Yuan-Yuan Liu

Shi Dl

Yan-Jun Zhang

Chong Zhang

Si-Yao Lin

De-Li Shi
email: de-li.shi@upmc.fr

Ming Shao
email: shaoming@sdu.edu.cn

Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Since its first application to induce mutations in mammalian cells [START_REF] Cong | Multiplex genome engineering using CRISPR/Cas systems[END_REF][START_REF] Mali | RNA-guided human genome engineering via Cas9[END_REF], CRISPR/Cas9 rapidly becomes a routine technique to perform genome editing in a variety of biological systems due to its facile, robust, and multiplexable features [START_REF] Hwang | Efficient genome editing in zebrafish using a CRISPR-Cas system[END_REF][START_REF] Guo | Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis[END_REF][START_REF] Wang | One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[END_REF]. In every system, Cas9 and sgRNA should co-express to induce mutations. This is typically achieved by injection of the sgRNA mixed with Cas9 mRNA or protein into the fertilized eggs in zebrafish. As the in vivo translation of capped RNAs delay the presence of sufficient Cas9 protein, the mRNA injection produces more mosaic animals and shows relatively lower efficiency when compared to Cas9 protein injection [START_REF] Burger | Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes[END_REF]. However, it is much more cost effective than using commercially available Cas9 protein, especially in the case of large-scale mutation screens.

To further simplify this procedure, lower the cost and maintain an acceptable high genome editing efficiency in zebrafish, we set out to generate a transgenic line with stable and specific Cas9 expression in the egg. zpc (zp3b) promoter (zpc0.5) was reported to drive robust and specific GFP expression in the oocyte [START_REF] Onichtchouk | Transgene driving GFP expression from the promoter of the zona pellucida gene zpc is expressed in oocytes and provides an early marker for gonad differentiation in zebrafish[END_REF], we thus fused it with a zebrafish codon optimized cas9 (zcas9) followed by an SV40 3' UTR (Fig. 1A and File S1; [START_REF] Liu | Efficient gene targeting in zebrafish mediated by a zebrafish-codon optimized cas9 and evaluation of off-targeting effect[END_REF], and introduced this construct to zebrafish genome by Tol2 mediated transgenesis [START_REF] Urasaki | Functional Dissection of the Tol2 Transposable Element Identified the Minimal cis-Sequence and a Highly Repetitive Sequence in the Subterminal Region Essential for Transposition[END_REF]. To screen female F0 fish harboring this transgene, the F0 female founders were outcrossed with the wild-type male, the resulting eggs were injected with an sgRNA targeting slc45a2 [START_REF] Moreno-Mateos | CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo[END_REF], a gene essential for melanin synthesis, and assayed for their pigmentation phenotype. We found that the offspring of one founder showed a high proportion of pigmentation defect. Some of them exhibited a homozygous mutant like albino phenotype, suggesting a robust Cas9 expression in the egg and a very high efficient gene disruption.

To examine the specificity of zcas9 expression in the transgenic line, the offspring (F1) of this F0 founder were raised to adulthood, and the ovaries of female F1 transgenic fish were dissected and subjected to in situ hybridization (ISH) employing the probe of zcas9. We found that the transcripts of zcas9 were specifically distributed in oocytes with a diameter of 40-200 µm, at around IB-II stages, while in WT embryos, no hybridization signals were detected (Fig. 1B,C). We did not detect zcas9 expression in stage IA oocytes or in larger ones after stage II. Accordingly, by RT-PCR, we also failed to find zcas9 transcripts in the fertilized eggs or early embryos of transgenic fish (Fig. 1D), suggesting that zcas9 transcripts were only present in certain stages of oogenesis, and that only maternally supplied Cas9 protein remained after fertilization. We also observed an ovary specific zcas9 expression among adult tissues, while its expression was absent in the brain, eye, gill, liver, tail fin, skin or muscle (Fig. 1D). We also injected the slc45a2 sgRNA into the fertilized eggs from a cross between transgenic male and wild-type female, and found no pigmentation phenotype (Fig. S1). These results, when put together, suggest that we have successfully generated an oocyte specific zcas9 transgenic zebrafish.

To establish a progeny with high genome editing efficiency, the embryos from female F1 fish were screened by injecting sgRNA targeting slc45a2 and bmp2b respectively, and then assessed the efficiency by analysing the resulting phenotype. Among the six transgenic F1 females, #3 fish showed a maximum efficiency, such that some of its embryos injected with slc45a2 sgRNA showed an albino-like phenotype in addition to high proportions of embryos with mosaic pigmentation in the retina (Fig. 1E; Fig. S2A). This high genome editing efficiency was also revealed by the severely dorsalized phenotype after injection of a sgRNA targeting bmp2b, a key ventralising signal during dorsoventral patterning (Fig. 1F, G; Fig. S2B-H). So we named this line originated from #3 F1 fish as Tg(zpc:zcas9 high ) and compared its efficiency of genome editing with injections of cas9 mRNA or protein.

To perform this comparison, the concentration of slc45a2 and bmp2b sgRNAs was fixed to a dose of 100 pg/embryo. They were mixed separately with three different doses of Cas9 Protein (1000, 333 and 111 pg/embryo) or zcas9 mRNA (200, 100, and 50 pg/embryo). After injection of these mixtures in 1-2 cell stage wild-type embryos, their genome editing efficiency was compared with the transgenic embryos injected with the same sgRNA dose by phenotypic analysis [START_REF] Shao | Vegetally localised Vrtn functions as a novel repressor to modulate bmp2b transcription during dorsoventral patterning in zebrafish[END_REF] and chromatogram quantification using the TIDE method [START_REF] Etard | Tracking of Indels by DEcomposition is a Simple and Effective Method to Assess Efficiency of Guide RNAs in Zebrafish[END_REF]. The results showed that the mutant phenotypes induced by either sgRNA in the embryos from the Tg(zpc:zcas9 high ) F1 fish is comparable to or slightly stronger than the wild-type embryos injected with the intermediate dose of either Cas9 protein or the zcas9 mRNA (Fig. 1H,I). The quantified efficiency of bmp2b sgRNA at this circumference was about 38% on average, better than that of 333 pg Cas9 protein and 100 pg zcas9 mRNA injection (Fig. 1J), both of which showed a 29% of mutation rate. This indicates that the genome editing efficiency of the embryos from a heterozygous Tg(zpc:zcas9 high ) mother is high enough to perform routine knockout experiment. It would be anticipated that the efficiency in its homozygous offspring should be even higher to approach a maximum efficiency.

As Tol2 transgenesis often causes multiple copy insertion [START_REF] Kawakami | Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage[END_REF], we next performed the copy number profiling in Tg(zpc:zcas9 high ) F1 transgenic fish by qPCR using primers targeting the zpc0.5 promoter. The copy number of zpc fragment in the heterozygous transgenic fish is about 1.5 folds higher than that of the wild-type, suggesting single copy insertion (Fig. 1K). To identify the insertion site and to establish a reliable genotyping method (Supplementary materials and methods, Table S1), we carried out nested inverse PCR using six different restriction enzymes, and finally obtained a specific PCR product in the EcoRI group (Fig. S3). In fact, the zpc:zcas9 cassette was inserted into the middle of the large third intron of gfra2a gene (Fig. 1L; File S2 for detailed sequence), which should not affect the splicing of this intron. In another aspect, the initiation of gfra2a expression is very late, at about Prim-15 stage, corresponding to 30 hpf or so (Fig. S4; [START_REF] White | A high-resolution mRNA expression time course of embryonic development in zebrafish[END_REF], and there is no phenotypic output of the gfra2a morpholino [START_REF] Shepherd | Roles for GFRalpha1 receptors in zebrafish enteric nervous system development[END_REF]. As a result, this insertion, even if it might affect gfra2a expression, should not have any effect on embryogenesis, especially during early development. Accordingly, all the offspring from a cross between heterozygous Tg(zpc:zcas9 high ) fish are viable, and no aberrant phenotype was observed (not shown).

In summary, we have generated an oocyte specific cas9 transgenic line with high genome editing efficiency after fertilization. This line exhibits obvious advantages over Cas9 mRNA or protein injections. In particular, it significantly simplifies the workflow and lowers the cost of conventional gene knockout procedure in zebrafish. To target a gene, only sgRNA is synthesized, while the mRNA and commercially available Cas9 protein are dispensable to this end. As a result, this modification will further increase the capacity of sgRNA dosage, and should be of help for simultaneously targeting multiple genes. In addition, as Cas9 is stably expressed in the oocyte, this line may also provide more stable and reproducible editing efficiency compared to mRNA and protein injections. It is worth to note that zcas9 expression in this transgenic line exhibits little leakage and is much "cleaner" than other cas9 transgenes driven by ubiquitous promoters [START_REF] Yin | Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs[END_REF]. After fertilization, only Cas9 protein remains and the transcription of zcas9 is silenced in the whole animal during a considerably long time.

This makes genome editing in this line almost equivalent to Cas9 protein injection. The construction of this transgenic line also makes it possible to directly target maternal factors, especially those with zygotic lethal or sterile mutant phenotypes, so it would be interesting to further test the efficiency of gene knockout in the oocytes using this transgenic line. Numbers on each column indicate embryos counted in each group. J: quantification of the editing efficiency of Cas9 protein, mRNA and the Tg(zpc:zCas9 high ) line at the bmp2b locus. The edited genomic region in each group was PCR amplified and Sanger sequenced. The chromatograms were subjected to quantification using the online software (http://iai-gec-server.iai.kit.edu/uploadFiles.php).

The experiment was performed independently at least 3 times. K: qPCR analysis of the zpc copy number in Tg(zpc:zcas9 high ) heterozygous fish. Three experimental repeats were performed in each group. Data in J and K are presented as mean ± standard deviation, * in K indicates P<0.05 (paired Student's t test). L: Diagram showing the inserted site and direction of zpc:zcas9 cassette in the third intron of gfra2a. Note that the length of the cassette is not in the same scale as the genome. See also supplementary data for detailed sequence. Scale bars: 100 µm in B and the left most inset of E, 400 µm in E and F.

Fig. 1 .

 1 Fig. 1. Generation of an oocyte specific cas9 transgenic zebrafish with high genome editing efficiency. A: schema showing the structure of the vector to transfer the zpc:zcas9 cassette to the genome via Tol2 mediated transgenesis. See supplementary Data for its sequence and annotation. B and C: in situ hybridization to detect zcas9 mRNA expression in the ovaries of wild-type (B) and the transgenic line (C). D: RT-PCR analysis of zcas9 expression in the early embryos and different adult tissues of the Tg(zpc:zcas9) fish. β-actin is used as a loading control. E: representative pigmentation phenotypes at 36 hpf generated by injection of slc45a2 sgRNA (200 pg) to the embryos from Tg(zpc:zcas9 high ) heterozygous female fish (#3 fish indicated in the text). Albino-like embryos corresponding to those without any pigmentation in the retina and the body. Mosaic II indicates embryos with less than 50%

Acknowledgments

We thank B. Zhang for providing the pGH-T7-zCas9 plasmid and its sequence; members of our laboratory for zebrafish care. This work was supported by grants from the National Natural Science Foundation of China (31101038 to M. Shao, 31471360 and 31671509 to D.-L. Shi),) and Shandong Provincial Natural Science Foundation (ZR2017BC068 to M. Shao).


Julie Castillo-Rogez

Pierre Vernazza

Kevin Walsh

Geophysical Evidence that Saturn's Moon Phoebe Originated from a C-Type Asteroid Reservoir

Keywords: Kuiper belt: general, Planetary Systems, planets and satellites: Interiors, Planetary Systems, minor planets, asteroids, general, Planetary Systems

Saturn's Moon Phoebe has been suggested to originate from the Kuiper Belt. However, its density is twice that of Kuiper Belt objects (KBOs) in the same size class, which challenges that relationship. Since the internal evolution of midsize planetesimals (100-300 km in diameter) is primarily driven by the amount of accreted short-lived radioisotopes, it is possible to constrain the relative times of formation of these bodies based on their bulk porosity content, hence their densities. From modeling the thermal evolution of KBOs, we infer a difference in formation timing between these bodies and Phoebe. This confirms prior suggestions for a delayed accretion timeframe with increasing distance from the Sun. This geophysical finding combined with spectral observations suggests Phoebe formed in the same region as C-type asteroids and support recent dynamical models for a C-type body reservoir between the orbits of the giant planets. On the other hand, the similarly low densities of midsize D-type asteroids, Trojan asteroids, and KBOs add to the growing evidence that these objects shared a common reservoir near or beyond the orbit of Neptune and were heat starved overall.

Introduction:

The main belt of asteroids is composed of a variety of bodies. About half of the mass of the main belt is encompassed in the four most massive asteroids (Ceres, Vesta, Pallas, Hygiea). The rest of the main belt mass is dominated by mid-sized (100-300 km diameter) asteroids, which are believed to be the source of the many fragments found in the belt [START_REF] Bottke | The fossilized size distribution of the main asteroid belt[END_REF][START_REF] Cuzzi | Towards initial mass functions for asteroids and Kuiper Belt Objects[END_REF] and the progenitors of asteroid families [START_REF] Dermott | The common origin of family and non-family asteroids[END_REF]. The current status quo is that these planetesimals accreted fast [START_REF] Morbidelli | Asteroids were born big[END_REF]) and thus represent the first generation of large bodies in the solar system [START_REF] Bottke | The fossilized size distribution of the main asteroid belt[END_REF][START_REF] Cuzzi | Towards initial mass functions for asteroids and Kuiper Belt Objects[END_REF]. That class of bodies is a common feature of all the reservoirs of small bodies, although these objects are most frequently found in the main belt of asteroids and the Kuiper Belt. They are scarcer among the Jupiter Trojan asteroid clusters and irregular satellites (Fig. 1).

In water-rich mid-sized bodies, long-lived radioisotopes, which deliver their heat over billionyear timescales, have little bearing on internal evolution, especially since ice thermal conductivity is very high (5-16 W/m/K, [START_REF] Andersson | Thermal conductivity of crystalline and amorphous ices and its implications on amorphization and glassy water[END_REF] at the temperatures of interest. Furthermore, in their detailed review of small body heat sources, [START_REF] Leliwa-Kopystynski | Evolution of porosity in small icy bodies[END_REF] showed that accretional energy is a marginal heat source in bodies less than 500 km diameter. Hence, this leaves short-lived radioisotopes as the primary driver of thermal evolution in these objects. Their impact is a function of the relative fractions of rock and ice. The role of short-lived radioisotopes, and especially aluminum-26 ( 26 Al), in driving aqueous alteration in the parent bodies of carbonaceous chondrites was recognized early on (e.g., [START_REF] Schramm | The isotopic abundance of 26 Mg and limits on 26 Al in the early solar system[END_REF]. In particular, the class of Ch asteroids, most within 100-300 km, show evidence for aqueous alteration [START_REF] Rivkin | The Ch-class Asteroids: Connecting a Visible Taxonomic Class to a 3 μm Band Shape[END_REF] and are believed to be parent bodies of the CM chondrites (e.g., [START_REF] Young | Fluid flow in chondrite parent bodies: deciphering the compositions of planetesimals[END_REF][START_REF] Young | Conditions for pore water convection within carbonaceous chondrite parent bodies -implications for planetesimal size and heat production[END_REF][START_REF] Mcadam | Aqueous alteration on asteroids: Linking the mineralogy and spectroscopy of CM and CI chondrites[END_REF][START_REF] Vernazza | Compositional homogeneity of CM parent bodies[END_REF]). Short-lived radioisotopes are also believed to have played a major role in the evolution of mid-sized Kuiper Belt objects (KBOs) and other transneptunian objects (TNOs) (e.g., [START_REF] Prialnik | Thermal and chemical evolution of comet nuclei and Kuiper Belt objects[END_REF]Shchuko et al. 2014). However, the density data returned for representatives of these reservoirs over the past decade instead suggest they have preserved a large fraction of porosity, which is evidence for weakly evolved objects. On the other hand, Saturn's moon Phoebe, which was suggested to come from the Kuiper Belt (e.g., [START_REF] Johnson | Saturn's moon Phoebe as a captured body from the outer Solar System[END_REF], has a density that is almost twice that of observed at KBOs. We summarize the state of knowledge of 100-300 km planetesimals in Section 2. We use porosity as a gauge of internal evolution, following the approach of [START_REF] Castillo-Rogez | Constraints on the time of formation of Ceres and Ceres-like asteroids -A joint reservoir with C-type irregular satellites?[END_REF], to compare the internal states of bodies formed with different amounts of (live) 26 Al. We focus on the modeling of KBO internal evolution for comparison with the Castillo-Rogez et al. ( 2012) modeling of Phoebe, as presented in Section 3. Since 26 Al has a short half-life (~0.71 My), the amount of 26 Al required to explain observed densities can been used as a chronometer, i.e., to constrain the time at which bodies fully assembled. Hence our results lead to new constraints on the relative formation timing of small bodies reservoirs found across the solar system and their genetic relationships, as discussed in Section 4.

Overview of Planetesimal Densities

Hydrated C-type asteroids (Ch and Cgh-types) have densities in the 1.4-2.5 g/cm 3 range [e.g., Hanus et al. 2017] with a marked dependence on size, as previously noted by Carry (2012). That density range may reflect a low fraction of free volatiles (ice) combined with a low porosity fraction, especially at increasing sizes. Densities between 1.6-2.2 g/cm 3 obtained for bodies 100-200 km in diameter matches their meteoritic analogs, namely CM chondrites (average density ~2.1 g/cm 3 , [START_REF] Consolmagno | The significance of meteorite density and porosity[END_REF]. Times of formation for these bodies believed to be parent bodies of CM chondrites [START_REF] Vernazza | Compositional homogeneity of CM parent bodies[END_REF]) is 3.5 My after CAIs [START_REF] Jogo | Mn-Cr ages and formation conditions of fayalite in CV3 carbonaceous chondrites: Constraints on the accretion ages of chondritic asteroids[END_REF]) assuming a canonical 26 Al/ 27 Al = 5.5x10 -5 . Densities of IDP-like C-type asteroids (these objects represent ~50% of C-complex asteroids and possess spectral properties unlike those of CM chondrites; [START_REF] Vernazza | Interplanetary Dust Particles as Samples of Icy Asteroids[END_REF]Vernazza et al. , 2017) ) show a large spread, beyond the large uncertainties, with densities ranging between 1.0-1.6 g/cm 3 for bodies in the 100-200 km size range.

Many irregular satellites share several similarities with C-asteroids. This is the case of Saturn's irregular satellite Phoebe, high-density body (1.67 g/cm 3 , [START_REF] Thomas | Shapes of the saturnian icy satellites and their significance[END_REF]). Outer solar system bodies in the same size range for which density data are available (besides icy moons) show much lower densities (Figure 1). Phoebe also displays expressions of aqueous alteration and geophysical evolution explained by an early phase of melting driven by 26 Al decay heat [START_REF] Castillo-Rogez | Evolution of Phoebe, a large planetesimal in the outer Solar system[END_REF]) and an ice-rich subsurface (below the regolith) [START_REF] Clark | Compositional maps of Saturn's moon Phoebe from imaging spectroscopy[END_REF][START_REF] Fraser | Phoebe: A Surface Dominated by Water[END_REF]. The density of another irregular satellite, Jupiter's moon Himalia was inferred to be also about 1.63 g/cm 3 , assuming a mean radius of ~85 km [START_REF] Cruikshank | Radii and albedos of four Trojan asteroids and Jovian satellites 6 and 7[END_REF], and even larger for the lower bound on radius determination of ~65 km [START_REF] Porco | Cassini imaging of Jupiter's atmosphere, satellites, and rings[END_REF]. Detailed analysis of Himalia's impact on the orbits of neighboring satellites by Brozovic and Jacobson (2017) yields a similar density, between 1.55 and 2.26 g/cm 3 .

On the other hand, Kuiper Belt objects smaller than 500 km in diameter have densities around 0.6 g/cm 3 and down to 0.3 g/cm 3 whereas those objects bigger than 800 km have densities >1 g/cm 3 [START_REF] Stansberry | Physical properties of trans-neptunian binaries (120347) Salacia-Actaea and (42355) Typhon-Echidna[END_REF][START_REF] Brown | The Density of Mid-sized Kuiper Belt Object 2002 UX25 and the Formation of the Dwarf Planets[END_REF]. Above 500 km diameter, KBOs show a quasi-linear density dependence on size, up to 2.5 g/cm 3 [START_REF] Brown | The Density of Mid-sized Kuiper Belt Object 2002 UX25 and the Formation of the Dwarf Planets[END_REF]. [START_REF] Brown | The Density of Mid-sized Kuiper Belt Object 2002 UX25 and the Formation of the Dwarf Planets[END_REF] pointed out that the density of 0.82±0.11 g/cm 3 he inferred for the 650-km large 2002UX25 reflects both substantial porosity and a deficit in rock. Larger KBOs have densities that do reflect a rock-rich interior. This diversity has been interpreted by [START_REF] Barr | Interpreting the densities of the Kuiper belt's dwarf planets[END_REF] as evidence for the partial removal of ice shells from differentiated KBOs. Hence rock scarcity cannot uniquely account for the large porosity preserved in midsize KBOs. P-and D-type asteroids found in the main belt have relatively low densities: ~1 g/cm 3 for the large (250 km) 65 Cybele and its family members: e.g., 87 Sylvia (1.2±0.15 g/cm 3 , [START_REF] Marchis | Multiple asteroid systems: Dimensions and thermal properties from Spitzer Space Telescope and ground-based observations[END_REF]. Furthermore, large D-and P-types do not show evidence for hydration, similarly to Trojan asteroids. The ~270 km P-type 65 Cybele's surface displays water ice [START_REF] Licandro | 65) Cybele: detection of small silicate grains, water-ice, and organics[END_REF]. Few density measurements have been obtained for Trojan asteroids: 0.8 +0.2 /-0.1 g/cm 3 for Patroclus [START_REF] Marchis | Shape, size and multiplicity of main-belt asteroids[END_REF]) and 1±0.2 g/cm 3 for Hektor [START_REF] Marchis | The Puzzling Mutual Orbit of the Binary Trojan Asteroid (624) Hektor[END_REF], although a much higher estimate of 2.43±0.35 g/cm 3 has been suggested for the latter [START_REF] Descamps | Dumb-bell-shaped equilibrium figures for fiducial contact-binary asteroids and EKBOs[END_REF]. [START_REF] Marchis | The Puzzling Mutual Orbit of the Binary Trojan Asteroid (624) Hektor[END_REF] pointed out that the difference between the densities of Patroclus and Hektor is consistent with the difference in compaction driven by the pressures that can be reached in these objects, assuming that they started with similar, volatile-rich compositions.

Density measurements are lacking for Centaurs, but indirect observations also suggest these objects have low densities. For example, an upper bound density of 0.9 g/cm 3 was derived for the ~200-km large Chiron [START_REF] Meech | Observations of Structures in the Inner Coma of Chiron with the HST Planetary Camera[END_REF]) and the ~190 km Pholus has a density of the order of 0.5 g/cm 3 (Tegler et al. 2004). A large transneptunian objects, the 920-km large Orcus has a density of 1.53 +0.15 /-0.13 g/cm 3 [START_REF] Fornasier | Aqueous alteration on main belt primitive asteroids: Results from visible spectroscopy[END_REF]. It displays potential spectral evidence for ammonium [START_REF] Delsanti | Methane, ammonia, and their irradiation products at the surface of an intermediatesize KBO? A portrait of Plutino (90482)[END_REF], a marker of aqueous alteration like in the case of Ceres [START_REF] De Sanctis | Ammoniated phyllosilicates with a likely outer Solar System origin on (1) Ceres[END_REF], although other types of nitrogen compounds can explain the Orcus spectra (e.g., NH 3 , [START_REF] Delsanti | Methane, ammonia, and their irradiation products at the surface of an intermediatesize KBO? A portrait of Plutino (90482)[END_REF].

These discrepancies show that midsize planetesimals across the solar system underwent different evolutionary pathways where composition alone cannot account for the disparities in densities.

Results: Internal Evolution of Objects Formed in the Transneptunian Region

We model porosity evolution and the possible occurrence of aqueous alteration in several examples of midsized bodies representing Phoebe-like bodies formed with various amounts of 26 Al. The approach is similar to [START_REF] Castillo-Rogez | Iapetus' Geophysics: Rotation Rate, Shape, and Equatorial Ridge[END_REF], 2012, 2017) and [START_REF] Marchis | The Puzzling Mutual Orbit of the Binary Trojan Asteroid (624) Hektor[END_REF] using porosity evolution data for various icy compositions (e.g., Leliwa-Kopystynski and Kossacki 2000; [START_REF] Durham | Cold compaction of water ice[END_REF]) and accounting for the insulating effect (i.e., lower thermal conductivity) of porosity [START_REF] Shoshany | Monte Carlo Modeling of the Thermal Conductivity of Porous Cometary Ice[END_REF]. We assume accretion is near instantaneous (Leliwa-Kopystynski and Kossacki 2000). It has generally been assumed that the relative fractions of ice and rock for objects formed in the solar nebula was determined by nebula temperatures and the form (reduced or oxidized) taken by carbon, with grain densities ranging from about 1.5 to 2.2 g/cm 3 (Wong et al. 2008; Castillo-Rogez et al. 2012). We use 1.5 g/cm 3 and 1.8 g/cm 3 as references densities for this modeling since the resulting models yields the best fits to the observations. The corresponding mass fractions of dry rock ranges from about 53% to 67%. We also assume a starting bulk porosity of 50% in all our models. While icy bodies in the far outer Solar system contain second-phase impurities that can promote low temperature compaction creep, their abundances are not constrained. Here, we assume that relaxation is only driven by ice. This endmember case yields a bound on the amount of compaction driven by thermal evolution. We track microporosity evolution but not macroporosity introduced by impacts or subcatastrophic collision and reaccretion that may contribute 10-15% of the overall porosity. Lastly, we account for the impact of aqueous alteration on material thermophysical properties, following the approach presented in Castillo-Rogez et al. (2019).

Results are presented in Figure 2. Short-lived radioisotopes represent a powerful heat source that can drive long-term geophysical evolution. Times of formation of 3 and 3.5 My after CAIs allows early ice melting, aqueous alteration, and separation of an ice rich shell (Fig. 2a and2b).

In these examples, compaction and most of the thermal evolution occurs during the first 10s My after formation and the body freezes by 100 My. The impact of long-lived radioisotopes is negligible because of the high thermal conductivity of ice at 30 K (~16 W/m/K, [START_REF] Andersson | Thermal conductivity of crystalline and amorphous ices and its implications on amorphization and glassy water[END_REF].

The most recent estimate for the lifetime of the solar nebula based on magnetic measurements of meteorites is about 4 My [START_REF] Wang | Lifetime of the solar nebula constrained by meteorite paleomagnetism[END_REF]. Weaker 26 Al decay heat resulting from later times of formation leads to partial compaction for 4 My after CAIs (Fig. 2c) or insignificant compaction if the time of formation is longer than ~4.5 My after CAIs (Figs 2e,f). Hence, just 1 My delay in the formation of a Phoebe-like body leads to a very different outcome. If Phoebe-sized TNOs accreted after 4 My, then a slightly lower density (e.g., 1500 kg/m3, Fig. 2d), greater initial porosity, and/or significant macroporosity are needed to reproduce a density of <1 g/cm 3 generally observed in that group of bodies (Fig. 1). Another example, for a body 250 km in radius also suggests a grain density lower than that required for Phoebe (Figure 3). A density of 1500 kg/m 3 yields a bulk density of ~1300 kg/m 3 , which appears consistent with observations (noting the small sample) (Fig. 3b).

For TNOs greater than 250 km in radius, the linear trend in the density versus size seen in Figure 1 can be ascribed to the combined effect of size and long-lived radioisotope decay heat. Macroporosity could shift the bulk density toward lower values, while material loss from impacts at differentiated bodies and redistribution of volatiles upon heating could increase it [START_REF] Barr | Interpreting the densities of the Kuiper belt's dwarf planets[END_REF]. A more detailed study is required to narrow down the original grain density of KBOs.

In summary, the preservation of >50% porosity in certain classes of small bodies can be directly ascribed to a scarcity in short-lived radioisotopes. The contrast in formation temperatures between these reservoirs is not sufficient to explain the difference in evolution. These results suggest that interior evolution of midsized KBOs was more limited than previously anticipated (e.g., [START_REF] Merk | Combined modeling of thermal evolution and accretion of trans-neptunian objects -Occurrence of high temperatures and liquid water[END_REF][START_REF] Desch | Thermal evolution of Kuiper belt objects, with implications for cryovolcanism[END_REF]. Similar evolution between Trojan asteroids, Pand D-type asteroids found in the main belt, Centaurs, and KBOs support a genetic relationship between these classes of bodies, as simulated by many studies [START_REF] Morbidelli | Chaotic capture of Jupiter's Trojan asteroids in the early Solar System[END_REF][START_REF] Marchis | The Puzzling Mutual Orbit of the Binary Trojan Asteroid (624) Hektor[END_REF]Brown 2015, Levison et al. 2009;[START_REF] Vernazza | Interplanetary Dust Particles as Samples of Icy Asteroids[END_REF]Beck 2017). This also suggests that P and D type asteroids found throughout the Solar system were barely chemically evolved and offer truly pristine material to sample below their weathered crust.

Discussion and Conclusions:

Phoebe was suggested to come from the Kuiper Belt based on the observation that its density matches the grain density inferred from cosmochemical models [START_REF] Johnson | Saturn's moon Phoebe as a captured body from the outer Solar System[END_REF][START_REF] Castillo-Rogez | Evolution of Phoebe, a large planetesimal in the outer Solar system[END_REF]. The density contrast between Phoebe and KBO questions that relationship. Thermal modeling confirms earlier work showing that most of Phoebe's original porosity could be removed provided that it accreted in ~3-3.5 My My after calcium-aluminum inclusions [START_REF] Castillo-Rogez | Evolution of Phoebe, a large planetesimal in the outer Solar system[END_REF]. Furthermore, Phoebe displays hydrated material on its surface, which is consistent with internal evolution involving aqueous alteration simulated for times of formation of 3-3.5 My after CAIs (Fig. 2a.b). Himalia and most of its family members display a flat spectrum in the 0.4-0.9 m range and similar spectral properties in the visible and near infra-red as certain C-type asteroids, for example 52 Europa [START_REF] Brown | The 3 m spectrum of Jupiter's irregular satellite Himalia[END_REF][START_REF] Bhatt | Composition of Jupiter irregular satellites sheds light on their origin[END_REF]. Phoebe and Himalia contrast with other irregular satellites showing a reddening that might link them to the classes of P and D asteroids (e.g., Themisto, Ananke). The latter have been interpreted as evidence for an origin of irregular satellites from a reservoir shared with KBOs. On the other hand, the similarities between the spectra of Phoebe, Himalia, and Ctype asteroids was interpreted as evidence that the two irregular satellites actually migrated from the main belt of asteroids (e.g., [START_REF] Hartmann | A satellite-asteroid mystery and a possible early flux of scattered C-class asteroids[END_REF]. As an alternative, early giant planet migration and/or planetary growth [START_REF] Walsh | A low-mass for Mars from Jupiter's early gas-driven migration[END_REF]Raymond and Izidoro 2017;[START_REF] Ronnet | Saturn's Formation and Early Evolution at the Origin of Jupiter's Massive Moons[END_REF]) could provide a scenario for a common origin for hydrated C-type asteroids and some irregular satellites from between the orbits of the giant planets. This model implies accretion was early and fast in that region, consistent with recent accretion scenarios (e.g., [START_REF] Johansen | Growth of asteroids, planetary embryos, and Kuiper belt objects by chondrule accretion[END_REF]. As a corollary, this model suggests bodies in the Jupiter-Uranus region formed fast enough for 26 Al to play a role in their geophysical evolution, as suggested for Iapetus [START_REF] Castillo-Rogez | Iapetus' Geophysics: Rotation Rate, Shape, and Equatorial Ridge[END_REF]). On the other hand, short-period comets, if they are connected to the Kuiper Belt, also formed with little or no 26 Al, contrary to previous assumptions (e.g., Gounelle et al. 2008). The long required time of formation for TNOs, potentially longer than 4 My needs to be reconciled with the current estimate for the solar nebula lifetime [START_REF] Wang | Lifetime of the solar nebula constrained by meteorite paleomagnetism[END_REF]. Certain processes, such as the latent heat of supervolatile ice vaporization, need to be accounted for in order to derive a most accurate timeline of events in the outer solar system.

New main belt asteroid densities are necessary to assess whether the scattering observed across that class of bodies in Figure 1 is real and can potentially reflect different origins across the solar system for the various types of C-type asteroids. Density constraints will become available from observations with the Very Large Telescope at mid-sized main belt asteroids in the 2019-2020 timeframe (HARISSA Project, [START_REF] Vernazza | The impact crater at the origin of the Julia family detected with VLT/SPHERE?[END_REF]) and by the Lucy mission at the Jupiter Trojans.

Lastly, Phoebe's evolved interior needs to be reconciled with the very high D/H ratio (0.0013±0.0003) recently inferred from the infrared observations returned by the Cassini mission [START_REF] Clark | Isotopic Ratios of Saturn's Rings and Satellites: Implications for the Origin of Water and Phoebe[END_REF]. This high value is an order of magnitude higher than that of Jupiter-Family-Comets and its meaning remains to be further understood, especially if Phoebe is not a pristine object.

Acknowledgements:

The authors are grateful to Sean Raymond for his comments, which significantly improved the quality of this manuscript. JCCR is thankful to Hap McSween, Driss Takir, and Orkan Umurhan for feedback. Part of this work was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under contract to NASA. Government sponsorship acknowledged. Copyright 2018. All rights reserved.

Figure 1. Bulk densities of minor planets less than 1500 km in diameter. Ice I density (solid line) and a mixture of 50vol.% rock/50vol.% ice with 40% porosity added (dashed line) are shown for reference. The spread in the 100-300 km region illustrates the contrast in remnant porosity and thus heat budget available to these various classes of bodies. Two clusters of bodies can be distinguished with densities <~1g/cm 3 and >~1.3 g/cm 3 . Additional data for large (>1000km) KBOs is provided to support the discussion. References for asteroids: [START_REF] Yeomans | Estimating the Mass of Asteroid 253 Mathilde from Tracking Data During the NEAR Flyby[END_REF]), Mueller et al. (2009), Fienga et al. (2010), [START_REF] Marchis | Shape, size and multiplicity of main-belt asteroids[END_REF]Marchis et al. ( , 2008[START_REF] Marchis | Multiple asteroid systems: Dimensions and thermal properties from Spitzer Space Telescope and ground-based observations[END_REF][START_REF] Marchis | Characteristics and large bulk density of the C-type main-belt triple asteroid (93)[END_REF], [START_REF] Baer | Astrometric masses of 21 asteroids, and an integrated asteroid ephemeris[END_REF], [START_REF] Baer | Astrometric masses of 26 asteroids and observations on asteroid porosity[END_REF], [START_REF] Goffin | Astrometric asteroid masses: a simultaneous determination[END_REF]), Hanus et al. (2016), Hanus (2017), [START_REF] Pajuelo | Physical, spectral, and dynamical properties of asteroid (107) Camilla and its satellites[END_REF]. References for TNOs: [START_REF] Meech | Observations of Structures in the Inner Coma of Chiron with the HST Planetary Camera[END_REF], Tegler et al. (2004), [START_REF] Stansberry | Physical properties of trans-neptunian binaries (120347) Salacia-Actaea and (42355) Typhon-Echidna[END_REF], [START_REF] Vilenius | TNOs are Cool": A survey of the trans-Neptunian region. VI. Herschel/PACS observations and thermal modeling of 19 classical Kuiper belt objects[END_REF], [START_REF] Brown | The Density of Mid-sized Kuiper Belt Object 2002 UX25 and the Formation of the Dwarf Planets[END_REF], [START_REF] Marchis | The Puzzling Mutual Orbit of the Binary Trojan Asteroid (624) Hektor[END_REF].

Figures

  Figures2a, b. Thermal and porosity evolution of Phoebe (~107 km radius) assuming a time of formation of (a) 3 My and (b) 3.5 My after CAIs, a grain density of 1.8 g/cm 3 , an initial bulk porosity of 50%, and a formation temperature of 30 K. The final porosity and bulk density are indicated in the boxes.In these two cases, the final bulk density is close to the observed density of 1.63 g/cm 3 .

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formation of (a) 3 My and (b) 3.5 My after CAIs, a grain density of 1.8 g/cm 3 , an initial bulk porosity of 50%, and a formation temperature of 30 K. The final porosity and bulk density are indicated in the boxes.In these two cases, the final bulk density is close to the observed density of 1.63 g/cm 3 .

Radius (km)