Alar Karis

Transcription Factor Sp1 Is Essential for Early Embryonic Development but Dispensable for Cell Growth and Differentiation

Transcription factor Sp1 has been implicated in the expression of many genes. Moreover, it has been suggested that Sp1 is linked to the maintenance of methylation-free CpG islands, the cell cycle, and the formation of active chromatin structures. We have inactivated the mouse Sp1 gene. Sp1-/- embryos are retarded in development, show a broad range of abnormalities, and die around day 11 of gestation. In Sp1-/- embryos, the expression of many putative target genes, including cell cycle-regulated genes, is not affected, CpG islands remain methylation free, and active chromatin is formed at the globin loci. However, the expression of the methyl-CpG-binding protein MeCP2 is greatly reduced in Sp1-/- embryos. MeCP2 is thought to be required for the maintenance of differentiated cells. We suggest that Sp1 is an important regulator of this process.

The POU Factor Oct-6 and Schwann Cell Differentiation

The POU transcription factor Oct-6, also known as SCIP or Tst-1, has been implicated as a major transcriptional regulator in Schwann cell differentiation. Microscopic and immunochemical analysis of sciatic nerves of Oct-6−/− mice at different stages of postnatal development reveals a delay in Schwann cell differentiation, with a transient arrest at the promyelination stage. Thus, Oct-6 appears to be required for the transition of promyelin cells to myelinating cells. Once these cells progress past this point, Oct-6 is no longer required, and myelination occurs normally.

Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear.

Patterning the vertebrate ear requires the coordinated expression of genes that are involved in morphogenesis, neurogenesis, and hair cell formation. The zinc finger gene GATA‐3 is expressed both in the inner ear and in afferent and efferent auditory neurons. Specifically, GATA‐3 is expressed in a population of neurons in rhombomere 4 that extend their axons across the floor plate of rhombomere 4 (r4) at embryonic day 10 (E10) and reach the sensory epithelia of the ear by E13.5. The distribution of their cell bodies corresponds to that of the cell bodies of the cochlear and vestibular efferent neurons as revealed by labeling with tracers. Both GATA‐3 heterozygous and GATA‐3 null mutant mice show unusual axonal projections, such as misrouted crossing fibers and fibers in the facial nerve, that are absent in wild‐type littermates. This suggests that GATA‐3 is involved in the pathfinding of efferent neuron axons that navigate to the ear. In the ear, GATA‐3 is expressed inside the otocyst and the surrounding periotic mesenchyme. The latter expression is in areas of branching of the developing ear leading to the formation of semicircular canals. Ears of GATA‐3 null mutants remain cystic, with a single extension of the endolymphatic duct and no formation of semicircular canals or saccular and utricular recesses. Thus, both the distribution of GATA‐3 and the effects of null mutations on the ear suggest involvement of GATA‐3 in morphogenesis of the ear. This study shows for the first time that a zinc finger factor is involved in axonal navigation of the inner ear efferent neurons and, simultaneously, in the morphogenesis of the inner ear. J. Comp. Neurol. 429:615–630, 2001.

Expression of the transcription factor GATA-3 is required for the development of the earliest T cell progenitors and correlates with stages of cellular proliferation in the thymus

GATA‐3 is a zinc‐finger transcription factor that is essential for both early T cell development and Th2 cell differentiation. To quantify GATA‐3 expression during T cell development in vivo in the mouse, the GATA‐3 gene was targeted by insertion of a lacZ reporter by homologous recombination in embryonic stem (ES) cells. Although we could detect GATA‐3+ cells throughout T cell development in the thymus, the proportions of GATA‐3+ cells varied considerably between the distinct differentiation stages. The two periods of TCR α and β gene recombination, which occur in quiescent or slowly dividing cells, were associated with low proportions of GATA‐3+ cells. Conversely, the stage of rapidly proliferating cells, which insulates these two waves of TCR rearrangement, was characterized by a large proportion of GATA‐3+ cells. In addition, we generated chimeric mice by injection of GATA‐3‐deficient, lacZ‐expressing ES cells into wild‐type blastocysts. In this in vivo competition analysis, no contribution of GATA‐3‐deficient cells to the T cell lineage was detected, not even in the earliest CD44+CD25− double‐negative (CD4−CD8−) cell stage in the thymus. These results parallel data implicating other GATA family members as key regulators of proliferation and survival of early hematopoietic cells. We therefore propose that GATA‐3 is required for the expansion of T cell progenitors, and for the control of subsequent proliferation steps, which alternate periods of TCR recombination in the thymus.

Transcription factor Sp3 is essential for post-natal survival and late tooth development

Sp3 is a ubiquitously expressed transcription factor closely related to Sp1 (specificity protein 1). We have disrupted the mouse Sp3 gene by homologous recombination. Sp3‐deficient embryos are growth retarded and invariably die at birth of respiratory failure. The cause for the observed breathing defect remains obscure since only minor morphological alterations were observed in the lung, and surfactant protein expression is indistinguishable from that in wild‐type mice. Histological examinations of individual organs in Sp3−/− mice show a pronounced defect in late tooth formation. In Sp3 null mice, the dentin/enamel layer of the developing teeth is impaired due to the lack of ameloblast‐specific gene products. Comparison of the Sp1 and Sp3 knockout phenotype shows that Sp1 and Sp3 have distinct functions in vivo, but also suggests a degree of functional redundancy.

Inactivation of Btk by insertion of lacZ reveals defects in B cell development only past the pre-B cell stage.

Brutons tyrosine kinase (Btk) is a cytoplasmic protein kinase that is defective in X‐linked agammaglobulinaemia in man and in X‐linked immunodeficiency in the mouse. There is controversy regarding the stages of B cell development that are dependent on Btk function. To determine the point in B cell differentiation at which defects in Btk become apparent, we generated a mouse model by inactivating the Btk gene through an in‐frame insertion of a lacZ reporter by homologous recombination in embryonic stem cells. The phenomenon of X‐chromosome inactivation in Btk+/− heterozygous female mice enabled us to evaluate the competition between B cell progenitors expressing wild‐type Btk and those expressing the Btk‐/lacZ allele in each successive step of development. Although Btk was already expressed in pro‐B cells, the first selective disadvantage only became apparent at the transition from small pre‐B cells to immature B cells in the bone marrow. A second differentiation arrest was found during the maturation from IgD(low)IgM(high) to IgD(high)IgM(low) stages in the periphery. Our results show that Btk expression is essential at two distinct differentiation steps, both past the pre‐B cell stage.

An intrinsic but cell-nonautonomous defect in GATA-1-overexpressing mouse erythroid cells

GATA-1 is a tissue-specific transcription factor that is essential for the production of red blood cells. Here we show that overexpression of GATA-1 in erythroid cells inhibits their differentiation, leading to a lethal anaemia. Using chromosome-X-inactivation of a GATA-1 transgene and chimaeric animals, we show that this defect is intrinsic to erythroid cells, but nevertheless cell nonautonomous. Usually, cell nonautonomy is thought to reflect aberrant gene function in cells other than those that exhibit the phenotype. On the basis of our data, we propose an alternative mechanism in which a signal originating from wild-type erythroid cells restores normal differentiation to cells overexpressing GATA-1 in vivo. The existence of such a signalling mechanism indicates that previous interpretations of cell-nonautonomous defects may be erroneous in some cases and may in fact assign gene function to incorrect cell types.

Enforced Expression of GATA-3 in Transgenic Mice Inhibits Th1 Differentiation and Induces the Formation of a T1/ST2-Expressing Th2-Committed T Cell Compartment In Vivo

The transcription factor GATA-3 is essential for early T cell development and differentiation of naive CD4+ T cells into Th2 effector cells. To study the function of GATA-3 during T cell-mediated immune responses in vivo, we investigated CD2-GATA3-transgenic mice in which GATA-3 expression is driven by the CD2 locus control region. Both in the CD4+ and the CD8+ T cell population the proportion of cells exhibiting a CD44highCD45RBlowCD62Llow Ag-experienced phenotype was increased. In CD2-GATA3-transgenic mice, large fractions of peripheral CD4+ T cells expressed the IL-1 receptor family member T1/ST2, indicative of advanced Th2 commitment. Upon in vitro T cell stimulation, the ability to produce IL-2 and IFN-γ was decreased. Moreover, CD4+ T cells manifested rapid secretion of the Th2 cytokines IL-4, IL-5, and IL-10, reminiscent of Th2 memory cells. In contrast to wild-type CD4+ cells, which lost GATA-3 expression when cultured under Th1-polarizing conditions, CD2-GATA3-transgenic CD4+ cells maintained expression of GATA-3 protein. Under Th1 conditions, cellular proliferation of CD2-GATA3-transgenic CD4+ cells was severely hampered, IFN-γ production was decreased and Th2 cytokine production was increased. Enforced GATA-3 expression inhibited Th1-mediated in vivo responses, such as Ag-specific IgG2a production or a delayed-type hypersensitivity response to keyhole limpet hemocyanin. Collectively, these observations indicate that enforced GATA-3 expression selectively inhibits Th1 differentiation and induces Th2 differentiation. The increased functional capacity to secrete Th2 cytokines, along with the increased expression of surface markers for Ag-experienced Th2-committed cells, would argue for a role of GATA-3 in Th2 memory formation.

Localization of Distant Urogenital System-, Central Nervous System-, and Endocardium-Specific Transcriptional Regulatory Elements in the GATA-3 Locus

ABSTRACT We found previously that neither a 6-kbp promoter fragment nor even a 120-kbp yeast artificial chromosome (YAC) containing the whole GATA-3 gene was sufficient to recapitulate its full transcription pattern during embryonic development in transgenic mice. In an attempt to further identify tissue-specific regulatory elements modulating the dynamic embryonic pattern of the GATA-3 gene, we have examined the expression of two much larger (540- and 625-kbp) GATA-3 YACs in transgenic animals. A lacZ reporter gene was first inserted into both large GATA-3 YACs. The transgenic YAC patterns were then compared to those of embryos bearing the identical lacZinsertion in the chromosomal GATA-3 locus (creating GATA-3/lacZ “knock-ins”). We found that most of the YAC expression sites and tissues are directly reflective of the endogenous pattern, and detailed examination of the integrated YAC transgenes allowed the general localization of a number of very distant transcriptional regulatory elements (putative central nervous system-, endocardium-, and urogenital system-specific enhancers). Remarkably, even the 625-kbp GATA-3 YAC, containing approximately 450 kbp and 150 kbp of 5′ and 3′ flanking sequences, respectively, does not contain the full transcriptional regulatory potential of the endogenous locus and is clearly missing regulatory elements that confer tissue-specific expression to GATA-3 in a subset of neural crest-derived cell lineages.

Enforced Expression of GATA-3 During T Cell Development Inhibits Maturation of CD8 Single-Positive Cells and Induces Thymic Lymphoma in Transgenic Mice

The zinc finger transcription factor GATA-3 is of critical importance for early T cell development and commitment of Th2 cells. To study the role of GATA-3 in early T cell development, we analyzed and modified GATA-3 expression in vivo. In mice carrying a targeted insertion of a lacZ reporter on one allele, we found that GATA-3 transcription in CD4+CD8+ double-positive thymocytes correlated with the onset of positive selection events, i.e., TCRαβ up-regulation and CD69 expression. LacZ expression remained high (∼80% of cells) during maturation of CD4 single-positive (SP) cells in the thymus, but in developing CD8 SP cells the fraction of lacZ-expressing cells decreased to <20%. We modified this pattern by enforced GATA-3 expression driven by the CD2 locus control region, which provides transcription of GATA-3 throughout T cell development. In two independent CD2-GATA3-transgenic lines, ∼50% of the mice developed thymic lymphoblastoid tumors that were CD4+CD8+/low and mostly CD3+. In tumor-free CD2-GATA3-transgenic mice, the total numbers of CD8 SP cells in the thymus were within normal ranges, but their maturation was hampered, as indicated by increased apoptosis of CD8 SP cells and a selective deficiency of mature CD69lowHSAlow CD8 SP cells. In the spleen and lymph nodes, the numbers of CD8+ T cells were significantly reduced. These findings indicate that GATA-3 supports development of the CD4 lineage and inhibits maturation of CD8 SP cells in the thymus.