Elliott W. Abrams

Tissue‐specific expression of murine Nkx3.1 in the male urogenital system

The molecular mechanisms involved in growth and morphogenesis of the mammalian urogenital system are largely undefined. In this study, we describe the cloning and characterization of a novel murine homeobox gene, Nkx3.1, which is expressed in the male urogenital system during late embryogenesis and adulthood. We show that Nkx3.1 encodes a 38 kDa homeoprotein that has DNA binding properties similar to those of other Nkx family members. By RNAse protection analysis, we demonstrate that Nkx3.1 is expressed in late‐gestation embryos and adults by tissues of the male urogenital system, including the testis, seminal vesicle, and the prostate. In adult males, expression of Nkx3.1 in the prostate increases during sexual maturation, and is significantly reduced following castration, suggesting that androgens are required for maintenance of Nkx3.1 expression. In situ hybridization analysis of mid‐ and late‐gestation male embryos shows that Nkx3.1 is expressed in the developing urogenital sinus, testis, and prostatic buds. In addition to its expression in the urogenital system, we also find that Nkx3.1 is expressed in the dorsal aorta and kidney. These results implicate Nkx3.1 in the growth and development of the prostate and/or other tissues of the male urogenital system, and suggest that Nkx3.1 may play a role in sexually dimorphic as well as non‐sexually dimorphic organogenesis. Dev. Dyn. 209:127–138, 1997.

Early zebrafish development: It’s in the maternal genes

The earliest stages of embryonic development in all animals examined rely on maternal gene products that are generated during oogenesis and supplied to the egg. The period of maternal control of embryonic development varies among animals according to the onset of zygotic transcription and the persistence of maternal gene products. This maternal regulation has been little studied in vertebrates, owing to the difficulty in manipulating maternal gene function and lack of basic molecular information. However, recent maternal-effect screens in the zebrafish have generated more than 40 unique mutants that are providing new molecular entry points to the maternal control of early vertebrate development. Here we discuss recent studies of 12 zebrafish mutant genes that illuminate the maternal molecular controls on embryonic development, including advances in the regulation of animal-vegetal polarity, egg activation, cleavage development, body plan formation, tissue morphogenesis, microRNA function and germ cell development.

CrebA regulates secretory activity in the Drosophila salivary gland and epidermis

Understanding how organs acquire the capacity to perform their respective functions is important for both cell and developmental biology. Here, we have examined the role of early-expressed transcription factors in activating genes crucial for secretory function in the Drosophila salivary gland. We show that expression of genes encoding proteins required for ER targeting and translocation, and proteins that mediate transport between the ER and Golgi is very high in the early salivary gland. This high level expression requires two early salivary gland transcription factors; CrebA is required throughout embryogenesis and Fkh is required only during late embryonic stages. As Fkh is required to maintain late CrebA expression in the salivary gland, Fkh probably works through CrebA to affect secretory pathway gene expression. In support of these regulatory interactions, we show that CrebA is important for elevated secretion in the salivary gland. Additionally, CrebA is required for the expression of the secretory pathway genes in the embryonic epidermis, where CrebA had previously been shown to be essential for cuticle development. We show that zygotic mutations in several individual secretory pathway genes result in larval cuticle phenotypes nearly identical to those of CrebA mutants. Thus, CrebA activity is linked to secretory function in multiple tissues.

Fork head and Sage maintain a uniform and patent salivary gland lumen through regulation of two downstream target genes, PH4αSG1 and PH4αSG2

(Fkh) is required to block salivary gland apoptosis, internalize salivary gland precursors, prevent expression of duct genes in secretory cells and maintain expression of CrebA, which is required for elevated secretory function. Here, we characterize two new Fkh-dependent genes: PH4αSG1 and PH4αSG2. We show through in vitro DNA-binding studies and in vivo expression assays that Fkh cooperates with the salivary gland-specific bHLH protein Sage to directly regulate expression of PH4αSG2, as well as sage itself, and to indirectly regulate expression of PH4αSG1. PH4αSG1 and PH4αSG2 encode α-subunits of resident ER enzymes that hydroxylate prolines in collagen and other secreted proteins. We demonstrate that salivary gland secretions are altered in embryos missing function of PH4αSG1 and PH4αSG2; secretory content is reduced and shows increased electron density by TEM. Interestingly, the altered secretory content results in regions of tube dilation and constriction, with intermittent tube closure. The regulation studies and phenotypic characterization of PH4αSG1 and PH4αSG2 link Fkh, which initiates tube formation, to the maintenance of an open and uniformly sized secretory tube.

Prolyl 4-hydroxylase α-related proteins in Drosophila melanogaster: tissue-specific embryonic expression of the 99F8-9 cluster

The extracellular matrix (ECM) is proposed to play critical roles in organ morphogenesis through the stabilization and/or sequestration of signaling factors and adhesion molecules, and by maintaining organ integrity. As a first step toward understanding molecules involved in ECM modification and maturation, we have examined the embryonic expression profiles of ten prolyl 4-hydroxylase alpha subunit (PH4alpha)-related genes. Prolyl 4-hydroxylases (PH4) catalyze the formation of 4-hydroxyproline in collagens, the major components of the ECM, and are implicated in the hydroxylation of proline in several other secreted proteins. To date, two alpha subunit proteins have been described in both humans (PHalphaI and PHalphaII) and worms (PHY-1/DPY-18 and PHY-2), whereas only a single Drosophila alpha subunit has been identified. The ten PH4alpha-related genes described in this study are clustered in a 183-kb region near the tip of chromosome arm 3R and include the previously described Drosophila alpha subunit gene. Six of the ten PH4alpha genes in the cluster have tissue-specific embryonic expression. PH4alphaSG1 and PH4alphaSG2 are expressed in the salivary gland, PH4alphaMP is expressed in mouth-part precursors, PH4alphaPV is expressed in the proventriculus, and CG9698-E is expressed in the epidermis. PH4alphaEFB is expressed more broadly, with expression in the anterior and posterior midgut primordia, the fat body, the hemocytes and the epidermis. The expression profiles of these PH4alpha-related genes suggest that tissue-specific ECM modifications may be critical to organ formation and/or function.

Constructing an organ: the Drosophila salivary gland as a model for tube formation.

Tubes are required in metazoans to transport the liquids and gases that sustain life. The conservation of molecules and mechanisms involved in tube formation suggests that what we learn by studying simple systems will apply to related processes in higher animals. Studies over the past 10 years have revealed the molecules that specify cell fate in Drosophila salivary gland and the cellular events that mediate tube morphogenesis. Here, we discuss how anterior-posterior and dorsal-ventral patterning information specifies both the position of salivary-gland primordia and how many cells they contain. We examine the transformation of a polarized epithelial sheet into an elongated, unbranched tube, and the intrinsic and extrinsic factors that influence the final position of the salivary gland.

Dynamic Assembly of Brambleberry Mediates Nuclear Envelope Fusion during Early Development

To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres.

The Chromosomal Passenger Protein Birc5b Organizes Microfilaments and Germ Plasm in the Zebrafish Embryo

Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs), which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC) component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation.

Mutations in vacuolar H+ -ATPase subunits lead to biliary developmental defects in zebrafish.

We identified three zebrafish mutants with defects in biliary development. One of these mutants, pekin (pn), also demonstrated generalized hypopigmentation and other defects, including disruption of retinal cell layers, lack of zymogen granules in the pancreas, and dilated Golgi in intestinal epithelial cells. Bile duct cells in pn demonstrated an accumulation of electron dense bodies. We determined that the causative defect in pn was a splice site mutation in the atp6ap2 gene that leads to an inframe stop codon. atp6ap2 encodes a subunit of the vacuolar H(+)-ATPase (V-H(+)-ATPase), which modulates pH in intracellular compartments. The Atp6ap2 subunit has also been shown to function as an intracellular renin receptor that stimulates fibrogenesis. Here we show that mutants and morphants involving other V-H(+)-ATPase subunits also demonstrated developmental biliary defects, but did not demonstrate the inhibition of fibrogenic genes observed in pn. The defects in pn are reminiscent of those we and others have observed in class C VPS (vacuolar protein sorting) family mutants and morphants, and we report here that knockdown of atp6ap2 and vps33b had an additive negative effect on biliary development. Our findings suggest that pathways which are important in modulating intracompartmental pH lead to defects in digestive organ development, and support previous studies demonstrating the importance of intracellular sorting pathways in biliary development.

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.