Nuclear lamina builds tissues from the stem cell niche

Abstract

Recent studies show that nuclear lamins, the type V intermediate filament proteins, are required for proper building of at least some organs. As the major structural components of the nuclear lamina found underneath the inner nuclear membranes, lamins are ubiquitously expressed in all animal cells. How the broadly expressed lamins support the building of specific tissues is not understood. By studying Drosophila testis, we have uncovered a mechanism by which lamin-B functions in the cyst stem cell (CySC) and its differentiated cyst cell, the cell types known to form the niche/microenvironment for the germline stem cells (GSC) and the developing germ line, to ensure testis organogenesis ¹. In this extra view, we discuss some remaining questions and the implications of our findings in the understanding of how the ubiquitous nuclear lamina regulates tissue building in a context-dependent manner.

Introduction

Each animal organ or tissue consists of different cell types that interact and form specific structures to support the function and homeostasis of the organism. For example, organs such as the testes and intestines consist of different post-mitotic cells that perform the reproductive and absorptive functions, respectively, and stem cells that support the self-renewal of the organ.² Therefore, studying how different cell types interact and support one another within an organ is critical to the understanding of organ building and maintenance. Rapid progress made in developmental and stem cell biology has led to the appreciation of how signaling pathways and transcriptional cascades regulate cell lineage specification, organogenesis, and tissue homeostasis. However, the morphological basis by which different cells come together to make distinct organs has remained poorly understood.

Considering that the interaction and function of a given cell type rely on the cell’s distinct morphology, understanding the contribution of cell’s structures toward organogenesis should provide important insights regarding how tissues achieve their distinct architectures. Among many common cellular structures, the nuclear lamina (NL), which consists of lamin polymers and other nuclear peripheral proteins, is known to connect the chromatin in the nucleus to various cytoskeletal elements in the cytoplasm. Since the cytoskeleton plays critical roles in establishing cellular structures and inter-cellular interactions, NL may function as an important node that connects cell/tissue organization to signal and transcriptional regulation, thereby ensuring proper organogenesis. In this context, it is not surprising that deletions or mutations in lamins or other NL proteins lead to either human diseases or developmental defects in animal models.^3-6 As the NL is a ubiquitous nuclear structure, any functions assigned to the NL based on the cell biological studies in tissue culture cells or assay systems in vitro have been perceived as general NL functions applicable in all cell types.^7-10 Yet studies of the role of lamins in development suggest that the NL has tissue-specific functions.^11-19 Therefore, it is important to understand how a given NL function established by in vitro studies participates in the development of a specific organ.

Among the model organisms, Drosophila melanogaster offers a wide range of tools to study cell-type specific functions during development and tissue homeostasis. The simplified tissue organization in Drosophila compared with mammals should facilitate the molecular dissection of cell-specific functions of lamins during development. Of the many tissues, the Drosophila testis is one of the best-characterized organs in which each of the three cell types has distinct morphologies and functions. The post-mitotic hub cells, found at the tip of the testis, directly interact with both the cyst stem cells (CySCs) and the germline stem cells (GSCs) (Fig. 1A). The communications among these cells at the tip of testis support the self-renewal and differentiation of CySCs and GSCs into cyst cells and germ line cells, respectively (Fig. 1A). Each pair of CySCs encloses a GSC and functions as the niche for GSC. The division and differentiation of the pairs of CySCs into pairs of cyst cells accompany the division and differentiation of the GSCs into gonialblasts. While the gonialblasts further divide to form spermatogonia, spermatocytes, and mature sperm, the pair of post-mitotic cyst cells undergo extensive expansion to enclose and support the developing germline cells (Fig. 1A). By studying the role of lamins in testis development, we have been able to demonstrate a role of NL in coupling the signal transduction pathway with nuclear organization in specific cell types to support organogenesis.¹

Figure 1. Cartoon illustration of the role of lamin-B in regulating spermatogenesis. (A) In wild-type testes, the CySCs interact with the hub and the GSC, whereas the cyst cells extend their cell bodies to encapsulate the developing germline cells. (B) In lamin-B mutants, excess expression of integrin in CySCs may disrupt the proper interaction of the CySCs with the hub and GSCs. These and other defects in the CySC lineage result in defects in cyst cell differentiation and morphogenesis, which lead to the failure of the encapsulation of germline cells by the cyst cells.

Lamin-B Regulates Nucleoporin Distribution in Specific Cell Lineages During Development

By analyzing the existing lamin mutants in Drosophila, we found that lamin-B is required for male and female fertility. Using cell-type specific lamin depletion and rescue, we show that lamin-B, but not lamin-C, is specifically required in the CySC lineage to support testis development. Lamin-B depletion disrupts the interactions of CySCs with the niche and the GSCs. The cyst cells also fail to extend their cell bodies and are displaced away from the germline cells. Consequently, the germline cells are not encapsulated by the cyst cells (Fig. 1B). We show that lamin-B ensures the even distribution of the Drosophila nucleoporins NUP153 and MTOR (homologous to the vertebrate Nup called Tpr) throughout the nuclear periphery in the CySC lineage. Since depletion of NUP153 in the CySC lineage phenocopies lamin-B-null testis defects, lamin-B functions, at least in part, through NUP153 in the CySC lineage to support testis development.

Interestingly, studies using vertebrate cultured cells and in vitro assays have shown that lamins can regulate either Nup153 incorporation into NPCs^20,21 or the even distribution of Nup153 and Nup98 throughout the nuclear periphery in neurons migrating out of the neurospheres in vertebrates,²² but the biological significance of the regulation has remained unclear. Our electron microscopy analyses reveal a normal assembly of NPCs in the CySC lineage in both the wild-type and CySC lineage-specific lamin-B depleted animals. Since both the wild-type and lamin-B depleted CySCs and cyst cells exhibit normal nuclear import of proteins and proper distribution of Nups such as NUP154 and NUP98, lamin-B appears to regulate the distribution of specific nucleoporins the CySC lineage.

Lamin-B Links Nucleoporin Distribution with Nuclear EGF Signaling and Cell/Tissue Morphogenesis

Previous studies in mammalian tissue cultured cells suggest that Nup153 is required for the nuclear retention of phosphorylated Erk (pErk).^23-26 Although nuclear pErk is essential for the nuclear signaling of the EGF pathway, the physiological significance of Nup153 in the context of development has remained unknown. The wealth of information regarding how various signaling pathways regulate testis development has allowed us to show that lamin-B functions to promote the nuclear EGF signaling, in part, through NUP153 in the CySC lineage. By taking advantage of the well-defined localization and interaction of different cells within the testis, we demonstrate that the lamin/nucleoporin-regulated nuclear EGF signaling in CySCs is critical for CySCs and GSCs to interact with one another and with the hub. Disruption of these interactions leads to the displacement of GSCs from the hub by CySCs such that CySCs can no longer support proper development of the germline.

An important question is how the lamin-B/nucleoporin-regulated nuclear EGF signaling controls cell-cell interactions and tissue building. By analyzing E-cadherin and integrin, 2 adhesion proteins known to mediate the interaction between CySCs and the hub,^27,28 we show that the level of integrin is upregulated in CySCs in the absence of lamin-B, which could explain the increased interaction between CySC and the hub at the expense of the GSCs (Fig. 1B). However, we still do not know how lamin-B represses integrin expression in CySCs. Since it has been reported that EGFR signaling can activate a repressor of integrin called SOCS36E in the Drosophila imaginal disc,²⁹ we speculate that lamin-B also regulates the expression of integrin through EGFR-regulated SOCS36E in CySCs (Fig. 2). Interestingly, the NL is known to facilitate gene repression but the mechanism remains unknown.^30-33 Further dissecting how the nuclear EGF signaling leads to gene repression should shed light on how lamins function in the NL to create a repressive environment for gene expression in different tissue contexts.

Figure 2. A hypothesis illustrating how Lamin-B represses the expression of integrin in CySCs. Lamin-B may function through EGFR to activate SOCS36E, which in turn represses integrin in CySCs. Lamin-B may also function through SOCS36E or may directly repress integrin in CySCs.

Another question is how lamin-B regulates NUP153 localization in the CySC lineage. If lamin-B regulates NUP153 incorporation into NPCs as suggested by a study using cell free assays,²¹ one may expect an absence of NUP153 around the nucleus upon lamin-B depletion. Yet NUP153 appears aggregated in the nuclear periphery in the lamin-B depleted CySCs and cyst cells. Perhaps NUP153 forms inappropriate interactions with other NPC and/or NL components in the absence of lamin-B, which leads to its aggregation.

Additionally, we found that forced nuclear expression of pErk in the lamin-B depleted CySCs only partially rescued the developmental defects of the testis. This indicates that lamin-B may also function independent of nuclear EGF signaling to regulate testis development. Considering that lamin-B associates with large stretches of chromatin,^31,33-36 it may regulate gene expression by collaborating with other transcription factors, chromatin regulators, and signaling pathways to control the CySC lineage (Fig. 3). Analyzing how depletion of lamin-B in CySCs affects gene expression by RNA-seq may help to discover the EGF-independent lamin-B functions in spermatogenesis. A genome-wide RNAi screen for suppressors and enhancers of lamin-B mutants may also reveal new contributors to testis development.

Figure 3. A cartoon illustration of the NL and chromatin organization in CySCs. As the structural component of the NL, lamin-B associates with long stretches of chromatin (lamina-associated domain) and the NPCs. These interactions may ensure the coupling of nuclear organization with gene expression and signaling pathways. The regulation of nuclear EGF signaling in CySCs via lamin-B and specific Nups we uncovered represents an example for the importance of the coupling during organogenesis development.

The Requirement for Lamins in Different Cell Types

Decades of studies using tissue culture cells and in vitro assays have assigned diverse functions to lamins. The challenge now is to dissect which lamin function is relevant in which cell type or tissue in vivo. Besides the CySC lineage, we found that the follicle cells, which provide the microenvironment for the female germline in Drosophila, also rely on lamin-B to regulate Nup153 distribution and nuclear p-Erk accumulation. Interestingly, both the CySCs and the follicle cells also express very little lamin-C. Therefore, the particularly striking phenotype we have observed upon lamin-B loss in these cells may not simply indicate a lamin-B-specific function in CySCs and follicle cells. Indeed, we show that the CySC lineage-specific expression of lamin-C can partially rescue the developmental defect of testes caused by lamin-B depletion. Based on our findings, further dissecting the role of lamin-B and lamin-C in CySCs and follicle cells during spermatogenesis and oogenesis, respectively, should shed light on how different lamins perform the shared and specific functions during development.

Our analyses suggest that lamin-B is not required in the GSCs for testis development. Since GSCs express no or very low levels of lamin-C, one interpretation is that lamins are not required in GSCs. Interestingly, lamins are also not required for the self-renewal and in vitro differentiation of the mouse embryonic stem cells (mESC).^13,37 If lamin’s function is to couple genome organization and gene expression to the elaborate cytoskeletal organization and cell morphology, there may not be an apparent need for lamins in GSCs and ESCs because these cells have very limited cytoplasm and do not assume elaborate cell architectures as those seen in the cyst cells and follicle cells. However, it is important to note that our studies have not completely ruled out the function of lamins in GSCs and ESCs. For example, lamins may be required for maintaining GSCs during aging or under stress conditions. Lamins may also be required to establish chromatin organization and epigenetic landscape in ESCs to ensure the establishment of transcription programs in different stem cell niches for organogenesis. Considering the intimate connections between NL to cytoskeleton and NL to chromatin, a combination of studies of NL using model organisms and in vitro differentiation systems should help to elucidate the morphological basis of transcriptional programs that underlies organogenesis.

Chen H, Chen X, Zheng Y. The nuclear lamina regulates germline stem cell niche organization via modulation of EGFR signaling. Cell Stem Cell. 2013;13:73–86. doi: 10.1016/j.stem.2013.05.003.

10.4161/fly.28063