Development of the mammalian gonad: the fate of the supporting cell lineage

Sex determination in mammals is mediated via the supporting cell lineage in the fetal gonad. In the very early stages of gonadal development, the fate of the supporting cell population is critically dependent on the expression of the male-determining gene on the Y chromosome. If this gene is absent or fails to be expressed, or is expressed too late or in too small a number of supporting cells, all supporting cells (XX or XY) differentiate as pre-follicle cells and development proceeds along the female pathway. Supporting cells in which the male-determining gene is expressed in a timely manner differentiate as pre-Sertoli cells; given sufficient such cells, testis cords form and development proceeds in a male direction. If XX supporting cells are also present, a few may be recruited into the pre-Sertoli population and participate in testis cord formation. The subsequent fate of pre-follicle cells depends critically on interaction with the germ cell population in the developing gonad: absence of germ cells may lead to partial masculinization of the gonad, and/or to disappearance of the supporting cell component.     

Caenorhabditis elegans germline patterning requires coordinated development of the somatic gonadal sheath and the germ line

Interactions between the somatic gonad and the germ line influence the amplification, maintenance, and differentiation of germ cells. In Caenorhabditis elegans, the distal tip cell/germline interaction promotes a mitotic fate and/or inhibits meiosis through GLP-1/Notch signaling. However, GLP-1-mediated signaling alone is not sufficient for a wild-type level of germline proliferation. Here, we provide evidence that specific cells of the somatic gonadal sheath lineage influence amplification, differentiation, and the potential for tumorigenesis of the germ line. First, an interaction between the distal-most pair of sheath cells and the proliferation zone of the germ line is required for larval germline amplification. Second, we show that insufficient larval germline amplification retards gonad elongation and thus delays meiotic entry. Third, a more severe delay in meiotic entry, as is exhibited in certain mutant backgrounds, inappropriately juxtaposes undifferentiated germ cells with cells of the proximal sheath lineage, leading to the formation of a proximal germline tumor derived from undifferentiated germ cells. Tumors derived from dedifferentiated germ cells, however, respond to the proximal interaction differently depending on the mutant background. Our study underscores the importance of strict developmental coordination between neighboring tissues. We discuss these results in the context of mechanisms that may underlie tumorigenesis.     

Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate

Relatively little is known about the developmental signals that specify the types and numbers of pancreatic cells. Previous studies suggested that Notch signaling in the pancreas inhibits differentiation and promotes the maintenance of progenitor cells, but it remains unclear whether Notch also controls cell fate choices as it does in other tissues. To study the impact of Notch in progenitors of the beta cell lineage, we generated mice that express Cre-recombinase under control of the Pax4 promoter. Lineage analysis of Pax4(+) cells demonstrates they are specified endocrine progenitors that contribute equally to four islet cell fates, contrary to expectations raised by the dispensable role of Pax4 in the specification of the alpha and PP subtypes. In addition, we show that activation of Notch in Pax4(+) progenitors inhibits their differentiation into alpha and beta endocrine cells and shunts them instead toward a duct fate. These observations reveal an unappreciated degree of developmental plasticity among early endocrine progenitors and raise the possibility that a bipotent duct-endocrine progenitor exists during development. Furthermore, the redirection of Pax4(+) cells from alpha and beta endocrine fates toward a duct cell type suggests a positive role for Notch signaling in duct specification and is consistent with the more widely defined role for Notch in cell fate determination.     

Attenuation of Notch and Hedgehog signaling is required for fate specification in the spinal cord

During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.     

Proliferation of germ cells during gonadal sex differentiation in medaka: Insights from germ cell-depleted mutant zenzai

The proliferation of germ cells becomes sexually dimorphic during gonadal sex differentiation, although the underlying dynamics of this are not well understood in vertebrates. By tracing GFP-labeled germ cells in vivo and analyzing the germ cell-depleted mutant, zenzai, we show that the proliferation and differentiation of germ cells are regulated in a sexually dimorphic manner in the teleost fish medaka. In the undifferentiated gonads, germ cells resume proliferation by slow intermittent division (type I), producing isolated daughter cells. While germ cells in the male gonads continue this mode of proliferation, some germ cell fractions in the female gonads initiate two to four rounds of continuous division (type II), forming cysts of four, eight, or sixteen cells, which subsequently enter meiosis synchronously. Thus, female germ cells become differentiated much earlier than do male germ cells. In the zenzai mutant, a defect in slow intermittent division eventually leads to the depletion of germ cells in the adult gonads in both sexes, despite the fact that cyst-forming division is unaffected. This argues that slow intermittent division is essential for the maintenance of germ cells. The proliferation and differentiation of germ cells are thus important components of gonadal sex differentiation in vertebrates.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling

Wiskott-Aldrich syndrome proteins, encoded by the Wiskott-Aldrich syndrome gene family, bridge signal transduction pathways and the microfilament-based cytoskeleton. Mutations in the Drosophila homologue, Wasp (Wsp), reveal an essential requirement for this gene in implementation of cell fate decisions during adult and embryonic sensory organ development. Phenotypic analysis of Wsp mutant animals demonstrates a bias towards neuronal differentiation, at the expense of other cell types, resulting from improper execution of the program of asymmetric cell divisions which underlie sensory organ development. Generation of two similar daughter cells after division of the sensory organ precursor cell constitutes a prominent defect in the Wsp sensory organ lineage. The asymmetric segregation of key elements such as Numb is unaffected during this division, despite the misassignment of cell fates. The requirement for Wsp extends to additional cell fate decisions in lineages of the embryonic central nervous system and mesoderm. The nature of the Wsp mutant phenotypes, coupled with genetic interaction studies, identifies an essential role for Wsp in lineage decisions mediated by the Notch signaling pathway.     

Sex chromosomes and brain gender

In birds and mammals, differences in development between the sexes arise from the differential actions of genes that are encoded on the sex chromosomes. These genes are differentially represented in the cells of males and females, and have been selected for sex-specific roles. The brain is a sexually dimorphic organ and is also shaped by sex-specific selection pressures. Genes on the sex chromosomes probably determine the gender (sexually dimorphic phenotype) of the brain in two ways: by acting on the gonads to induce sex differences in levels of gonadal secretions that have sex-specific effects on the brain, and by acting in the brain itself to differentiate XX and XY brain cells.     

Continuing primordial germ cell differentiation in the mouse embryo is a cell-intrinsic program sensitive to DNA methylation

The initial cohort of mammalian gametes is established by the proliferation of primordial germ cells in the early embryo. Primordial germ cells first appear in extraembyronic tissues and subsequently migrate to the developing gonad. Soon after they arrive in the gonad, the germ cells cease dividing and undertake sexually dimorphic patterns of development. Male germ cells arrest mitotically, while female germ cells directly enter meiotic prophase I. These sex-specific differentiation events are imposed upon a group of sex-common differentiation events that are shared by XX and XY germ cells. We have studied the appearance of GCNA1, a postmigratory sex-common germ cell marker, in cultures of premigratory germ cells to investigate how this differentiation program is regulated. Cultures in which proliferation was either inhibited or stimulated displayed a similar extent of differentiation as controls, suggesting that some differentiation events are the result of a cell-intrinsic program and are independent of cell proliferation. We also found that GCNA1 expression was accelerated by agents which promote DNA demethylation or histone acetylation. These results suggest that genomic demethylation of proliferative phase primordial germ cells is a mechanism by which germ cell maturation is coordinated.     

FGF9 promotes survival of germ cells in the fetal testis

In addition to its role in somatic cell development in the testis, our data have revealed a role for Fgf9 in XY germ cell survival. In Fgf9-null mice, germ cells in the XY gonad decline in numbers after 11.5 days post coitum (dpc), while germ cell numbers in XX gonads are unaffected. We present evidence that germ cells resident in the XY gonad become dependent on FGF9 signaling between 10.5 dpc and 11.5 dpc, and that FGF9 directly promotes XY gonocyte survival after 11.5 dpc, independently from Sertoli cell differentiation. Furthermore, XY Fgf9-null gonads undergo true male-to-female sex reversal as they initiate but fail to maintain the male pathway and subsequently express markers of ovarian differentiation (Fst and Bmp2). By 14.5 dpc, these gonads contain germ cells that enter meiosis synchronously with ovarian gonocytes. FGF9 is necessary for 11.5 dpc XY gonocyte survival and is the earliest reported factor with a sex-specific role in regulating germ cell survival.     

Effects of estradiol-17beta on germ cell proliferation and DMY expression during early sexual differentiation of the medaka Oryzias latipes

Sex reversal of XY male to functional females was induced by estrogen treatment during the embryonic period in the medaka Oryzias latipes. The present study aimed to examine whether exogenous estrogen (estradiol-17beta; E(2)) affects early sex differentiation, paying particular attention to DMY expression and proliferation activity of germ cells in estrogen treated XY individuals. Our results showed that germ cell number was not affected by E(2) treatment at hatching, and that DMY expression was not suppressed under conditions of sex reversal. Therefore, male differentiation of germ cells, which is triggered by the expression of DMY in the supporting cell lineage, proceeds even in E(2) treated XY individuals until hatching, and early sex differentiation is not altered by estrogen. However, sex reversal occurred after hatching probably because of estrogen remaining in the yolk. Interestingly, DMY expression was also detected in the large follicle layer of E(2 )treated XY ovary. These results suggested that DMY regulates male determination in early embryonic stage but does not suppress female follicle development.     

The blood contains multiple distinct progenitor populations with clonogenic B and T lineage potential

The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.     

Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos

The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation.     

Specification of ectodermal teloblast lineages in embryos of the oligochaete annelid Tubifex: involvement of novel cell-cell interactions

In embryos of clitellate annelids (i.e. oligochaetes and leeches), four ectodermal teloblasts (ectoteloblasts N, O, P and Q) are generated on either side through a stereotyped sequence of cell divisions of a proteloblast, NOPQ. The four ectoteloblasts assume distinct fates and produce bandlets of smaller progeny cells, which join together to form an ectodermal germ band. The pattern of the germ band, with respect to the ventrodorsal order of the bandlets, has been highly preserved in clitellate annelids. We show that specification of ectoteloblast lineages in the oligochaete annelid Tubifex involves cell interaction networks distinct from those in leeches. Cell ablation experiments have shown that fates of teloblasts N, P and Q in Tubifex embryos are determined rigidly as early as their birth. In contrast, the O teloblast and its progeny are initially pluripotent and their fate becomes restricted to the O fate through an inductive signal emanating from the P lineage. In the absence of this signal, the O lineage assumes the P fate. These results differ significantly from those obtained in embryos of the leech Helobdella, suggesting the diversity of patterning mechanisms that give rise to germ bands with similar morphological pattern.