The specification and maintenance of renal cell types by epigenetic factors

The specification of cell lineages and patterning in the embryo occurs sequentially as specific regions are increasingly restricted in their developmental fates. When and how this occurs is still not entirely clear. Nevertheless, the roles of epigenetic regulatory genes in partitioning the genome into active and inactive domains is evident in a variety of organisms and is highly conserved through evolution. The function of Pax2 in the kidney has been inferred by the phenotypic analysis of loss-of-function mutants in mice, fish and humans. Although Pax2 and the related gene, Pax8, are essential for early intermediate mesoderm specification and are found in the epithelial lineage arising from that mesoderm, how these proteins regulate cell lineage restriction and gene expression patterns has remained obscure. Our recent data, suggests that Pax proteins help establish chromatin domains within cell lineages by providing the locus and tissue specificity for epigenetic imprinting complexes that modify histones. The novel protein PTIP is a key adaptor that links Pax proteins and possibly many other types of DNA binding proteins to a histone H3K4 methyltransferase complex. Given the prevalence of Pax2 expression in kidney development and in kidney disease, we now need to address the effects of epigenetics on renal disease states, on the stability of the terminal epithelial phenotype, and in the aging cell.Key words: kidney development, Pax2, PTIP, histone methyltransferase     

The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage

As the result of early specification processes, sea urchin embryos eventually form various mesodermal cell lineages and a gut consisting of fore-, mid- and hindgut. The progression of specification as well as the overall spatial organization of the organism is encoded in its gene regulatory networks (GRNs). We have analyzed the GRN driving endoderm specification up to the onset of gastrulation and present in this paper the mechanisms which determine this process up to mid-blastula stage. At this stage, the embryo consists of two separate lineages of endoderm precursor cells with distinct regulatory states. One of these lineages, the veg2 cell lineage, gives rise to endoderm and mesoderm cell types. The separation of these cell fates is initiated by the spatially confined activation of the mesoderm GRN superimposed on a generally activated endoderm GRN within veg2 descendants. Here we integrate the architecture of regulatory interactions with the spatial restriction of regulatory gene expression to model the logic control of endoderm development.     

Stepwise renal lineage differentiation of mouse embryonic stem cells tracing in vivo development

The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24(+) cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III.     

Investigating divergent mechanisms of mesoderm development in arthropods: the expression of Ph-twist and Ph-mef2 in Parhyale hawaiensis

The evolution of mesoderm was important for the development of complex body plans as well as key organ systems. Genetic and molecular studies in the fruitfly, Drosophila melanogaster, have provided the majority of information concerning mesoderm development in arthropods. In Drosophila, twist is necessary for the specification and correct morphogenesis of mesoderm and myocyte enhancing factor 2 (mef2) is involved downstream of twist to activate muscle differentiation. In Drosophila, mesoderm is defined by positional cues in the blastoderm embryo, while in another arthropod group, the amphipod crustaceans, cell lineage plays a greater role in defining the mesoderm. It is not known how different mechanistic strategies such as positional information vs. cell-lineage-dependent development affect the timing and use of gene networks. Here we describe the development of the mesoderm in a malacostracan crustacean, Parhyale hawaiensis, and characterize the expression of Parhyale twist and mef2 orthologues. In Parhyale, the mesoderm of the post-mandibular segments arises mainly through the asymmetric division of mesoteloblasts as the germband elongates. Ph-twist expression is seen in a subset of segmental mesoderm during germband development, but not during early cleavages when the specific mesodermal cell lineages first arise. ph-mef2 expression starts after the segmental mesoderm begins to proliferate and persists in developing musculature. While the association of these genes with mesoderm differentiation appears to be conserved across the animal kingdom, the timing of expression and relationship with different mechanisms of mesoderm development may give us greater insight into the ancestral use of these genes during mesoderm differentiation.     

Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.     

Paraxial mesoderm contributes stromal cells to the developing kidney

The development of most, if not all, tubular organs is dependent on signaling between epithelial and stromal progenitor populations. Most often, these lineages derive from different germ layers that are specified during gastrulation, well in advance of organ condensation. Thus, one of the first stages of organogenesis is the integration of distinct progenitor populations into a single embryonic rudiment. In contrast, the stromal and epithelial lineages controlling renal development are both believed to derive from the intermediate mesoderm and to be specified as the kidney develops. In this study we directly analyzed the lineage of renal epithelia and stroma in the developing chick embryo using two independent fate mapping techniques. Results of these experiments confirm the hypothesis that nephron epithelia derive from the intermediate mesoderm. Most importantly, we discovered that large populations of renal stroma originate in the paraxial mesoderm. Collectively, these studies suggest that the signals that subdivide mesoderm into intermediate and paraxial domains may play a role in specifying nephron epithelia and a renal stromal lineage. In addition, these fate mapping data indicate that renal development, like the development of all other tubular organs, is dependent on the integration of progenitors from different embryonic tissues into a single rudiment.     

Two novel EGFP insertion alleles reveal unique aspects of Pax2 function in embryonic and adult kidneys

The Pax2 gene encodes a DNA binding protein with multiple functions in the developing intermediate mesoderm and urogenital tract. Loss of Pax2 in mice results in the complete absence of kidneys, ureters, and sex specific epithelial structures derived from the intermediate mesoderm in both males and females. In this report, we describe two new alleles of Pax2 created by inserting the enhanced green fluorescent protein coding region into the 5' untranslated leader sequence. One allele is a hypomorph that generates less protein and exhibits structural defects in kidneys and ureters upon homozygosity. A second allele is a true null that can be used to image Pax2 expressing cells in a mutant background. Organ culture and embryo analyses point to a loss of epithelial cell polarity and increased mobility in cells that have deleted Pax2 function. These experiments provide new insight into the role of Pax2 protein levels in determining correct renal architecture and cell fate. These new Pax2 alleles are valuable genetic reagents for in vivo studies of urogenital development.     

The forkhead genes, Foxc1 and Foxc2, regulate paraxial versus intermediate mesoderm cell fate

During vertebrate embryogenesis, the newly formed mesoderm is allocated to the paraxial, intermediate, and lateral domains, each giving rise to different cell and tissue types. Here, we provide evidence that the forkhead genes, Foxc1 and Foxc2, play a role in the specification of mesoderm to paraxial versus intermediate fates. Mouse embryos lacking both Foxc1 and Foxc2 show expansion of intermediate mesoderm markers into the paraxial domain, lateralization of somite patterning, and ectopic and disorganized mesonephric tubules. In gain of function studies in the chick embryo, Foxc1 and Foxc2 negatively regulate intermediate mesoderm formation. By contrast, their misexpression in the prospective intermediate mesoderm appears to drive cells to acquire paraxial fate, as revealed by expression of the somite markers Pax7 and Paraxis. Taken together, the data indicate that Foxc1 and Foxc2 regulate the establishment of paraxial versus intermediate mesoderm cell fates in the vertebrate embryo.     

nessy, an evolutionary conserved gene controlled by Hox proteins during Drosophila embryogenesis.

From a library of DNA fragments associated with Ultrabithorax protein in vivo, we have isolated nessy, a new Drosophila gene that encodes a putative transmembrane protein conserved in evolution from Caenorhabditis elegans, to human. Zygotic expression occurs transiently in mesectodermal cells at gastrulation, proceeds in mesoderm and endoderm lineages during germ band movements and becomes then restricted to anterior and posterior domains in the visceral mesoderm. The Hox proteins Ultrabithorax, Antennapedia and AbdominalA are likely acting simultaneously to repress nessy in the other parts of the visceral mesoderm.     

Functions of Huntingtin in Germ Layer Specification and Organogenesis

Huntington’s disease (HD) is a neurodegenerative disease caused by abnormal polyglutamine expansion in the huntingtin protein (Htt). Although both Htt and the HD pathogenic mutation (mHtt) are implicated in early developmental events, their individual involvement has not been adequately explored. In order to better define the developmental functions and pathological consequences of the normal and mutant proteins, respectively, we employed embryonic stem cell (ESC) expansion, differentiation and induction experiments using huntingtin knock-out (KO) and mutant huntingtin knock-in (Q111) mouse ESC lines. In KO ESCs, we observed impairments in the spontaneous specification and survival of ectodermal and mesodermal lineages during embryoid body formation and under inductive conditions using retinoic acid and Wnt3A, respectively. Ablation of BAX improves cell survival, but failed to correct defects in germ layer specification. In addition, we observed ensuing impairments in the specification and maturation of neural, hepatic, pancreatic and cardiomyocyte lineages. These developmental deficits occurred in concert with alterations in Notch, Hes1 and STAT3 signaling pathways. Moreover, in Q111 ESCs, we observed differential developmental stage-specific alterations in lineage specification and maturation. We also observed changes in Notch/STAT3 expression and activation. Our observations underscore essential roles of Htt in the specification of ectoderm, endoderm and mesoderm, in the specification of neural and non-neural organ-specific lineages, as well as cell survival during early embryogenesis. Remarkably, these developmental events are differentially deregulated by mHtt, raising the possibility that HD-associated early developmental impairments may contribute not only to region-specific neurodegeneration, but also to non-neural co-morbidities.     

Identification and expression of the lamprey Pax6 gene: evolutionary origin of the segmented brain of vertebrates

The Pax6 gene plays a developmental role in various metazoans as the master regulatory gene for eye patterning. Pax6 is also spatially regulated in particular regions of the neural tube. Because the amphioxus has no neuromeres, an understanding of Pax6 expression in the agnathans is crucial for an insight into the origin of neuromerism in the vertebrates. We have isolated a single cognate cDNA of the Pax6 gene, LjPax6, from a Lampetra japonica cDNA library and observed the pattern of its expression using in situ hybridization. Phylogenetic analysis revealed that LjPax6 occurs as an sister group of gnathostome Pax6. In lamprey embryos, LjPax6 is expressed in the eye, the nasohypophysial plate, the oral ectoderm and the brain. In the central nervous system, LjPax6 is expressed in clearly delineated domains in the hindbrain, midbrain and forebrain. We compared the pattern of LjPax6 expression with that of other brain-specific regulatory genes, including LjOtxA, LjPax2/5/8, LjDlx1/6, LjEmx and LjTTF1. Most of the gene expression domains showed conserved pattern, which reflects the situation in the gnathostomes, conforming partly to the neuromeric patterns proposed for the gnathostomes. We conclude that most of the segmented domains of the vertebrate brain were already established in the ancestor common to all vertebrates. Major evolutionary changes in the vertebrate brain may have involved local restriction of cell lineages, leading to the establishment of neuromeres.     

Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells

Heparan sulfate (HS) has been implicated in regulating cell fate decisions during differentiation of embryonic stem cells (ESCs) into advanced cell types. However, the necessity and the underlying molecular mechanisms of HS in early cell lineage differentiation are still largely unknown. In this study, we examined the potential of EXT1(-/-) mouse ESCs (mESCs), that are deficient in HS, to differentiate into primary germ layer cells. We observed that EXT1(-/-) mESCs lost their differentiation competence and failed to differentiate into Pax6(+)-neural precursor cells and mesodermal cells. More detailed analyses highlighted the importance of HS for the induction of Brachyury(+) pan-mesoderm as well as normal gene expression associated with the dorso-ventral patterning of mesoderm. Examination of developmental cell signaling revealed that EXT1 ablation diminished FGF and BMP but not Wnt signaling. Furthermore, restoration of FGF and BMP signaling each partially rescued mesoderm differentiation defects. We further show that BMP4 is more prone to degradation in EXT1(-/-) mESCs culture medium compared with that of wild type cells. Therefore, our data reveal that HS stabilizes BMP ligand and thereby maintains the BMP signaling output required for normal mesoderm differentiation. In summary, our study demonstrates that HS is required for ESC pluripotency, in particular lineage specification into mesoderm through facilitation of FGF and BMP signaling.