assembly of higher-order fibers within anlagen. Pitx2 therefore appears to regulate muscle anlagen development by appropriately balancing expression of cytoskeletal and adhesion molecules.

How to build transcriptional network models of mammalian pattern formation.

Background Genetic regulatory networks of sequence specific transcription factors underlie pattern formation in multicellular organisms. Deciphering and representing the mammalian networks is a central problem in development, neurobiology, and regenerative medicine. Transcriptional networks specify intermingled embryonic cell populations during pattern formation in the vertebrate neural tube. Each embryonic population gives rise to a distinct type of adult neuron. The homeodomain transcription factor Lbx1 is expressed in five such populations and loss of Lbx1 leads to distinct respecifications in each of the five populations. Methodology/Principal Findings We have purified normal and respecified pools of these five populations from embryos bearing one or two copies of the null Lbx1GFP allele, respectively. Microarrays were used to show that expression levels of 8% of all transcription factor genes were altered in the respecified pool. These transcription factor genes constitute 20–30% of the active nodes of the transcriptional network that governs neural tube patterning. Half of the 141 regulated nodes were located in the top 150 clusters of ultraconserved non-coding regions. Generally, Lbx1 repressed genes that have expression patterns outside of the Lbx1-expressing domain and activated genes that have expression patterns inside the Lbx1-expressing domain. Conclusions/Significance Constraining epistasis analysis of Lbx1 to only those cells that normally express Lbx1 allowed unprecedented sensitivity in identifying Lbx1 network interactions and allowed the interactions to be assigned to a specific set of cell populations. We call this method ANCEA, or active node constrained epistasis analysis, and think that it will be generally useful in discovering and assigning network interactions to specific populations. We discuss how ANCEA, coupled with population partitioning analysis, can greatly facilitate the systematic dissection of transcriptional networks that underlie mammalian patterning.