Austin Cooney | Baylor College of Medicine (original) (raw)

Papers by Austin Cooney

Scientific reports, Jan 19, 2016

Mesp1 directs multipotential cardiovascular cell fates, even though it's transiently induced ... more Mesp1 directs multipotential cardiovascular cell fates, even though it's transiently induced prior to the appearance of the cardiac progenitor program. Tracing Mesp1-expressing cells and their progeny allows isolation and characterization of the earliest cardiovascular progenitor cells. Studying the biology of Mesp1-CPCs in cell culture and ischemic disease models is an important initial step toward using them for heart disease treatment. Because of Mesp1's transitory nature, Mesp1-CPC lineages were traced by following EYFP expression in murine Mesp1(Cre/+); Rosa26(EYFP/+) ES cells. We captured EYFP+ cells that strongly expressed cardiac mesoderm markers and cardiac transcription factors, but not pluripotent or nascent mesoderm markers. BMP2/4 treatment led to the expansion of EYFP+ cells, while Wnt3a and Activin were marginally effective. BMP2/4 exposure readily led EYFP+ cells to endothelial and smooth muscle cells, but inhibition of the canonical Wnt signaling was require...

Obstetrical & Gynecological Survey, 2011

ABSTRACT During normal mating in mammals between males and females, fertilization of oocytes by s... more ABSTRACT During normal mating in mammals between males and females, fertilization of oocytes by spermatozoa produces progeny with alleles inherited from both parents. Pluripotent embryonic stem (ES) cells can differentiate into all adult cell types, including oocytes and sperm. In culture, genetically male mouse ES lines spontaneously lose the Y chromosome at a frequency of about 1%, resulting in genetically female XO cell lines that can produce viable fertile females. Previous studies have not shown that such XY-derived XO pluripotent stem cells can generate viable progeny with genetic information from 2 distinct fathers. This present study exploited this stem cell strategy (in vitro sex reversal) to generate viable male and female mice that combine the haploid genomes from 2 fathers. Manipulation of fibroblasts from somatic cells of a male (XY) mouse fetus produced induced pluripotent stem cell lines. Injection of these XY-derived XO ES cells into blastocysts from donor female mice produced functional oocytes in female chimeras. Natural mating of the female chimeras (from father 1) with genetically distinct males (father 2) resulted in live progeny with haploid genomes that were equally derived from both fathers. These findings demonstrate that use of induced pluripotent stem cell technology can generate male and female progeny by combining the alleles from 2 male mice. This new method of mammalian reproduction may have major implications for improving livestock breeds and preserving endangered species as well as for advancing assisted reproductive technology in humans.

Biology of Reproduction, 2011

In sexual species, fertilization of oocytes produces individuals with alleles derived from both p... more In sexual species, fertilization of oocytes produces individuals with alleles derived from both parents. Here we use pluripotent stem cells derived from somatic cells to combine the haploid genomes from two males to produce viable sons and daughters. Male (XY) mouse induced pluripotent stem cells (Father #1) were used to isolate subclones that had spontaneously lost the Y chromosome to become genetically female (XO). These malederived XO stem cells were used to generate female chimeras that were bred with genetically distinct males (Father #2), yielding progeny possessing genetic information that was equally derived from both fathers. Thus, functional oocytes can be generated from male somatic cells after reprogramming and spontaneous sex reversal. These findings have novel implications for mammalian reproduction and assisted reproductive technology.

Molecular and Cellular Biology, 2001

The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, ... more The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the GCNF gene in mice. Germ line mutation of the GCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF ؊/؊ embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF ؊/؊ embryos suffer significant defects in posterior development. Unlike GCNF ؉/؉ embryos, GCNF ؊/؊ embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF ؊/؊ embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF ؊/؊ embryos is 13 instead of 25 as in the GCNF ؉/؉ embryos. Interestingly, the tailbud of GCNF ؊/؊ embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF ؊/؊ embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.

Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate ... more Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that Germ Cell Nuclear Factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly reprogrammed , allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not reprogrammed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal-reprogrammed cells re-capitulated the phenotype of Gcnf knock out (Gcnf −/−) ES cells. Re-introduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf −/− ES cells, rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely reprogrammed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells.

， Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds ... more ， Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however, the mechanisms that regulate the levels of human Oct4 expression remains poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF), an orphan nuclear receptor, in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells.

Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression ... more Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse, however, it remains unknown how cyclin D1 expression is regulated during early embryonic development. Here we define the role of germ cell nuclear factor (GCNF) in the activation of cyclin D1 expression during embryonic stem (ES) cell differentiation as a model of early development. During our study of GCNF knock out (GCNF) ES cells, we discovered that loss of GCNF leads to the repression of cyclin D1 activation during ES cell differentiation. This was determined to be an indirect effect of deregulation Mir302a, which is a cyclin D1 suppressor via binding to the 3`UTR of cyclin D1 mRNA. Moreover, we showed that Mir302 is a target gene of GCNF that inhibits Mir302 expression by binding to a DR0 element within its promoter. Inhibition of Mir302a using Mir302 inhibitor during differentiation of GCNF ES cells restored cyclin D1 expression. Similarly over-expression of GCNF during differentiation of GCNF ES cells rescued the inhibition of Mir302a expression and the activation of cyclin D1. These results reveal that GCNF plays a key role in regulating activation of cyclin D1 expression via inhibition of Mir302a.

PLoS ONE, 2014

Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor ge... more Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.

Stem cells (Dayton, Ohio), 2011

The pluripotency gene Oct4 encodes a key transcription factor that maintains self-renewal of embr... more The pluripotency gene Oct4 encodes a key transcription factor that maintains self-renewal of embryonic stem cell (ESC) and is downregulated upon differentiation of ESCs and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, mediates Oct4 gene expression. Here, we show that the orphan nuclear receptor germ cell nuclear factor (GCNF) initiates Oct4 repression and DNA methylation by the differential recruitment of methyl-CpG binding domain (MBD) and DNA methyltransferases (Dnmts) to the Oct4 promoter. When compared with wild-type ESCs and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in retinoic acid (RA)-differentiated GCNF(-/-) ESCs and GCNF(-/-) embryos. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified MBD3, MBD2, and de novo Dnmts as GCNF interacting factors. Upon differe...

Nature, 2001

The discovery of stem cells that can generate neural tissue has raised new possibilities for repa... more The discovery of stem cells that can generate neural tissue has raised new possibilities for repairing the nervous system. A rush of papers proclaiming adult stem cell plasticity has fostered the notion that there is essentially one stem cell type that, with the right impetus, can create whatever progeny our heart, liver or other vital organ desires. But studies aimed at understanding the role of stem cells during development have led to a different view - that stem cells are restricted regionally and temporally, and thus not all stem cells are equivalent. Can these views be reconciled?