Tissue morphogenesis and vascular stability require the Frem2 , product of the mouse myelencephalic blebs

John R. Timmer*, Tracy W. Mak, Katia Manova, Kathryn V. Anderson, and Lee Niswander†‡

Developmental Biology Program, Memorial Sloan–Kettering Cancer Center, New York, NY 10021

Contributed by Kathryn V. Anderson, June 29, 2005 Adhesive properties of cells undergoing morphogenetic rearrange- carry a mutation in the related Frem1 gene, which has also been ments can be regulated either at the cellular level or by altering the proposed to be an ECM component (8). Mutations in two other environment in which rearrangements occur. Here, we describe the related , Frem2 and Frem3, have not been reported. The eb identification of a mutation (myF11) in the mouse extracellular mutation is a deletion in the multiPDZ protein GRIP1, which matrix component Frem2, and provide evidence that suggests interacts with Fras1 and Frem2 and promotes their Frem2 expression creates an environment conducive to morpho- transport to the basal cell surface of epithelial cells (9). genetic events. Loss of Frem2 function results in defects in devel- The Fras1 and Frem1 proteins have been proposed to act as opmental events associated with morphogenetic rearrangements general mediators of epithelial adhesion during development. of the vasculature and of tissues arising from all germ layers. The Fras1 is uniformly expressed in all epithelial tissues (4, 7), Frem2 transcript is restricted both spatially and temporally whereas Frem1 is expressed in a more restricted pattern in the and appears in advance of cell rearrangement events. Thus, ex- underlying dermis (8). Loss of either gene is sufficient to cause pression of Frem2 may dynamically alter the to provide a substrate for cell migration and rearrangements breaks in the ECM underlying epithelial cells, resulting in a during embryogenesis. blistering of the embryonic ectoderm analogous to the adult skin of dystrophic epidermolysis bullosa patients (4, 7, 8). Given the extracellular matrix ͉ hemorrhage ͉ Frem ͉ CALX␤ ͉ neural tube defect widespread expression of Fras1 and Frem1, it is unclear why relatively few epithelial structures are affected and what controls the onset of defects. The only other data relevant to the orphogenetic events, which include both coordinated mi- ͞ Mgrations and cellular rearrangements, are essential to the Fras Frem family comes from studies of the sea urchin Frem2 development of multicellular organisms. The coordinated orthologue, ECM3. During gastrulation, the ECM3 protein is changes in cell shape and position necessary for such events localized in a fibrous mesh; primary mesenchyme cells appear to suggest an equivalent coordination of changes in gene expres- associate with and reorder this structure during their dorsal sion, directional cues, cytoskeletal structure, and cell adhesion. migration (10). Thus, ECM3 appears to act as a substrate for cell Typically, cell adhesion changes are viewed in terms of changes migration. in the expression of cell-surface adhesion molecules in the Here, we describe the spectrum of morphological defects migrating cells against the backdrop of a static environment. caused by a previously uncharacterized mutation in the my gene Here, we describe a previously uncharacterized allele of the and demonstrate that the mutation results in a premature stop classical mouse mutant myelencephalic blebs (my) and show that codon in the transcript which produces the Frem2 protein. In it is caused by a mutation in the Frem2 gene, a proposed contrast to the other characterized family members, the Frem2 extracellular matrix (ECM) component. We show that my mu- transcript is expressed in a spatially and temporally dynamic tants develop severe defects in tissues as they undergo morpho- fashion in tissues derived from all three germ layers. It is genetic changes and that Frem2 is expressed in a dynamic fashion expressed in those tissues that show my phenotypes, appearing in in these tissues, appearing in advance of any overt phenotypes. advance of any overt morphological defects. Based on the These results suggest Frem2 functions in the extracellular space expression pattern and phenotypic analysis, we propose that to enable morphogenetic rearrangements. Frem2 functions like its orthologue, ECM3, to provide a sub- The classic mouse mutant my was first described in 1923 and strate for morphogenetic tissue rearrangements during embry- named for the hemorrhagic blisters, or blebs, that develop during ogenesis rather than functioning in general adhesion. This model embryogenesis. Depending on genetic background, this muta- accounts for the specificity and timing of the defects seen in my tion causes additional phenotypes, including exencephaly, agen- esis or defective development of the kidneys, and eye defects mice. (1–3). Neither the mechanism(s) underlying these varied defects nor the affected gene have been identified. Three additional Materials and Methods unlinked mouse mutations, blebs (bl), eye blebs (eb), and head Details of the genetic screen and complete protocols for geno- blebs (heb), cause a similar spectrum of defects, suggesting that typing, immunofluorescence, and in situ analysis are available at a common developmental pathway or process exists that de- http:͞͞mouse.ski.mskcc.org. pends on the function of these genes (4–7). The recent identification of the genes mutated in bl, eb, and heb mice suggests that the bleb phenotypes are due to defects in Freely available online through the PNAS open access option. the ECM, resulting from the loss of molecules containing repeats Abbreviations: E, embryonic day; ECM, extracellular matrix. of the chondroitin sulfate proteoglycan core domain (8). bl *Present address: Cell and Developmental Biology Department, The Weill Medical College results from a mutation in the Fras1 gene, named for Fraser of Cornell University, New York, NY 10021. Syndrome, the genetic disease caused by mutations in the human †Present address: Howard Hughes Medical Institute, Department of Pediatrics, University of orthologue of Fras1. Human and mouse Fras1 encode a large Colorado Health Sciences Center, Aurora, CO 80045-0511. protein proposed to be either an ECM component (4, 7) or a ‡To whom correspondence should be addressed. E-mail: [email protected]. cell-surface protein that interacts with the ECM (9). heb mice © 2005 by The National Academy of Sciences of the USA

11746–11750 ͉ PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505404102 Downloaded by guest on September 26, 2021 Generation and Mapping of Mutations. Mutations were generated by using N-ethyl-N-nitrosourea and isolated in a mixed C57BL͞ 6-C3H background as described in ref. 11. Phenotypic analysis was performed either on adults identified by phenotype or by using timed matings and genotyping of embryonic tissues. Recombination-mapping used simple-sequence length polymor- phism markers to test for association between phenotypes and regions of the C57BL͞6 genome. The myF11 mutation was found to reside between the markers D3Mit184 and D3Mit275. Further refinement of this position used markers from the SKI series (see http:͞͞mouse.ski.mskcc.org͞marker͞SKI͞intro.php).

Reconstruction of the my Transcript. The ECM3 homologue Frem2 was identified as a predicted transcript in the . Probable transcript structure was determined by using human͞ mouse homology and partial mouse EST sequences. Primers directed to these exons were used with RT-PCR to determine the actual intron͞exon structure. Two base differences between this sequence and the National Center for Biotechnology (NCBI) m30 genome assembly were confirmed by sequencing genomic Fig. 1. Blistering and hemorrhages in homozygous my embryos and mice. (A) DNA from both wild-type and mutant embryos. Exencephaly (denoted by arrowheads) in E13.5 my mutant. (B and C) Earliest visible hemorrhages in the choroid plexuses. Hemorrhage in the rostral (B) and Histological Analysis. In situ analysis and immunofluorescence caudal (C) choroid plexus of two representative E13.5 embryos. (Insets) Loca- were performed on cryosections with embryos fixed in 4% tions of the defects). (D) Blood-filled blisters on the surface of an E13.5 paraformaldehyde (PFA) at 4°C for 1 h. In situ analysis was embryo, with an internal hemorrhage (arrowhead) in the caudal choroid performed by using published techniques (12). DIG labeled plexus. (E) Hemorrhage on the E15.5 palate. (Inset) Lower magnification view probes were generated from three regions of the my transcript, of head. (F) Two hemorrhages on the head of an E15.5 embryo; the outer and identical results were obtained with each. Hematoxylin͞ hemorrhage (arrowhead) appears to be nearly resolved, whereas the inner, eosin staining and immunohistochemistry were performed on subdermal one (arrow) appears to be recently formed. (G) Hemorrhage on the head of a newborn animal. (H) Adult head, showing reduced or absent eyes paraffin sections from embryos fixed overnight in 4% PFA at (arrows), reduced ear (arrowhead), and discoloration of fur (white asterisk). (I) 4°C. Immunohistochemistry was performed by using a Discovery Adult my mutant with discolored fur on the flank (the arrowhead denotes the System from Ventana Medical Systems (Tucson, AZ). Antibod- border of discoloration). ies were from Sigma, integrin-␣V from BD Biosciences, and PECAM and FLK-1 from Santa Cruz Biotechnology.

to altered limb patterning or growth (data not shown). The BIOLOGY

Results penetrance of these defects was variable, and bilaterally sym- DEVELOPMENTAL Phenotypic Analysis of myF11. The myF11 mutation was identified in metric tissues were often differentially affected. an ongoing screen for N-ethyl-N-nitrosourea-generated muta- Other defects, such as exencephaly, did not clearly associate tions, which cause recessive developmental defects in mouse with developmental hemorrhages. Small regions of fur fre- embryos. myF11 mutants on a mixed C57Bl6͞C3H background quently lacked pigmentation, suggesting a defect in neural crest fail to close the neural tube in the midbrain (Fig. 1A) and die at differentiation or migration (Fig. 2F). Internal organs were also birth. The exencephalic phenotype was largely suppressed on a affected. Mutants frequently had malformed septa and valves of mixed C3H͞castaneus background, and a small percentage of the heart, including complete absence of the septa (Fig. 2G). The homozygotes survived to adulthood. Many of the defects dis- lungs often displayed aberrant septation (Fig. 2H), although played by surviving animals were associated with embryonic sections showed that the internal structure, formed through hemorrhages. Hemorrhages first appear at embryonic day branching morphogenesis, was largely normal (Fig. 2I). Many (E)12.5 in the choroid plexuses (Fig. 1 B and C). At E13, blisters neonates were significantly reduced in weight compared with and hemorrhages form on the surface of the head, often over the their wild-type littermates [average 81%, with some mutants eye (Fig. 1D). Hemorrhages became apparent in the palate at weighing as little as 25% (data not shown)] and died soon after E15.5 (Fig. 1E). Further hemorrhages could occur in the head birth. at any stage, and, in some samples, multiple hemorrhages were detected (Fig. 1F). Most hemorrhages resolved, but some in Identification of the Gene Mutated in my Mice. We mapped myF11 by these regions persisted to birth (Fig. 1G). recombination to a 1.2-Mb region of mouse 3, These hemorrhages appear to cause localized tissue necrosis which includes the classical mutation myelencephalic blebs (13). that leads to structural defects in adults. The eyes and͞or ears of The original my mutation causes developmental phenotypes mutants were often reduced to a rudiment or were entirely similar to those seen in myF11, including embryonic blisters and absent (Fig. 1H). Fur on the surface of the head and trunk was hemorrhages, exencephaly, craniofacial defects, degeneration of occasionally sparse or discolored (Fig. 1 H and I), and the the eyes, limb malformations, and renal agenesis (1–3, 6). Based morphology of the head suggested a malformation of the un- on the similarity of the phenotypes and map position, we derlying skeletal elements. Developmental hemorrhages also conclude that F11 is a new allele of my. affected the limbs, primarily the hind limb. In the distal limb, Genome sequence databases indicate that 6–11 transcripts are blistering and hemorrhage was apparent at E14 and ranged in present in the 1.2-Mb region containing myF11, including the severity from a slight loss of adhesion to a complete blistering Frem2 gene. We used a combination of RT-PCR, ESTs, and and hemorrhage (Fig. 2 A and B). Adult limbs displayed hem- computer analysis to assemble the mouse transcript (see Fig. 5, orrhages and malformations, ranging from soft tissue syndactyly which is published as supporting information on the PNAS web and alterations in digit flexure to the loss of digits and truncation site). myF11 mice have a C-to-A substitution that generates a stop of the limb (Fig. 2 C–E). Normal skeletal patterning suggests that codon and produces a truncated protein of 1,880 amino acids of malformations are attributable to the hemorrhages rather than the normal 3,100 and lacks four of the five CALX␤ motifs (see

Timmer et al. PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11747 Downloaded by guest on September 26, 2021 Fig. 2. Limb and organ defects in my embryos and mice. (A–E) Limb Defects. Shown are a fluid-filled blister on an E14.5 hind limb (A); a blood-filled blister Fig. 3. my͞Frem2 shows dynamic spatial and temporal gene expression. (A) encompassing distal structures of an E14.5 hind limb (B); a hemorrhage Frem2 RNA expression at E9.5, including in the roofplate (RP) of the dien- associated with malformation and dorsal flexure of the newborn hind limb cephalon, the midbrain͞hindbrain boundary (MB͞HB), and the branchial arch (C); a malformed adult limb, showing reversal of flexure and soft tissue ectoderm (BAE). (B) Frem2 expression at E10.5, including the nephric duct syndactyly (D); and a severely malformed adult limb (E). (F) Adult with unpig- (ND), precursor to the kidney, the ventral spinal cord (SC), and notochord (NC). mented fur (arrow), typical of neural crest defects. (G) Section of an E15.5 (C) Dynamic expression of Frem2 transcript in the eye, including the lens vesicle heart, showing failure of atrial septation (arrow) and a defect in ventricular (LV) and border of the retinal pigmented epithelium (RPE) at E10.5, the optic valves (arrowhead). Note that both ventricles drain into a single large cham- nerve (ON) at E11.5, and the conjunctival epithelium (CE) at E13.5. (D–G) ber. (H) Newborn lungs, showing failure of proper lung septation. The arrows Expression at E11.5. Shown are expression in the head in the rostral dienceph- point to lobe fusion. (I) Section of newborn my lung, showing normal mor- alon (D) and future choroid plexuses (CP) of the telencephalon, the forming phology. The arrows denote regions where the lung lobes have fused, and the nasal epithelium (NE), and future palate (P) (D); expression in the trunk in the arrowhead denotes a region with a limited adhesion between the two lobes. myotome (M), esophagus (E), the site where the heart is attached to the trunk (H), and bronchi in advance of the onset of branching morphogenesis that will generate the lung (L) (the arrows denote regions of expression in the limb Fig. 6, which is published as supporting information on the PNAS ectoderm and mesenchyme; note that expression in the ventral spinal cord has web site). Although it is possible that this transcript could been extinguished (E); in more caudal sections, expression is present in the produce a truncated protein that is folded and processed prop- developing kidneys (F); and expression is also present in other endodermal erly, the loss of several conserved regions suggests that any such derivatives, such as the stomach (G). product would have limited function. We therefore conclude that the myF11 mutation severely impairs or abrogates the function of Frem2. In addition to the features previously discussed for a variety of endodermal derivatives, such as the stomach, esoph- Frem2 (8), we noted a furin protease consensus cleavage site agus, and gut (Fig. 3 E and G). (RRXKR) 26 aa from the transmembrane domain. This suggests that, following GRIP1-mediated basal trafficking (9), the pro- Characterization of Hemorrhages in my Embryos. Frem2 is expressed tein may be liberated from the cell surface for inclusion in the in the head and limb several days in advance of hemorrhagic blisters, ECM. We have also noted that, between the CALX␤ domains suggesting that a specific developmental event may trigger the and the transmembrane domain, there are regions of conserva- hemorrhage. Indeed, hemorrhages correlated with the onset of tion among Fras1, Frem2, and ECM3, including 11 conserved major vascular rearrangements in the head and limb (see Fig. 8, cysteines (see Fig. 7, which is published as supporting informa- which is published as supporting information on the PNAS web tion on the PNAS web site). ECM3 contains 12 cysteines in this site). This finding suggested that either the process of angiogenesis, region). which normally results in the leakage of fluids from rearranging blood vessels, might trigger a failure of adhesion in cells lacking Dynamic Temporal and Spatial Expression of my. The Frem2 gene is FREM2 protein, or vascular stability itself may be defective in the expressed in rapidly changing temporal and spatial patterns in absence of FREM2 protein. Although Frem2 was not observed in cells derived from all germ layers (Fig. 3 A–G). my expression is the embryonic vasculature, it is expressed by many tissues that consistently observed in tissues before the appearance of devel- contact the developing vasculature. opmental defects. Frem2 is absent from neural and ectodermal To examine the developmental hemorrhages in detail, we tissues during neural plate folding, but, as the neural tube closes, focused on the formation of the choroid plexuses, heavily Frem 2 is expressed specifically at the site of future exencephaly vascularized structures that project into the ventricles of the in my mutants (Fig. 3A, the dorsal midbrain). Expression is brain and generate cerebrospinal fluid. These structures form by present at discrete times and places during development of the the elaboration of the roofplate of the telencephalon and eye (Fig. 3C), a site of recurrent hemorrhages (Fig. 1F). Ex- hindbrain at E12.5. Expression of roofplate markers such as pression is seen in each tissue that is defective in my mice, BMP7 and Msx-1͞2 was identical in wild type and mutant (Fig. including the limb ectoderm and mesenchyme, developing kid- 4A and data not shown), and morphology of the elaborating neys, palate, and lungs (Fig. 3 B and D–F), and this expression neural tissue in mutants was normal, except where distorted by is tightly regulated in time. For example, Frem2 is expressed at hemorrhage. Thus, although Frem2 is expressed by the neural E11.5 in neural cells that will give rise to the choroid plexus (Fig. component of the choroid plexuses, this tissue appears to 3D and data not shown), but expression is extinguished by E12.5, develop normally. A bed of capillaries surrounds the brain of when hemorrhages occur in the choroid plexus. Frem2 RNA is developing mice, and, as the choroid plexuses elaborate, the present throughout kidney development (Fig. 3 B and F) and in capillaries remain closely associated with the neural tissue,

11748 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505404102 Timmer et al. Downloaded by guest on September 26, 2021 Fig. 4. Vasculature defects at sites of hemorrhage in my embryos and developmental gene and protein expression at sites of choroid plexus hemorrhages in E12.5 embryos. (A) Patterning of the choroid plexus is normal in my mutants, as indicated by appropriate BMP-7 RNA expression in neural cells adjacent to a hemorrhage (H) (magnification, ϫ10). (B) Expression of PECAM in a wild-type choroid plexus shows close association of the vasculature with the elaborating neural tissue (magnification, ϫ10). PECAM expression at hemorrhages (H) in the rostral (C) and caudal (D) choroid plexus of my embryos (the arrow denotes disorganized endothelial cells in the area of hemorrhage, and the arrowhead shows a normal vascular structure at the hemorrhage edge (magnification, ϫ20). (E–G) Markers of angiogenic signaling and vascular structure are expressed normally in regions of hemorrhages in my embryos. Flk-1, a VEGF receptor, remains expressed by endothelial cells that have failed to form proper vascular structures in the area of a hemorrhage (H) (magnification, ϫ20). The arrow indicates disorganized endothelial cells in close proximity to normal blood vessels (arrowhead) (E). The expression of Integrin-␣V at the surface of neural and endothelial cells in the choroid plexus is shown in close proximity to a hemorrhage (H) (magnification, ϫ20). The arrowhead indicates a region of normal blood vessels. Cytoplasmic localization of this protein in some neural cells of the choroid plexus occurs in both mutant and wild-type samples (F). Laminin is deposited in the of both neural and vascular cells at the choroid plexus. This expression is retained in areas where hemorrhaging (H) occurs (magnification, ϫ20). The arrow indicates the region of disrupted ECM at a hemorrhage, and the arrowhead indicates nearby blood vessels that are unaffected (G).

resulting in a high concentration of small blood vessels, revealed Role of Frem Proteins in Development. Frem2 is a member of a small by the endothelial markers PECAM and FLK-1 (Fig. 4 B–E). In family of extracellular calcium-binding proteins that share a mutants, vascular cells have lost contact with the neural tissue in series of common structural motifs, including homology to the regions of hemorrhage, and the endothelial cells are disorga- NG2͞AN2 chondroitin sulfate protein and repeats of a CALX␤ nized, with no tubular structures visible (Fig. 4 C–E). In contrast, motif (8). Proteins with this combination of motifs are absent ␣ the vasculature appears normal in nearby tissues. Integrin- V, from the completed genome sequences of Caenorhabditis elegans BIOLOGY which both mediates vessel adhesion to the ECM and acts as a and Drosophila melanogaster, suggesting that these proteins are DEVELOPMENTAL receptor for pro- and antiangiogenic signaling (14–17), was unique to the deuterostome lineage. Differences among these expressed identically in wild-type and mutant embryos (Fig. 4F). proteins in structure, expression, and mutant phenotype, how- Laminin, a major component of the ECM surrounding the ever, suggest a significant specialization. Frem2 (my) mutants vasculature, appears disorganized at the site of hemorrhages have several phenotypes (choroid plexus hemorrhages and heart (Fig. 4G); yet, it is localized normally in mutant embryos without and lung defects) not described in bl or heb mutants. Frem2 RNA choroid plexus hemorrhages (data not shown), suggesting that is expressed in a highly dynamic temporal and spatial pattern in the disorganization is a result, rather than a cause, of the tissues of ectodermal, mesenchymal, and endodermal origin, and hemorrhage. its transient expression in tissues before the onset of overt defects Thus, in the choroid plexus, both vascular and neural tissue suggests a developmentally restricted requirement for this pro- types are properly specified, and the morphogenetic movements tein in the extracellular space for proper tissue and vascular of the neural tissue begin properly. Because no defect in the morphogenesis. Frem1 is also expressed in a dynamic manner but vasculature is observed before the elaboration of the neural is restricted to mesenchymal lineages, primarily the dermis (8), tissue, these data suggest that vascular failure and hemorrhaging whereas Fras1 is expressed uniformly in all epithelial lineages (4, is initiated by the onset of normal tissue morphogenesis. Similar 7). The clear homology between the sea urchin ECM3 protein vascular defects were observed in the palate, where Frem2 is and Frem2 suggest that functional differences among these expressed in the ectoderm. These data point to a defect in proteins may have arisen early in deuterostome evolution. vascular stability when the FREM2 protein is absent from The similarity in overall gene structure and phenotype, how- surrounding tissues, ultimately resulting in hemorrhages. ever, suggest that at least some functions of this protein family may overlap or that their gene products act in concert in some Discussion tissues. These phenotypes suggest that their products act to Our results describe a recently isolated mutation in the my structure the ECM. Mutations in Fras1 and Frem1 cause frac- locus and the identification of the my gene, which encodes tures within the ECM, indicating that they mediate ECM Frem2, a proposed ECM component. We show that Frem2 is cohesion, perhaps as ECM components (4, 7, 8). As such, it is dynamically expressed during development, and that expres- unlikely that they are direct mediators of cell attachment. sion precedes morphogenetic rearrangements in many of these Because there appear to be no defects in tissue specification or tissues. We found that loss of Frem2 leads to defects during differentiation in these mutants, it is unlikely that Fras1, Frem1, these rearrangements, causing severe developmental abnor- and Frem2 act as signaling molecules or influence the activity of malities, such as failure to close the neural tube, heart and lung known signaling pathways. Although the fracture of the ECM in defects, and developmental hemorrhages. Our data suggest the these mutants could clearly affect adhesion-mediated signaling, possibility that transient production of Frem2 may dynamically any such effect would be secondary to the loss of ECM cohesion. alter the ECM to provide a conducive substrate for cellular Thus, the phenotypes of these mutations suggest that the pro- rearrangement. teins act as structural components of the ECM. This function is

Timmer et al. PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11749 Downloaded by guest on September 26, 2021 consistent with the proposed activity of Frem2 as a mediator of surface and͞or within the ECM. Thus, Frem2 protein is posi- morphogenesis. Frem2 is an extracellular protein that is traf- tioned so as to allow it to mediate tissue cohesion during specific ficked to the basal cell surface (9), where it is likely to be rearrangements. Its closest known homologue, the sea urchin liberated by furin cleavage and, thus, would be expected to reside protein ECM3, appears to play precisely this role, because its in the ECM. Frem2’s apparent contributions to an ECM con- localization within the ECM is reorganized by migrating cells, ducive to tissue rearrangement and the general ECM structure which then continue to associate with the resulting ECM3- promoted by Fras1͞Frem1 may both be important at sites of containing fibers (10). major cellular rearrangements, such as the closure of the dorsal Many of the phenotypes seen in my mice support a general role neural tube. Consistent with this expectation, we have seen for Frem2 in embryonic morphogenesis. Although neural tube exencephaly in bl (Fras1) embryos on the C3H background (J.T., closure requires the coordination of a variety of cellular pro- unpublished data). The breeding of animals carrying mutations cesses (19, 20), Frem2 expression at the dorsal midline during in several of these genes should indicate whether the function(s) closure suggests that the protein may act to facilitate the cellular of these genes are discrete or overlapping. rearrangements that occur as the neural tissue and ectoderm separate from one another and fuse to form the neural tube and Vascular Defects in my Mutants. Our studies indicate that Frem2 is its overlying ectoderm. Pigmentation defects seen in my animals not expressed in the vascular endothelial cells but rather in the are likely due to neural crest abnormalities. Frem2 is not surrounding tissues, suggesting that the primary vascular defect expressed in the dorsal neural tube during neural crest formation in my mouse embryos is due to failure of endothelial-cell or exit from the neural tube, but it does appear in the myotome, adhesion to its surroundings in the absence of Frem2. Within the which is on the route of neural crest migration. choroid plexuses of mutant embryos, the ECM structure has a There is a clear morphogenetic component to the development normal appearance before hemorrhaging, and localization of of other tissues that are defective in my embryos. The dynamic several proteins that mediate both neural and vascular develop- expression of Frem2 in the eye as it undergoes several periods of ment is indistinguishable in mutant and wild-type embryos, morphogenesis and the defects in eye formation in my mutants suggesting that there is no gross disruption of this tissue. These highlight the importance of Frem2 in the generation of this organ. findings point to a role for Frem2 protein in mediating tissue– The lung forms through a process of branching morphogenesis, and tissue or tissue–ECM cohesion. the lung defects in my are very similar to those reported for the The vascular requirement for Frem2, however, must be limited, knockout of the ECM component laminin-␣5 (21), suggesting a link because most of the embryonic vasculature is stable in my mice, and between the ECM and morphological defects in this system. many tissues that express Frem2 do not suffer hemorrhages. We Although we have not seen kidney defects in myF11, they have been suggest two possible explanations for this limited phenotype. In reported for the original my allele (1, 3). Frem2 transcript is present tissues such as the limb and head, the onset of hemorrhagic from the earliest stages of kidney development. Failure of early blistering also correlates with times of major vascular rearrange- morphogenetic events may account for the complete absence of ments. The choroid plexus is also a site of ongoing angiogenesis kidneys in some genetic backgrounds (22). Formation of the heart (18). Thus, the Frem2 protein may assist in stabilizing the vascu- septae requires the recruitment of migratory neural crest cells and lature during periods of angiogenic rearrangement. Alternately, the mesenchyme from surrounding tissues and coordinated rearrange- hemorrhages occur during periods of major morphogenetic rear- ments of these cells and cells of the cardiac lineage. Because Frem2 rangements, such as outgrowth of the limb, fusion of the palate, and expression was detected in both the heart and nearby mesenchyme, elaboration of the choroid plexus. It is possible that hemorrhages defects in any one of these processes may be sufficient to generate are secondary to failures in morphogenesis. A combination of these the defects seen in my mice. Combined, these data strongly suggest two models is also possible: the Frem2 protein may be required for that the mechanism underlying the defects seen in my embryos is vascular stability only when the vasculature is under stress because a deficiency in specific morphogenetic rearrangements due to the of morphogenesis of neighboring tissues. In this sense, Frem2 may absence of the Frem2 protein. be a specific mediator of tissue cohesion during morphogenetic rearrangements, stabilizing angiogenic rearrangements within this We thank members of our lab, the staff of the Memorial Sloan–Kettering context. Cancer Center (MSKCC) Molecular Cytology facility, Aaron Daluiski, Richard Lang, David Lyden, and John Martignetti for their assistance and suggestions throughout these studies. This work was supported by Morphogenetic Defects in my Mice. The role of Frem2 as a more National Institutes of Health Grant U01 HD43478 and the MSKCC general mediator of morphogenesis is supported by expression of Cancer Center Support Grant. L.N. is an Investigator of the Howard the Frem2 transcript and localization of its product at the cell Hughes Medical Institute.

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