Formation of the zebrafish midbrain hindbrain boundary constriction requires laminin-dependent basal constriction

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MECHANISMS OF DEVELOPMENT 125 (2008) available at journal homepage: Formation of the zebrafish midbrain hindbrain boundary constriction requires
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MECHANISMS OF DEVELOPMENT 125 (2008) available at journal homepage: Formation of the zebrafish midbrain hindbrain boundary constriction requires laminin-dependent basal constriction Jennifer H. Gutzman a,1, Ellie G. Graeden a,b,1, Laura Anne Lowery a,b, Heidi S. Holley a,b,2, Hazel Sive a,b, * a Whitehead Institute for Biochemical Research, Nine Cambridge Center, Cambridge, MA 02142, USA b Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA ARTICLE INFO ABSTRACT Article history: Received 20 May 2008 Received in revised form 27 June 2008 Accepted 9 July 2008 Available online 18 July 2008 Keywords: Basal constriction Apical expansion Zebrafish morphogenesis Laminin Brain ventricle Midbrain hindbrain boundary Midbrain hindbrain boundary constriction (MHBC) Cell shape The midbrain hindbrain boundary (MHB) is a highly conserved fold in the vertebrate embryonic brain. We have termed the deepest point of this fold the MHB constriction (MHBC) and have begun to define the mechanisms by which it develops. In the zebrafish, the MHBC is formed soon after neural tube closure, concomitant with inflation of the brain ventricles. The MHBC is unusual, as it forms by bending the basal side of the neuroepithelium. At single cell resolution, we show that zebrafish MHBC formation involves two steps. The first is a shortening of MHB cells to approximately 75% of the length of surrounding cells. The second is basal constriction, and apical expansion, of a small group of cells that contribute to the MHBC. In the absence of inflated brain ventricles, basal constriction still occurs, indicating that the MHBC is not formed as a passive consequence of ventricle inflation. In laminin mutants, basal constriction does not occur, indicating an active role for the basement membrane in this process. Apical expansion also fails to occur in laminin mutants, suggesting that apical expansion may be dependent on basal constriction. This study demonstrates laminin-dependent basal constriction as a previously undescribed molecular mechanism for brain morphogenesis. Ó 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction During development of the vertebrate brain, the neural tube assumes a complex structure that includes formation of the brain ventricles and the appearance of conserved folds and bends. These folds and bends delineate functional units of the brain and are likely to shape the brain such that it can pack into the skull. The midbrain hindbrain boundary (MHB) is the site of one of the earliest bends in the developing brain. In the embryo, the MHB functions as an embryonic organizing center (Brand et al., 1996; Joyner, 1996; Puelles and Martinez-de-la-Torre, 1987; Sato et al., 2004) and later becomes the cerebellum and part of the tectum (Louvi et al., 2003). We have called the deepest point in the MHB the midbrain hindbrain boundary constriction (MHBC). In the present study, we ask what processes are necessary for MHBC morphogenesis, using the zebrafish as a model. In the * Corresponding author. Address: Whitehead Institute for Biochemical Research, Nine Cambridge Center, Cambridge, MA 02142, USA. Tel.: ; fax: address: (H. Sive). 1 These authors contributed equally to this work. 2 Present address: University of Wisconsin, Madison, WI 53706, USA /$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi: /j.mod MECHANISMS OF DEVELOPMENT 125 (2008) zebrafish, the MHBC forms between 17 and 24 hours post fertilization (hpf), concomitant with formation of the brain ventricles. At this stage of development, the neuroepithelium is a pseudostratified-columnar epithelium where apical cell surfaces face the brain ventricle lumen, and basal cell surfaces, on the outside of the tube, abut the basement membrane. Fig. 1 Zebrafish MHB morphogenesis occurs between 17 and 24 hpf, and requires laminin but not ventricle inflation. (A C) Brightfield and fluorescent images and schematics of wild type (WT) MHBC formation. (D) WT embryo at 24 hpf was stained with Laminin 1 antibody (green); nuclei were stained with propidium iodide (red). Laminin lines the basal surface of the neuroepithelium. (E) Brightfield image of snk, a ventricle inflation mutant, at 24 hpf. (F) snk embryo at 24 hpf stained as in (D). (G) Brightfield image and schematic of nok, a ventricle inflation mutant, at 24 hpf. (H) nok embryo at 24 hpf stained as in as (D). (I K) Brightfield and fluorescent images and schematics of MHBC formation in the laminin mutant, sly. (L) gup embryo at 24 hpf stained as in (D). Arrowheads indicate MHB at 21 hpf and MHBC at 24 hpf. F, forebrain; M, midbrain; H, hindbrain. Scale bars: A C, E, G, I K = 100 lm, D, F, H, L = 6 lm. 976 MECHANISMS OF DEVELOPMENT 125 (2008) Thus, interestingly, the MHBC forms by bending the basal side of the neuroepithelium. This is unusual, since essentially all epithelial bends have been described at the apical surface, via apical constriction. A single report mentions that Drosophila salivary gland morphogenesis may involve bending of the basal side of the epithelium (Fristrom, 1988). The organization of the neuroepithelium, and correlation with brain ventricle inflation led us to consider three factors that may drive MHBC morphogenesis: (1) fluid pressure on the inside of the neural tube as the brain ventricles inflate (Lowery MECHANISMS OF DEVELOPMENT 125 (2008) and Sive, 2005), (2) changes in cell shape during bending, and (3) interactions of the basal side of the neuroepithelium with the basement membrane. We show here that MHBC morphogenesis involves two processes, cell shortening at the MHB and basal constriction of the neuroepithelial cells at the MHBC. Basal constriction is dependent upon laminin function, but not upon inflation of the brain ventricles. These data indicate that the MHBC forms through changes in cell shape, dependent on the extracellular matrix, which have not previously been described during brain morphogenesis. 2. Results and discussion 2.1. Zebrafish MHBC morphogenesis occurs soon after neural tube closure In the zebrafish, brain morphogenesis begins after neural tube closure at 17 hpf (Kimmel et al., 1995; Lowery and Sive, 2005). At this stage, a slight indentation is visible at the MHB anlage on the basal side of the neuroepithelium (Fig. 1A). Beginning at 18 hpf, the opposing apical sides of the neuroepithelium separate along the midline and inflate to form the fore-, mid-, and hindbrain ventricles (Lowery and Sive, 2005). However, cells at the MHB remain closely apposed at the midline. At 21 hpf, after the midbrain and hindbrain ventricles have opened further, the indentation at the MHB outside the tube is more prominent, but still shallow (Fig. 1B). By 24 hpf, the MHB is bent acutely at the basal surface creating a sharp point on the outside of the tube (Fig. 1C). This is clearly visible in all wild type embryos by staining the outside of the neural tube with a laminin antibody (Fig. 1D). We have called this sharp point, at the deepest point of this fold, the midbrain hindbrain boundary constriction (MHBC). This constriction is highly conserved amongst the vertebrates (Rhinn and Brand, 2001) A sharp MHBC forms in ventricle inflation mutants In order to determine the mechanisms regulating MHBC morphogenesis, we asked whether brain ventricle inflation plays a role in this process. We hypothesized that pressure from the embryonic cerebrospinal fluid (ecsf) within the brain ventricles is required to form the MHBC, through a passive pushing mechanism (Lowery and Sive, 2005). Supporting this hypothesis, blood flow modifies heart chamber morphology and stimulates valve morphogenesis (Berdougo et al., 2003; Hove et al., 2003; Seidman and Seidman, 2001). We analyzed MHBC morphogenesis in two zebrafish mutants lacking inflated brain ventricles, snakehead (snk), with a mutation in atp1a1 encoding a Na + K + ATPase (Lowery and Sive, 2005) and nagie oko (nok), a mutant allele of the MAGUK scaffolding protein, Mpp5 (Wei and Malicki, 2002). snk and nok embryos were imaged at 24 hpf to examine the overall outline of the neural tube and shape of the MHBC. The abnormal refractility in snk embryos prevented visualization of the MHBC by brightfield microscopy (Fig. 1E). However, laminin staining of all snk embryos analyzed revealed that the MHBC does define a sharp point, although the angle at the MHB is less acute than that of wild type embryos (Fig. 1F). In nok mutants laminin, staining indicated that the MHBC also defined a sharp point in all embryos observed (Fig. 1G and H). The acuteness of the MHBC in both snk and nok is clearly reduced compared to wild type embryos. Thus, ventricle inflation may be required to push together the neuroepithelium to form an extremely acute angle, but it is not required to form a sharp point at the MHBC Laminin is required for MHBC formation We also hypothesized that the basement membrane, which lines the MHBC on the outside of the brain primordium, may play a role in its formation. Laminin is a major component of the basement membrane, that interacts with integrins to mediate adhesion to the cytoskeleton of overlying cells (Miner and Yurchenco, 2004). A role for laminin has been demonstrated during mouse salivary gland branching, axon pathfinding in multiple organisms, and zebrafish notochord development (Bernfield et al., 1984; Garcia-Alonso et al., 1996; Karlstrom et al., 1996; Parsons et al., 2002; Paulus and Halloran, 2006). Laminin has not previously been implicated in brain morphogenesis in any system, although it has been shown to play a role in b Fig. 2 MHBC formation requires cell shortening and basal constriction. (A C 0 ) Live laser-scanning confocal imaging of wild type embryos injected with memgfp mrna at the one cell stage and imaged at 17, 21, and 24 hpf. Boxed regions from (A) to (C) are enlarged for (A 0 C 0 ). Individual cells in the MHB are outlined, and a dividing cell is indicated by an arrow in (B). Asterisks in (A C) mark cells outlined in (A 0 C 0 ). Cells with two asterisks are outside the MHB. (A 0 ) At 17 hpf, cells at the MHB are similar in length to the cells in the surrounding tissue. (B 0 ) At 21 hpf, cells at the MHB are shorter than the cells in the surrounding tissue. (C 0 ) At 24 hpf, cells at the MHBC are constricted basally and expanded apically. (A C 0 ) Some green fluorescence is visible within outlined cells since the plane of section contains the surface of the cell membrane. (D I) Timecourse of MHB morphogenesis beginning at 17 hpf. A single cell is outlined and followed through the time course. Cells at the MHB shorten relative to those surrounding (n = 6 embryos). (J) Relative cell lengths at and outside the MHB in 21 hpf wild type embryos. Cells at the MHB were 0.76 times the length of those outside the MHB (+/ 0.06 s.d.) (n = 8 embryos, 3 cells at the MHB and 4 cells outside the MHB were measured per embryo). (K) Relative apical width of unwedged cells (those outside the MHBC) and basally constricted cells (at the MHBC) in wild type embryos at 24 hpf. Cells at the MHBC had 1.6 times the apical width of those outside the MHBC (+/ 0.29 s.d.) (n = 6 embryos, 2 cells at the MHBC and 3 cells outside the MHBC were measured per embryo). (L) Numbers of basally constricted cells at the MHBC in wild type embryos at 24 hpf (n = 9 embryos). Arrowheads indicate the MHBC. M, midbrain. Scale bars: A C = 20 lm, A 0 C 0 =9lM, D I = 30 lm. 978 MECHANISMS OF DEVELOPMENT 125 (2008) development of the eye, which is derived from neuroepithelium (Svoboda and O Shea, 1987). We tested the requirement for laminin by examining the MHBC in the sleepy mutant (sly m86 ) that has a mutation in the gamma1 laminin gene (lamc1) (Parsons et al., 2002) and in the grumpy mutant (gup hi1113b ), which has a viral insertion in the first intron of the laminin beta1 gene (lamb1), (Amsterdam et al., 2004 and A. Amsterdam, personal communication). By brightfield imaging, sly mutants showed an initially normally shaped neural tube (Fig. 1I and J), MECHANISMS OF DEVELOPMENT 125 (2008) but by 24 hpf, the MHBC remained a shallow indentation (Fig. 1K). Similar results were observed with gup mutants (data not shown). Consistent with brightfield imaging, at 24 hpf, a shallow MHBC was observed in gup mutant embryos stained with the Laminin 1 antibody (Fig. 1L). This angle was consistently shallow in all embryos, observed either by brightfield imaging or by laminin staining. We used the gup hi1113b viral insertion mutants for Laminin 1 antibody staining, because the Laminin 1 antibody is not immunoreactive in the allele of sly used in this study (sly m86 ), nor in the other gup allele previously described (gup m186 )(Parsons et al., 2002). Although the mechanism by which this antibody recognizes Laminin 1 in gup hi1113 is not known, the viral insertion may result in a recognizable, but non-functional protein, whereas point mutation alleles of sly m86 and gup m186 result in the introduction of a premature stop codon and likely severely truncated proteins (Parsons et al., 2002). These data show that laminin function is essential for the sharp point normally seen at the MHBC and define a new role for laminin in brain morphogenesis Cells shorten and basally constrict at the MHBC Bends or folds in epithelial sheets are driven by changes in cell length and formation of wedge-shaped cells, such as the cell shortening and apical constriction during neurulation in Xenopus, optic vesicle formation in mice, and ventral furrow invagination in Drosophila (Lee et al., 2007; Smith et al., 1994; Svoboda and O Shea, 1987; Sweeton et al., 1991). We, therefore, hypothesized that wedge-shaped cells would be required to form the MHBC. However, based on the orientation of the MHBC, we hypothesized that such wedge-shaped cells would be basally, rather than apically, constricted. In order to test this hypothesis, we analyzed cell shape at the MHBC in wild type embryos by expressing membrane-localized green fluorescent protein (memgfp) and imaging live embryos by laser-scanning confocal microscopy. At 17 hpf, cells in the midbrain, hindbrain, and MHB are uniform in length and are both spindle and columnarshaped, with some rounded dividing cells visible (Fig. 2A and A 0 ). In contrast, by 21 hpf, MHB cells are shorter in length (0.76 the apical-basal length) than those in either the midbrain or hindbrain (Fig. 2B, B 0 and J). Do these MHB cells shorten relative to surrounding cells, or do they fail to lengthen in concert with the rest of the neuroepithelium? By imaging wild type embryos, using spinning-disk confocal microscopy, to generate a live time-lapse data series, between 17 and 21 hpf, we showed that single cells at the MHB shorten relative to surrounding cells (Fig. 2D I). While the shortening event appears to be graded along the MHB, the uneven nature of the pseudostratified neuroepithelium makes quantification of subtle changes in cell length in regions flanking the future MHBC difficult to measure. In conclusion, a first step in MHBC formation is the shortening of cells at the MHB. Subsequent to cell shortening, we found that, by 24 hpf, a group of cells at the MHBC had become wedge-shaped, with constriction at their basal surface (Fig. 2C and C 0 ). Within a single plane (Z-section) three to four wedge-shaped cells meet at a sharp point to form the MHBC in wild type (Fig. 2C 0 and L). Basally constricted cells were defined as those with a clear wedge-shaped morphology such that their basal surface was constricted to a point. We found that the apical width of the wedge-shaped cells at the MHBC had expanded to 1.6 times that of cells outside the MHBC (outlined cells in Fig. 2C 0 and K). Interestingly, although the midline in the MHB does not separate, we found that the basally constricted MHBC cells were not apposed at the midline, but instead were oriented with their apical surfaces exposed to the midbrain ventricle lumen (Fig. 2C and C 0 ). These data demonstrate that cells at the MHBC undergo basal constriction and apical expansion Basal constriction at the MHBC occurs without ventricle inflation, but requires laminin Since the MHBC forms a sharp point in the ventricle inflation mutants snk and nok, we asked whether basally con- b Fig. 3 Basal constriction at the MHBC is laminin-dependent and not dependent on ventricle inflation. (A C 0 ) Live laserconfocal imaging of wild type, snk and nok embryos at 24 hpf after injection with memgfp. Boxed regions in (A C) are enlarged in (A 0 C 0 ). Cells at the MHBC in (A A 0 ) wild type, (B B 0 ) snk and (C C 0 ) nok undergo basal constriction (see cell outlines). (D F 0 ) Imaging of sly mutants injected with memgfp mrna at the one cell stage and imaged at 17, 21, and 24 hpf. Boxed regions in (D F) are enlarged for (D 0 F 0 ). (D 0 ) At 17 hpf, MHB and surrounding cells are similar in length (see outlined cells). (E 0 ) At 21 hpf, cells at the MHB are shorter than those surrounding. One cell at and one cell outside the MHB are outlined in yellow. Some cells are visible outside the neural tube. (F 0 ) At 24 hpf, cells at the MHBC fail to basally constrict. For (A C) and (D F) asterisks indicate the cell that is outlined in the image below. Cells with two asterisks are outside MHB. Arrowheads indicate the MHBC. Dotted lines delineate the outside of the neural tube. Some green fluorescence is visible within outlined cells since the plane of section contains the surface of the cell membrane. Anterior is to the left in all images. Scale bars: A C=22lM, A 0 C 0 =12lM, D F = 18 lm, D 0 F 0 =9lM. (G) Length of cells at the MHB relative to those outside the MHB in WT and sly mutants. At 21 hpf, cells at the MHB (at MHB) in sly mutants are 0.76 (+/ s.d.) the length of those outside the MHB (outside MHB), as in WT embryos (WT data is the same as from Fig. 2)(n = 6 embryos, 2 cells at MHB, 3 cells outside MHB were measured per embryo). (H) Graph compares the apical width of cells outside MHBC to cells at MHBC in WT, sly, snk, and nok embryos at 24 hpf. Basally constricted cells at the MHBC do not apparently show corresponding apical expansion snk and nok (n = 3 embryos each mutant, 2 cells at MHBC, 3 cells outside MHBC were measured per embryo). Cells at the MHBC in sly mutants that have shortened, but are not constricted basally, are also not expanded apically (n = 6 embryos, 3 cells at MHBC, 4 cells outside MHBC were measured per embryo). 980 MECHANISMS OF DEVELOPMENT 125 (2008) stricted cells formed in these mutants. Confocal imaging indicated that cells at the MHBC in both mutants demonstrated basal constriction (Fig. 3A C 0 ). However, unlike wild type, the basally constricted cells in these mutants did not show apical expansion relative to adjacent cells in the same embryo (Fig. 3H). This may be because apical expansion requires that cells have an unconstrained apical surface, which occurs when wild type MHBC cells abut the midbrain lumen. Where the ventricles do not inflate and the midline of the brain primordium does not separate, the mutant cells may be constrained in their ability to expand apically. Therefore, the reduced bend angle formed at the MHBC in nok and snk may be due to failure of the cells at the MHBC to expand apically, in response to ventricle inflation. These data show that the basal constriction in the MHBC can occur independent of brain ventricle inflation, and moreover, that basal constriction is independent of apical expansion. In order to determine what aspect of MHBC formation is disrupted in laminin mutants, we analyzed sly embryos for changes in cell length and shape (Fig. 3D F 0 ). At 17 hpf, the cells at the MHB of sly mutants appeared similar to wild type (compare Fig. 2A 0 with Fig. 3D 0 ). By 21 hpf, cells at the MHBC in sly mutants were 0.76 the length of cells on either side, similar to wild type (Fig. 3E, E 0 and G). However, by 24 hpf, cells at the MHBC in sly mutants ha
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