Differential Cytokeratin Gene Expression Reveals Early Dorsal-Ventral Regionalization in Chick Mesoderm
Total Page：16
File Type：pdf, Size：1020Kb
Development 110, 417-425 (1990) 417 Printed in Great Britain © The Company of Biologists Limited 1990 Differential cytokeratin gene expression reveals early dorsal-ventral regionalization in chick mesoderm TIMOTHY S. CHARLEBOIS*, JONATHAN J. HENRYt and ROBERT M. GRAINGER Department of Biology, University of Virginia, Charlottesville, VA 22901, USA Present addresses: •Genetics Institute, One Bum Rd, Andover, MA 01810, USA t Department of Biology, Indiana University, Bloomington, IN 47405, USA Summary The induction and spatial patterning of early mesoderm even while the mesoderm layer is being formed, and are known to be critical events in the establishment of persists during the extensive cellular movements and the vertebrate body plan. However, it has been difficult tissue remodelling associated with morphogenesis. These to define precisely the steps by which mesoderm is results point to an early step in which two fundamentally initially subdivided into functionally discrete regions. distinct states are established along the presumptive Here we present evidence for a sharply defined dorsal-ventral axis in the mesoderm, and suggest that distinction between presumptive dorsal and presumptive determination in this germ layer occurs in a hierarchical ventral regions in early chick mesoderm. Northern blot manner, rather than by direct specification of individual and in situ hybridization analyses reveal that transcripts types of histological differentiation. The differential corresponding to CKsel, a cytokeratin gene expressed expression of CKsel represents the earliest molecular during early development, are present at high levels in index of dorsoventral regionalization detected thus far in the presumptive ventral mesoderm, but are greatly the mesoderm. reduced or undetectable in the future dorsal region of mesoderm, where the formation of axial structures Key words: chick embryo, mesoderm regionalization, occurs later in development. This distinction is present cytokeratin gene expression. Introduction histological differentiation, and examining their devel- opment in isolation. In the amphibian embryo, isolated The formation of vertebrate mesoderm has recently cultures of prospective dorsal mesoderm differentiate been the subject of intensive scrutiny because this germ primarily into notochord and muscle (the most dorsal layer plays a central role in the establishment of mesodermal derivatives), but very little mesothelium or embryonic pattern (Slack, 1983). Even at very early blood (ventral mesoderm). Isolates removed from stages, the mesoderm appears not to be homogeneous progressively more ventral locations form much more but instead possesses region-specific characteristics mesothelium and blood, with the amount of muscle or along the dorsal-ventral axis of the embryo. This notochord becoming greatly reduced, particularly in the regional specialization of early mesoderm has particu- most ventral isolates (Dale and Slack, 1987). larly important implications for the formation of the While the above data argue that regional specializ- basic vertebrate body plan, as recognized by Spemann ation occurs along the dorsal-ventral axis in the early and Mangold (1924), who first demonstrated the unique mesoderm, it has been difficult to define the steps by ability of the dorsal lip mesoderm to direct the which cell diversity is first established in this germ layer. formation of a second set of axial structures when One possibility is that local, possibly graded signals placed in a ventral location. In addition to this striking could establish a series of positional states along the regional difference in inductive ability, the mesoderm dorsal-ventral axis. Individual states could then specify becomes subdivided with respect to the types of the various types of histological differentiation, such as phenotypic differentiation that are specified at different blood, mesothelium, muscle or notochord. Alternately, positions along the dorsoventral axis (Slack and the mesoderm might be subdivided in a progressive, Forman, 1980; Dale and Slack, 1987). Such differences hierarchical manner starting perhaps with fundamen- can be detected by removing portions of the prospective tally distinct dorsal and ventral states. Cells within the mesoderm at very early stages, before any sign of dorsal and ventral regions could then interact to 418 T. S. Charlebois, J. J. Henry and R. M. Grainger generate various intermediate grades of mesoderm. dehyde gel, transferred to nylon membrane, and UV- This latter idea is supported by data indicating that crosslinked at 1200 ^Wcm~2 for 2 min. Equivalency of gel loading was checked by ethidium bromide staining of rRNA interactions within the mesoderm can influence the final 6 1 1 pheriotypic outcome of cells in this germ layer (Dale bands. Filters were hybridized to 4.5xl0 ctsmin~ ml~ of ^P-labelled antisense RNA transcribed from pCKsel (see and Slack, 1987). However, there is considerable Charlebois et al. 1990), in 50% formamide, 0.05% ficoll, overlap in the types of differentiation obtained in 0.05% polyvinylpyrollidone, 0.1% sodium dodecyl sulfate induction and explant studies, and no direct means of (SDS), 5xSSC (lxSSC is 0.15M NaCl, 0.015M sodium detecting regional specialization has been available for citrate), 100/igml"1 salmon sperm DNA at 62°C. Blots were the period before the activation of cell type-specific washed in 2xSSC, 5% SDS, at room temperature, followed genes. Consequently, it has not been possible to by O.lxSSC, 1 % SDS at 67 °C. Exposures were carried out establish the existence or location of a boundary using X-Omat AR film at —70°C with intensifying screens. delineating fundamentally distinct regions of dorsal and Autoradiograms were scanned using a Biolmage Visage 2000 ventral mesoderm. computerized image analysis system. Here we present evidence for an early and sharply In situ hybridization defined distinction between presumptive dorsal and ventral regions of chick mesoderm. Previously, we Embryos were fixed in 4% paraformaldehyde, dehydrated and embedded in Paraplast. 8fim sections were prepared, isolated a chick cDNA clone (CKsel) on the basis of dried on slides, paraffin was removed and sections were differential hybridization within the ectoderm of the treated with proteinase K and acetic anhydride prior to early embryo (Charlebois et al. 1990). This cDNA clone hybridization. [35S]RNA (antisense) probes were prepared by encodes a putative type II cytokeratin which exhibits transcription of pCKsel using T7 RNA polymerase, and approximately 75 % identity with a number of keratins subjected to partial hydrolysis with 0.2 M NaHCO3/Na2CO3 characteristic of embryonic and simple epithelia, and is (pHIO). Hybridization was carried out for 16 h at 61 °C in most closely related to cytokeratin number 7 of the 50% formamide, 0.3 M NaCl, 20mM Tris-HCl (pH8.0), 5mM human catalog (Moll et al. 1982; Glass et al. 1985). EDTA, 0.02% bovine serum albumin, 0.02% ficoll, 0.02% polyvinylpyrollidone, 10% dextran sulfate, 10 mM dithiothrei- CKsel was found to be expressed at high levels in body 35 ectoderm beginning at neurulation, but transcript levels tol to which [ S]RNA was added to a final concentration of approximately 1.3xl07ctsmin-1 ml"1. Slides were washed in were seen to be greatly reduced in ectoderm of the head 4XSSC, 10 mM dithiothreitol at room temperature and treated and in neural ectoderm (Charlebois et al. 1990). In the with RNase A (20/^gml"1) at 37°C for 30min. Slides were present work we report on our finding that this gene is then washed in 50% formamide, 2xSSC at 50°C for 45min, expressed differentially in the mesoderm as well. The followed by O.lxSSC at 50°C for 45min. Dried slides were pattern of CKsel gene expression in this germ layer dipped in Kodak NTB-2 emulsion and exposed for approxi- appears to delineate two distinct states that are defined mately 3 days at 4°C. Control sections were hybridized under with respect to cellular position along the future identical conditions to a sense probe prepared from the 3' Pstl dorsal-ventral axis. fragment of a chick 6-crystallin cDNA clone (Bhat and Piatigorsky, 1979). No specific hybridization to this control probe was observed; only a very low and uniform level of signal could be seen throughout these sections (Charlebois, Materials and methods 1988). Embryos and tissue dissections Fertile White Leghorn chicken eggs (Truslow Farms, Chester- Results town, MD) were incubated at 38 °C, 60% relative humidity, and staged according to Hamburger and Hamilton (1951). For In the course of carrying out preliminary in situ the isolation of presumptive dorsal and ventral mesoderm, the hybridization studies to determine the regional distri- trunk region (denned as the portion of the body between the first and last somite; see Fig. 1A) of stage 11-13 embryos was bution of CKsel transcripts in early ectoderm, we placed in 2.25% trypsin (Gibco, 1:250), 0.75% pancreatin observed that in the trunk region of stage 10 embryos (Difco) in phosphate-buffered saline (PBS) on ice for (stages are according to Hamburger and Hamilton, 15-30 min, and transferred to 20% fetal bovine serum 1951, for primitive streak and later stage embryos), (Sigma; heat inactivated at 56°C) in PBS for dissection. CKsel was also expressed in the mesoderm and Endoderm and ectoderm were removed and discarded, and endoderm (Charlebois et al. 1990). However, CKsel mesoderm was separated into presumptive dorsal and ventral transcripts were seen only in the more lateral regions of components by making a longitudinal cut at the ventrolateral mesoderm and did not appear to be detectable in the margin of the somites (see Fig. IB). Tissue fragments were mesoderm immediately flanking the midline. The three rinsed in PBS, and frozen in a dry ice/ethanol bath pending primary germ layers at this stage are still largely RNA isolation, which was performed as described elsewhere (Charlebois et al. 1990). Embryos used for in situ hybridiz- discrete, laying flat on the large yolk mass in the order ation were trimmed at the outer margin of the area pellucida (from the yolk) endoderm, mesoderm, ectoderm (see (stages 4-6) or within 3—4 somite widths of the midline (stage Fig.